Synthetic Biology of Yeasts Tools and Applications (2022)

Farshad
Darvishi Harzevili Editor
Synthetic
Biology
of Yeasts
Tools and Applications
Synthetic Biology of Yeasts
Farshad Darvishi Harzevili
Editor
Synthetic Biology
of Yeasts
Tools and Applications
Editor
Farshad Darvishi Harzevili
Department of Microbiology
Alzahra University
Tehran, Iran
ISBN 978-3-030-89679-9 ISBN 978-3-030-89680-5 (eBook)

https://doi.org/10.1007/978-3-030-89680-5
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Preface
Yeasts are one of the most attractive microbial cell factories for the production
of a wide range of valuable products, including pharmaceuticals, nutraceuticals,
cosmetics, agrochemicals, biofuels, and so on. Tools of synthetic biology have
been developed to improve the metabolic engineering of yeasts in a faster and
more reliable manner. Nowadays, synthetic biology tools have been used to make
synthetic pathways and rewiring metabolism to produce valuable products at high
titer, rate, and yield.
This book covers recent advances and future trends in the synthetic biology
of yeasts. The book consists of two parts. The first part opens with an intro-
duction to rational metabolic pathway prediction and design using computational
tools and their applications for yeast systems and synthetic biology (Chap. “Ratio-
nal Metabolic Pathway Prediction and Design: Computational Tools and Their
Applications for Yeast Systems and Synthetic Biology”). After rational metabolic
pathway design, synthesis, and engineering tools for assembly of standardized bio-
bricks, powerful genome editing and optimization of synthetic pathways are very
important in metabolic engineering of yeasts. Hence, construction and assem-
bly of standardized biobricks for synthetic pathways engineering in yeasts were
summarized in Chap. “Construction and Assembly of Standardized Biobricks
for Synthetic Pathways Engineering in Yeasts”.
Chapter “Cellular Engineering of Yarrowia lipolytica for Biomanufacturing
of High-Value Products from Oils and Fats” presented an example about cellular
engineering of yeasts. A combined cell morphology engineering and metabolic
pathway optimization of non-conventional yeast Yarrowia lipolytica is used to
biomanufacturing of high-value products from oils and fats as hydrophobic sub-
strates. Chapter “Whole Cell Yeast-Based Biosensors” provided an overview about
engineering of yeast cells to act as whole-cell yeast-based biosensors. The highly
modular nature of yeast-based biosensors suggests that they can be a useful tool for
detecting a wide range of biologically relevant analytes, including pharmaceuticals,
toxins, and chemically undefined or complex mixtures ranging from environmental
mutagens to bioavailable phosphorous.
The second part, Chaps. “Yeast Synthetic Biology Approaches for the Produc-
tion of Valuable Polyphenolic Compounds”–“Synthetic Biology in the Candida
v
vi Preface
(CTG) Clade”, covers applications of synthetic biology to produce diverse and

attractive products by some well-known yeasts. Chapter “Yeast Synthetic Biology
Approaches for the Production of Valuable Polyphenolic Compounds” summa-
rized yeast synthetic biology approaches for the production of valuable phenolic
compounds including hydroxycinnamic acids, flavonoids, stilbenoids, coumarins,
curcuminoids, and polyphenolic amides. Production of artemisinin as an anti-
malarial drug with anti-cancer, anti-inflammation, anti-viral, and anti-SARS-CoV-2
activities via yeast metabolic engineering was discussed in Chap. “Yeast Synthetic
Biology for Production of Artemisinin as an Antimalarial Drug”. Chapter “Yeast
Synthetic Biology for the Production of Terpenoids Derived from Traditional
Chinese Medicinal Plants” reviewed yeast synthetic biology applications for the
production of terpenoids derived from traditional Chinese medicinal plants. The
synthetic biology of yeasts for the production of fragrant compounds like citrus
flavors was reviewed in Chap. Application of Yeast Synthetic Biology for the Pro-
duction of Citrus Flavors. Synthesis of polyols and organic acids by metabolically
engineered Yarrowia lipolytica strains is summarized in Chap. “Synthesis of Poly-
ols and Organic Acids by Wild-Type and Metabolically Engineered Yarrowia
lipolytica Strains”. Chapter Recent Advances in Synthetic Biology Applications
of Pichia Species reviewed recent advances in synthetic biology applications for
Pichia species. Kluyveromyces marxianus as a platform in synthetic biology for the
production of useful materials was considered in Chap. “Kluyveromyces marxianus
as a Platform in Synthetic Biology for the Production of Useful Materials”. In the
last chapter, synthetic biology in the Candida (CTG) clade was summarized.
Overall, this book will serve as a suitable reference for students, scientists,
and researchers at universities, industries, corporations, and government agencies
interested in yeast synthetic biology, systems biology, and metabolic engineering
as well as all disciplines related to biotechnology, microbiology, bioengineering,
and chemical engineering.
Harzevil, Gilan, Iran Farshad Darvishi Harzevili

Contents
Synthetic Biology Tools and Cellular Engineering

Rational Metabolic Pathway Prediction and Design: Computational
Tools and Their Applications for Yeast Systems and Synthetic
Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Pedro A. Saa
Construction and Assembly of Standardized Biobricks for Synthetic
Pathways Engineering in Yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Paulina Korpys-Woźniak, Monika Kubiak, Monika Borkowska,
and Ewelina Celińska
Cellular Engineering of Yarrowia lipolytica for Biomanufacturing
of High-Value Products from Oils and Fats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Na Liu, Ya-Hue Valerie Soong, Andrew Olson, and Dongming Xie
Whole Cell Yeast-Based Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Heather A. M. Shepherd, Emilia-Maria A. Bondarenko,
Katherine M. Jennings, Rachel A. Miller, and Holly V. Goodson
Applications of Yeast Synthetic Biology

Yeast Synthetic Biology Approaches for the Production of Valuable
Polyphenolic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Daniela Gomes, João Rainha, Ligia R. Rodrigues,
and Joana L. Rodrigues
Yeast Synthetic Biology for Production of Artemisinin
as an Antimalarial Drug . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Arman Beyraghdar Kashkooli, Karim Farmanpour-Kalalagh,
and Alireza Babaei
Yeast Synthetic Biology for the Production of Terpenoids Derived
from Traditional Chinese Medicinal Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Yongjun Wei
vii
viii Contents
Application of Yeast Synthetic Biology for the Production of Citrus

Flavors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Karim Farmanpour-Kalalagh, Arman Beyraghdar Kashkooli,
and Alireza Babaei
Synthesis of Polyols and Organic Acids by Wild-Type
and Metabolically Engineered Yarrowia lipolytica Strains . . . . . . . . . . . . . . . 227
Chong Li, Weichao Lin, Khai Lun Ong, Jinhua Mou,
Carol Sze Ki Lin, and Patrick Fickers
Recent Advances in Synthetic Biology Applications of Pichia Species . . . 251
Wan Sun, Yimeng Zuo, Zhanyi Yao, Jucan Gao, Zengyi Shao,
and Jiazhang Lian
Kluyveromyces marxianus as a Platform in Synthetic Biology
for the Production of Useful Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
Noppon Lertwattanasakul, Mochamad Nurcholis,
Nadchanok Rodrussamee, Tomoyuki Kosaka, Masayuki Murata,
and Mamoru Yamada
Synthetic Biology in the Candida (CTG) Clade . . . . . . . . . . . . . . . . . . . . . . . . . . 337
Dalal Kasir, Sébastien Besseau, Marc Clastre, Audrey Oudin,
Monzer Hamze, Vincent Courdavault, Marwan Osman,
and Nicolas Papon
Synthetic Biology Tools and Cellular
Engineering
Rational Metabolic Pathway
Prediction and Design:
Computational Tools and Their
Applications for Yeast Systems
and Synthetic Biology
Pedro A. Saa
Abstract
Continuous progress in metabolic engineering of microbial cell factories like
yeast requires the support of computational tools for finding novel unintuitive
biotransformations routes. In this chapter, a succinct overview is provided of the
most relevant computational tools for pathway prediction by retro-biosynthesis,
and pathway design through stoichiometry-based optimization methods. Illus-
trative case studies are also presented showcasing different strategies for
pathway optimization in yeast, namely redox cofactor balancing, improved pre-
cursor supply, and heterologous expression of carbon fixation pathways. Finally,
challenges and limitations hindering the broad adoption and implementation of
these tools for metabolic engineering will be discussed.
Keywords
Pathway design • Pathway analysis • Metabolic engineering • Yeast
1 Introduction
Advancements in metabolic engineering and synthetic biology have enabled accel-

erated engineering of microbial factories for the production of valuable chemicals
(Smolke and Tyo 2012; Lee and Kim 2015; Isaacs et al. 2011), realizing the
P. A. Saa (B)
Departament of Chemical and Bioprocess Engineering, Pontificia Universidad Católica de Chile,
Santiago, Chile
e-mail: [email protected]
Institute for Mathematical and Computational Engineering, Pontificia Universidad Católica de
Chile, Santiago, Chile
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 3

F. Darvishi Harzevili (ed.), Synthetic Biology of Yeasts,
https://doi.org/10.1007/978-3-030-89680-5_1
4 P. A. Saa
promise of a more sustainable (bio)economy (Voigt 2020). To keep pace with

these expectations, pathway prediction and design play a crucial role in finding
novel pathways for various applications like drug discovery (Galanie et al. 2015;
Moura et al. 2016; Hafner et al. 2021) and value-added biochemical production
(Yim et al. 2011; Tokic et al. 2018; Henry et al. 2010a, b). In this scenario,
metabolic workhorses like yeast could be greatly benefited by broadening their
product spectrum and improving their metabolic capabilities and performance in
terms of their yields, titers, and productivities (Nielsen and Keasling 2016; Ko
et al. 2020). For this task, progress in computational tools and methods capable of
guiding experimental efforts is crucial for the optimization of cellular metabolism
and incorporation of synthetic designs for the production of unnatural heterologous
compounds.
In this chapter, an overview of the most relevant retro-biosynthesis and pathway
optimization methods is provided with a focus on tools with direct application in
metabolic engineering tasks. Starting with the reconstruction of a comprehensive
reaction network from public databases and resources, both retro-biosynthesis tools
for de novo pathway prediction and stoichiometry-based pathway optimization
methods for metabolic redesign are described. Particularly in the latter case, conve-
nient engineering objectives taking into consideration product yield, cofactor use,
thermodynamic plausibility, and enzyme cost are discussed. Additionally, several
relevant pathway engineering case studies in yeast are also presented, highlighting
the improvement potential from the implementation of rational pathway designs.
Finally, perspectives on the increasing adoption of these tools for metabolic
engineering as well as limitations reducing their effectiveness are discussed.
2 In Silico Pathway Prediction and Design
Retro-biosynthesis and stoichiometry-based optimization methods have been

established for pathway design and prediction, differing mostly in their scope and
methodology. While both tools generate metabolic pathways producing the tar-
get metabolite, they do so by applying fundamentally different reaction network
representations (i.e., graph or stoichiometric matrix) and search algorithms (i.e.,
optimization-based enumeration or retro-synthetic search) (the reader is referred to
Wang et al. (2017) for a comprehensive review). Furthermore, their computational
complexity and efficacy can vary significantly depending on the product of inter-
est, and thus, careful selection of the appropriate tool for the case at hand is a must
(Saa et al. 2019). In the following, the most relevant data- and knowledge-bases for
reconstructing and parameterizing reaction networks are presented, which consti-
tute the starting point for the application of any of these tools (Fig. 1a). Then, the
most relevant retro-biosynthesis (Fig. 1b) and stoichiometry-based optimization
methods for pathway prediction and design (Fig. 1c) are described.
Rational Metabolic Pathway Prediction and Design … 5
Fig. 1 Workflow for reaction network reconstruction and application of metabolic pathway pre-
diction and design tools. a Assembly of accumulated metabolic reaction data into a comprehensive
reaction network is a requirement for the application of the reviewed tools. Depending on the
application objective, different network representations are employed for either predicting de novo
pathways (i.e., retrosynthesis typically using a graph representation), or (re)designing pathways
for higher metabolic performance (i.e., optimization-based pathway design using an stoichiometric
representation). b Retro-biosynthetic tools explore a substrate graph seeking to connect the target
molecule with some predefined precursors. Starting with the target molecule and moving back-
wards, these tools can generate several possible pathways that are typically ranked using different
criteria (e.g., length, enzyme availability, thermodynamics, among others). c Stoichiometry-based
pathway prediction methods employ a reaction network with known and fixed reactions to enu-
merate mass-balanced pathways that optimize a desired objective such as product yield, pathway
length, thermodynamic favorability, and enzyme cost. In this case, different types of constraints
can be defined to restrict the feasible solution space and narrow the search upfront
2.1 Data- and Knowledge-Bases for Metabolic Reaction

Network Reconstruction
Databases for pathway search are an absolute requirement for exploring the fea-
sible reaction space, as they contain the critical information of how metabolites
are connected to others through biochemical reactions. There are numerous pub-
lic data- and knowledge-bases populated with metabolic reaction data. Among
the most popular, KEGG (Kanehisa et al. 2016), MetaCyc (Caspi et al. 2016),
BIGG (King et al. 2016), KBase (Arkin et al. 2018), ModelSEED (Henry et al.
2010a, b), MetRxn (Kumar et al. 2012), and MetaNetX (Ganter et al. 2013; Moretti
et al. 2021) stand out to name a few (for a more details refer to Wang et al.
(2017)). Some of these databases (KEGG, MetaCyc, and Kbase) integrate multi-
ple sources of biological information, e.g., genetic, molecular, physicochemical,
6 P. A. Saa
and experimental, which makes them not only useful for metabolic pathway pre-
diction purposes but also data integration (Lewis et al. 2012). The rest of the
databases are mostly devoted to metabolic network reconstruction, offering either
highly curated reconstructions for specific organisms (e.g., BIGG) or broader albeit
possibly less curated biochemical reaction networks (e.g., MetRxn, ModelSEED,
and MetaNetX). Ultimately, the modeling purpose will dictate the most convenient
source of information considering their specific scope, breadth, and informa-
tion quality. Complementary databases like BRENDA (Jeske et al. 2019) (kinetic
information) and eQuilibrator (Flamholz et al. 2012) (thermodynamic informa-
tion) also constitute valuable resources for parameterizing different optimization
formulations.
The aforementioned databases contain information for known reactions, which
may restrict the pathway search considering the current enzymatic knowledge
gaps. Resources like the ATLAS of Biochemistry (Hadadi et al. 2016) (derived
from the BNICE tool (Hatzimanikatis et al. 2005)) and MINE (Jeffryes et al.
2015) offer larger networks including hypothetical reactions and metabolites that
can expand the reachable chemical space and allow higher complexity. Briefly,
these resources exploit user-defined reaction rules that can act on chemically sim-
ilar compounds, thereby yielding new hypothetical reactions. The latter reactions
have recently been shown to enable filling some of the gaps in current enzyme-
reaction associations (Hadadi et al. 2019). Lastly, another significant and recent
tool for proposing hypothetical reactions that has been employed for pathway
prediction is rePrime used by the novoStoic tool (Kumar et al. 2018) (see sub-
section 2.3 for more details). The former method extracts reaction rules from
molecular signatures found in annotated reactions—defined by the presence of a
set of chemical ‘moieties’—for proposing hypothetical enzymatic transformations
with a high structural encoding fidelity. Unfortunately, this tool currently lacks an
associated open database for its use.
2.2 Pathway Prediction Using Retro-Biosynthesis Tools
Firstly, a distinction is made between retro-biosynthesis and classical retro-

synthesis, as the latter is focused on the design of chemical reaction pathways,
typically without relying on enzyme catalysis (Lin et al. 2019). Retro-biosynthesis
tools seek to identify de novo biosynthetic pathways for the production of valuable
compounds from inexpensive precursors using known and hypothetical enzyme
activities (Wang et al. 2017; Lin et al. 2019). Another—though less explored—
application of these tools involves the opposite, that is, the prediction of novel
enzymatic routes for the degradation of recalcitrant compounds, e.g., for biore-
mediation purposes (Finley et al. 2009, 2010; Ellis et al. 2006). For pathway
prediction, retro-biosynthesis tools explore the full chemical space for synthetic
pathways toward the target compound. For this task, these tools typically represent
the network as a (substrate) graph that can be readily traversed using known enu-
meration algorithms. Graph traversal is possible by connecting the substrates using
various criteria based on structural (chemical) similarity, reaction promiscuity, and

defined reaction rules. In the following, a relevant subset of retro-biosynthesis tools
employed for metabolic engineering/synthetic biology applications is described
(Table 1).
One of the most established tools for de novo pathway retro-biosynthesis is
BNICE (Hatzimanikatis et al. 2005). This framework employs predefined ‘general-
ized enzymatic reaction rules’ (encoded in a bond-electron matrix) that are applied
to precursor molecules on their reactive sites to yield new product molecules.
BNICE uses a substrate graph representation of the chemical network, which can
be traversed using graph search algorithms starting from the target compound and
moving backwards until connecting with one of the defined precursors. Different
pruning criteria are employed to keep the search breadth computationally tractable.
Table 1 Retro-biosynthesis tools for metabolic pathway prediction

Tool Database Pathway scoring Applications and references Source
BNICE KEGG, Pruning criteria In silico identification of https://
ATLAS assessment novel pathways and enzyme lcsb-databa
(thermodynamics, candidates validated ses.epfl.ch/
pathway length, etc.) experimentally for the pathways/
production of GraphList
(S)-tetrahydropalmatine
derived from intermediates
of the known
benzylisoquinoline alkaloid
biosynthetic pathway
(Hafner et al. 2021)
In silico evaluation of
putative feasible
biodegradation pathways for
1,2,4-trichlorobenzene
(Finley et al. 2010)
Enzyme annotation for
orphan and novel reactions
from KEGG (Hadadi et al.
2019)
Discovery of novel
metabolic pathways for the
biosynthesis of
3-hydroxypropanoate
(Henry et al. 2010a)
PathPred KEGG, Compound similarity Prediction of putative https://
RPAIR and pathway scores biodegradation pathways for www.gen
xenobiotics (e.g., ome.jp/
1,2,3,4-tetrachlorobenzene) tools/pat
as well as biosynthetic hpred/
pathways for plants
secondary metabolites (e.g.,
gentiodelphin)
(continued)
8 P. A. Saa
Table 1 (continued)
Tool Database Pathway scoring Applications and references Source
SimPheny BiGG Pathway length, In silico prediction and -
(BioPathway thermodynamics, subsequent experimental
Predictor) product yield, validation of an
number of known unprecedented synthetic
metabolites/enzymes pathway for 1,4-butanediol
production in E. coli (Yim
et al. 2011)
RetroPath MetaCyc, Pathway Prediction of https://
BioCyc thermodynamics, successfully-implemented www.mye
sequence similarity, biosynthetic pathways for xperiment.
pathway length, 146 compounds in metabolic org/workfl
number of putative engineering projects. 81% of ows/4987.
enzyme steps, and the compounds were html
product yield predicted with at least one
pathway (Delépine et al.
2018)
At the end of the algorithm, pathways are ranked by features such as pathway
length, thermodynamics, among others. Methodologically close to BNICE, Path-
Pred (Moriya et al. 2010) uses instead RDM patterns consisting of reaction center
atoms (R), atoms of different regions (D), and atoms of the matched region (M) for
exploring the substrate graph. Pruning of the network is executed using structural
similarity criteria, and pathway ranking is performed using compound similar-
ity and pathway scores. SimPheny (Yim et al. 2011; Schilling et al. 2005) uses
reactions rules from the third Enzyme Commission (EC) number level for gen-
erating reaction rules that enable reaction promiscuity for broader explorations.
In this case, a retro-synthetic search is employed for enumerating feasible routes
that produced intermediates of reasonable size (i.e., below a predefined size), and
later they are ranked based on various criteria. Another retro-biosynthesis tool
with recent important applications is RetroPath (Delépine et al. 2018). This tool
uses a retro-synthetic search, albeit combined with MILP formulations for the
application of various ranking criteria, such as thermodynamics, gene prediction,
pathway length, number of putative steps, and product yield. In contrast to BNICE,
RetroPath maintains a stoichiometric representation of the network (as opposed to
a substrate graph) that enables computation of various scores. Moreover, molecular
signatures are used to generate reaction rules based on a substructure of adja-
cent atoms, enabling the generation of substantially more and flexible reaction
rules (Duigou et al. 2018). A recent implementation of reinforcement learning in
RetroPath (RetroPath RL) has yielded promising results in the retro-biosynthetic
prediction of biologically relevant pathways (Koch et al. 2020).
2.3 Stoichiometry-Based Optimization Methods for Pathway

(Re)design
Given a universal metabolic reaction network, the ‘pathway design’ problem

seeks to identify ‘optimal’ route(s) for the production of the target compound.
As opposed to the retro-biosynthesis problem, possible connecting reactions are
fixed and known upfront. Construction of the reaction network knowledge base
is achieved by combining metabolic data from curated databases and/or from
databases that also include putative reactions derived, for example, from gener-
alized reaction rules (Hadadi et al. 2016; Hatzimanikatis et al. 2005; Jeffryes et al.
2015) or molecular signatures (Kumar et al. 2018). Regardless of the source of
the data, reaction data must be charge- and mass-balanced to yield correct results,
which typically is ensured in a manual curation step. Metabolite/reaction name
inconsistencies are also an important source of issues that affect network con-
nectivity and consistency, which often have to be resolved manually. While there
have been attempts to standardize reaction and metabolite identifiers (King et al.
2016; Kumar et al. 2012; Alcántara et al. 2012), name reconciliation is challeng-
ing due to the incessant annotation of new metabolites and enzymatic activities,
albeit important progress has been made in recent database versions (Moretti et al.
2021).
Once the reaction network has been assembled and mathematically formulated
into a stoichiometric matrix, prediction of different pathway designs can be read-
ily computed using optimization-based methods provided a convenient objective
function. Among the most relevant objectives, one can name the minimization
of the pathway length ensuring a minimum product yield (e.g., by fixing the
overall stoichiometry) (Pharkya et al. 2004), maximization of the product yield
observing thermodynamic constraints (Kumar et al. 2018; Kamp and Klamt 2020;
Chowdhury and Maranas 2015), maximization of the thermodynamic favorability
of the pathway (Flamholz et al. 2013; Noor et al. 2014; Hädicke et al. 2018; Yang
et al. 2020; Ng et al. 2019), and minimization of the pathway´s enzymatic cost
(Flamholz et al. 2013; Ng et al. 2019; Court et al. 2015; Bar-Even et al. 2010).
For each of these objectives, different optimization problems must be formulated
and solved, often requiring various parameters (e.g., thermodynamic and kinetic)
from other sources for computing the optimal solution(s). In the following, the
most relevant stoichiometry-based optimization methods for metabolic pathway
prediction are presented. Further details about the methods and applications can
be found in Table 2.
2.3.1 Pathways with Desired Stoichiometric Properties

Constraint-based modeling methods (Edwards and Palsson 2000) can be readily
adapted for the computation of pathways exhibiting a desired stoichiometry (i.e.,
yield) (Kumar et al. 2018; Chowdhury and Maranas 2015; Ng et al. 2019), shortest
length (Chowdhury and Maranas 2015; Ng et al. 2019), convenient precursor use
(Kamp and Klamt 2020), and if using an organism’s reaction network as metabolic
chassis, minimum addition of exogenous reactions (Pharkya et al. 2004; Kim et al.
10
Table 2 Optimization-based methods for metabolic pathway design

Pathway design objective Method Goal(s) Applications and references
Desired stoichiometric properties OptStrain Maximization of product yield Prediction of in silico production pathways for hydrogen in
(e.g., yield, length, among others) using the minimum number of E. coli, C. acetobutylicum, and M. extorquens AM1, as
heterologous reactions in a well as exploration of interventions strategies for achieving
production host growth-coupling (Pharkya et al. 2004)
In silico prediction of heterologous vanillin production
pathways in E. coli and computation of their maximum
theoretical yields and number of non-native enzymes to be
included (Pharkya et al. 2004)
SimOptStrain Maximization of growth-coupled Computation of optimal succinate and glycerol production
production including heterologous pathways using E. coli as metabolic chassis in terms of
reactions in a production host gene deletions and non-native reaction additions (Kim
et al. 2011)
OptStoic Optimization of overall reaction In silico verification of the non-oxidative glycolysis
stoichiometry (e.g., max. product pathway design (Chowdhury and Maranas 2015)
yield, co-factor use, etc.), and Computation of optimal conversions and novel pathways
minimization of the length or total for co-utilization of carbon dioxide and methanol for the
flux through the pathway capable production of C2+ metabolites (e.g., malonate, citrate,
of sustaining the desired succinate, acetate, among others) (Chowdhury and
stoichiometry Maranas 2015)
Evaluation of feasible glycolytic alternatives and
optimality analysis of trade-offs in ATP yield, enzymatic
cost, and maximum-minimum driving force (Ng et al.
2019)
(continued)
P. A. Saa
Table 2 (continued)
MEMO Minimization of the size of the Enumeration of optimal regeneration modules capable of
regeneration pathway (module) sustaining the operation of the synthetic
needed for sustaining cofactor(s) crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA
requirement(s) of a production (CETCH) cycle for in vitro carbon dioxide fixation in
pathway cell-free systems (Kamp and Klamt 2020)
GEM-Path Maximization of growth-coupled Computation of 245 unique synthetic pathways for the
production including putative production of bulk chemicals and integration of these
(promiscuous) enzymatic reactions pathways into E. coli metabolism for yield analysis and
in a production host metabolic optimization (Campodonico et al. 2014)
novoStoic Minimization of the pathway In silico verification of synthetic pathways for
length 1,4-butendiol (BDO) production, identification of putative
Or pathways for phenylephrine synthesis and benzo[a]pyrene
Maximization of profit degradation (Kumar et al. 2018)
MapMaker/ Minimization of carbon transfers Identification of possible conversion pathways to
Rational Metabolic Pathway Prediction and Design …
PathTracer between reactants and products putrescine from glutamate, and CO2 fixation pathways in
using the shortest pathway E. coli (Tervo and Reed 2016)
(continued)
11
12
Table 2 (continued)
Thermodynamic favorability OptMDFpathway Maximization of the minimum Computation of all substrate-product combinations in E.
driving force of the production coli that enable net CO2 assimilation via
pathway thermodynamically feasible pathways (Hädicke et al.
2018)
In silico design of multi-strain communities of a
single-species (E. coli) that achieve maximum
thermodynamic driving force for product synthesis under
the assumption of pathway specialization (i.e., parts of the
production pathway are performed by different community
members) (Bekiaris and Klamt 2021)
Enzymatic cost ECM Minimize enzyme cost or In silico computation of putative efficient CO2 fixation
or burden metabolic burden pathways into C2-C4 compounds (Bar-Even et al. 2010)
Optimality analysis of enzyme cost minimization in central
carbon metabolism of E. coli (Noor et al. 2016)
Prediction of rate/yield trade-offs in metabolic pathways
under oxygen-limited conditions in E. coli (Wortel et al.
2018)
P. A. Saa
2011). In practice, these pathway enumeration methods rely on the solution of

various LP and/or MILP optimization problems that optimize some of the afore-
mentioned objectives subject to not only stoichiometric constraints but possibly to
thermodynamic and/or economic constraints.
OptStrain, SimOptStrain, and OptStoic are classical tools for pathway predic-
tion, although they differ in their scope. While the first two seek to predict optimal
pathways and metabolic interventions for the production of a target metabo-
lite leveraging the microbial host reaction network (Pharkya et al. 2004; Kim
et al. 2011), the latter aims to find complete mass-balanced conversion pathways
that yield a desired stoichiometry from precursors to product(s) using metabolic
databases as the input reaction network (Chowdhury and Maranas 2015). Addi-
tional constraints related to a minimum guaranteed product yield, thermodynamic
plausibility of the pathway, and/or substrate costs can be readily included to obtain
more convenient designs. Recently, a computational method called MEMO (Kamp
and Klamt 2020) has been proposed for identifying the smallest metabolic mod-
ules with specified stoichiometric and thermodynamic properties. For instance, this
approach has been employed to find small cofactor regeneration (e.g., ATP/ADP,
NAD(P)H, NAD(P), among others) modules that can sustain bioconversions in the
context of cell-free applications under defined thermodynamic conditions.
The aforementioned methods rely on existing annotated enzymatic reactions
for metabolic conversions. However, as mentioned in the previous subsection,
promiscuous enzymatic activities are characteristic features of metabolic reaction
networks, likely playing an evolutionary role as a starting point in enzyme func-
tions (Khersonsky and Tawfik 2010). Importantly, the existence of this feature
suggests that there is still untapped potential for a broader chemical reaction space
to be explored. By using various extraction techniques for learning putative reac-
tions from known enzymatic reactions, it is possible to populate and assemble
larger databases for pathway prediction. An example of these methods is Map-
Maker/PathTracer (Tervo and Reed 2016), which employs precomputed carbon
transfer maps (CTMs) based on chemical and stoichiometric information (Map-
Maker) for the prediction of short, carbon-balanced pathways from substrates to
products (PathTracer). GEM-Path (Campodonico et al. 2014) is another frame-
work that, using a genome-scale metabolic reconstruction of E. coli as base
reaction network, combines heterologous pathway integration (similar to OptStoic)
with constraint-based growth-coupled methods for the computation of metabolic
designs. Increased biochemical reaction exploration is achieved through the intro-
duction of a chemical similarity measure to assess enzyme-catalyzed reaction
promiscuity. Lastly, the novoStoic/rePrime framework (Kumar et al. 2018) enables
exploration of a far greater chemical transformation space through the imposition
of chemical ‘moiety’ conservation (refer to Sect. 2.1) that is particularly suited
for the prediction of optimal pathways with maximum yield or length. Impor-
tantly, this mathematical treatment avoids chemical reaction information loss (e.g.,
stereoselectivity) as opposed to other approaches like MapMaker/PathTracer.
14 P. A. Saa
2.3.2 Pathways with Maximum Thermodynamic Favorability

Pathway thermodynamics exerts a fundamental control in metabolic flux with
seemingly important consequences for microbial fitness (Du et al. 2018). While
there have been different methods for combining stoichiometric-based analysis
with reaction thermodynamics (Henry et al. 2007; Kummel et al. 2006), it has
not been until recently that thermodynamic favorability has been mathematically
formalized. For this task, the Max-min Driving Force (MDF) index (Noor et al.
2014) has been proposed for quantifying the smallest absolute Gibbs free energy
(or driving force) of a given pathway under the most favorable metabolic condi-
tions. As the latter captures the driving force of the most unfavorable conversion
step (i.e., thermodynamic bottleneck), its maximization yields the most favorable
operating conditions for a given pathway. More recently, the OptMDFpathway
method (Hädicke et al. 2018) was introduced to identify the most thermodynami-
cally favorable pathways in a given reaction network, thereby enabling exploration
of thermodynamically plausible production pathways in the context of microbial
metabolism (Hädicke et al. 2018; Yang et al. 2020), and more recently, in microbial
communities (Bekiaris and Klamt 2021).
2.3.3 Pathways with Minimum Enzymatic Cost

Cellular metabolism incurs a metabolic cost when committing to the synthesis
of a particular set of proteins (enzymes). As seemingly similar enzymes can
still display large differences in their catalytic properties (Bar-Even et al. 2011),
it is natural to seek pathways that can yield the maximum return of invest-
ment (flux) per protein (enzyme) mass synthesized. For this task, the Enzyme
Cost Minimization (ECM) (Noor et al. 2016)—and later termed the Enzyme-Flux
Cost Minimization (EFCM) (Wortel et al. 2018)—formulation computes the min-
imum enzyme load (i.e., the aggregated enzyme mass allocated) required for a
metabolic pathway to yield a given flux (Flamholz et al. 2013; Bar-Even et al.
2010). While this formulation originally required a thermodynamically consistent,
fully parameterized kinetic model for this calculation (Saa and Nielsen 2017),
increasingly enzymatically-constraint GSMMs (Sánchez et al. 2017) and ME-
models (metabolic and expression) (Lerman et al. 2012) are being considered
and employed for these calculations under the optimistic scenario of enzymatic
catalysis at capacity. Finally, the ECM/EFCM does not support performing path-
way enumeration, although it can be readily employed as a ranking index when
combined with the previous approaches.
3 Case Studies of Metabolic Pathway Prediction

and Optimization in Yeast
In this section, selected case studies illustrate different pathway engineering

aspects required for improving metabolic performance overall, and particularly, in
yeast. These examples showcase strategies for redox cofactor balancing, increased
precursor supply, and engineering of central pathways for carbon fixation. The
impact of the latter applications is especially highlighted in the context of harness-

ing the metabolic potential of yeast for industrial bioproduction. Figure 2 illustrates
the details of the revised strategies.
3.1 Balancing Redox Cofactor Supply for Improving Substrate

Utilization and Isoprenoids Production
Regeneration of either redox and/or energy cofactors often limits the production of
high-value metabolites. In order to increase the availability of the required cofac-
tor(s), central carbon metabolism must be intervened and engineered in such a
way that it favors bioproduction without extremely affecting microbial growth (Lee
et al. 2013). This challenge is particularly relevant for many NAD(P)H-expensive
valuable compounds that are being produced in yeast (Cataldo et al. 2020; López
et al. 2020, 2019) and other microbes (Ko et al. 2020).
Increased supply of redox cofactors can be achieved by either overexpressing
key enzymes involved in cofactor generation (Lee et al. 2007; San et al. 2002;
Lim et al. 2002) or by increasing the expression of alternative redox partner
systems. A recent application of the latter has proved effective for enhancing
the unprecedented heterologous production of violaxanthin in S. cerevisiae by
approx. two-fold (Cataldo et al. 2020). However, the success of these approaches
is likely limited due to the presence of different intrinsic balancing mechanisms
for maintaining homeostasis in yeast (Hou et al. 2010). An illustrative example of
the latter can be found in the study of Nissen et al. (2001). Here, heterologous
expression of the pyridine nucleotide transhydrogenase system (sth gene, absent
in yeast) that transfers reducing equivalents from NADPH to NADH (and vice
versa), did not improve ethanol formation in anaerobic conditions. On the contrary,
ethanol production was reduced concomitantly with the increase of fermentation
by-products (glycerol and 2-oxoglutarate) required for redox rebalancing. Another,
less intuitive and possibly more effective, strategy for (re)balancing redox cofac-
tors supply and demand involves cofactor swapping (Verho et al. 2003; Martínez
et al. 2008). Computational studies in S. cerevisiae and E. coli support this strategy
as a promising intervention for forcing higher metabolic performance (King and
Feist 2014). Simply put, this approach seeks to replace native (redox-consuming)
enzymes with heterologous counterparts with a different cofactor specificity (e.g.,
NAD(P)H—for a NA(D)H-dependent enzyme).
The first application of the latter strategy involved the optimization of D-
xylose utilization for ethanol production in S. cerevisiae (Verho et al. 2003).
This carbon source is assimilated through the pentose phosphate pathway (PPP)
as D-xylulose-5-phosphate and then incorporated as glyceraldehyde 3-phosphate
in glycolysis. In theory, D-xylose should produce CO2 and ethanol in a 1:1
molar ratio under redox-neutral anaerobic conditions (Kötter and Ciriacy 1993).
However, D-xylose assimilation requires extra NADPH and NAD+ that must be
regenerated by other separate processes, which are very inefficient in yeast, ren-
dering D-xylose fermentation slow. To overcome this bottleneck and force higher
16 P. A. Saa
Fig. 2 Illustration of selected reported strategies for achieving improved cofactor balancing,
increased acetyl-CoA supply and engineering CO2 fixation in yeast. The details of each
strategy are discussed in Sect. 3. Relevant metabolite names are represented by uppercase
bold fonts, whereas enzyme names are represented by uppercase italics fonts. Abbreviations:
13DPG, 1,3-diphosphoglycerate; 3PG, 3-phosphoglycerate; 6PGL, 6-phospho-D-glucono-1,5-
lactone; ACCOA, acetyl-CoA; ACE, acetate; ACETAL, acetaldehyde; AcP, acetyl phosphate;
ACS, acetyl-CoA synthetase; AKG, alpha-ketoglutarate; ALD, aldehyde dehydrogenase; DHAP,
dihydroxyacetone phosphate; ETOH, ethanol; F6P, D-fructose 6-phosphate; FDP, D-fructose 1,6-
disphosphate; FOR, formate; G3P, glycerol 3-phospate; G6P, D-glucose 6-phosphate; G6PD,
glucose 6-phosphate dehydrogenase; GAP, glyceraldehyde 3-phosphate; GAPDH, glyceralde-
hyde 3-phosphate dehydrogenase; GDH, glutamate dehydrogenase; GLC, D-glucose; GLU, glu-
tamate; GLY, glycerol; HMGCOA, 3-hydroxy-3-methyl-glutaryl-CoA; HMGCOAR, HMG-CoA
reductase; MEOH, methanol; MEV, mevalonate; NH4, ammonia; PDH, pyruvate dehydrogenase;
PFL, pyruvate formate lyase; PK, phosphoketolase; PRK, phosphoribulokinase; PTA, phospho-
transacetylase; PYR, pyruvate; R5P, D-ribose 5-phosphate; Ru15P, ribulose 1,5-disphosphate;
Ru5P, D-ribulose 5-phosphate; STH, transhydrogenase; Xu5P, D-xylulose 5-phosphate; XYL, D-
xylose
NADPH supply and flux through lower glycolysis, the native NAD-dependent
GAP dehydrogenase (GAPDH) was replaced by an NADP-dependent GAPDH and
the NADPH-dependent glucose-6-phosphate dehydrogenase (G6PD) was knocked
out, which also prevented carbon loss as CO2 (Verho et al. 2003). This strat-
egy almost doubled the ethanol yield on D-xylose (from 18 to 41%) and reduced
the CO2 /ethanol molar ratio close to the theoretical 1:1 (from 2.5 to 1.3). Later,
expression of the heterologous phosphotransacetylase (PTA) and phosphoketolase
(PK) for improving NADH reoxidation in the D-xylose utilization pathway gen-
erated an increase in ethanol yield (25% higher) without affecting the growth rate
(Sonderegger et al. 2004).
Cofactor rebalancing and swapping strategies for the synthesis of NADPH-
expensive isoprenoid-derived compounds have shown to be particularly effective
in yeast. For instance, α-santalene production yields a net production of NADH
and consumption of NADPH, which calls for the rebalancing of the cofactor sup-
ply (Scalcinati et al. 2012). By deleting known reactions involved in glutamate
metabolism (ammonium assimilation) that consume NADPH (GDH1) and acti-
vating NAD-dependent counterparts (GDH2) (Nissen et al. 2000), the production
of α-santalene was substantially improved. Similarly in a different study of pro-
topanaxadiol production—another isoprenoid-derived compound—the availability
of NADPH was enhanced by replacing the native NADH-generating acetaldehyde
dehydrogenase (ALD2) with a functionally equivalent NADPH-generating enzyme
(ALD6), resulting in a 11-fold increase in titer (Kim et al. 2018). Lastly, swap-
ping of the native NADP-dependent 3-hydroxy-3-methyl-glutaryl-CoA reductase
(HMG-CoA reductase)—third committed step of the mevalonate pathway respon-
sible for the production of isoprenoid precursors—has also shown to increase the
overall pathway flux in E. coli (Ma et al. 2011). This result was leveraged by
Meadows et al. (2016) whereby an NADH-consuming HMG-CoA reductase from
Silicibacter pomeroyi was employed for the overproduction of the sesquiterpene
18 P. A. Saa
farnesene. Implementation of other computationally predicted major metabolic

cofactor swaps like the alcohol dehydrogenase (ALCD2) and GAPD for the
improved production of isoprenoids remains to be tested experimentally (King
et al. 2016), as they could be potentially beneficial for boosting production as
shown in other microorganisms (Martínez et al. 2008).
3.2 Increasing Cytosolic Acetyl-CoA Availability for Metabolic

Production
Cytosolic acetyl-CoA is a key metabolite for the production of a range of valuable

compounds in yeast (Rossum et al. 2016). Native production of this compound
requires 2 mol of ATP and yields 2 mol of acetyl-CoA and 4 mol of NAD(P)H
per mol of glucose (Rossum et al. 2016). To improve the availability of this precur-
sor and lower the ATP cost, different heterologous enzymes have been introduced
to either bypass the native aldehyde dehydrogenase (ALD) and acetyl-CoA syn-
thetase (ACS) system using bacterial counterparts, i.e., A-ALD and PFL, that do
not incur such high cost (Kozak et al. 2014a, b), or to enable acetyl-CoA biosyn-
thesis in situ by expressing the pyruvate dehydrogenase (PDH) complex in the
cytoplasm (Kozak et al. 2014a, b). While the former application showed mixed
results in terms of growth and yield (mainly due to by-product accumulation),
the second approach along with a knock-out of the native ACS reaction exhibited
similar metabolic performance to the control, but at a lower ATP cost.
A different approach for improving acetyl-CoA availability relies on increasing
its yield. For this task, the phosphoketolase pathway (PKP) was early suggested
for the conversion of 1 mol of F6P into 3 mol of acetyl-P without carbon
loss (Schramm and Racker 1957). Conversion of acetyl-P to acetyl-CoA can be
later achieved by the reversible phosphotransacetylase (PTA) reaction (Rossum
et al. 2016). This was initially implemented in yeast for improving D-xylose fer-
mentation (Sonderegger et al. 2004) (refer to previous section). More recently,
Bogorad et al. (2013) implemented the full PKP in E. coli and demonstrated
almost stoichiometric conversion of C5 and C6 sugars into acetate under anaero-
bic conditions. A similar approach was replicated in yeast accompanied by several
genetic interventions to increase acetyl-CoA-derived farnesene (Meadows et al.
2016). This non-native pathway increased carbon utilization by 25%, decreased
oxygen consumption by 75%, and reached 15% v/v of farnesene. As illustrated
here, increasing acetyl-CoA availability may be critical not only for maximizing
production but also for overall improving metabolic performance.
3.3 Engineering a Heterologous CBB Cycle for CO2 Fixation
There is a growing interest in the field for engineering carbon assimilation path-
ways in heterotrophs for improving product yields—e.g., by reducing carbon loss
as CO2 –, and most notably, for implementing one-carbon (C1) compounds (e.g.,
CO2 ) fixation pathways to develop more sustainable fermentation bioprocesses.
In an early effort from Guadalupe-Medina et al. (2013), a heterologous Calvin–

Benson–Bassham (CBB) cycle was implemented in S. cerevisiae seeking to
improve ethanol yield by reducing carbon loss under anaerobic conditions. The
authors noted that by expressing the CBB enzymes phosphoribulokinase (PRK)
and ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO), a working path-
way could be realized where CO2 is effectively used as an electron acceptor for
NADH oxidation, thereby coupling CO2 fixation by RuBisCO with the fermen-
tation redox balance. Importantly, this mechanism rendered NADH reoxidation
through the native glycerol formation pathway unnecessary (90% reduction in
glycerol titer), increasing ethanol yield by 14% (Guadalupe-Medina et al. 2013).
While the latter strategy was successful in increasing product yield, it did so by
reducing carbon loss as glycerol and not by significantly increasing CO2 assimi-
lation (Guadalupe-Medina et al. 2013). A more radical approach is to engineer a
CO2 assimilation pathway capable of sustaining growth and production. In a pio-
neer work from Antonovsky et al. (2016), E. coli was transformed and evolved to
grow solely on CO2 as a carbon source and pyruvate as an electron source. Again,
expression of the missing CBB enzymes PRK and RuBisCO, and knock-out of
the phosphoglycerate mutase (PGM)—revealed by Flux Balance Analysis (Lewis
et al. 2012)—forced CBB operation by decoupling carbon fixation from energy
production. This metabolic phenotype was termed hemi-autotrophic growth, and it
has since been implemented in other bacteria like the methanol-consuming bacteria
Methylobacterium extorquens AM1 through the expression of the previous CBB
enzymes and deletion of essential genes for methanol assimilation (Borzyskowski
et al. 2018). Building on these strategies, a recent study reported the conversion of
the yeast P. pastoris into an autotroph that grows on CO2 as the sole carbon source
and methanol as an energy source (Gassler et al. 2020). Briefly, P. pastoris can use
methanol as both energy and carbon sources. By decoupling the formaldehyde—
the assimilated product of methanol oxidation—dissimilatory (carbon-fixating) and
assimilatory (energy-producing) pathway branches, one can force CO2 assimilation
by blocking the dissimilatory branch through the deletion of the dihydroxyacetone
synthase (DAS1 and DAS2) and alcohol oxidase 1 (AOX1). Then, complemen-
tation of the native peroxisomal xylose monophosphate (XuMP) cycle with six
enzymatic steps enables operation of the CBB cycle allowing growth on CO2 .
In stoichiometric terms, 1 mol of oxidized methanol produces 2 mol of NADH,
which can be used to fuel the CBB cycle though not in stoichiometric proportions
with CO2 (3 mol of ATP and 2 mol of NADH are needed to fix 1 mol of CO2 ).
The resulting mutant strain reached a maximum specific growth rate of 0.018 h−1
(Gassler et al. 2020) and constitutes an unprecedented advance for compartmental-
ized C1 carbon fixation in yeast differing from seemingly similar efforts in bacteria
(Antonovsky et al. 2016; Bang and Lee 2018).
20 P. A. Saa
4 Challenges and Outlook
During the past decade, yeast metabolic engineering has shown great progress and
promise (Smolke and Tyo 2012; Nielsen and Keasling 2016), quickly becoming
one of the preferred microbial factories for realizing the bioproduction of new
chemicals or improving the production of traditional ones. This success has been
largely driven by the continuous advances in the development of genetic and
molecular tools (Smolke and Tyo 2012), as well as novel computational frame-
works for pathway discovery and optimization (Wang et al. 2017; Saa et al.
2019). The latter has brought not only new possibilities for the evaluation of
novel biochemical synthesis routes but also has provided more rational meth-
ods for designing metabolic pathways with superior performance by rewiring
metabolism at a whole-cell scale (Saa et al. 2019). In time, such capabilities will
become increasingly essential for arriving at designs that scale industrially and
meet commercial expectations.
Pathway discovery is supported by the use of retro-synthesis tools that generate
putative routes connecting substrates to products. A comprehensive exploration of
the chemical space typically rests on the availability of reaction rules, which fills
the gaps between the metabolic precursors and target chemical(s). Generation and
application of such rules must be carefully performed, as they may provide infea-
sible pathways that may obscure results interpretation (Wang et al. 2017). Atom
mapping information can be of great aid for validating the application and gen-
eration of certain reaction rules, see e.g., RouteSearch (Latendresse et al. 2014)
and ReTrace (Pitkänen et al. 2009), which can be further completed with enzyme
promiscuity knowledge if available (Mazurenko et al. 2020). Another incipient
alternative for learning novel chemical reaction routes rests on machine learn-
ing techniques (Koch et al. 2020), which can potentially increase exponentially
the size of the reachable chemical space as shown elsewhere (Coley et al. 2019;
Mikulak-Klucznik et al. 2020). Efficient navigation of such vast space would nec-
essarily have to rely on the introduction of pathway scores and rankings to focus
the attention on the most promising and realizable designs. For this task, eval-
uation of the objectives reviewed here along with others—e.g., use of enzymes
with known promiscuous activity or cofactor specificity—constitutes a natural way
for prioritizing and selecting desired pathways. Rational integration of the vari-
ous objectives can be achieved by leveraging mature multi-decision multi-criteria
techniques (Bonissone et al. 2009), which remains largely unexplored in the field.
Notably, the latter techniques are also transferable to optimization-based methods
for pathway prediction, which could enable a more holistic evaluation of pathway
performance and robustness.
While the revised computational methods and tools for pathway prediction have
provided unintuitive and useful insights, their experimental application and vali-
dation remain still limited. Although there have been recent applications in yeast
(Hafner et al. 2021) and other model organisms (Yim et al. 2011) where some
of the tools have proven to be critical for finding effective in vivo metabolic
designs, there is still resistance to their broad adoption. Indeed, in vivo implemen-
tation of complex in silico metabolic designs is not trivial, typically demanding
great amounts of experimentation time before arriving at a working pathway
(Antonovsky et al. 2016; Schwander et al. 2016; Savile et al. 2010). Such efforts
could gain from recent computational frameworks for kinetic model construction
(Saa and Nielsen 2017) that could help to predict a priori the expected performance
of the pathway (see, for example, (Theisen et al. 2016)), greatly reducing the time
and resources needed. As the metabolic prediction capabilities of current models
increase (Foster et al. 2021), it is expected that the use of these tools for ratio-
nal pathway engineering in yeast and other microbial factories will progressively
become part of the basic toolbox for metabolic engineering.
Acknowledgements The author acknowledges the financial support of ANID Fondecyt Iniciacion
Grant No 11190871 and FONDAP Grant No 15090007 of the Center for Genome Regulation (CGR).
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Construction and Assembly
of Standardized Biobricks
for Synthetic Pathways Engineering
in Yeasts
Paulina Korpys-Woźniak, Monika Kubiak, Monika Borkowska,

and Ewelina Celińska
Abstract
Over the past years, a tremendous variety of modular cloning and DNA assem-
bly strategies has been developed. By definition, they enable one-step fusion of
multiple DNA modules, arranged in a defined position and orientation. They
differ in terms of flexibility, robustness and precision, but they all overcome
limitations of time-consuming traditional cloning. Irrespectively of technical
details, modular cloning and standardized bioparts assembly strategies con-
tribute to great progress within the field of genetic engineering of yeast. Their
exploitation allows the researchers to focus on addressing the scientific ques-
tions, rather than on execution of tedious laboratory procedures. Modularity
and standardization facilitate wide-spread collaboration within the yeast com-
munity and speed-up accomplishing of set goals. This chapter provides an
overview of synthetic biology tools for assembly of large DNA constructs (in
many cases—from predesigned modules) to construct and fine-tune complex
DNA assemblies for yeasts. The principles of the techniques are presented, and
then followed by examples of yeast species-specific implementations for Sac-
charomyces cerevisiae, Komagataella phaffii and Yarrowia lipolytica. A general
division into restriction digestion/recombination-based techniques and overlap
extension assembly strategies were adopted.
Paulina Korpys-Woźniak and Monika Kubiak—These Authors contributed equally to the work and
should be considered as the two first Authors.
P. Korpys-Woźniak · M. Kubiak · M. Borkowska · E. Celińska (B)

Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, ul.
Wojska Polskiego 48, 60-637 Poznań, Poland

https://doi.org/10.1007/978-3-030-89680-5_2
28 P. Korpys-Woźniak et al.
1 General Concept of Modularity
Gene cloning has gone a long way since its origin in ’70 of the XX century. For
many years, the DNA fragments to be cloned were processed with specific and
unique Restriction Enzymes (RE or ER—Endonuclease Restriction) and individu-
ally ligated with a vector that enabled maintenance of the fragment in the host cell,
and in some cases—its expression. The procedure had to be individually tailored
to a given sequence’s specificity, i.e., its nucleotide sequence and the presence of
ER Recognition Sites (RS) at a specific location. Along with time and complex-
ity of generated DNA constructions, the need for streamlined cloning procedures
has been growing. Traditional Restriction Digestion (RD) with specific RE, and
cloning of the fragments one-by-one did not meet growing demands. Until the con-
cept of modularity has been transplanted to molecular biology from engineering
and programming fields.
Modularity dissects a system (= complex DNA construction) into individual
modules (= biobricks/bioparts), each having a specific position and task to exe-
cute, in order to develop the desired functionality of the whole system. A given
module (e.g., promoter/ORF) may have different variants (e.g., strong, weak,
inducible/fluorescent, enzymatic) that can be interchangeably shuffled. Impor-
tantly, in a specific design of the system, each module has a strictly defined location
and function that cannot be exchanged with another module (only its variants can
be exchanged at the same position). A given module’s location (and function)
is predefined by a scaffold, which is a system of linking and docking adjacent
modules with each other. By organizing the modules within the scaffold, they
are correctly ordered and oriented, and cannot be translocated/shifted to another
location (only to another variant of the same module). Modularity gives the advan-
tage of an open upgrading option, provided that the new module/variant will fit
into the scaffold. This quality is not available for cloning through the traditional,
sequence-specific RE-based strategies.
To put the modularity concept into a molecular biology perspective, we need to
translate a term “module” into “a function at a specific location” within a multi-
part DNA construction (Fig. 1). This can be, for example, a promoter element at
the second T ranscription Unit (TU; TU is a system of promoter-ORF-terminator)
within a two TU-bearing DNA construction (P2 in Fig. 1).
This (P2) element is defined by specific linking/docking elements, flanking the
module. These may be specific ER RS, Recombinase (Rec) RS, OverLapping
protruding elements (OL; overlaps) generated by PCR or in vitro DNA synthe-
sis—anything that facilitates correct assembly (hybridization and ligation) of the
subsequent DNA fragments. They are specific for a given element (as P2 in our
example), and unique within the whole DNA construction. These elements guar-
antee correct positioning and orientation of the (P2) element within the multipart
DNA assembly. Depending on the linking/docking system adapted for a particular
technique, the final assembly may bear scars or be scarless.
Construction and Assembly of Standardized Biobricks …
Fig. 1 General outline of bioparts assembly and combinatorial cloning

29
Box 1
A scar is a short nucleotide sequence, present in the final assembly, that is
not a functional part of any of the subsequent bioparts, but was necessary at
the assembly stage for correct hybridization of the modules. Typical scars, in
traditional and in BioBrick clonings, are ER RS, in GoldenGate—4 nt over-
hangs, in the OL extension methods—depends on the technique design. The
ER-based techniques typically lead to scar-bearing assemblies. On the other
hand, OLs may be designed to leave a scarless assembly, when each OL is
composed solely of the 3’ and 5’ sequence of the two adjacent bioparts. But,
OL-based methods may leave the scars—when the OLs bear standardized
but unique short sequences, altogether generating a scaffold.
The (P2) module may have different variants, as stated above—e.g., strong,
weak, constitutive, inducible, etc., provided that they are fitted into the scaffold
by flanking with specific linking/docking elements. Such systematic exchange of
different variants of a given module, to test their functionality, is termed combina-
torial cloning. The process of transforming a given DNA fragment into a module
requires endowing it with the flanks, compatible with the system’s scaffold, and
ensuring that the nucleotide sequence of the fragment will remain inert upon the
enzymatic reaction taking place during the assembly reaction (restriction diges-
tion, recombination, ligation, amplification, etc., depending on the method). Thus,
the module must not contain any nucleotide sequence recognized and modified by
the enzymes used in the following enzymatic reaction, that physically assemble the
modules into a single DNA construction. The collection of such elements (DNA
fragments transformed into modules) is called a library; and, together with the
predefined scaffold, they can be also termed a standard. Sometimes, the term
“standard” refers only to the scaffold itself. In the majority of modular cloning
strategies, it is possible to generate extensive, multi-element DNA construction
in a single-tube and one-step reaction. It is facilitated by careful design of the
scaffold and the modules that are precisely positioned in the pre-designed order
and orientation; even if >10 elements are assembled (put into the same tube and
processed by the enzymes) at the same time.
Box 2
Once assigned to a given module functionality can be later modified. Consid-
ering our example, if a gene (ORF; G2) cloned under control of P2 must be
transcriptionally fused to an element (signal peptide or fusion partner) at its
5’ terminus, and this element will be shuffled in the following cloning (dif-
ferent variants will be tested), two different strategies could be followed: (i)
expanding the scaffold by modifying the linking/docking elements of P2 at 3’
terminus and G2 at 5’ terminus (adding additional fusion site) so that another
module can be inserted; (ii) functionality of all the elements upstream of the
Construction and Assembly of Standardized Biobricks … 31
G2 can be changed, but the linking/docking sites remain unchanged—this

approach would require the generation of a modified library of all elements
upstream from the modified site.
By definition, entering a modular cloning approach is a long-term strategy of

DNA construction assembling. To take full advantage of benefits it offers, one
must first make an effort to design the standard, the system of nomenclature and
cataloging, and to expand the library. Once entered, working within a given sys-
tem offers a multitude of profits. Laboratories working in the same standard may
easily exchange modules, expanding their libraries. Additionally, standardization
of the methods greatly simplifies the design of cloning strategies and minimizes
the number of steps required to obtain a desired construct. Most techniques enable
combinatorial construction of complex DNA constructions (cassettes) using DNA
parts from libraries, as well as from open registries of modular components, avail-
able for the synthetic biology community. These standards facilitate the reuse of
fragments between experiments and their exchange between research groups. Apart
from constructing expression cassettes of a defined number of TUs, DNA assembly
is useful to build for example whole chromosomes.
Box 3
Modular cloning may have different formats in DNA construction assembly.
The number of shuffled modules in a single reaction may differ signifi-
cantly—from a single biopart (when all the remaining elements necessary
for the gene expression in yeast cell are already present in the destina-
tion/acceptor vector), up to all the “yeast elements” (when only the “bacterial
part” in a shuttle vector* remains unchanged). Technically, the approach
depends on the scaffold’s structure. An extensive and highly specific scaf-
fold (with a large number of unique and specific linking/docking elements)
accepts more elements to be shuffled in a single reaction. Limited scaffold,
allow shuffling one or two bioparts at a time. In the latter case, all the remain-
ing elements, necessary for full functionality of the expression cassette must
be already present in the acceptor vector. (* typically the DNA bioparts
assembly is conducted in a shuttle vector—bearing a “bacterial part”, to
be assembled and amplified in E. coli, and a “yeast part”—composed of
yeast-specific regulatory elements and the target genes, to be transformed
and expressed in a yeast host cell).
Over the past years, a tremendous variety of modular cloning and DNA assem-
bly strategies has been developed. They differ in terms of flexibility, robustness
and precision of cloning, but they all overcome limitations of time-consuming
traditional cloning using RD and DNA ligation, typically joining only two, “single-
use” DNA parts simultaneously. DNA assembly (incl. modular cloning) typically
enables one-step fusion of multiple DNA modules, arranged in a defined position
and orientation. This chapter provides an overview of “state-of-the-art” synthetic
biology tools for assembly of large DNA constructs (in many cases—from pre-
designed modules) to construct and fine-tune complex DNA assemblies for yeasts.
For the sake of clarity and comprehensiveness, the principles of the techniques are
presented and then followed by yeast species-specific examples. Saccharomyces
cerevisiae, Komagataella phaffii and Yarrowia lipolytica were chosen as the case
studies, due to their fundamental role or high interest in the yeast community.
Selection of methods was an arbitrary choice of the authors, based on their pop-
ularity in research by the yeast community. General division into RE-dependent
and OL assembly methods was adopted.
2 Historical Outline
The evolution of modular cloning and DNA bioparts assembly strategies spans
the last 30 years. General trend of the techniques’ evolution leads from founding
the basic principles of a method, through demonstration of its applicability in a
given yeast species, up to development of the so-called, toolkit (or toolbox), built
of species-specific standardized modules and/or DNA constructions and recipi-
ent strains. In order to put the techniques into a research community perspective
and illustrate dynamics of the methods’ evolution, in this section we summarize
timeline of development of the most popular techniques, according to that key—
establishing the technical concept, through “the first mention” for a given yeast
species, up to a toolkit development (where applicable; Fig. 2a, b). A more detailed
description of the techniques with deeper insight into current state of the art is
given in the following sections of this chapter.
Technical implementation of the DNA bioparts assembling concept was at first
addressed by Overlap Extension PCR (OE-PCR). The method was initially adopted
for assembly of relatively short elements (300–400 bp), incl. gene’s exons, or
parts located upstream and downstream of the mutation site (two parts) (Ho et al.
1989; Horton et al. 1989). The technique was later adopted for creating chimeric
genes or gene alleles in S. cerevisiae (Anthony and Liebman 1995), K. phaffii
(Eldin et al. 1997) and Y. lipolytica (Zhang et al. 2010). In many cases, due to
simplicity of the method, lack of spectacular technical background behind it and
low efficiency when compared to the other assembly method, the authors tend
to use it as a routine laboratory technique for simple assemblies, without special
mention. Nevertheless, later elaborated variants of OE-PCR, combined with “in
yeasto” assembly, demonstrated the great potential of that simple method, which
ultimately enabled assembly of extensive DNA constructions (Shao et al. 2009;
Gao et al. 2014).
Fig. 2 Historical outline of DNA bioparts assembly and modular cloning strategies invention and
implementation. a Inventions and implementations are given chronologically. b Inventions and
implementations are sorted by the principal technique
Box 4
The term “in yeasto” refers to phenomena or reactions taking place within
the living yeast cell. It may be considered as an abbreviation of stating
“in vivo in the yeast cell”. It can be sometimes found in the research papers
issued by/dedicated to the yeast community.
In contrast, an assembly technique developed only 2 years later, USER®

(Uracil-Specific Excision Reaction; Nisson et al. 1991; Smith et al. 1993) has
gained immediate, great attention by the yeast research community. The princi-
ples were developed and commercialized by New England Biolabs in 2003, and
the toolkit is commercially available. The USER standard was introduced to a
molecular biology tools portfolio for (i) K. phaffii (Nour-Eldin et al. 2006), with
several advancing modifications improving flexibility of the method, (ii) S. cere-

visiae (Mikkelsen et al. 2012), demonstrating great robustness of the technique
by “USER cloning” of an 8-gene pathway—in 4 assemblies each bearing 9 mod-
ules, and (iii) for Y. lipolytica (Holkenbrink et al. 2018). In the latter study, the
authors presented a USER-cloned EasyCloneYali genetic toolbox comprising 27
modular vectors for site-specific integrations assisted by CRISPR-Cas9 elements
available from Addgene (Holkenbrink et al. 2018). Likewise, standardized biolog-
ical parts assembly according to the BioBrick concept, proposed in 2003 (Knight
2003; Shetty et al. 2008), has gained great popularity in the yeast community.
BioBrick-based approach was reported as a convenient way for generating expres-
sion cassettes with multiplied copies of a specific TU for K. phaffii (Shen et al.
2016), or was used as a basic principle for the development of advanced modular
cloning system for Y. lipolytica (Wong et al. 2017), where new, recyclable modules
with reporter genes and regulatory elements were presented. While the latter modu-
lar cloning system in its final version operates according to principles of BioBrick
standard, it was mainly constructed using another technique of large DNA con-
struction assembly, namely the Gibson method. Since the first, model experiments,
demonstrating a native capacity of S. cerevisiae to an assembly in vivo (or “in
yeasto”) nearly 40 DNA fragments of >1 kbp in length (Gibson 2009), and, even
more importantly, demonstration of mastering of recombination reaction in vitro
(Gibson et al. 2009), the Gibson assembly method has gained tremendous inter-
est. The technique is widely used, e.g., combinatorial cloning and testing of large
libraries of regulatory elements for K. phaffii (Vogl et al. 2015), generation of a
complete toolkit for gene disruption and protein epitope tagging for Candida albi-
cans (Dueñas-Santero et al. 2019), or as a routine cloning strategy, used aside from
the other cloning methods, for Y. lipolytica (Rodriguez et al. 2016). The in vitro
recombination has also gained much attention due to invention and implementation
into laboratory practice of Gateway™ cloning technique, first proposed in 2000
(Hartley et al. 2000). Only two years later, a rich library of Gateway™ bioparts
and destination vectors was developed for constitutive or inducible expression of
(various) epitope-tagged proteins in S. cerevisiae (Funk et al. 2002). Gateway™
standard was successfully adopted to Y. lipolytica by developing a broad-use desti-
nation vector, a platform strain and several modules with reporter proteins (Leplat
et al. 2015). Earlier, Gateway™—assembled constructions, merging modules bear-
ing signal peptides and secretory proteins, were tested in K. phaffii (Esposito et al.
2005) with positive outcome. Gateway™ combinatorial cloning was also used for
efficient generation of a panel of deletion cassettes dedicated for C. albicans (Lebel
et al. 2006).
Extensive and widely available toolkits for a generation of multiple, highly
complex DNA constructions were developed using another modular cloning
method, namely GoldenGate. The principles of modular cloning according to
GoldenGate strategy were first reported in 2008 (Engler et al. 2008; Weber et al.
2011), and it took 7 years to develop a first comprehensive toolkit for Golden-
Gate cloning in yeast (S. cerevisiae) (Agmon et al. 2015). That first toolkit set the
standards and paved the way for further species-specific libraries of bioparts and
destination vectors. In 2017, customized collections of standardized bioparts for

modular cloning in Y. lipolytica (Celińska et al. 2017) and K. phaffii (Prielhofer
et al. 2017) were reported. The new standards were further developed and intro-
duced to open repositories of bioparts (Prielhofer et al. 2017; Larroude et al. 2019)
and are now widely available for the yeast community.
3 Restriction Digestion/Recombination-Based Assembly

Strategies
Restriction digestion/recombination-assisted bioparts assembly relies on flanking

the modules with short sequences, specifically recognized by the enzymes used
in the next step. By definition, these methods generate small scars at the fusion
sites. Cloning according to the ER/Rec-based strategies requires elimination of
unintended ER/Rec RS from the module’s sequence, to maintain it intact during
the assembly reaction. Moreover, a scaffold of linking/docking elements must be
developed in advance. On the other hand, exploitation of the predesigned scaffolds
facilitates combinatorial cloning, which is usually limited in OL-based techniques.
Additionally, these techniques are considered more precise when compared to OL-
dependent methodologies (discussed hereafter).
3.1 BioBricks
BioBrick assembly (BB) (Knight 2003) of standardized bioparts, otherwise known

as BBF RFC 9, was first proposed by Tom Knight in 2003 (version RFC 10 in
2007). The principle of this method relies on the use of two pairs of standard
Res—(i) Unique REs located outside of a biopart (URE1/2) and (ii) IsoCaudoMers
(IsoCM1/2)—located in direct proximity of the element (Fig. 3). As in any method
relying on RE—their RS must not be present in the bioparts to be assembled. It
might be thus necessary to remove the RS by site-directed mutagenesis before the
assembly reaction.
Box 5
Isocaudomers are pairs of RE that recognize slightly different RS but upon
cleavage, they generate compatible overhangs. After ligation of such pro-
truding elements, an asymmetrical sequence is generated and thus cannot be
recognized by any of the ERs—it is lost, but in this way, the sequence is sta-
ble during the next cycle of assembly. Examples of IsoCMs: 1: BamHI/BglII,
2: NdeI/MseI, 3: XbaI/AvrII/NheI/SpeI, 4: XhoI/SalI.
There is no specific scaffold of linking/docking in BB technique. While it saves

the initial design effort, only two parts can be assembled in a single reaction. To
get a multipart assembly, the cycles of the two-part-joining must be repeated. The
Fig. 3 General strategy for BioBrick parts assembly (e.g.: promoter, ORF and terminator). Each of
the modules is first equipped with prefix and suffix bearing RS for a single URE (unique restriction
endonuclease recognition site) and a single IsoCM (isocaudomer restriction endonuclease recog-
nition site). Full description is given in the text. “Scar” site is shown as a purple circle in the final
assembly. Circular objects indicate the bacterial ori of replication (black) and ampicillin resistance
gene AmpR (orange) contained in the “bacterial” part of the assembly, which is typically discarded
before yeast cell transformation
modules are prepared by adding specific prefixes and suffixes in the PCR to a
biopart, each bearing RS for URE at 5’ and IsoCM at 3’. The two modules to
be assembled are then cleaved in a specific pattern each with URE and IsoCM to
generate compatible overhangs at the linking/docking site. The overhangs at the
fusion site are generated by different IsoCM for each of the modules. RS of URE
flank the assembly, and compatible RS are present in the destination vector. As
a result of the following ligation reaction, the sequence between the fused DNA
fragments bears a 6 bp scar, but the URE-bearing prefix and suffix sequences
remain unchanged, hence the other BB parts can be assembled in the next step. In
this technique, the destination vector bearing two assembled bioparts becomes an
entry vector in the next cycle of assembly.
The reaction conditions are standard for ER and ligation (typically 1 h at 37 °C
and 1 h 16–22 °C). Most of compatible BBs for yeasts are listed on the website
of the Registry of Standard Biological Parts (http://partsregistry.org/ Main_Page),

and the bioparts are available from Addgene and iGEM.
As compared with the traditional cloning, several advantages of the BB stan-
dard assembly can be highlighted, such as utilization of only four RE, “endless”
number of cycles of pairwise assembly, practically limited only by the plasmid
size, and no necessity of recloning or even re-PCR amplification. Despite facilitat-
ing automation and reuse of parts, the method is not truly high-throughput, as the
fusion is always only pairwise, and the scar left at the fusion site can affect the
coding sequence or mRNA’s secondary structure.
The BB standard has been subjected to specific adjustments, relying on the
same principle (including the BglBrick standard (Anderson et al. 2010) and 2ab
assembly (Leguia et al. 2013)) but with improved flexibility and efficiency. A
collection of several thousands of S. cerevisiae-specific modules, compatible with
BB standard, is available from the iGEM Registry of Standard Biological Parts.
Specific implementations of BB parts assembly in the selected yeast species
cover several relatively recent reports. In 2012, Schneider et al. (Schneider et al.
2012) presented at annually held iGEM competition (International Genetically
Engineered Machine) a Yeast BioBrick Assembly toolkit for S. cerevisiae, com-
patible with BB Standard. Presented cloning system facilitated cloning of seven
modules in an acceptor vector, bearing already auxotrophic selection marker, one
promoter and one terminator, flanking the cloning site. Hence, altogether three
complete TUs could be cloned. A mevalonate pathway, composed of three TUs
served as a proof-of-concept. BB standard was implemented for multicopy cloning
of expression cassettes encoding enzymes: protease from Trichoderma koningii
(Shu et al. 2016), organophosphorus hydrolase from Pseudomonas pseudoalcali-
genes (Shen et al. 2016) and two L-amino acid oxidases (Rao et al. 2020) in K.
phaffii. In the two former reports, the BB cycle was repeated four times, to gener-
ate four TU-bearing assemblies of ~15 kbp. A single module was built by a gene
flanked with regulatory elements (three elements already subcloned through tradi-
tional methods). In the latter report, the authors repeated the BB cycle up to three
times, obtaining up to three copies of TUs. This time, each TU was built of five
fragments: a promoter, a gene-encoding enzyme flagged at N- and C-termini, and
a terminator. The generated vector was more than 18 kbp. Altogether, the principal
aim of the multicopy cloning in K. phaffii was to increase gene dosage and produce
more of the heterologous proteins. As another implementation of the BB cloning
standard, (Wong et al. 2017) proposed a complete toolkit for modular cloning in Y.
lipolytica. The authors generated reusable and ready-to-use modules of 12 native
promoters of 1 kbp, intronic sequences (as they proposed and validated operon-
type cloning system in Y. lipolytica, instead of traditional TUs) and terminators.
Additionally, four modules bearing reporter genes (fluorescent and chromogenic)
were constructed in accordance with the BB standard. Robustness of the toolkit
was validated by a generation of 5-gene violacein biosynthetic pathway of 12 kbp.
Thanks to BB modular cloning assisted by rapid DNA assembly technique (Gib-
son assembly was adopted), the extensive pathway was constructed in one week.
Thanks to the modularity of the system, combinatorial cloning of the pathway was
executed, and positional effects and different genes’ configurations could be stud-
ied. The system of reusable modules compatible with the BB standard is available
from Addgene.
3.2 GoldenGate
Introduction of GoldenGate (GG) cloning strategy to molecular biology portfolio

is dated back to 2008 (Engler et al. 2008). The technique relies on the use of
type IIS RE (e.g., BsaI, BsmBI) that recognizes a 6 bp sequence but cleaves 5 bp
downstream of the RS, leaving a 4 bp overhang. But the key element is that the
cleaved region may be of any nucleotide sequence.
Box 6
Type IIS RE belongs to a specific group of RE that recognize asymmetric
DNA sequences (BsaI: GGTCTC) and cleave at a defined distance outside
of their RS, (1–20 bp) but the combination of nucleotides at the cleavage
site is not defined. Since the sequence at the cutting site is indifferent to
the RE, it can be modified/designed. It is possible to generate a system of
unique sequences, that will form overlaps after the cleavage with type IIS
RE, which is the essence of designing a scaffold for GG cloning.
In this way, the user may design overhangs that will be generated upon cleavage.
A set of such unique overhangs, serving as a linking/docking system, consti-
tutes a scaffold of the GG standard defining position and orientation of assembled
parts (Fig. 4). To assure compatibility of the modules, the scaffold must remain
unchanged. Adjacent bioparts share the same overhang at the junction points. Mod-
ules are prepared by PCR amplification with primers adding ER IIS RS at 5’,
followed by the 4 bp overhang in the middle of the primer, and a sequence com-
patible with the biopart at its 3’ end. Such construction allows to “loose” the type
IIS RE RS after cleavage, leaving only the 4 bp overhang after digestion and a scar
after fusion. To generate a library of GG modules, the PCR-amplified bioparts are
cloned in the entry vectors. The entry vectors with the modules are pooled together
with a destination vector in the assembly reaction. The destination vector typically
bears some chromophore gene that serves as a negative control after assembly and
cloning in E. coli. The chromophore region in the destination vector is flanked with
type IIS RE RS cloned inwardly, so the cleavage leaves 4 bp overhangs at the vec-
tor’s termini, and the chromophore cassette with the type IIS RE RS is discarded.
The reaction is conducted in a cycling profile of subsequent incubations at 37 °C
for ER action and 16 °C for the action of T4 ligase. With each ER/ligation cycle,
new fusions are generated, devoid of type IIS RE RS—disallowing any further
modifications. The number of elements that can be fused in a single cycling reac-
tion in a single tube is limited by the scaffold design. More than ten modules are
Fig. 4 General outline of GoldenGate parts assembly (e.g., promoter, ORF and terminator). Each
of the modules is first equipped with RS for type IIS ER and 4 bp overhang sequences (indicated
as red, purple, orange and green XXXX), according to a predesigned scaffold. Full description is
given in the text. 4-bp scar sites are shown as purple and orange rectangles in the final assembly.
Circular objects indicate the bacterial ori of replication (black) and bacterial resistance gene: KanR
(blue) and AmpR (orange) contained in the “bacterial” part of the assembly, which is typically
discarded before yeast cell transformation
regularly fused (examples are given hereafter) with a cloning efficiency of about
90% (Engler et al. 2008).
The GG concept-based variation, called MoClo (Weber et al. 2011), allows gen-
erating large multigene construction (up to 33 kb) in three cloning steps, from basic
modules, through pre-assembled TUs, finally merged into an expression construct.
Likewise, the GG standard was modified to generate a cloning system GoldenBraid

(Sarrion-Perdigones et al. 2011), particularly useful for extensive DNA constructs
assembly. The approach relies on a set of four destination vectors designed to
incorporate multipart assemblies, consisting of standard DNA parts, and to com-
bine them binarily to build a highly complex multigene construct (by introducing
double loop, “braid” topology).
The first implementation of GG modular cloning to the yeast molecular biology
tools portfolio relied on combinatorial cloning of 3- and 5-module DNA assem-
bly in S. cerevisiae (Agmon et al. 2015). Scaffolds covering respectively 4 and 6
overhangs were developed, and the elements to be assembled were converted into
modules by flanking with type IIS RE RS and the predesigned overhangs. Shuttle
destination vector contained the “bacterial part” with a chromogenic reporter, and
auxotrophic selection marker for selection in yeast. Generated assemblies were
of relatively small length—1,5 kbp and 2,2 kbp. Importantly, a panel of destina-
tion vectors with different selection markers and maintenance options (ARS/CEN,
2μ and integration) for S. cerevisiae is available for the yeast community from
Addgene. At nearly the same time, another GG-based toolkit (YeastFab) for S.
cerevisiae was proposed by another research group (Guo et al. 2015). Here, the
scaffold was much more extensive (10 overhangs) facilitating the cloning of 3 TUs.
A 3-gene carotenoid synthesis pathway was chosen as a proof-of-concept. First,
the authors tested 151 different promoters, mined from S. cerevisiae’s genome,
in a single TU assembly, to determine their transcriptional efficiency. Three of
them were chosen as modules for further combinatorial cloning, and fine-tuning
of the pathway. Altogether 27 different assemblies were generated (each ~6,5 kbp)
by shuffling three promoters, with three genes, and a single terminator. The opti-
mal combinations for this particular pathway were indicated. All the elements are
available from Addgene for the yeast community.
Instead of completely in vitro assembly of multiple DNA elements via the typ-
ical GG method, (Chuang et al. 2018) proposed a combination of GG technique
with in vivo assembly by VEGAS (VErsatile Genetic Assembly System).
Box 7
VEGAS is a molecular biology technique exploiting the innate capacity of a
microbial cell to combine DNA fragments with terminal complementarity by
homologous recombination using orthogonal adapter sequences (VAs). The
adapters are introduced to the DNA fragments as terminal modules flanking
the TUs in the core. Their location and orientation direct desired order of
the core pathway genes.
First, five individual DNA assemblies (violacein synthesis pathway), each com-
posed of five modules in a 6-overhang scaffold, were constructed in vitro in a
typical GG reaction. In each assembly, central TU (promoter, gene, terminator)
was flanked with terminal adapters, facilitating in vivo homologous recombina-
tion. The five DNA assemblies were transformed into E. coli and correctly fused
in a complete construction of ~13 kbp by the native recombination machinery of

a cell.
Another interesting example of GG cloning usage in S. cerevisiae’s genetic engi-
neering is assembling of CRISPR-Cas9-sgRNA cassette, for multiplexed genome
editing (Zhang et al. 2019). The authors exploited modular cloning to assemble a
CRISPR-Cas9 cassette made of 4 up to 11 modules in a scaffold composed of 5
to 12 predesigned overhangs. All the generated assemblies bear a gene for Cas9,
auxotrophic marker, a promoter and a different number of sgRNAs, to facilitate
simultaneous disruption of up to 6 genes at 96% efficiency.
Several comprehensive GG-based systems were proposed for K. phaffii. One of
the most extensive toolkits was developed and provided to the yeast community by
(Prielhofer et al. 2017). The system GoldenPiCS (available from Addgene) covers
ready-to-use, multi-use modules bearing 20 different promoters, 10 terminators, 4
integration loci and 4 dominant selection markers. The modules can be cloned in a
scaffold composed of 13 fusion sites. The authors developed a hierarchical system
of three destination vectors (BB1-3; 3 different variants of BB1, 8—BB2, 21—
BB3), each dedicated for specific cloning—BB1 serves as the entry vectors with
single, individually cloned modules, BB2 is for cloning of individual TUs, and
BB3—for merging up to 8 TUs in a single DNA assembly. Each of the vectors’
type is prepared to be assembled via a specific type IIS RE, and bears some specific
traits, like incompatible RS between subsequent levels of hierarchical cloning, or
elements targeting genomic integration (BB3). Based on provided information,
the largest DNA assembly generated via hierarchical cloning from BB1 to BB3
bears 27 modules of total length ~23 kbp. The GoldenPiCS technique was recently
used for construction of a 10-modules-bearing, very convenient CRISPRi system
for genome editing in K. phaffii (Gassler et al. 2019). The system requires only
one-step cloning of sgRNA sequence in a ready-to-use BB3 vector to a get fully
operable construction (also available from Addgene).
MoClo Pichia Toolkit (termed Yeast Toolkit; YTK) for hierarchical, modular
cloning was extended in a work on standardized regulatory elements and secretory
tags for fine-tuning of heterologous secretory proteins in K. phaffii (Obst et al.
2017). Twenty-one new bioparts, compatible with the standard, were added to
the existing library; these were 4 promoters, 10 secretion tags, 1 terminator and
2 ori. All the DNA assemblies were composed of 8 modules in a 9-fusion sites
scaffold. With the new modules, added to existing elements, it was possible to
overexpress a given gene in 264 different ways. The authors built and characterized
the expression and secretion efficiency of 124 constructs that combined different
regulatory elements with two fluorescent reporter proteins, of approximate length
4 kbp. The authors pointed that it was possible to cross-use S. cerevisiae’s YTK
bioparts in K. phaffii, which, when combined, give >4000 different possibilities of
a given gene cloning.
Schreiber et al. (2017) developed a 7-fusion sites GG scaffold to perform
combinatorial cloning of chimeric proteins in K. phaffii. The study comprised con-
struction of 11 new modules, incl. 2 promoters, 3 pre-sequences, 1 pro-sequence,
1 protease cleavage site, 3 genes of interest and a single terminator. Shuffling of all
the module variants allowed to rapid construction and test of 18 DNA assemblies.
A very similar approach was undertaken in a very recent study by (Püllmann et al.
2020) aiming at overexpression of eight enzyme-encoding genes. In that study, the
authors prepared destination vectors with a pre-cloned dominant selection marker,
promoter and terminator for the gene of interest transcription, and ARS for episo-
mal maintenance. Thus, only a 4-fusion sites GG scaffold was used for the modules
shuffling. The three shuffled modules were: (i) signal peptide (one of 17 variants),
(ii) target gene (out of 8), (iii) protein tag for purification (7 variants available).
The authors deposited all the modules in Addgene for the yeast community.
A remarkable variety of GG modular cloning implementations were reported for
Y. lipolytica, with the first mention published in 2017 (Celińska et al. 2017). In that
report, a scaffold composed of 13 fusion sites was developed for modular cloning
of 12 bioparts, equipped with specific 4 bp overhangs. The authors proposed 48
new modules, to be fitted into the scaffold, covering different promoters, termina-
tors, selection markers and elements directing targeted genomic integration. Soon
after that first mention, a report on biotechnologically relevant exploitation of that
scaffold and the modules was published (Larroude et al. 2018). The authors shuf-
fled promoters of different strengths to optimize expression of a 3-gene carotenoids
synthesis pathway in Y. lipolytica. The shuffling process was conducted in a single-
tube, single-step reaction to assembly 12-module pathway of >12 kb. The initial
scaffold was later on modified by the addition of a new overhang into the scaffold
(Celińska et al. 2018). The additional fusion site was used to enable combinatorial
cloning of different signal peptides, to be tested with different heterologous secre-
tory proteins. The initial scaffold was designed to enable the rapid fusion of 12
bioparts—3 TUs + selection marker +2 integration targeting sites. Introduction of
this additional fusion site enabled the reuse of the previously developed modules
(the problem is addressed in Box 1) and insertion of an additional one for a sig-
nal peptide (between promoter and target gene). The site was carefully designed
to maintain the reading frame unchanged and to minimize amino acid changes
within the encoded secretory proteins. The authors shuffled ten novel signal pep-
tides with two reporter proteins to indicate optimal fusions. In the following study
(Celińska et al. 2020), the best fusions (signal peptide + gene) were amplified as
single modules and could be cloned in the former scaffold, as multiple TUs bear-
ing DNA assembly. In that study, the authors investigated positional effect on the
genes’ expression, by combinatorial cloning of optimized fusions (signal peptide
and the genes) in either the first or the second TU. Thanks to the modularity of
the GG method, it was possible to rapidly generate the necessary set of DNA con-
structions. An improved scaffold with an enriched library of modules (altogether
64 modules, validated with three different fluorescent reporters) was finally made
available to the yeast community through Addgene (Larroude et al. 2019). The
same scaffold, either in its full length or limited to a single/double TUs was later
exploited in different applications, like rapid single genes cloning under strong
promoter (Korpys-Woźniak et al. 2020) or testing the strength of newly described
promoters (Park et al. 2019).
Recently, (Liu et al. 2019) exploited the GG assembly method to construct

chimeric pathways of lycopene synthesis in Y. lipolytica. The authors selected
nine pathways from S. cerevisiae, generating a panel of 100 heterologous genes,
to be transformed into the modules and fit into GG scaffolds of up to 5 TUs.
The heterologous genes, cloned in GG assemblies, operated together with native
lipophilic terpene synthesis genes in Y. lipolytica generating a variety of different
phenotypes. Stochastic, combinatorial approach, facilitated through efficient mod-
ular cloning, enabled rapid optimization of the pathway expression and >150-fold
enhancement in lycopene synthesis. Additionally, new knowledge on molecular
background behind this biotechnologically relevant trait was gained.
Finally, the GG assembly technique was exploited to generate a CRISPR-Cas9
acceptor vector for genome editing in Y. lipolytica (Larroude et al. 2020). The
acceptor vector bears a typical bacterial part, ARS for episomal maintenance in
Y. lipolytica, an excisable selection marker gene, an optimized Cas9 gene under
a strong hybrid promoter, and a BsmBI RS flanking RFP chromophore, for con-
venient selection of positive (white) clones. The BsmBI RS site was designed
for cloning of the targeting sgRNA. A collection of CRISPR-Cas9 vectors with
different selection markers is now available from Addgene.
3.3 Gateway
Gateway™ cloning technology (GW) relies on a system of short att sequences

recognized by a viral DNA recombinase. The mechanism was discovered in bac-
teriophage lambda where it facilitates the integration of the phage’s DNA into E.
coli’s genome (Hartley et al. 2000). The cloning system (vectors and enzymes
mixes) was developed and commercialized by Invitrogen (http://www.invitrogen.
com/; now ThermoFisher Scientific). The technology is routinely used to transfer
DNA fragments between vectors containing compatible att recombination sites.
Box 8
There are four different variants of att sites (B, P, L, R). All of them con-
tain a core 25-bp “recognition region” (black box) whereas L, R/P contain
“arms” on either/both sides of the core, acting as interaction sites for the
recombination enzymes (left arm—white box; right arm—lined box). The
core region bears a central 7-bp “asymmetric overlap” (yellow box) that is
recognized by clonases and thus determines where DNA is cut and rejoined.
In its basic format, GW is used for single-fragment subcloning, but can be

expanded to a multipart format for simultaneous assembly of multiple fragments
in desired sequence and orientation (Fig. 5). For the latter, a scaffold of specific
att sites is required. The modules are thus generated by flanking the bioparts with
specific attB sequences (only 25-bp core). In this system, the modules must be
first cloned into the donor vectors (which could be de facto omitted for BB and
GG). Cloning in the donor vectors is executed by BP clonase that recombines
attB sites from PCR-amplified modules with attP sites, present in the vectors.
Upon recombination, the BP clonase generates attL (or attR—depends on the
scaffold design) sites, which are further processed in the next step, conducted by
LR clonase. In the multipart format, it is necessary to carefully design subtypes
of the flanking regions (e.g., attB1 compatible with attP1, but not with attP2 or
attP3; Fig. 5), so that only the border modules (the most 5’ and the most 3’ in the
multipart assembly) bear attL(1) sites compatible with attR(1) sites present in the
destination vector (Fig. 5).
Box 9
Definition of att sites’ subtype is executed by making nucleotide changes
within the 25-bp region. Setting different subtypes of att sites facilitates
cloning of each DNA fragment in only one position and orientation.
In the final cloning stage, the LR clonase executes the reverse reaction to BP
clonase, generating attB sites at the fusion sites. The destination vector bears attR
sequences flanking ccdB toxic cassette, which, when maintained, disable clones
growth. The reaction is conducted in vitro, over short incubation (1 h) at 25 °C.
The multipart format allows for one-step cloning of up to five modules. As the
other RD-/Rec-based assembly techniques, GW leaves (relatively long—25-bp)
scars at the fusion sites. One of the biggest advantages of GW techniques is a wide
array of destination vectors available, which can be either purchased or obtained
from Addgene/iGEM collections.
Apart from the GW assembly system, some other recombination-based cloning
systems have been issued, like Univector (Liu et al. 1998) and Creator (Siegel
et al. 2004) relying on the use of Cre recombinase and loxP sites, or In-Fusion
Fig. 5 General outline of Gateway cloning strategy (e.g., promoter, ORF and terminator). Each
of the modules is first equipped with RecRS by the introduction of attB sites (indicated as red,
purple, orange and green arrows), according to a predesigned scaffold. The reaction is two-stage as
described in detail in the text. 25-bp scar sites are shown as red, green, purple and orange rectangles
in the final assembly. Circular objects indicate the bacterial ori of replication (black) and bacterial
resistance gene: KanR (blue) and AmpR (orange) contained in the “bacterial” part of the assembly,
which is typically discarded before yeast cell transformation
(Berrow et al. 2007), MAGIC (Li and Elledge 2005) and SEFC (Zhu et al. 2010)
systems using homologous recombination either in vitro or within E. coli cells.
Soon after setting the principles of the GW method, first reports on its imple-
mentation into yeasts systems were published. Funk proposed and validated a
system of 32 acceptor vectors for att-based cloning in S. cerevisiae with four
different promoters (two constitutive and two inducible), four different epitopes
for protein tagging and four auxotrophy selection markers (Funk et al. 2002).
The destination vectors are compatible with donor vectors bearing elements to
be cloned flanked with attB sites. Operability of the system was demonstrated
using a fluorescent reporter protein. Similarly, at approximately the same time, a
set of 20 destination vectors for recombinant protein production in S. cerevisiae
was designed and constructed in accordance with GW cloning (Van Mullem et al.
2003). In that toolkit, the vectors differ in the selection marker modules (2 variants
available) and the epitope modules for protein tagging (four variants available).
Operability of the system was validated with two entry vectors bearing differ-
ent genes. In 2007, an extensive library of 285 S. cerevisiae- and GW-compatible
destination vectors was developed, and made available to the yeast community
by Addgene (Alberti et al. 2007). The vectors were constructed based on com-
monly used pRS shuttle vector series. Variants covered by that collection bear
either constitutive or inducible promoters and one of several options for epitope
tagging (with a short tag or a fluorescent fusion partner). Specific implementations
of the GW toolkits in S. cerevisiae metabolic engineering cover, e.g., enhanced
synthesis of phenylalanine for improved production of styrene (McKenna et al.
2014), or engineering acetyl-CoA synthesis by introducing alternative acetyl-CoA
synthetases (out of 4 modules prepared) (Kozak et al. 2014).
All the mentioned above GW vectors were intended for a single biopart (gene)
modular cloning, but the multisite GW system was also used in S. cerevisiae.
The first GW system for multipart assembly was designed for 3-modules fusion
(Nagels Durand et al. 2012). The authors presented a set of 3 destination vectors
for gene expression in S. cerevisiae, enabling fast assembly of any promoter, open
reading frame, and epitope tag in combination with any of 4 auxotrophic markers
and 3 distinct replication mechanisms. The system was proved operable based on
an example of a 3-domain protein complex. A set of GW shuttle vectors for 2-
biopart assemblies was presented by (Giuraniuc et al. 2013). The authors used the
system to develop 2-parts fusions to make a new, synthetic regulatory element, and
to generate combinatorial fusions between five different promoters and 12 ORFs.
The first implementation of GW standard to K. phaffii system was reported by
(Esposito et al. 2005). The authors generated a GW destination vector by mod-
ification of a popular pPICZ vector for secretory overproduction of proteins in
a methanol-inducible manner. Interestingly, the authors used modified attB sites,
which were reported to give 4-times more clones after transformation, than the
original attBs. Of key importance was the fact, the authors evidenced a lack
of negative impact of the scar (att site) left between N-terminal fusion and the
mature polypeptide-encoding sequence. With the same aim and highly correspond-
ing approach, two GW-/K. phaffii-compatible destination vectors were developed
based on pPICZ and pBGP1 (inducible/constitutive promoter) (Sasagawa et al.

2011). The vectors were used for single biopart cloning of secretory proteins
tagged with an epitope and an additional 6xHis tag. Operability of the new vectors
was tested with different hydrolase-encoding proteins.
A comprehensive toolkit for high-throughput gene cloning according to the GW
principle was proposed for Y. lipolytica (Leplat et al. 2015). The authors devel-
oped a destination vector with a species-specific promoter, selection marker and
terminator, enabling att-recombination cloning under control of a strong constitu-
tive promoter. Moreover, a docking platform within Y. lipolytica strain’s genome
was constructed. The platform was located within ura3 locus (generating auxotro-
phy for the following GW-cassettes cloning), bearing a fluorescent reporter and
zeta-docking sites for homologous recombination with the GW-generated cassette.
The system was validated with a series of fluorescent and enzymatic reporters.
In the following study by the same Research Team, the GW system was used
for optimization of heterologous reporter genes overproduction by adopting pro-
moter shuffling approach (Dulermo et al. 2017). The authors took advantage of
modularity offered by GW cloning and prepared modules for 7 promoters of
different strengths and 3 reporter proteins (fluorescent and enzymatic). Random
shuffling of different promoters was conducted for each of the reporters indi-
vidually, which allowed for interesting observations. In the following study, the
authors cloned over 150 genes in the entry vectors and, using a newly developed
high-throughput cloning protocol, subcloned them to a pre-developed destination
vector, and ultimately—transformed the assemblies to a compatible Y. lipolytica
host strain (Leplat et al. 2018). It was possible to conduct large-scale screens
for the desired trait. That study is an excellent demonstration of GW cloning’s
high-throughput character and its pronounced capacity. The proposed system and
developed protocols are very convenient and efficient in terms of high-throughput
testing of different genes overexpression in Y. lipolytica.
4 Overlap Assembly Strategies
Overlap assembly strategies rely on exploitation of binary primers, partly comple-

mentary to DNA sequences of the two fused bioparts. The primers can be designed
to leave no scar between the two modules if they are composed solely of the
adjacent bioparts sequences. No scaffold per se is needed, as the primers contain
only the adjacent modules’ sequences. On the other hand, all the elements must
be specifically designed, and no generic elements are typically used. When used
without a scaffold, the system lacks a modularity option. Depending on the spe-
cific approach, the bioparts termini are processed by different enzymes to generate
single-strand DNA overhangs, that are ligated in the next step. In terms of the
assembly reaction precision, OL assembly techniques are considered to be less
efficient and precise when compared to RE / Rec-based techniques. For the classic
experiment published by (Gibson 2009) on “in yeasto” assembly of short overlap-
ping oligomers, frequently reported efficiency is 64.3%. However, it relates to the
number of correctly assembled full-length products, but—bearing mutations. The

actual number of full-length clones, with an identical sequence as designed was
approx. 10%. Showing that OL-based techniques tend to be more susceptible to
errors. On the other hand, the modules preparation stage is much simplified, as no
elimination of internal RE RS step is needed.
4.1 Gibson
The principle of Gibson (GB) assembly technique relies on exploitation of specific

binary primers covering sequences of adjacent modules to be fused and a mix of
three enzymatic activities processing termini of the modules. So, by definition, the
modularity of this technique is impaired as no scaffold is used. On the other hand,
GB does not leave any scars at the fusion sites. During amplification reaction, the
binary primers introduce 15–40-bp OLs between the adjacent elements and it is
the only step required for the preparation of the modules (Fig. 6). Complementary
Fig. 6 General outline of Gibson cloning (e.g., promoter, ORF and terminator). Each of the mod-
ules is first amplified using binary primers covering sequences of adjacent elements (indicated as
red, purple, orange and green OL XXX). The OLs are typically 40 nucleotides in length and define
the final arrangement of the construct. The reaction is two-stage as described in detail in the text.
No scars are left at the fusion sites. Circular objects indicate the bacterial ori of replication (black)
and ampicillin resistance gene AmpR (orange) contained in the “bacterial” part of the assembly,
which is typically discarded before yeast cell transformation
OLs must be also present in the destination vector (OL compatible with 5’ of the
first module and 3’ of the last module within the assembly). Overlapping bioparts
are mixed in a single tube with the destination vector and subjected to the action of
three different enzymes. The 5’-exonuclease generates single-stranded DNA OLs
by acting from the 5’-end so that the complementary regions are exposed and can
be annealed. High-fidelity DNA polymerase fills the gaps and Taq DNA ligase
covalently joins fragments. The reaction is a single step and isothermal (50 °C,
over 15–60 min). Typically, with the use of a commercial mix of enzymes, up
to 5 modules can be assembled simultaneously in a single-tube reaction using a
one-step master mix of enzymes, whereas a two-step reaction allows combining as
many as 15 bioparts. The size limit for this in vitro DNA assembly is unknown.
Typically, efficient cloning of the cassettes reaching 20-kbp is reported, but the
largest demonstrated construct has reached 900-kbp (Gibson 2009; Gibson et al.
2009). The GB method was a basis for the development of other techniques, like
MODAL (Casini et al. 2013) and UNS (Unique Nucleotide Sequences)-guided
isothermal assembly (Torella et al. 2014).
The GB method’s principles were first described in a paper on the enzymatic
assembly of DNA bioparts in vitro (Gibson et al. 2009), where an isothermal,
single-step reaction for assembling multiple overlapping DNA molecules by the
concerted action of the three enzymes was described. As in the other OL-based
techniques, no sensu stricte scaffold is needed (as it is for the GG method).
Recently, (Cataldo et al. 2020) constructed an expression cassette for S. cerevisiae
via the so-called “full in vitro Gibson assembly.” The authors observed a toxic
effect of the cloned genes on E. coli (the subcloning host). The in vitro-assembled
GB cassette was composed of 8 bioparts (2 genes, bidirectional promoter, 2 ter-
minators, up and down elements for integration, and a selection marker) bearing
40 bp OLs. To bypass the bottleneck of subcloning in E. coli, the full-length assem-
bly was used as a template in a PCR. The amplified cassette was then transformed
into the final host—S. cerevisiae.
Several interesting implementations of the GB method to complex DNA assem-
blies construction for K. phaffii were described. Combination of OE-PCR and
GB assembly methods was adopted to construct an extensive, 12-bioparts bear-
ing DNA assembly in 12 different variants (Vogl et al. 2016). A 4-gene pathway
for carotenoids synthesis was used as a model. At first, the authors generated a
library of 12 methanol-dependent promoters of different strengths and a differ-
ent mechanism of induction; and an additional collection of 4 terminators. They
assembled a complete expression cassette in a step-wise manner. First, 3 indi-
vidual elements building individual TUs (promoter, gene, terminator) were fused
using OE-PCR. Subsequently, 4 such TUs were assembled via the GB method to
generate a complete DNA construction. A combinatorial approach was followed to
construct different variants of the complete DNA assembly, each bearing 4 TUs.
This way, in the initial OE-PCR, 3 bioparts were assembled, and in the follow-
ing GB reaction, 4 modules (each bearing individual TU) were shuffled to build a
complete carotenoid pathway, but controlled by different regulatory elements. The
strategy enabled fine-tuning of the model pathway.
GB assembly was used to optimize vectors for CRISPR-Cas9-based genome

editing in K. phaffii (Weninger et al. 2016). In that study, full advantage of the
GB method’s efficiency and modularity was taken. The authors shuffled multiple
elements in the CRISPR-Cas9 vectors (constructed on a pPpT4_GAP shuttle vec-
tor’s backbone), including gRNA, ribozymes and structural components of sgRNA,
promoters for Cas9 and sgRNA expression, as well as different variants of codon-
optimized Cas9 with different fusions. From among 95 DNA assemblies, only 5
were operable. The optimized system was proved highly efficient in multiplexed
gene deletions and genomic integration of DNA expression cassettes.
Based on conducted literature search, it seems, that the yeast community work-
ing with K. phaffii widely uses GB technology for both: more complex cloning
strategies (Royle and Polizzi 2017) and routine single-gene assemblies (Torres
et al. 2019). Likewise, GB is broadly used for the generation of various DNA
constructions dedicated to cloning in Y. lipolytica. By the simple introduction
of specific OLs in the primers used during DNA fragments amplification and
the following GB reaction, multiple different constructions were generated in
work on glycogen synthase modification in Y. lipolytica (Bhutada et al. 2017) or
xylose utilization pathway engineering (Rodriguez et al. 2016); where it enabled
rapid incorporation of sgRNA into modular pCRISPRyl, or assembly of multipart
overexpression cassettes.
4.2 USER®
Uracil-Specific Excision Reaction (USER®; NEB, New England Biolabs)-based

cloning is a RE- and ligase-independent technique that enables assembly of mul-
tiple DNA fragments by the means of short OLs of 7–12 bp in length (Nour-Eldin
et al. 2006). The basic principle is to use binary primers (as in GB), complemen-
tary to both adjacent elements, to generate the bioparts. But the key distinguishing
element is to replace a single deoxythymidine (dT ) nucleotide with one deoxyuri-
dine (dU) nucleotide at the 3’ terminus of the primers (Fig. 7). The primers are
used at the stage of the preparation of the modules, so the DNA polymerase used
here must be tolerant to dU (e.g. PfuX7 (Nørholm 2010)). The presence of dU at
the borders of the modules precisely marks the site for an uracil DNA glycosylase
and endonuclease VIII, contained in the Uracil-specific excision reagent. By the
action of the two enzymes, the uracil residue is excised together with upstream
sequence, releasing single-stranded 3’OL of 7–12-bp, permitting the directional
assembly of several modules (up to 7). The modules are assembled in a single-
tube reaction incubated at 37 °C for 20 min, and 25 °C for 20 min, reaching up to
90% efficiency (Lund et al. 2014). While no scar is present between the two fused
modules, a 13-bp fragment of the USER cassette is present between the vector
backbone and the assembly. A panel of yeast species-specific modules compatible
with USER cloning is available from Addgene.
Fig. 7 General outline of USER® cloning (e.g., promoter, ORF and terminator). Each of the mod-
ules is first amplified using binary primers covering sequences of adjacent elements (indicated as
red, purple, orange and green XXX). The primers include a single deoxyuracil residue (dU) flank-
ing the 3’ ends of the homology region. The OLs are typically 7–12 nucleotides in length and define
the final arrangement of the construct. The reaction details are given in the text. No scars are left
at the fusion sites. Circular objects indicate the bacterial ori of replication (black) and ampicillin
resistance gene AmpR (orange) contained in the “bacterial” part of the assembly, which is typically
discarded before yeast cells transformation
Several interesting implementations of the USER cloning strategy to the

selected yeast species genetic engineering have been published. In 2012, the tech-
nique was exploited for the construction of a comprehensive cloning system for
complex pathway assembly and their site-specific genomic integration in S. cere-
visiae (Mikkelsen et al. 2012). The authors generated a chassis for cloning up
to 22 genes: 2 genes under bidirectional promoter per DNA assembly and 11
validated, individual integration sites within S. cerevisiae’s genome. The inte-
gration sites in the yeast genome were validated by tracking growth rate and
reporter enzyme synthesis after a reporter cassette integration. The operability of
the system was demonstrated by the heterologous expression of an 8-gene path-
way for indolylglucosinolate synthesis. The generated destination vector comprised
genomic integration targeting sites (up and down), 2 terminators, auxotrophic
selection marker, and the “bacterial part.” Three modules were fused in each reac-
tion to obtain operable cassettes—two genes flanking a bidirectional promoter.
The 8-gene pathway was thus generated by construction of 4 expression cassettes,
each bearing 2 genes under a bidirectional promoter. Each time, 3 modules were
fused at 4 fusion sites, resulting in expression cassettes of 3–4-kbp (without the
backbone vector). The whole pathway was assembled in the yeast host by 4 con-
secutive transformations with the selection marker recycling in-between. In that
report, marker recycling was facilitated by flanking the gene by direct repeats.
Soon after that report, the same Research Group evolved a previously developed
USER-based system and developed EasyClone system for iterative integration of
DNA constructions assisted with a selection marker recycling system based on a
loxP-Cre mechanism (Jensen et al. 2014). The USER-constructed system covered
both episomal and integrative destination vectors. Their construction was iden-
tical as previously (Mikkelsen et al. 2012) with the difference in the selection
marker gene flanks—in the updated version—loxP elements. USER-reaction was
each time conducted with a constitutive promoter and a fluorescent reporter protein
(out of 3 available). The authors sequentially transformed the host strain, recycled
the selection marker, reaching up to 3 reporters coexpression.
USER cloning was combined with the CRISPR-Cas9 genome-editing system
to create the EasyClone-MarkerFree vector toolkit for S. cerevisiae (Jessop-Fabre
et al., 2016). The new system was developed on a backbone of EasyClone 2.0
vectors, by amplifying a fragment with bacterial elements, specific homology-
integration sites, and the USER-cloning site flanked with terminators. Most
importantly – the yeast selection markers were not included in the new backbone
vector. The expression cassettes (two genes flanking a bidirectional promoter) to be
cloned in such a new backbone vector were first assembled (via USER) and then
fused with the marker-less vector. To precisely direct genomic integration of these
cassettes, a gRNA-expressing vector was prepared. In the proof-of-concept exam-
ple, the authors prepared three USER-cloned assemblies, each bearing two genes
under bidirectional promoter and specific homology-integration regions, and a vec-
tor bearing 3 gRNAs, compatible with the homology regions. The three expression
cassettes were then cotransformed with the triple gRNA vector into a S. cerevisiae
strain already synthetizing Cas9 from an episomal vector. The cassettes are then
integrated into loci specified by gRNA and the 500-bp homology regions. The
vector expressing gRNAs is then lost by subculturing the strains in a non-selective
medium. Similarly, Cas9-bearing helper vector could be lost, if no further engi-
neering was planned. The developed system enables simultaneous introduction of
up to three integration cassettes into the genome of S. cerevisiae without the use
of any yeast selection markers. Using standardized USER-bioparts, the integration
cassettes can be constructed for overexpression of one or two genes per integra-
tion site. The authors suggested that for better stability, the selected integration
sites should not be in direct proximity to each other. The system was validated not
only in laboratory strain (CEN.PK) but also in an industrial diploid (EthanolRed),
which is of great interest. The EasyClone-MarkerFree vector toolkit is available to
the yeast community from Addgene.
The USER® assembly system was used in multiple following studies by that
Research Team. One of the interesting examples is a paper presenting a method
enabling simultaneous disruption of multiple genetic targets, where a combina-
tion of CRISPR/Cas9, in vivo recombination, USER assembly, and RNAi was
developed (Kildegaard et al. 2019). USER cloning was used there for assembly
of multipart expression cassettes. Interestingly, the authors improved the modular-
ity of USER by introducing standardized spacers/overhangs of 60-bp at the fusion
sites, flanking individual TUs. These were used in combination with standard over-
hangs adopted for USER cloning, for fusions inside the TUs. Such specific design
allowed reusing a standard set of primers for amplification of different TUs, so
the genes and regulatory elements could be cloned in a combinatorial manner. By
adopting that sophisticated system of cloning, the authors constructed an exten-
sive assembly, covering 3 TUs, with a selection marker element, and 2 genomic
integration regions flanking the whole assembly.
The USER-based EasyClone system was also adopted for Y. lipolytica in a
form of a comprehensive toolbox (Holkenbrink et al. 2018). The system com-
prises a broad set of integrative expression vectors, compatible with USER cloning,
which allows cloning up to 2 genes. As in the case of the EasyClone toolkit for
S. cerevisiae, the “Yali” system was extensively studied in terms of the selection
of intergenic and highly transcribed integration loci. As for the former system,
stringent requirements were set to propose candidate integration sequences, and
the presumptions were carefully verified using a reporter protein system. Within
the EasyClone Yali toolbox, the Authors proposed 5 integrative, marker-less vec-
tors, precisely directed for integration by CRISPR/Cas9 system, and 11 vectors
to be integrated and maintained using auxotrophic/resistance selection mark-
ers. Additionally, the vectors and protocols for marker-less deletion of up to 2
genes simultaneously using the CRISPR/Cas9 system were provided. Using 90-bp
double-stranded oligos as the DNA repair templates, the authors obtained efficien-
cies of up to 90% and 66% for individual and double knockouts, respectively.
The expression/integration cassette construction in EasyClone Yali is facilitated
by USER cloning and a rich collection of standardized and reusable bioparts. The
EasyCloneYali vectors can be obtained via Addgene. Operability and robustness of
the system were evidenced in another paper by that research Team on cloning and
optimization of astaxanthin synthesis pathway in Y. lipolytica (Kildegaard et al.
2017). In that study, a great variety of diverse combinations of 6 genes of differ-
ent origins was shuffled and cloned in both laboratory auxotrophs and native Y.
lipolytica strains. Thanks to intensive engineering of several side-branches of the
terpenes synthesis pathway, the flux through the target astaxanthin was enhanced.
Among the other, efficient cloning techniques were the enabling factor for that
study.
4.3 OE-PCR
Overlap Extension Polymerase Chain Reaction (OE-PCR) is a method that enables

extending DNA fragments of interest to obtain assembled DNA constructions. In
its standard form, OE-PCR is not a typical module assembly strategy, as no scaf-
fold and no specific fusions system is needed. But the technique was proven to
be useful for site-directed mutagenesis (Ho et al. 1989) and gene splicing (Horton
et al. 1989). In principle, the assembly takes place solely by PCR (DNA ampli-
fication by DNA polymerase). First, the bioparts are individually amplified using
binary primers partly complementary to the adjacent elements. 20–50-bp of com-

plementarity was proved sufficient (Anthony and Liebman 1995; Nelson and Fitch
2011). In the next step, the bioparts are pooled in a single tube for PCR. The
denaturation step renders single-stranded DNA and the complementary regions of
the adjacent fragments act as priming sites for DNA polymerase at the annealing
step (so-called “auto-priming”). Hence, the fragments can be fused in a PCR with-
out additional, flanking oligonucleotide primers (Fig. 8). Typical primers, flanking
a complete assembly from 5’ and 3’ termini, are added to the reaction after sev-
eral rounds of auto-priming. As reported, the OE-PCR allows assembling up to 8
elements of different lengths in a single in vitro reaction (Kadkhodaei et al. 2016).
The reaction is conducted under a standard PCR cycling profile, adjusted to the
properties of the assembled elements. Due to its simplicity and wide availability,
this method has gained popularity, but rather for assembling a limited number of
small elements. The biggest drawback in terms of the complex assemblies gener-
ation is low efficiency of fusion, and that typical OE-PCR is not amenable neither
for standardization nor modularity, as no specific scaffold is generated and no des-
tination vectors are necessary. It was demonstrated that even upon using 40-bp OLs
Fig. 8 General outline of OE_PCR assembly strategy. Each of the modules is first amplified using
binary primers (represented as letters A-H) covering sequences of adjacent elements: promoter
(solid lines), open reading frame (ORF, dotted lines), terminator (dashes and dots) and destina-
tion vector. Complementary regions to the adjacent parts are marked in colors (red, purple, orange
and green squares). Product AB is generated using “A” and “B” primers on the promoter template.
Parts “CD” “EF” and “GH” are amplified in the same way. Restriction enzyme sites are intro-
duced in primers (black boxes). The reaction details are given in the text. No scars are left at the
fusion sites. Circular objects indicate the bacterial ori of replication (black) and ampicillin resis-
tance gene AmpR (orange) contained in the “bacterial” part of the assembly, which is typically
discarded before yeast cells transformation
between adjacent fragments, the fusion product may not be correctly assembled
(Cha-aim et al. 2009). However, several specific improvements and modifications
were incorporated to the techniques, to address these issues (Xiao and Pei 2011;
Guo et al. 2019). For example in 2007, (Dong et al. 2007) improved the OE-PCR
technique by introducing into the protocol several elements, like primers design
by DNA Works software instead of manual design, provided the Melting Temper-
atures™ of the OLs to ensure their specificity, reduced the length of OLs down
to < 5 bp, and promoted the use of high fidelity DNA polymerase, which was
specifically designed for OE-PCR conditions. The technique in the new format
was validated with several genes of ~1–1.5 kbp.
While in its basic format OE-PCR technique may seem inferior to the other
modular cloning and DNA assembly techniques, several interesting modifications
advanced the technique and scope of its exploitation. To more easily apply OE-
PCR to the generation of DNA assemblies and improve its specificity, a system
of short, GC-rich OLs at the fusion sites was proposed (Cha-aim et al. 2009).
The authors demonstrated that introduction of short G/C stretches can significantly
improve specific annealing of compatible DNA fragments in a fusion PCR. Pre-
cisely, C(15) OL (but not GC repeat) turned out to be highly efficient in directing
specific hybridization between the fusion elements under all examined tempera-
tures. Such stretch cannot be used for fusion of functionally related parts (signal
peptide and mature polypeptide or promoter and ORF) but could be used for fusion
of independent bioparts—integration sites, selection markers, TUs. The authors
demonstrated operability of their method on 3 bioparts fusion in a single reaction.
In 2009 (Shao et al. 2009) reported on “DNA Assembler” strategy for “in
yeasto” assembly of complex, multi-element DNA constructions. In fact, the
method highly corresponds to the initial, model experiment described by (Gibson
2009), when a 1170-bp DNA fragment was dissected into 38 short oligomers (60-
bp long) and flanked with 20-bp complementarity regions to adjacent fragments,
that were assembled “in yeasto.” The key difference between the two reports is
the size of the assembled bioparts. Gibson assembled ~1 kb fragment from 38
synthetic oligos, and (Shao et al. 2009) executed complex pathway assembly of
>10-kpb. Indeed, both techniques should be classified in the same sort of cloning
strategies, as they rely on identical principles. Basically, the method relies on
designing short OLs flanking adjacent fragments to be assembled. These can be
either short oligos (Gibson 2009), or modules bearing regulatory elements and
heterologous genes, as in the case of (Shao et al. 2009). For the latter, all proof-
of-concept cassettes were assembled step-wise. The cassettes were as follows: (i)
xylose utilization pathway (∼9 kb DNA consisting of 3 genes), (ii) zeaxanthin
biosynthesis pathway (∼11 kb DNA consisting of 5 genes) and (iii) combined
xylose utilization and zeaxanthin biosynthesis pathway (∼19 kb consisting of 8
genes). Basically, all TUs were assembled in vitro by standard OE-PCR. Adja-
cent TUs were already flanked with specific OLs, enabling correct assembly in
desired orientation upon transformation into the yeast cell. The length of OLs
was individually adjusted to the complexity of the DNA construction—less com-
plex assembly (3–5 genes, ~10 kb) could be correctly fused with only 50-bp OLs
reaching very high efficiency of 80–100%, while for more complex DNA construc-
tions (8 genes, ~19 kb) the efficiency of 40–70% could be achieved with longer
OLs (~125–430 bp). It was also demonstrated that by increasing the amount (in
ng) of the modules per the same amount of the backbone vector, the efficiency
of assembly can be increased. The authors stressed simplicity and rapid character
of the DNA assembly method, which actually requires only appropriate design of
PCR and exploitation of native recombination system of the yeast cell.
Many other, aim-specific implementations of OE-PCR to S. cerevisiae genetic
engineering have been described. Just to give several different examples, OE-PCR
was exploited for high-throughput production of linear DNA templates, that were
later processed by cell-free transcription-translation system from S. cerevisiae (Gan
and Jewett 2014), or for generation of chimeric genes involved in phenylpropanoid
pathway (Jiang et al. 2005) or assembly of resveratrol synthesis pathway (Wang
et al. 2011).
Likewise, multiple literature reports demonstrate wide applicability of OE-PCR
for the assembly of synthetic DNA constructions that are later used for K. phaffii
modifications. OE-PCR was used to adjust standard pGAPZα vector for removing
unnecessary elements, and high-throughput mutagenesis of Candida rugosa lipase
to be overproduced in K. phaffii (Chang et al. 2005). The scientific aim of that
study was to exchange a rare codon for a frequent codon, which improved enzyme
production. OE-PCR was also used in K. phaffii-related applications to conduct
in vitro splicing (Jeya et al. 2009), or generate chimeric DNA templates encoding
a gene with a fusion partner and cell-surface anchor (Yang et al. 2017).
The “DNA Assembler” technique reported by (Shao et al. 2009) was later
“transplanted” to Y. lipolytica system to rapidly assemble multipart DNA cassettes
(Gao et al. 2014). While the authors termed their cloning strategy as one-step
assembly, in fact the approach was executed in a step-wise manner. First, the TUs
and the flanking regions homologous to rDNA sequences in Y. lipolytica genome
were assembled by OE-PCR in groups of 3–4 bioparts. Terminal elements of indi-
vidual OE-PCR-assembled TUs were overlapping adjacent bioparts according to
a design. The OLs between the adjacent parts were 45–60-bp, depending on the
constructed variant. Such pre-assembled TUs were then mixed and transformed
into the recipient strains which assembled them. The efficiency of the assembly
reaction of a 4-gene pathway ranged between 11 and 21%, but the most impor-
tant advancement relates to a decrease in the time necessary for the procedure
completion. According to the authors calculations, the total procedure could be
completed in less than one week, as compared to a previously reported sequential
gene integration method that required n weeks for n genes (Celińska and Grajek
2013).
In 2020, (Guo et al. 2020) used OE-PCR to assemble 9 elements, altogether
forming the first completely synthetic yeast artificial chromosome for Y. lipolytica
(Yl-YAC), with centrally located ARS and flanked with telomeres. As the authors
mainly aimed at optimization of Yl-YAC construction, they adopted combinatorial
approach to testing different combinations of chromosomal elements (ARS and
TEL) and reporter gene-encoding modules, which were transformed into Y. lipoly-
tica cells as free DNA elements. Each module contained 50-bp flanking regions
compatible with adjacent sequence, according to a design. Importantly, as in the
case of previous works with S. cerevisiae, Y. lipolytica native recombination mech-
anisms correctly assembled pre-designed DNA construction. After examining the
replicative character and transferability of the Yl-YAC, the authors set at trans-
ferring both xylose and cellobiose utilization pathways to Y. lipolytica within the
newly constructed Yl-YAC. A complete construction comprised three key genes for
xylose (XYL1, XYL2 and XKS1) and three genes for cellobiose utilization (CBP1,
CDT1 and scPGM2), of total length 23 kb.
5 Summary
A great variety of other modular cloning and DNA parts assembly methods have
been developed, that were not covered by the main part of this chapter. For specific
reasons, those methods did not gain popularity in the yeast research community.
One of such methods is ligase cycling reaction (LCR) that employs single-stranded
bridging oligomers (partly complementary to adjacent bioparts) that guide correct
hybridization of DNA fragments. The bioparts to be fused must be phosphorylated
at 5’ends (either by kinase or by amplification with 5’phosphorylated primers).
The method is amenable to modularity by possible use of a 40-bp scaffold of
oligonucleotide connectors (complementary to adjacent elements) which can be
reused in alternative assembly design (Kok et al. 2014). The LCR is executed by
hot-start thermostable ligase and polymerase under cycling profile of denaturation,
annealing of the fragments and ligation at 66 °C. As reported, up to 20 bioparts of
total length 20 kbp can be assembled in a single reaction without leaving scars at
the fusion sites. The other such technique is circular polymerase extension cloning
(CPEC) conducted with DNA polymerase solely (Quan and Tian 2009). The frag-
ments must bear 15–25 bp OLs to anneal correctly. CPEC was reported to be
highly efficient, but only up to 4 bioparts.
Irrespectively of technical details, modular cloning and standardized bioparts
assembly strategies contribute to great progress within the field of genetic engi-
neering of yeast. Their exploitation allows the researchers to focus on addressing
the scientific questions, and to expand the scope of experiments to accurately test
hypotheses, rather than execution of tedious laboratory procedures. Modularity and
standardization facilitate wide-spread collaboration within the yeast community
and speed-up accomplishing of new goals.
Acknowledgements PKW was financially supported by the Ministry of Sciences and Higher Edu-
cation in Poland project: DI 2017 000947. MK was financially supported by the Ministry of Sciences
and Higher Education in Poland project: DI2017 001047.
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Cellular Engineering of Yarrowia
lipolytica for Biomanufacturing
of High-Value Products from Oils
and Fats
Na Liu, Ya-Hue Valerie Soong, Andrew Olson, and Dongming Xie
Abstract
Yarrowia lipolytica is a safe and robust yeast to efficiently use lipid as the sole
carbon source, which provides us opportunities for biomanufacturing of a series
of high-value products from cost-effective agriculture feedstocks such as plant
oils and animal fats. Y. lipolytica has a unique propensity for high flux through
tricarboxylic acid (TCA) cycle intermediates and biological precursors such as
acetyl-CoA and malonyl-CoA that can be diverted into a variety of heterolo-
gous value-added bioproducts. With recent advances in metabolic engineering
and synthetic biology tools, the potential of using Y. lipolytica for biomanufac-
turing of high-value products has been expanded. Examples include industrial
enzymes, extracellular proteins, fatty alcohols, wax esters, long-chain diacids,
omega-3 fatty acids, and carotenoids. For large-scale biomanufacturing using
oils/fats as substrate, the poor mixing and mass transfer caused by the insolu-
bility of substrates in an aqueous medium is one of the major challenges that
have to be addressed in addition to the pathway engineering and optimization
for both fatty acid biosynthesis and conversion. The multi-phase computational
fluid dynamics (CFD) simulation can be used as a powerful tool for analy-
sis of mixing and mass transfer behaviors in bioreactors and further guide the
bioreactor design and optimization of operating conditions. Cell morphology
has a profound effect on cell growth, oil substrate uptake, and product forma-
tion. Both PKA and cAMP-dependent signaling pathways are involved in the
dimorphic transition in Y. lipolytica. Maintaining the dimorphic yeast shape via
Na Liu and Ya-Hue Valerie Soong are contribute equally to the paper.
N. Liu · Y.-H. V. Soong · A. Olson · D. Xie (B)

Department of Chemical Engineering, University of Massachusetts Lowell, Lowell, MA 01854,
USA

https://doi.org/10.1007/978-3-030-89680-5_3
64 N. Liu et al.
morphology engineering strategies has been explored. This chapter also intro-
duced several examples of how we combined cell morphology engineering,
metabolic pathway optimization, and bioreaction engineering to significantly
improve the production of intracellular lipids, citric acid, and wax esters from
plant oils. Potential strategies for further improving the biosynthesis efficiency
via transporter engineering in yeast were also introduced.
Keywords
Biomanufacturing • Metabolic engineering • Cell morphology • CFD

simulation • Oils and fats • Yarrowia lipolytica
1 Introduction
Yarrowia lipolytica is one of the most intensively studied “non-conventional” yeast

which possesses the potential to act as a biotechnological workhorse. It has a wide
range of biotechnological applications that include degradation of hydrophobic
substrates such as fatty acids (FAs), oils, fats, n-alkanes, production of organic
acids, and secretion of homologous and heterologous proteins (Morales-Vargas
et al. 2012). Y. lipolytica has a unique propensity for high flux through tricarboxylic
acid (TCA) cycle intermediates and biological precursors such as acetyl-CoA
and malonyl-CoA that can be diverted into a variety of heterologous products.
Y. lipolytica is generally regarded as safe (GRAS) by the American Food and
Drug Administration (FDA) (Soong et al. 2019). This safety approval is particu-
larly important for broadening the range of possible applications of the products
derived from the fermentation of Y. lipolytica.
The dimorphic yeast Y. lipolytica has been considered an adequate model for
dimorphism studies, as it can grow as yeast-like (single oval cells) or in the form of
a mycelium (pseudohypha and hypha) depending on the environmental conditions,
and to be reversible between each one (Kawasse et al. 2003). Under nutrient-
rich conditions, the organism grows as a mixture of yeast-like and short mycelial
cells (Ruiz-Herrera and Sentandreu 2002). It is believed that yeast dimorphism
is related to a defense mechanism to adverse conditions (Kawasse et al. 2003).
Different effectors have been reported to be implicated in the dimorphic transi-
tion of Y. lipolytica. There are a number of genes that have been identified as
apparent regulators of the dimorphic transition of Y. lipolytica, the genes YlMHY1,
YlHOY1, YlBEM1, YlCLA4, YlRPK1, YlTPK1, YlRAS2, and YlSTE11 have been
reported responsible for yeast-to-hypha transition in Y. lipolytica while the genes
YlTPK1 and YlZNC1 have been reported as negative regulators of Y. lipolytica fil-
amentation (Bankar et al. 2018; Martinez-Vazquez et al. 2013; Pomraning et al.
2018; Soong et al. 2019).
Y. lipolytica is often found naturally in environments rich in hydrophobic sub-
strates, such as oils, fats, fatty acids, or n-alkanes. The unique physiological
features that Y. lipolytica has developed to grow in oils and fats have made this
Cellular Engineering of Yarrowia lipolytica … 65
yeast an important biotechnological yeast, which has been proposed for the treat-
ment of petroleum oil-polluted soil or water (Ledesma-Amaro and Nicaud 2016b).
In addition, Y. lipolytica has been efficiently cultivated on oils or fats for pro-
ducing many intra- or extracellular metabolites of industrial significance (Fickers
et al. 2005a, b; Papanikolaou et al. 2007). Vegetable oils and animal fats were
reported as promising substrates for biosurfactant production, as the oily or fatty
carbon sources are consumed by microorganisms could work as a building block
for biosurfactant synthesis (Goncalves et al. 2014). Various studies have indicated
that the production and secretion of lipases in Y. lipolytica strains are stimulated
by the presence of long-chain fatty acids (LCFAs) (Fickers et al. 2003), plant oils
(Braga et al. 2012; Deive et al. 2010; Kebabci and Cihangir 2012; Najjar, et al.
2011), and animal fats (Kamzolova et al. 2005) in the culture medium. Conversely,
glucose in the medium might repress the production of lipases (Liu et al. 2021).
The oleaginous yeast Y. lipolytica has been reported capable of accumulating a
significant amount of intracellular lipid, stored in lipid bodies, during growth on
vegetable oil (Najjar et al. 2011) and animal fats or their industrial derivatives
(Papanikolaou et al. 2002, 2007). With recent improvements in the synthetic biol-
ogy tools, the industrial potential of Y. lipolytica has been expanded to include
organic acids such as citric acid, α-ketoglutaric acid, and itaconic acid, enzymes
such as lipases, RNase, and esterase, lipids and lipid-derived compounds such as
biodiesel, dicarboxylic acids, and biosurfactants (Liu et al. 2021).
The great potential for industrial applications of Y. lipolytica has driven the
development of metabolic engineering tools for it as well as the basic research to
understand its physiological features. In recent years, different metabolic engineer-
ing tools and strategies have been created and applied in Y. lipolytica, which has
further expanded the application of this yeast.
This chapter summarizes the degradation of oil and fat substrates by Y. lipoly-
tica, fermentation and cellular engineering efforts of the Y. lipolytica metabolic
engineering program to improve the utilization efficiency and bioconversion of
oils and fats. The distribution of oil–water mixing in stirred bioreactor was studied
by computational fluid dynamics (CFD) simulation. The CFD results were further
used to guide the optimization of the bioreactor design and operation conditions
during bioreactor fermentation experiments. The effect of cell morphology on the
fermentation with lipid substrates was examined by creating the hyphal strain and
the yeast-like strain through overexpression and disruption of the gene YlMHY1
in the wild-type Y. lipolytica ATCC20362, respectively. The strategies of biore-
actor design and operation conditions and cell morphology engineering can also
be used for the production of other high-value products (e.g., fatty alcohol, wax
esters). The metabolic engineering for the biosynthesis of wax esters by Y. lipolyt-
ica from oils and fats is introduced as a case study. This chapter also summarizes
the engineering of transporters in yeast that are used to enhance bioproduction.
66 N. Liu et al.
2 Oils- or Fats-Substrate Utilization
Y. lipolytica is often found in environments with the presence of plant oils or ani-
mal fats, being able to assimilate FAs, oils, and fats efficiently. The route of oils
or fats substrates into the cells induces several modifications of the substrates to
improve their accessibility. The initial challenge is a contact between the poorly
water-miscible substrate, and the cell surface. Y. lipolytica can produce surfactants,
amphiphilic compounds consist of hydrophilic and hydrophobic moieties, which
can reduce surface and interfacial tensions in aqueous media and hydrophobic sub-
strates and also reduce the size of the oils/fats droplets, thus increasing the contact
between substrates and cell surface (Fickers et al. 2005a, b). In bioreactor fermen-
tation, this step could be additionally facilitated by powerful agitation. Moreover,
surfactants can also facilitate cell adhesion to oils/fats droplets (Beopoulos et al.
2009).
The main component of oils and fats is triglycerides (TAGs) which cannot be
directly uptake by Y. lipolytica, as no TAGs transporters have been identified (Liu
et al. 2021). Y. lipolytica secretes extracellular lipases (LIP2, LIP7 and LIP8),
which help hydrolyze TAGs to form glycerol and the respective free fatty acids
(FFAs). Grown in oils/fats leads to structural changes on the cells surface of Y.
lipolytica and results in the formation of protrusions that enable cells to take up
FFAs from the medium (Mlickova et al. 2004). The action of biosurfactant and
lipase occurs progressively, the formation of numerous small-sized droplets facil-
itating the surface-mediated substrate transport (Beopoulos et al. 2009). Once in
the cytoplasm, FFA becomes activated by conversion into a fatty acyl-CoA by
the enzyme acyl-CoA synthetase FAA1. Then the fatty acyl-CoA can enter the
Kennedy pathway to be stored into the lipid body as TAGs (formed by DGA1 and
DGA2) (Abghari and Chen 2014), as precursors for the synthesis of fatty-acid-
derived products, or be transported into the peroxisome to carry out β-oxidation
(POX1-6, MFE1, and POT1). FFAs can be released from the TAGs through intra-
cellular lipases (TGL3 and TGL4), and thereafter, they can be activated to form
fatty acyl-CoA then transported into the peroxisome (Ledesma-Amaro et al. 2016).
The β-oxidation pathway, the main pathway for the breakdown of these fatty acyl-
CoA esters, is a four-reaction cycle. After each cycle, the CoA ester of FA gets
two carbons shorter and one molecule of acetyl-CoA is released. In Y. lipolytica,
the first and most important step of β-oxidation is carried out by six acyl-CoA
oxidases (POX1-6). The strain with POX1-6 genes knock-out is unable to degrade
FFAs resulting in a lipid accumulation (Wang et al. 1999). The second and third
steps in β-oxidation are catalyzed by the multi-functional enzyme (MFE). In con-
trast to acyl-CoA oxidases, which are encoded by six genes, MFE is encoded by
a single gene MFE1. The deletion of the MFE1 gene has been extensively studied
in Y. lipolytica for lipid production due to its technical simplicity (Blazeck et al.
2014). The fourth and last step is carried out by a peroxisomal thiolase POT1. As
β-oxidation takes place in the peroxisome, this pathway has been blocked by dis-
rupting the genes, PEX3, PEX10, and PEX11, involved in peroxisome biogenesis
Fig. 1 a Culture medium (blue) and oil (yellow) in the flask, oil stay on the top of the aque-
ous medium due to hydrophobic nature and light density. b Flask culture of Y. lipolytica with oil
substrate. c The assimilation of oils by Y. lipolytica: (i) Oil–water mixing was facilitated by surfac-
tant from Y. lipolytica; (ii) TAGs (oils/fats) are cleaved by extracellular lipases to give FFAs; (iii)
oils/FAAs droplets bind onto the cell surface; (iv) FAAs enter into cell interior via transport/export
mechanisms; (v) FAAs in the cytoplasm can be activated by cytoplasmic fatty acyl-CoA synthase
(FAA1); (vi) the activated fatty acid (fatty acyl-CoA) directly transported into the peroxisome for
β-oxidation degradation, (vii) converted into fatty acids derived products, or (viii) store into lipid
bodies as TAGs; (ix) lipid bodies could work as storage pools for fatty acids derived products,
TAG in lipid bodies could be hydrolyzed by lipases to release FFAs. Dash lines: Putative route,
not confirmed
(Ledesma-Amaro and Nicaud 2016b). The cycle is repeated several times, the-
oretically until the fatty acyl-CoA been completely breakdown. The acetyl-CoA
formed in β-oxidation is a key intracellular metabolite, which plays a major role
in various metabolic pathways that link catabolism and anabolism such as TCA
cycle, biosynthesis of acetyl-CoA-derived products (e.g., polyphenol, carotenoids)
(Fig. 1) (Chen et al. 2012).
3 CFD Modeling and Fermentation Engineering

for Improving Biosynthesis from Oils and Fats
In biomanufacturing processes, the growth of microorganism requires contact

between the substrate and cell surface. However, due to their relative immisci-
bility in water and the lighter density, oils/fats substrates tend to stay as a single
layer on the top of aqueous media. Y. lipolytica can produce some surfactants to
help emulsify oils/fats in the aqueous phase which is helpful in flask-scale culture.
However, in bioreactors fermentation, the transport of oils/fats from the organic
phase to the oils-aqueous medium and the delivery of the oils/fats droplets from
68 N. Liu et al.
the aqueous medium to the individual cells surface is still the critical and limiting
step. Hence, optimization of bioreactor design (e.g., impeller types, baffled) and
operation conditions (e.g., agitation speed, gas flow rate) is regarded as the promis-
ing solution to the mixing and mass transfer issues in oils/fats substrate involved
bioprocesses.
3.1 Investigation of Oil–Water Mixing in Stirred Bioreactor

by CFD Simulation
In addition to traditional fermentation experiments, the optimization of bioreactor

and operation conditions for more efficient oil/fat–water mixing and mass trans-
fer can be achieved by using CFD technology. CFD simulation has been used to
analyze the fluid flow and simulate the gas dispersion and water in oil mixing
performance in agitated reactors. This enables a better understanding of the inner
mechanisms of the multi-phase flow and the factors that modulate the hydrodynam-
ics (Hutmacher and Singh 2008; Wang et al. 2014). CFD modeling is a relatively
low cost and less time-consuming methodology that allows more efficient predic-
tion and possibly optimization of bioreactor design and operation conditions in
fermentation experiments.
In our research group, three-dimensional CFD models have been developed to
simulate the distribution of oil droplets in an aqueous medium in agitated biore-
actors with two standard Rushton turbines. The bioreactors were filled with water
with 5% (v/v) of vegetable oil as the dispersed phase. Simulations were performed
at the agitation speed of 500 rpm. The entire vessel was considered as the com-
putational domain. The simulation results showed that most larger oil droplets
gathered in the upper and central region of the vessel. This result suggests that
pitched-blade impellers with the blades set at a certain angle (e.g., 45◦ ) should
be applied in the fermentation experiment, as they could more efficiently facilitate
the mixing of the fluid from between the upper and bottom regions of the vessel
as well as from between the center to and the side regions (Fig. 2b), thus being
able to overcome the oil droplets’ buoyance forces to improve dispersion in an
aqueous medium. The Rushton turbines used in the CFD model usually produce
unidirectional radial flow (Fig. 2a). In addition, in the CFD simulation, oil droplets
around the impeller blades were broken into smaller droplets, of which some were
pushed into side regions of the bioreactor. From this result, it can be postulated
that increasing the agitation speed increases the shearing forces on the oil droplets,
which would lead to the formation of smaller oil droplets from larger ones and an
improvement in the distribution of the droplets to other regions of the vessel. The
CFD results and predictions were validated in 1-L fermentation experiments.
(b) A combinaƟon of 2 pitched-blades

(a) Rushton Impellers for FermentaƟon impellers (top & middle) and 1 Rushton
impeller (boƩom) for fermentaƟon
Fig. 2 Two typical impellers used for fermentation. a The blades on Rushton impellers are flat and
set vertically along an agitation shaft, which produces a unidirectional radial flow. b The blades on
pitched-blade impellers (top and medium) are flat and set at ∼45° angles, which produces a simul-
taneous axial and radial flow. The combination of pitched-blades and Rushton impellers provides
better overall mixing and creates a higher mass transfer rate
3.2 Fermentation Engineering for Improved Biosynthesis

from Oils and Fats
In our recent bioreactor fermentation experiments, the cell growth and citric acid
production of wild-type Y. lipolytica ATCC20362 on soybean oil substrate were
studied as a representation of the overall mixing and mass transfer, since better
mixing and mass transfer usually led to faster cell growth and/or higher prod-
ucts formation. To compare the effect of impellers on mixing and mass transfer,
two sets of impellers were tested in 1-L bioreactors, with one set of impellers con-
sisted of three evenly-spaced Rushton impellers (Fig. 2a), while the other consisted
of two pitched-blade impellers on top and one Rushton impeller at the bottom
(Fig. 2b). The agitation speed through fermentation was controlled between 500
and 1400 rpm. The bioreactor is equipped with three impellers, which has one
more impeller as compared to the bioreactor geometry in CFD simulation. The
third impeller was added to the top region of the bioreactor to break up and dis-
perse the oil droplets accumulated in the upper region. Furthermore, two maximal
agitation speeds, 1000 rpm and 1400 rpm, were tested in the bioreactor equipped
with two pitched-blade impellers on top and one Rushton impeller at the bot-
tom to examine if more powerful input provides better mixing and improves the
70 N. Liu et al.
(a) 70 (b) 140

3 Rushton, 1400 rpm 3 Rushton, 1400 rpm
60 2 pitched-blades + 1 Rushton, 1400 rpm 120 2 pitched-blades + 1 Rushton, 1400 rpm
Dry cells weight (g/L)
Citric acid Ɵter (g/L)

2 pitched-blades + 1 Rushton, 1000 rpm 2 pitched-blades + 1 Rushton, 1000 rpm
50 100
40 80
30 60
20 40
10 20
0 0
(c) 2.4 (d) 0.6

3 Rushton, 1400 rpm 3 Rushton, 1400 rpm
Specific citric acid (g/g DCW)
2 pitched-blades + 1 Rushton, 1400 rpm 0.5 2 pitched-blades + 1 Rushton, 1400 rpm
Citric acid yield (g/g)

1.8 2 pitched-blades + 1 Rushton, 1000 rpm 2 pitched-blades + 1 Rushton, 1000 rpm
0.4
1.2 0.3
0.2
0.6
0.1
0.0 0.0
Fig. 3 Fed-batch fermentation of Y. lipolytica ATCC20362 with soybean oil substrate in biore-
actors equipped with different impellers and under different maximal agitation speed. a Dry cells
weight (DCW), b citric acid titer, c specific citric acid, d citric acid yield at 144 h
fermentation performance, as suggested by the CFD model. The agitation speed

through fermentation was controlled within 500–1000 rpm and 500–1400 rpm,
respectively. Other than the impeller setup or agitation speed, conditions for each
fermentation were the same.
As shown in Fig. 3a, the dry cell weight (DCW) from the fermentation with
different impellers and maximal agitation speeds were similar, indicating that the
oil–water mixing condition with any set of the impellers and agitation speed
met the minimal requirements for cell growth. However, citric acid production
from soybean oil was significantly impacted by impeller type and agitation speed
(Fig. 3b, c, and d). With three Rushton impellers and the maximal agitation speed
of 1400 rpm, a citric acid titer of 84 g/L, a specific citric acid titer of 1.4 g/g DCW,
and a citric acid conversion yield of 0.32 g/g from soybean oil was obtained at
144 h. By replacing the two top Rushton impellers with two pitched-blade ones,
the titer, the specific titer and the yield of citric acid at 144 h increased to 95 g/L,
1.6 g/g DCW, and 0.35 g/g soybean oil, respectively. This confirmed the hypothe-
sis that the pitched-blade impellers are more beneficial for overall mixing and mass
transfer rate so that the oil droplets were dispersed into the whole bioreactor to be
uptake by the yeast cells to convert to citric acid. In addition, when the maximal
agitation speed decreased from 1400 to 1000 rpm, the titer of citric acid at 144 h
decreased from 95 g/L to 68 g/L, which also validated what the CFD simulation
results suggested, i.e., more powerful agitation could break down the oil droplets
into smaller ones and disperse them into the regions farther from the center so that
the cells were able to assimilate them and convert them into citric acid.
4 Cell Morphology Engineering in Dimorphic Yeast Y.

lipolytica
Understanding microbial dynamics is crucial in response to the interaction of

the gene expression and environmental conditions during cell growth course and
product formation (Guan et al. 2017; Timoumi, et al. 2018). The impacts of cell
microenvironment on metabolism, such as the supplementation of nutrients, vari-
ations in temperature (Arsène, et al. 2000; Barria et al. 2013) and pH, are known
factors to affect the cellular behavior (Arsène et al. 2000; Barria et al. 2013; Ruiz-
Herrera and Sentandreu 2002; Yuzbashev et al. 2010). Among the changes in biotic
responses of microorganisms, the morphological change provides important infor-
mation about the cell differentiation process, cell function, and signal responses.
Microorganisms are capable of altering their cellular structure dynamically, includ-
ing the shape and size, to adapt to a new environment and optimize the interactions
physicochemically and biologically (Okano et al. 1995). However, most previ-
ous studies have focused on the construction of novel biosynthetic pathways and
their metabolic regulatory processes. Increasing attention has recently been paid
to morphology engineering as a new strategy for constructing efficient microbial
cell factories, the purpose of which is to control cell shape and cell growth pat-
tern by manipulating cell morphology-related genes (Huo et al. 2020). Therefore,
improving tolerance of microbial species to various environmental stresses in a
predictable manner has been proposed to achieve optimal product titer, rate, and
yield at a large scale.
Among the microorganisms of biotechnological interest in the production of
metabolites with high values, the non-conventional oleaginous yeast Y. lipolytica
has been successfully used in recent years as microbial cell factories due to its
unique biochemical characteristics suitable for large-scale biomanufacturing and a
wide range of potential applications (Liu et al. 2015; Papanikolaou et al. 2020).
Y. lipolytica is considered as one of the most attractive hosts capable of assimilat-
ing an extremely broad range of raw substrates, including both hydrophilic (e.g.,
glucose, fructose, mannose, glycerol, ethanol, and organic acids) and hydropho-
bic (e.g., fatty acids, alkanes, triacylglycerols, plants oils, and animal fats) carbon
sources (Fickers et al. 2005a, b; Ledesma-Amaro and Nicaud 2016a; Soong et al.
2019; Spagnuolo et al. 2018).
4.1 Regulation of Yeast-To-Hyphal Transition in Y. lipolytica
Y. lipolytica is emerging as a model organism for dimorphism studies. It can grow

in two distinct phenotypes, oval-shaped yeast single cells or filamentous form
(i.e., pseudohyphae or hyphae) (Berman and Sudbery 2002; Dominguez et al.
2000; Palecek et al. 2002; van der Walt and von Arx 1980). Morphologic switch-
ing is dependent on the environmental and physiological conditions together with
genetic characteristics. Induction of yeast-to-hyphal transition is triggered by the
signal transduction from the cell surface sensors to signal transducer and then
72 N. Liu et al.
launches a series of intracellular signaling pathways, which activate the expres-

sion of morphology-related genes and result in cellular response (Berman and
Sudbery 2002; Lengeler et al. 2000; Su et al. 2018). The external signals in the
environmental conditions, such as physicochemical (i.e., temperature, pH, and dis-
solved oxygen levels), mechanical (i.e., pressure and mixing), or nutritional (i.e.,
carbon/nitrogen sources and concentration of metal ions and salts) can initiate the
dimorphic transition in Y. lipolytica (González-López et al. 2006; Ruiz-Herrera
and Sentandreu 2002). For example, in the presence of N-acetylglucosamine and
blood serum, organic sources of nitrogen (i.e., amino acid and peptone), thermal
shock and hypoxia could contribute to the filamentous phenotype, whereas in the
presence of glutamine and glutamate, exposure of the osmotic stress (i.e., 0.2 M
NaCl) and sufficient aeration have been reported to suppress the hypha formation
(Guevara-Olvera et al. 1993; Kim et al. 2000; Pérez-Campo and Domínguez 2001;
Szabo and Štofanı́ková 2002; Zinjarde et al. 2008).
4.2 The MAPK Pathway in Y. lipolytica
Two major signal transduction pathways are involved in the regulation of the yeast-
to-hyphal transition in dimorphic yeast. One is the mitogen-activated protein kinase
(MAPK) pathway (Fig. 4). MAPK signaling cascades are multi-functional path-
ways that are evolutionarily conserved in all eukaryotic cells (Ligterink and Hirt
2001; Xu et al. 2017). The MAPK pathway is initiated by specific extracellular
cues, amplifies, and integrates the signals through the activation of a particular
MAPK following the consecutive of a MAPK kinase kinase (MAPKKK) and a
MAPK kinase (MAPKK), and consequently elicits an appropriate physiological
response. Typically, the MAPKKK is activated by interactions with a small GTPase
and/or phosphorylation by protein kinases downstream from cell surface receptors
transducing signal downstream (Cuevas et al. 2007; Zhang and Liu 2002). Sev-
eral types of protein kinases (i.e., YlCLA4p, YlSTE11p, YlSTE7p) contributed
to the MAPK pathway in Y. lipolytica have been characterized (Cervantes-Chávez
and Ruiz-Herrera 2006; Martínez-Soto and Ruiz-Herrera 2017; Martinez-Vazquez
et al. 2013; Szabo 2001). YlCLA4p is highly homologous to CLA4 protein kinase
of Candida albicans and Saccharomyces cerevisiae, which are members of the
p21-activated kinase (PAK) family containing conserved internal Cdc42p-binding
regions (Bartholomew and Hardy 2009; Lai et al. 2012). Deletion of YlCLA4 in
Y. lipolytica is not lethal but completely loses the ability to form filaments or
invade agar (Szabo 2001). Disruption of the YlSTE11, which exhibits high homol-
ogy to fungal MAPKKK, loses the capacity to mate and grows constitutively in
the yeast-like form (Cervantes-Chávez and Ruiz-Herrera 2006). The expression
level of YlSTE7 encoding MAPKK is increased during yeast-to-hyphal transition
(Chaleff and Tatchell 1985; Leberer et al. 1996; Martinez-Vazquez et al. 2013).
Transcription factor is a downstream effector of upstream signaling pathway.
YlZNC1p contains a Zn(II)2 C6 fungal-type zinc finger DNA-binding domain and
a leucine zipper domain that acts as a transcription factor repressing hyphal forma-
tion. YlZNC1p is involved in the MAPK pathway via the regulation of YlCLA4,
Fig. 4 Simplified scheme describing the dimorphic transition in Y. lipolytica. Both MAPK and
cAMP-dependent signaling pathways are involved in the yeast-to-hyphal transition but have oppos-
ing actions. The activation of the MAPK pathway induces filamentation. The activation of the
cAMP-dependent pathway represses the filamentous growth. Abbreviations: PAK, p21-activated
kinase; MAPKKK, MAP kinase kinase kinase; MAPKK, MAP kinase kinase; MAPK, MAP
kinase; TCS, TEC1 consensus binding sequence; FRE, filamentation response element; GPCR,
G-protein coupled receptor; PKA, protein kinase A
YlSTE11, and YlSTE7 gene expressions (Martinez-Vazquez et al. 2013). Further-

more, both YlBMH1p and YlBMH2p are closely related to the S. cerevisiae
proteins ScBMH1p and ScBMH2p, which have been found to associate with
ScSTE20p (PAK family kinase) required for the MAPK signaling cascade that
regulate the hypha-specific genes (Costa et al. 2007; Hurtado and Rachubinski
2002). Therefore, by regulating MAPK cascades, cells can respond to a range of
stresses and lead to change in gene expression, and eventually adapt to changing
environmental conditions.
74 N. Liu et al.
4.3 The cAMP-Dependent PKA Pathway in Y. lipolytica
The other is the cAMP-dependent protein kinase A (PKA) pathway (Fig. 4). In
yeast and filamentous fungi, this pathway takes part in the pathogenesis, cellular
morphogenesis, nutrient sensing and acquisition, sexual reproduction, and stress
responses (D’Souza and Heitman 2001; Hogan and Sundstrom 2009; Kronstad
et al. 2011). Ras subfamily proteins, the small monomeric GTP-binding proteins,
activate adenylyl cyclase CYR1 by interaction with its Ras associating domain to
synthesize cAMP, which binds to the regulatory subunits of PKA and releases the
catalytic subunits. The unleashed active kinases then phosphorylate a diverse set of
substrates resulting in corresponding biological responses (Cao et al. 2017; Weeks
and Spiegelman 2003).
Unlike other yeast species, which usually has one or two Ras proteins, Y.
lipolytica possesses three Ras proteins, particularly YlRAS1p and YlRAS2p, are
implicated in the control of dimorphism. In comparison with YlRAS1p, YlRAS2p
plays a major role in the yeast-to-hyphal transition. The expression of YlRAS2
is increased dramatically at the transcription level during mycelial development.
Additionally, mutants in the YlRAS2 exhibit a severe defeat in pseudohyphal or
hyphal growth (Li, et al. 2014). The second messenger cAMP is an important
mediator to regulate the PKA activation. The increase of cytosolic cAMP concen-
tration, either by adenylyl cyclase activation or by entry of exogenous nucleotide
into the cell, inhibits the PKA pathway and the dimorphic transition in Y. lipolyt-
ica (Cervantes-Chávez and Ruiz-Herrera 2007; Johnson et al. 2001; Taylor et al.
2004). The core component of this pathway is PKA, which is a heterotetramer
consisting of two catalytic (cPKA) and two regulatory subunits (rPKA). The cat-
alytic subunits are encoded by the YlTPK1 gene and the regulatory subunits are
encoded by the YlRKA1 (homologous to S. cerevisiae BCY1) (Cervantes-Chávez,
et al. 2009; Toda et al. 1987). It has been previously reported that the YlRKA1
gene was up-regulated at the transcriptional level under conditions that induce
mycelial morphology, indicating that dimorphic transition is regulated by the PKA
pathway via YlPKA1p in Y. lipolytica (Cervantes-Chávez and Ruiz-Herrera 2007).
Expression of YlPKA1p in PKA cascade was also mediated by YlZNC1p. Hence,
YlZNC1p acting through both MAPK and PKA pathways represses the yeast-to-
hyphal transition. Mutant strains deleted in the YlTPK1 grew constitutively in the
mycelial form, whereas YlSTE11 and YlTPK1 double mutants grew constitutively
in the yeast form, suggesting that the default growth pattern of the Y. lipolytica is a
yeast-like form (Cervantes-Chávez et al. 2009). Surprisingly, all these data provide
evidence that the opposite actions of the MAPK and PKA pathways in regulating
Y. lipolytica dimorphism, in contrast to S. cerevisiae and C. albicans where both
pathways act together to regulate the dimorphic switching (Biswas et al. 2007).
Multiple genes, including YlHOY1 and YlMHY1, are also responsible for the
filamentous growth of Y. lipolytica. The YlHOY1 encoding a putative nuclear pro-
tein with a homeodomain function as transcriptional regulatory protein. Disruption
of YlHOY1 results in a defect in filamentation (Torres-Guzman and Domínguez
1997). Like the YlHOY1 gene, YlMHY1p is localized to the nucleus during fila-
mentous growth and acts as a transcription factor. YlMHY1 encoding a C2 H2 -type
zinc finger protein, YlMHY1p, exhibits strong homology to the S. cerevisiae stress
response factors MSN2p and MSN4p, which is involved in the regulation of mor-
phogenesis (Kobayashi and McENTEE 1993; Marchler et al. 1993). Like these
factors, YlMHY1p specifically recognizes and binds to putative cis-acting DNA
stress response elements (STREs) located on the upstream of a number of genes
conferring tolerance to a variety of stress conditions, such as heat shock, car-
bon source starvation, osmotic stress, and oxidative shock (Treger et al. 1998).
Transcription of YlMHY1 is dramatically increased during the dimorphic transi-
tion in Y. lipolytica. Deletion of YlMHY1 is unable to undergo mycelial growth,
indicating that YlMHY1p promotes hyphal development. Interestingly, overexpres-
sion of YlMHY1 in YlRAS2 mutant cells form abundant pseudohyphae or hyphae,
while overexpression of YlRAS2 in YlMHY1 mutant cells fail to induce filamentous
growth (Li et al. 2014). YlMHY1 is a gene whose overexpression could restore
hyphal growth and is required for Y. lipolytica YlRAS2p function, like S. cere-
visiae Ras protein does to MSN2p and MSN4p. Therefore, the transcription factor
YlMHY1p may function as a signal transducer downstream of YlRAS2p in the
control of the dimorphic transition.
Besides, the transcriptome analysis reveals the downstream target regulated
by YlMHY1p. These downstream genes encode proteins that are similar to the
flocculin FLO11, cell wall mannoprotein TIR3 and agglutinin AGA1 in S. cere-
visiae and HYR1 in C. albicans (Bailey et al. 1996; Rupp et al. 1999). Therefore,
YlMHY1p mediates the expression of a large group of cell wall proteins and
enzymes involved in cell wall maintenance. In addition to cell wall-related genes,
YlMHY1p also regulates genes involved in nutrient uptake, protein processing,
and lipid metabolism (Wu et al. 2020). YlMHY1p has multiple cellular functions.
4.4 Cell Morphology Engineering for Enhanced Utilization

of Oils and Fats
Morphology engineering strategies have been previously explored for improving

bacterial growth rate, enlarging cell size, and simplifying downstream separation. It
has also become an effective method to maintain the dimorphic yeast shape (Jiang
and Chen 2016; Zakhartsev and Reuss 2018). The manipulation of morphology-
related genes allows changing the cell shape from a sphere to a hyphal form
(Hurtado et al. 2000; Hurtado and Rachubinski 2002; Jiménez-Bremont et al.
2012; Ruiz-Herrera and Sentandreu 2002). Improving stress tolerance in a pre-
dictable manner in yeast cell factories should facilitate their widespread utilization
in the biobased economy and extend the range of products successfully produced
on large scale in a sustainable and economically profitable way.
In the dimorphic yeast Y. lipolytica, the MSN2p/MSN4p-like protein YlMHY1p
is a key positive regulator of yeast-to-hyphal transition. Both YlMHY1 knock-out
and overexpression affect filamentation (Konzock and Norbeck 2020; Wu et al.
76 N. Liu et al.
2020). Unlike the Y. lipolytica ATCC20362 wild-type strain, cells of the YlMHY1
deletion are unable to form filaments (Fig. 5a). Cells with YlMHY1 overexpression
form filaments longer than those of control cells (Fig. 5a), higher dry cell weight
(DCW) (Fig. 5b), decreased citric acid titer (Fig. 5c), and low lipid accumulation
(Fig. 5d) in liquid medium using glucose, soybean oil or waste cooking oil as
main carbon source. Citric acid is defined as a key precursor in lipid production.
Deletion of YlMHY1 in Y. lipolytica exhibits larger lipid bodies (LB), higher citric
acid titer (Fig. 5c), and increased lipid/DCW (Fig. 5d) in 1-L fed-batch fermenta-
tion, suggesting that the morphology arresting in yeast-like form has a beneficial
effect on the lipid-derived product formation and accumulation (Liu et al. 2021).
In comparison with glucose, lipid-derived feedstock (soybean oil and waste cook-
ing oil) benefits both Y. lipolytica cells’ growth and lipid production. Mutations in
YlMHY1 are shown to be more efficient in lipid feedstock utilization. Consistent
with this finding, Wang et al. reported that YlMHY1p regulates lipid biosynthesis
since the Y. lipolytica with YlMHY1 deletion increased carbon flux through lipid
biosynthesis and accumulated more intracellular oil than the wild-type strain dose
(Wang et al. 2018). Therefore, in combination with morphology engineering and
metabolic engineering strategies together with lipid-derived feedstock utilization
is expected to become an efficient and economical approach for constructing Y.
lipolytica cell factories.
(a) (b)
80
Glucose Soybean Oil Waste Cooking Oil
Wild-type YlMHY1- YlMHY1+ 70
Dry Cell Weight (g/L)
60
LB
50
LB 40
LB
30
20
10
0
Wild-type YlMHY1- YlMHY1+
(c) (d)
140 100%
Glucose Soybean Oil Waste Cooking Oil Glucose Soybean Oil Waste Cooking Oil
120
Citric Acid Titer (g/L)
80%
100
Lipid/DCW
80 60%
60 40%
40
20%
20
0 0%
Wild-type YlMHY1- YlMHY1+ Wild-type YlMHY1- YlMHY1+
Fig. 5 Cellular responses to different carbon sources for Y. lipolytica strains with YlMHY1 deletion
and overexpression. a Cell morphology under microscope: Cells of strains ATCC20362 (wild-
type), YlMHY1 knock-out (YlMHY1−), and YlMHY1 overexpression (YlMHY1+ ) were grown
at 30 °C for 120 h in liquid media containing glucose as the main carbon source. b dry cell
weight (DCW), c citric acid titer, and d lipid/DCW of Y. lipolytica wild-type, YlMHY1- and
YlMHY1+ strains after 144 h cultivation in 1-L fed-batch fermentation using glucose, soybean oil,
or waste cooking oil as the main carbon sources
5 Biosynthesis of Wax Esters from Oils and Fats
Wax esters are widely distributed nature compounds that are found in high evo-
lution plants, algae, microorganisms, and even insects and mammals. Natural
occurring waxes, consisting of fatty acids esterified to long-chain alcohols, are a
group of highly hydrophobic neutral lipids but structurally diverse. Physical prop-
erties and applications of wax esters are varied due to different chain lengths of
the fatty acid and the fatty alcohol components as well as the degree of unsat-
uration that affect melting temperature, oxidation stability, and pressure stability.
Wax esters have a variety of biological functions that provide the protective coat-
ing on the surface so that they are resistant to dehydration, ultraviolet rays, and
pathogens (Jetter and Kunst 2008; Wältermann et al. 2005). Wax esters are used
commercially to serve as a wide range of applications, such as cosmetics, printing
inks, lubricants, coatings, pharmaceuticals, and the food industries (Doan et al.
2017; Fiume et al. 2015; Petersson et al. 2005). Although the wax esters are found
in nature universally, the abundance of wax ester source is still low because only
a few organisms such as Jojoba plant (Simmondsia chinensis) and sperm whale
(Physeter macrocephalus) are capable of accumulating a considerable amount of
intracellular wax esters. Currently, wax esters are in a short supply due to the hunt-
ing ban for sperm whale, the high extraction cost, and the harsh requirement of
agriculture system for Jojoba (Miwa 1971). Therefore, establishment of an efficient
expression platform will be expected to advance the economic feasibility of wax
esters from low-cost substrates as carbon sources, especially from waste cooking
oil.
5.1 Characterization of the Wax Ester Biosynthesis Pathway
The biosynthesis and accumulation of TAG and wax esters (WEs) have been
reported in some soil and marine bacteria including Acinetobacter (Ishige et al.
2002; Santala et al. 2014), Marinobacter (Willis et al. 2011), Mycobacterium
(Sirakova et al. 2012), Streptomyces (Röttig et al. 2016), Euglena (Tomiyama
et al. 2017), and Rhodococcus (Round et al. 2019) genera under nitrogen-limited
conditions. In prokaryotes, mechanism of WE synthesis proceeds via sequen-
tial reactions (Fig. 6a): First, the fatty acyl-CoA or fatty acyl-ACP substrate is
reduced to a respective long-chain fatty aldehyde by fatty acyl-CoA reductase
(FAR) in an NADPH-dependent manner; the aldehyde is further reduced to corre-
sponding fatty alcohol by uncharacterized fatty aldehyde reductase (Alvarez 2016;
Mcdaniel et al. 2011). Second, the fatty alcohol is esterified with acyl-CoA through
the existence of a CoA-dependent acyltransferase enzyme known as wax ester
synthase/diacylglycerol acyltransferase (WS/DGAT).
In Y. lipolytica, biosynthesis of fatty alcohols by FAR has been intensively
studied (Madzak 2018; Zeng et al. 2018). Heterologous expression of FAR from
Marinobacter hydrocarbonoclasticus strain VT8 produced 5.75 g/L fatty alcohols
in Y. lipolytica when grown on modified YPD medium containing 91 g/L glucose
78 N. Liu et al.
Fig. 6 Introducing wax ester biosynthesis pathway in Y. lipolytica. a Biosynthesis pathway of wax
esters in Y. lipolytica during the growth on oils/fats as carbon source. b Fatty alcohol and wax ester
titers in the engineered Y. lipolytica Po1f strain, which contained the fatty acyl CoA reductase
(ScFAR, MmFAR or MhFAR) and wax ester synthase (AbWS) expression via plasmid transfor-
mation. c Phenotype and d wax ester titer of engineered Y. lipolytica ATCC20362 strains with
co-expression of MhFAR and AbWS via chromosomal integration grown on glucose, oleic acid,
soybean oil or waste cooking oil as a carbon source for 120 h in a shaking flask. ScFAR from S.
chinensis; MmFAR from Mus musculus; MhFAR from M. hydrocarbonoclasticus strain VT8; AbWS
from A. baylyi ADP1; LB, Lipid bodies
in shaking flask scale (Zhang et al. 2019). A recent report also showed that Y.
lipolytica possessing two genes coding for MhFAR achieved at titers of 5.8 g/L
fatty alcohol production in a minimal glucose media under fed-batch fermentation
(Cordova et al. 2020). Also, Y. lipolytica was engineered to produce fatty acid
methyl esters (FAME) and fatty acid ethyl esters (FAEE) by introducing wax ester
synthase (Gao et al. 2018; Xu et al. 2016).
5.2 Introducing Wax Ester Biosynthesis Pathway into Yarrowia

lipolytica
Recently, several studies have been conducted to achieve significantly greater cell
growth rate and higher production yield of microbial-derived biodiesel (Blazeck
et al. 2014; Darvishi et al. 2017). Other research efforts include metabolic engi-
neering of microorganisms to enhance the conversion of hydrophobic substrates
into value-added, lipid-derived products (Sabirova et al. 2011; Xie 2017; Xue et al.
2013; Yang et al. 2019). In Y. lipolytica, co-expression of MhFAR (M. hydrocarbon-
oclasticus strain VT8) and the AbWS (A. baylyi ADP1) via plasmid transformation
produced the most fatty alcohols and wax esters as compared to the strains co-
expressing the ScFAR (S. chinensis) or the MmFAR (Mus musculus) together with
the AbWS (Fig. 6b). Oil-based substrates have been employed for the improving
production of lipid-derived products by Y. lipolytica (Enshaeieh et al. 2014; Li
et al. 2008). In particular, oleic acid (C18:1) is the major fatty acid present in
Y. lipolytica, whereas palmitic (C16:0) and linoleic (C18:2) acids have also been
detected in high content in the cells (Dobrowolski et al. 2016; Magdouli, Guedri
et al. 2020).
In our recent study (Soong et al. 2021), as shown in Fig. 6c, the enlarged
lipid bodies were produced when the engineered Y. lipolytica (integrating the
MhFAR and AbWS into the chromosome) cells grown on the oleic acid or oil-
containing media, indicating that the accumulation of lipid-derived products could
be improved. In addition, by switching the main carbon source from glucose to
waste cooking oil, the wax ester titer was increased by 70-fold, reaching a maxi-
mum of 7.58 g/L after 120 h of cultivation in a shaking flask (Fig. 6d). Thus, in
the presence of exogenous oleic acid or oil-based substrates (soybean oil or waste
cooking oil) to the culture medium, the engineered Y. lipolytica strain tends to
rely on an external source of fatty acids rather than endogenous de novo fatty acid
biosynthesis for wax ester production. This work opens a door toward the econom-
ical production of lipids, lipid-derived, and lipid-assisted products for applications
as fuels, chemicals, nutraceuticals, and pharmaceuticals.
6 Transporter Engineering in Yeasts
Transporters are key components for efficient import and export activities. The
transport of the substrates, such as sugars and fatty acids, across the cytoplasmic
membrane into the cell, which links extracellular substrates utilization and intra-
cellular metabolic pathways, is a critical step for consolidated yeast bioprocessing.
Furthermore, the transport of the produced products out of the cell is also critical
for maintaining a high production rate (Hara et al. 2017). This section summa-
rizes the engineering of specific transporters that are used in yeast to enhance
bioproduction.
80 N. Liu et al.
6.1 Sugar Transport
Yeast can utilize varieties of sugars including monosaccharides and disaccharides,

but sugars are not able to freely transport across the cell membrane. Transporter
proteins exist at the interface of the cell membrane that can selectively trans-
port sugars across cell membranes into the cytoplasm (Spagnuolo et al. 2018).
Monosaccharides (e.g., hexose, pentose) are widely used as substrates in biotech-
nology since they are readily assimilated by microorganisms to convert them into
desired products (Ledesma-Amaro and Nicaud 2016a).
Hexose (C6 ) sugars (e.g., glucose, galactose, fructose, mannose) are the favored
carbon and energy sources for most biotechnological microorganisms. There are
24 sugar transporters have been identified in Y. lipolytica (e.g., W29, H222), among
them at least 6 proteins (e.g., YALI0C06424p, YALI0F19184p, YALI0E23287p,
YALI0B06391p, YALI0C08943p, and YALI0C08943p) can function as hexose
transporters in a study of the heterologous host S. cerevisiae (Lazar et al.
2017). Generally, one transporter is capable of transporting more than one hex-
ose but with a preference for one (Spagnuolo et al. 2018). Hexose transporters
YALI0C06424p, YALI0F19184p, and YALI0E23287p appear to transport a wider
range of sugars, efficiently importing four hexoses (e.g., glucose, galactose, fruc-
tose, mannose) into S. cerevisiae, demonstrated with the drop-test assays. The
transporter YALI0B06391p is seemingly dedicated only to the transport of galac-
tose and mannose. YALI0C08943p did not transport glucose, and YALI0B01342p
did not enable fructose uptake at any concentration (Hara et al. 2017; Spagnuolo
et al. 2018). Sugar transporters have been well-studied in the conventional yeast S.
cerevisiae (Luyten et al. 2002; Rintala et al. 2008). Transport of hexoses in S. cere-
visiae is through facilitated diffusion mediated by several transporters (Leandro
et al. 2009). There are 17 hexose transporters, HXT1p-HXT17p, and one galactose
permease, GAL2p, that have been identified in S. cerevisiae (Leandro et al. 2009).
Wieczorke et al. (1999) demonstrated that all 18 proteins, except HXT12, can
transport hexoses. HXT1p-HXT7p are the main hexose transporters (Reifenberger
et al. 1997). Kim et al. (2015) have enhanced glucose uptake by overexpression of
5 hexose transporters including HXT1p, HXT2p, HXT3p, HXT4p, and HXT7p,
and overexpression of HXT7p was most effective in increasing the glucose uptake
rate. Wieczorke et al. (1999) restored growth of the HXT1-17 and GAL2 deletion S.
cerevisiae on glucose, fructose, and mannose but not on galactose by overexpress-
ing the individual HXP5p, HXP8p, HXP13p, HXP15p, HXP16p, and HXP17p,
while HXT9p, HXP10p, and HXP11p were also identified to transport galactose.
Pentose (C5 ) sugars (e.g., xylose, arabinose) are the second most prevalent
carbon and energy sources for biotechnological microorganisms (Ryu and Trinh
2018). The uptake of pentose sugars in Y. lipolytica has been debated. The wild Y.
lipolytica has been known to be very inefficient at utilizing pentose sugars. Xylose
transporters are an important rate-limiting step that causes poor xylose assimila-
tion (Ryu and Trinh 2018). Xylose could be weakly transported into Y. lipolytica
by endogenous hexose transporters YALI0C06424p and YALIB06391p (Young
et al. 2014). More recently, two pentose-specific transporters, YALI0C04730p and
YALI0B00396p, have been identified in Y. lipolytica (Ryu and Trinh 2018). The
yeast S. cerevisiae lacks pentose-specific transporters; it is known to uptake D-
xylose and L-arabinose via endogenous hexose/glucose transporters. The efficiency
of hexose transporters for D-xylose uptake was as follows: HXT7 > HXT5 >
GAL2 HXT1 > HXT4 (Sedlak and Ho 2004). Overexpression of endogenous
hexose transporters and/or heterologous xylose transporters have effectively facil-
itated D-xylose uptake into engineered S. cerevisiae strains (Hector et al. 2008).
Because D-xylose uptake is competitively inhibited by D-glucose, cofermentation
of D-glucose and D-xylose, such as in cellulosic and hemicellulose hydrolysates,
is not cost-efficient (Farwick et al. 2014). Many studies have focused on effec-
tive D-xylose fermentation from mixed sugars by developing more xylose-specific
transporters. Farwick and coworkers (Farwick et al. 2014) engineered hexose
transporters HXT7p and GAL2p and have achieved glucose-insensitive xylose
transporters. These engineering efforts have facilitated the economic production
of value-added products from renewable feedstocks.
6.2 Fatty Acid Transport
Cells cannot directly uptake oils or fats in the format of triacylglycerides (TAGs),
as no known TAG transporters have been identified (Beopoulos et al. 2009). After
hydrolysis of TAGs catalyzed by extracellular lipases, the released free fatty acids
(FFAs), usually are long-chain fatty acids (LCFAs), can be transported into cells by
membrane-bound FA-transporters. In S. cerevisiae, the transport system of LCFAs
has been well characterized. In this yeast, exogenous LCFAs traverse the mem-
brane via the FA-transporter ScFAT1p. Once transported across the membrane,
LCFAs are activated by conversion into a fatty acyl-CoA by the fatty acyl-CoA
synthetases ScFAA1p and ScFAA4p (DiRusso and Black 1999). Y. lipolytica has
LCFAs transport and activation proteins similar to those of S. cerevisiae, but the
transporter YlFAT1p had a different function than ScFat1p, and it is also involved
in the export of FAs from lipid bodies after the TAGs hydrolysis under the catalyza-
tion of intracellular lipases (Dulermo et al. 2014). Once FAs have been exported
from intracellular lipid bodies and activated, they can enter the peroxisomes and
be degraded as a result of β-oxidation cycle. In S. cerevisiae, the membrane-bound
transport heterodimer ScPXA1p/ScPXA2p is responsible for transporting fatty
acyl-CoA into peroxisomes (Shani and Valle 1996). Disruption of the peroxisomal
fatty acyl-CoA transporter ScPXA1p increased the intracellular TAGs accumula-
tion by 14% (Ferreira et al. 2018). In Y. lipolytica, there is a membrane-bound
transport protein pair YlPXA1p (YALI0A06655) and YlPXA2p (YALI0D04246)
corresponding to transporters in S. cerevisiae, which were responsible for the trans-
port of fatty acyl-CoA into peroxisome (Dulermo et al. 2015). In Y. lipolytica,
the utilization of FAs is largely dependent on transporters YlFAT1p, YlPXA1p,
and YlPXA2p. The deletion of YlPXA1and YlPXA2 in Y. lipolytica has increased
the accumulation of FAs when cells were grown in an oleate (C18:1) medium
(Dulermo et al. 2015).
82 N. Liu et al.
6.3 Product Export
In bioproduction, the accumulation of intracellular products is often toxic to the

host cells and impairing cell growth and the production of target materials. Trans-
porting the products to the extracellular space is an effective strategy to release
the toxicity. In addition, the export of target products also could avoid the nega-
tive feedback regulation, thus resulting in more efficient fermentation processes.
So far, transporter engineering has been employed in a few cases for improving
yeast bioproduction. In yeasts, the transporter MAE1p from fission yeast S. pombe
has been applied to increase the export of various organic acids, including malate,
maleic, oxaloacetic, and succinic (Darbani et al. 2019). The tolerance limit of S.
cerevisiae against alkanes has been improved by about 80-fold through harnessing
Y. lipolytica ATP-binding cassette (ABC) transporters pump alkanes out of the cell
(Chen et al. 2013). The expression of human transporter FATP1p, which has been
known as a fatty acid importer, resulted in a higher biomass yield and a 4.5-fold
higher fatty alcohol production in S. cerevisiae (Hu et al. 2018).
Engineering transporters to facilitate substrate uptake and reduce products
cytotoxicity then increase the production is important for yeast bioproduction.
However, the transporters and their mechanisms are still unclear, and further stud-
ies on developing efficient expression methods for membrane transporters and
improving specific functional properties of the transporter are required.
7 Conclusions
The GRAS status of Y. lipolytica and its metabolic traits, such as the ability to uti-
lize diverse hydrophobic substrates, high flux through acetyl-CoA, has made the
oleaginous yeast an important host for the production of fuels, commodity chem-
icals, nutraceuticals, and pharmaceuticals that can be derived from acetyl-CoA,
FAs, and lipids. Plant oils and animal fats, especially the low-cost waste oils and
fats, can be the preferred substrates for the biomanufacturing of these products
at low cost and high yield. However, due to the insolubility of oils and fats in
water, poor oil–water mixing and mass transfer is one of the major challenges to
be addressed for the fermentation processes using plant oils or animal fats as sub-
strates. CFD model and simulation were applied to analyze the distribution of oil
droplets in water in a stirred bioreactor, which further guided the bioreactor design
and optimization of operating conditions to significantly improve the fermentation
performance. Controlling cell morphology in a yeast-like form via morphology
engineering enhanced the cell growth on oils/fats and improved the production
of lipid-derived products. However, compared to the hydrophilic substrates, more
work in both cellular engineering and bioreaction engineering of Y. lipolytica
should be conducted in the future to facilitate the fatty acid transport for more effi-
cient extracellular substrate uptake and intracellular bioconversion. In a summary,
with advances in metabolic engineering in various promising microorganisms, oils
or fats can be used as a great substitute for sugars for biomanufacturing a series
of high-value products.
Acknowledgements This research work presented in this chapter was supported by NSF
(#1911480) and UML-WPI seed grant (2019–2020). The authors would also like to thank Dr.
Carl Lawton and Massachusetts Biomanufacturing Center for providing experimental facilities and
technical support.
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Whole Cell Yeast-Based Biosensors
Heather A. M. Shepherd, Emilia-Maria A. Bondarenko,

Katherine M. Jennings, Rachel A. Miller, and Holly V. Goodson
Abstract
Analyte detection is a major component of fieldwork, environmental surveil-
lance, and health protection, but lack of resources or access to instruments can
create challenges in technology-limited environments such as remote research
sites or low- and middle-income countries (LMICs). Whole-cell yeast-based
biosensors provide potential solutions to these barriers. Here, we discuss the
components of whole-cell yeast-based biosensors, emphasizing the process by
which they are designed for their analyte of interest, approaches for harnessing
them to function in particular research environments, and their advantages and
disadvantages relative to other analytical tools. We provide examples of the var-
ious purposes for which existing whole-cell yeast-based biosensors have been
used, focusing most on those appropriate for detecting externally-generated ana-
lytes in technology-limited settings. The further development of field-friendly
whole-cell yeast-based biosensors still faces challenges, including the need to
reduce the time from contact with the analyte to signal readout of the biosen-
sor. Regardless, the inexpensive, robust, portable, environmentally friendly,
and highly modular nature of yeast-based biosensors suggests that they could
become useful tools for a range of analytical tasks.
Heather A.M. Shepherd and Emilia-Maria A. Bondarenko are Co-first authors.
H. A. M. Shepherd · E.-M. A. Bondarenko · K. M. Jennings · H. V. Goodson (B)

Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556,
USA
H. V. Goodson
Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA
R. A. Miller
Department of Biology and Chemistry, Bethel University, Mishawaka, IN 46545, USA

https://doi.org/10.1007/978-3-030-89680-5_4
92 H. A. M. Shepherd et al.
1 Introduction
Sensors are tools used for analyte detection, analyte quantification, and/or gather-
ing information relating to biological activity. One subset of sensors is biosensors.
Though this term has varied meanings, here we define a biosensor as an analytical
tool that utilizes biological components (e.g., proteins, nucleic acids, whole cells
or even animals) to detect the analyte(s) of interest (Ostrov et al. 2017). Whole-cell
biosensors are a subset of biosensors that utilize living cells for sensing tasks. One
advantage of whole-cell biosensors relative to many other types of sensors is that
whole-cell biosensors can provide information on the biological relevance of the
analyte. This information is particularly important in cases where the sensing task
is performed in a complex environment (e.g., wastewater) or where compounds of
interest have varied composition (such as with endocrine disruptors) or unknown
identity (e.g., as might occur with environmental mutagens). Additionally, whole-
cell biosensors have the potential to provide detection with both high sensitivity
and high specificity without need for expensive equipment or analytical-grade stan-
dards. These attributes suggest that whole-cell biosensor detection systems could
be a useful alternative to spectral and mass-based detection systems in areas that
lack sufficient equipment or resources (Martin-Yken 2020; Miller et al. 2020a, b).
Like most other sensors (biological and not), whole-cell biosensors are mod-
ular devices that can be viewed as being composed of standard components. As
typically described, the fundamental parts of a whole-cell biosensor are the recep-
tor (an element that interacts in a specific way with the analyte), the reporter (an
element that produces a detectable signal in response to the receptor-analyte inter-
action), and the transducer (an element or process that connects the state of the
receptor to the state of the reporter, sometimes with signal amplification) (Conroy
et al. 2009). However, in considering the full spectrum of whole-cell biosensors
and thinking about how they could be designed through synthetic biology, we
suggest that it is useful to view these systems as being composed of five com-
ponents: an analyte, a detection process (which often but not always involves
a specific receptor), a transduction process (often with signal amplification), a
response process (which often but not always involves production of an identifiable
reporter), and a readout process (which involves quantifying or otherwise detecting
the output of the response process) (Fig. 1, Table 1). In addition, most whole-cell
biosensors also require a sixth component: some type of external system housing
to contain the cells both during exposure to the analyte and periods of extended
storage (Fig. 1). The reason for breaking the full process down in this way is that
it enables a better understanding of how different modules can be rearranged,
especially for cases that don’t fit neatly into the “receptor-transducer-reporter”
paradigm.
Whole Cell Yeast-Based Biosensors 93
Analyte Readout Process

Whole Yeast Cell
• Chemical Analytes DetecƟon TransducƟon Response

• OpƟcal
- PharmaceuƟcals Process Process Process - Color
- Heavy Metals - Absorbance
- PesƟcides Produce
••Produce
reporterProteins - Fluorescence
- Toxins • TranscripƟon • TranscripƟon
•TranscripƟ reporter
- Mutagens factors &on &
TranslaƟon proteins in cell
•Changes • Olfactory
• GPCRs • Signaling
TranslaƟon Changes inorcell
•populaƟon - Scent
• Biological Analytes • Enzymes populaƟon,
metabolic
pathways
•Metabolis
- Nutrients • Nucleic Acids metabolic
acƟvity or • Physical Probe
• Metabolism
m
- Hormones acƟvity, or
[metabolite] - pH
- Metabolites [metabolite] - redox
- Endocrine - [metabolites]
Disruptors
System Housing
Fig. 1 Elements of whole-cell yeast-based biosensors. Whole cell biosensors are made up of five
key components: analyte, detection process, transduction process, response process, and readout
process. Here we have provided examples of each of these components featured in this review.
These five components are then contained within the system housing, which is selected based on
the environment in which the biosensor is intended to be operated. In technology-limited settings,
paper-based assays or tubes read by a portable reader or the human senses (eyes, nose) would be
ideal. In some settings, preloaded microtiter plates could work depending on the availability of
suitable plate-reading technology
In a whole-cell biosensor, the detection, transduction, and response processes

occur within or on the cell. Though the detection process often involves biological
components (particularly proteins) imported from other biological systems, the
transduction/amplification and response typically utilize endogenous machinery.
Thus, a major challenge to designing and building effective whole-cell biosensors
is figuring out how to properly integrate imported and endogenous components.
To illustrate these six components, consider a whole-cell yeast-based biosensor
recently developed in our lab, one which produces a detectable smell in response
to the hormone estrogen (Miller et al. 2020b):
94
Table 1 Examples of specific whole-cell yeast-based biosensors for exogenously-generated analytes

Analyte Detection process Transduction Response Readout Housing References
(Receptor)
Estrogen Human ER Transcription/translation Luciferase Light level Microtiter plate Leskinen et al.
(2003)
Estrogen Chimeric GEV Transcription/translation Banana smell Ordorant detection via Capped 50 mL Miller et al.
(isoamyl acetate) human nose or gas tube (2020b)
chromatography
Fungal C. albicans GPCR Signalling ➞ mCherry Red lycopene pigment Paper Ostrov et al. (2017)
pathogens Ste2 transcription/translation
Melatonin human GPCR Signalling ➞ GFP Fluorescence Microtiter plate Shaw et al. (2019)
MTNR1A in heavily transcription/translation
modified yeast
DNA damage Yeast chromosome DNA damage ➞ YFP, RFP Fluorescence Liquid cultures Burrill and Silver
transcription/translation (2011)
Bioavailable Multiple proteins Metabolism Cell growth Absorbance Microtiter plate Shepherd et al.
phosphorus or 50 mL tube (2021)
Chemical Varied; potentially Metabolism Reduced rate of Absorbance as a Microtiter plate Hung et al. (2018)
sensitivity unknown cell growth function of time
For a more comprehensive list see Martin-Yken (2020)
H. A. M. Shepherd et al.
• The analyte is estrogen.

• The detection process is accomplished by binding estrogen to the cytosolic
protein estrogen receptor alpha, imported from humans. More specifically, the
yeast cells are exposed to a test sample in liquid media, and the estrogen (if
present) diffuses across the membrane and binds to the estrogen receptor in the
yeast cytoplasm.
• The transduction process involves several steps. In a simplified proposed mech-
anism, the estrogen/estrogen receptor complex translocates to the nucleus to
bind to the DNA, activating transcription and translation of a reporter gene that
encodes an enzyme which catalyzes production of an odorant detectable by the
human nose.
• The response process consists of accumulating the enzyme that produces the
odorant and the odorant itself.
• The readout process consists of a human using their nose to provide a yes/no
answer about the presence of odorant, or a machine assessing its quantity.
• The housing device is a simple capped 50 mL tube containing the biosensor
yeast and liquid growth media, to which test water or pharmaceutical samples
have been added.
While the list above seems straightforward, people who are familiar with this
or similar systems will realize that there are some additional steps implicit in
the description above, and it is important to recognize these additional complica-
tions in considering how to design or improve particular whole-cell biosensors.
For example, the transduction process for induction of transcription in response to
estrogen is not as simple as implied above: it appears to involve ligand-mediated
release from the estrogen receptor of the cellular component HSP90, with atten-
dant translocation to the nucleus, though aspects of this process remain uncertain
(Fuentes and Silveyra 2019). Moreover, there are implicit amplification steps in
the process above. These amplification steps are important because there is no
way that a human nose would be able to detect the signal if there were a 1:1
relationship between analyte (estrogen) and response (produced odorant).
The involvement of additional (and potentially unknown) eukaryote-specific
cellular components and/or processes during transduction (as illustrated by the
estrogen sensor above) is a challenging aspect of harnessing exogenous receptors
and is a major reason why eukaryotic whole-cell biosensors are preferable to anal-
ogous prokaryotic systems for many applications. An additional issue with using
prokaryotes for whole-cell biosensors is that they are often poorly suited to express
eukaryotic receptors including the aforementioned estrogen receptor because they
lack many protein folding factors, post-translational modification enzymes, and
internal organelles specific to eukaryotic cells (Zhang et al. 2000; Vieira Gomes
et al. 2018). Biosensors based on mammalian cells provide a potential solution to
these problems, but they are expensive to maintain and require specialized equip-
ment to do so, as they are sensitive to environmental conditions (Jarque et al.
2016a).
In considering these challenges, it becomes apparent that the baker’s yeast

Saccharomyces cerevisiae offers some significant advantages for construction of
whole-cell biosensors relative to these systems. For example, yeast is recognized
by the public as safe, and they are easy to work with due to their robustness, short
doubling time, and extensive genetic toolbox (Redden et al. 2015). Whole-cell
yeast-based biosensors have already been used to detect a wide range of chemi-
cal and biological analytes in the lab, and some have even been commercialized
(e.g., the yeast estrogen (YES) and yeast androgen (YAS) tests as provided by
Xenometrix.ch). The rapid development of synthetic biology tools and approaches
in yeast (see other articles in this book) makes yeast an even more attractive
organism for whole-cell biosensor development.
This review focuses on basic principles of whole-cell yeast-based biosensors,
and it gives particular attention to those that would be appropriate for detect-
ing externally-generated analytes in technology-limited environments. To improve
access for readers less familiar with yeast molecular biology and avoid redun-
dancy with other chapters in this book, we will avoid detailed discussions of the
experimental technicalities of biosensor construction, though we do point out some
challenges and pitfalls in biosensor development. For a review that goes more into
depth in the technical side of whole-cell yeast-based biosensors, see (Adeniran
et al. 2015). For more comprehensive reviews including discussions of biosensors
for internally generated analytes and recent high-tech biosensor developments, see
(Adeniran et al. 2015; Jarque et al. 2016a; Martin-Yken 2020).
Below we discuss in turn each of the components of a whole-cell yeast-based
biosensor system: the analyte, a detection process, a transduction process (perhaps
with signal amplification), a response process, a readout process, and a device in
which to house the yeast and expose them to analyte (Fig. 1). As will become
apparent, each of these components can be considered as a separate module that
can potentially be mixed and matched to create a range of sensor functionalities.
Though this text focuses specifically on yeast whole-cell biosensors, many of the
principles discussed could apply to any whole-cell biosensor.
2 Initial Technical Considerations
In the discussion below, there is frequent reference to utilizing biological compo-

nents (proteins, nucleic acids) imported from other organisms, enabling the yeast
to detect analytes or produce reporters for which they have no natural machin-
ery. Most often, genes encoding these components are provided on one or more
plasmids, but protein-coding genes or other genetic elements can also be inserted
directly into a yeast chromosome (see e.g., (Vopálenská et al. 2015; Yu et al.
2018)). The discussion below assumes a basic understanding of these tools and
approaches (as well as yeast cell biology more generally), but it should still be
understandable to those new to working with yeast. For information on working
with yeast plasmids, see (Gnügge and Rudolf 2017). For a discussion of standard
approaches to engineering genes onto yeast chromosomes and other aspects of
yeast molecular biology, see (Gardner and Jaspersen 2014). For information about
recent advances in yeast genetic engineering that may be relevant to biosensor
engineering, see reviews including (Malcı et al. 2020; Schindler 2020) and the
other chapters in this book.
3 Analytes Suitable for Whole-Cell Yeast-Based Biosensors
In theory, yeast can be harnessed to make a biosensor suitable for any analyte to
which yeast naturally respond (e.g., nutrients, chemical toxins, metals, mutagens,
temperature, generic stress) or anything for which an exogenous detection system
can be imported from another organism (e.g., medicines, human hormones, light).
In order to maintain a living sensor (and thus maintain the cell machinery), the
analyte of interest must be within a concentration range compatible with yeast via-
bility. For the purposes of detecting general hazards, one could use a sensor based
on cell death. Whole-cell yeast-based biosensors can be particularly appropriate
for situations where a biologically-active analyte (e.g., a drug or toxin) is present
in low concentrations under conditions where detection by typical analytical tech-
niques (e.g., mass spectrometry) is not feasible; they can also be particularly useful
when the goal is to detect a biological activity (e.g., mutagenicity) instead of a par-
ticular chemical, or when the analyte is of unknown or of mixed composition. Note
that these last two applications stretch the typical definition of “analyte”.
In trying to design a whole-cell yeast-based biosensor, a key consideration is
where the analyte is expected to be when it is detected: on the cell surface, in
the cytoplasm, or in some other cellular compartment? It is important to recog-
nize that some analytes can cross the membrane spontaneously (e.g., hydrophobic
molecules such as steroid hormones), while others cross by dedicated transporters
(for example, nutrients such as sugars), and still others become spontaneously con-
centrated in organelles such as the mitochondria (e.g., hydrophobic cationic dye
molecules). However, many remain outside the cell or at best sequestered in inter-
nal vesicles because they can’t cross membranes spontaneously. Considering the
expected localization of the analyte is essential for proper design of any whole-
cell biosensor because one needs to make sure that the necessary detection system
components are in the same compartment as the analyte.
In our discussion of detection systems, we provide some examples of specific
analytes for which whole-cell yeast-based biosensors have been developed. For a
more comprehensive list, see (Adeniran et al. 2015; Jarque et al. 2016a; Martin-
Yken 2020).
4 Detection Systems for Whole-Cell Yeast-Based

Biosensors
4.1 General Considerations
The detection system of many familiar whole-cell biosensors involves a specific

receptor (e.g., a locally-produced protein) that binds directly and specifically to the
analyte and then undergoes a conformational change in response to the binding
event. As discussed more below, this conformational change then activates, by
one of a number of possible mechanisms, a downstream sequence of events (the
transduction/amplification process) that eventually leads to a detected signal.
In theory, one might be able to design a receptor for a given analyte, but in prac-
tice, a much more effective approach (at least thus far) has been to find a natural
receptor for the analyte of interest. Because the range of chemicals/environmental
components detected by the diversity of the living world is enormous, there is a
reasonable chance that a receptor exists for the analyte of interest, or if not, some-
thing similar to it. If the desired receptor is unavailable, similar natural receptors
can then be used as a starting point for developing improved (e.g., more sensitive
or altered specificity) receptors through approaches such as genetic engineering
(see e.g., (Urlinger et al. 2000; Roney et al. 2016)) or directed evolution (see
(Zhou et al. 2006; Stainbrook et al. 2017; Adeniran et al. 2018)).
To harness an exogenous receptor for a yeast whole-cell biosensor applica-
tion, there are several issues to consider. First, one must understand the receptor’s
structure and its function in the context of the source organism. Just as whole-
cell biosensors are modular, so are the receptor proteins themselves. However, the
specific functionalities of a given receptor’s modules depend on the natural signal
transduction process it connects to. For example, some receptors work by binding
DNA; others change the concentration of a second messenger, interface with the
signal transduction machinery, or produce a metabolite. Knowing how the recep-
tor works in its natural setting is essential for determining how straightforward
it will be to integrate it with the endogenous yeast transduction machinery. This
need to communicate properly with endogenous systems makes it challenging to
utilize exogenous receptors. Another key point to consider in harnessing receptors
is where the receptor is expected to interact with the analyte: on the cell sur-
face, in the cytoplasm, or in some other cellular compartment. Making sure that
the receptor has access both to the analyte and the downstream signal transduction
machinery is essential. This can involve modifying the receptor to localize it to the
relevant part of the cell (e.g., the nucleus), though it is important to recognize that
such targeting does not always work as expected (e.g., (Emr et al. 1984)). Finally, it
is important to recognize that some additional endogenous cellular processes (e.g.,
amplification) are important for the proper function of a receptor-based signaling
process, while others such as sensory adaptation can interfere.
While the detection modules of whole-cell biosensors often utilize particular
receptors that bind in a specific way to individual analyte molecules, this is not
always the case. For example, as discussed more below, some detect activities such
as the ability to induce DNA damage (Paetkau et al. 1994; Billinton et al. 1998;
Burrill and Silver 2011; Lu et al. 2015; Bui et al. 2015), cause cell stress (Hollis
et al. 2000; Hung et al. 2018; Gong et al. 2020), or serve as nutrients (Shepherd
et al. 2021; Trentman et al. 2021).
4.2 Detectors for Cytoplasmic Analytes
Most whole-cell biosensors developed thus far detect analytes that can make their
way into the cytoplasm. Many of these utilize specific receptors that interface
with the transcription/translation system for the transduction part of the sensing
process. Thus, these receptors typically have an analyte binding domain and a
DNA binding domain; those that work as activators (not repressors) generally also
have a transcription activator domain. Generally speaking, binding of analytes to
these receptors changes their ability to bind DNA, thus altering the amount of
transcription of a reporter gene and ultimately the amount of reporter protein.
An example of an endogenous DNA-binding receptor used in biosensors is
provided by the copper-response system; the yeast CUP1 promoter is activated
in response to copper through the action of endogenous machinery including the
DNA-binding Ace1p protein (Dameron et al. 1991; Smith et al. 2017). One par-
ticularly well-characterized exogenous DNA-binding intracellular receptor is the
bacterial tetracycline receptor (TetR), which can either activate or repress tran-
scription in yeast in response to binding tetracycline; the specific effect depends
on the details of how the system is set up (Baron and Bujard 2000). TetR is a
very “portable” type of gene response element (i.e., it can work in diverse organ-
isms) because it needs nothing other than itself and a target sequence placed near
a promoter to cause production of a reporter in response to TetR.
Another example of an imported DNA-binding receptor is provided by the
human estrogen receptor (ER), mentioned earlier. However, while the DNA bind-
ing ability of Ace1 and TetR are directly regulated by binding to their ligands
(copper and tetracycline), the effect of estrogen on the DNA-binding activity of
ER is more indirect and involves the participation of some additional cellular fac-
tors. Ultimately, binding of estrogen to ER causes increased transcription from
promoters controlled by “estrogen-response elements” (EREs), similar to what
happens with TetR (Gruber et al. 2004). However, the change in transcription in
response to estrogen cannot be explained simply by differential binding to EREs
in the presence of estrogen. Instead, it appears to involve regulation of ER nuclear
localization through differential binding to chaperone HSP90 and also involves
the participation of co-receptors (Smith and Toft 1993). When such additional
elements are involved in the downstream transduction process, the detection ele-
ment is much less portable, and use of compatible eukaryotic systems that contain
these downstream components (either naturally or by engineering) can be essen-
tial. Indeed, the compatibility of yeast with the estrogen receptor (as well as other
steroid receptors, see (Ito-Harashima et al. 2020)) is an advantage of yeast over
bacteria for whole-cell biosensor applications (Routledge and Sumpter 1996).
Interestingly, the modularity of these DNA-binding intracellular receptors sug-

gests that one might be able to “mix and match” analyte-binding and DNA-binding
domains (Aranda-Díaz et al. 2017). Indeed, this logic has led to generation of
functional chimeric receptors where the presence of estrogen turns on a galactose-
responsive promoter (Louvion et al. 1993; Gao and Pinkham 2000; Miller et al.
2020b). However, this strategy generally depends on utilizing protein domains
that fold and act independently (i.e., that do not require interactions with distant
components of the original protein for activity).
One type of intracellular analyte receptor that has been used in bacteria but
much less so in yeast is riboswitches, which are self-regulating mRNA sequences.
The binding of an analyte to a riboswitch changes the shape of the riboswitch,
affecting whether the sequence is translated into a protein (Zhang et al. 2015).
Most riboswitches are found in prokaryotes in nature and will not work if directly
imported into yeast because of differences in translation between prokaryotes and
eukaryotes. Nevertheless, harnessing of riboswitches remains an interesting idea
that might have utility for yeast-based biosensors. As a related idea, ribozyme-
based switches controlling gene expression have been utilized in yeast as sensors
for the detection of aminoglycosides (Klauser et al. 2015).
While many detection systems for intracellular analytes use specific recep-
tors for detection and the transcription/translation system for transduction, other
designs exist. In particular, some specific receptors for cytoplasmic analytes uti-
lize metabolism for their transduction process, with the response being increased
(or decreased) cell division. Note that increased transcription may occur as part
of these responses, but the effect is indirect. Often these metabolism-based sen-
sors detect nutrients. For example, one could imagine a whole-cell yeast-based
biosensor designed to detect a specific sugar; in this biosensor, the receptor would
be an enzyme that starts the process of metabolizing that sugar, and the response
would be cell growth when the sugar is present (assuming that carbon is other-
wise limiting). In some examples of metabolism-based transduction systems, there
is no specific receptor: the detector is the cell as a whole because multiple pro-
teins are involved in parallel in the detection process. An excellent example of
this type of biosensor is provided by the use of yeast for "BOD" (biological oxy-
gen demand) assays, which are effectively assays for bioavailable carbon in all its
guises (Renneberg et al. 2004; Seo et al. 2009; Yudina et al. 2015). Similarly, a
yeast assay for bioavailable phosphorous has recently been developed (Shepherd
et al. 2021; Trentman et al. 2021). The response for these nutrient-detection assays
is typically increased cell growth, and readout of these systems can be accom-
plished through measurement of parameters ranging from turbidity as measured
by a spectrophotometer (Shepherd et al. 2021; Trentman et al. 2021) to oxygen
utilization as measured by potentiometry (Yudina et al. 2015). Using similar logic,
researchers have developed assays for toxins, where the toxin is detected through
decreased cell growth (Paetkau et al. 1994; Billinton et al. 1998; Hung et al. 2018).
However, it is important to note that systems utilizing cell division (increased or
decreased) as an output can be less specific than other typical reporting systems,
so it is essential to include appropriate controls.
Using a related set of ideas, researchers have developed yeast-based assays for
stress-inducing agents. Though these agents can overlap chemically and biologi-
cally with toxins, the transduction process in these assays occurs through induction
of cellular stress-response pathways. A typical response for these stress-detecting
sensors would be an accumulation of reporters placed under control of stress
response promoters (Hartner and Glieder 2006; Adeniran et al. 2015; Zhao et al.
2019). Similarly, there are yeast-based sensors for mutagens in which the yeast
genome is the detector, the transduction process is provided by the machinery
that senses and responds to DNA damage, and the response is increased tran-
scription/translation of reporters placed under control of DNA-damage-induced
promoters (Burrill and Silver 2011). An even more sensitive example of a muta-
gen detector is provided by a biosensor where the detector is a yeast CAN1 gene.
Mutations in CAN1 make yeast resistant to the toxin canavanine; the response to
mutagen is thus an increase in the number of yeast cells that are resistant to cana-
vanine, and the readout process is to count the number of canavanine-resistant
colonies (Ong et al. 2021).
4.3 Detectors for Analytes that Remain Outside of Cells
Extracellular analytes are typically detected by specific receptors. In contrast to the

intracellular-analyte-detecting receptors discussed above, a receptor that directly
detects an extracellular analyte needs to have an extracellular domain, one or more
transmembrane regions, and an intracellular domain (Fig. 2). The purpose of the
extracellular domain is to bind to the analyte, while the transmembrane domain
fixes the protein in the membrane, and the intracellular domain interfaces with one
of various possible transduction systems to convey to the rest of the cell that the
analyte is present. In most cases, the intracellular domain communicates with the
endogenous signal transduction machinery to trigger a downstream response. For
example, the intracellular domain could be an enzyme that catalyzes production
of a second messenger (a small molecule that diffuses away and turns on other
proteins), or a kinase that phosphorylates other proteins (which then diffuse away)
to turn them on or off (Fig. 2a).
Thus far, the field of biosensors based on heterologous transmembrane recep-
tors in yeast is under-developed, likely because of the challenges presented by the
need to integrate the heterologous receptors with the endogenous yeast downstream
transduction machinery and hook this machinery up to a useful response system.
As yet, much of the whole-cell biosensor work done in yeast with transmembrane
receptors has focused on G-protein coupled receptors (GPCRs, Fig. 2b), with a
few exceptions including antibody display for point of care devices (Venkatesh
et al. 2015). GPCRs are found throughout the eukaryotic world and are involved
in detecting the presence of compounds ranging from peptide hormones to tastants,
and even light (Hilger et al. 2018; Ahmad and Dalziel 2020). Yeast cells have two
endogenous classes of GPCRs: one type consists of two proteins that respond to
yeast mating factors, while the other responds to glucose (Versele et al. 2001).
Fig. 2 Examples of biosensor pathways for extracellular analytes, where endogenous signaling
pathways are typically used as part of the transduction process. The response can involve activa-
tion of transcription of a reporter gene or activation of other enzymes. a Analytes can either diffuse
across the membrane (red) or remain outside and bind to extracellular receptors (orange). Ligands
that can freely diffuse across the membrane typically bind to a receptor protein in the cytoplasm,
enabling the complex to enter the nucleus and alter transcription. For analytes that remain extra-
cellular, potential detection mechanisms include kinase receptors and second-messenger systems;
these bind ligands (orange) and then can either activate transcription, alter the activity of other
enzymes, or both. b Specific example of a medium-chain fatty acid biosensor detected through a
GPCR. A heterologous GPCR (yellow) detects a medium-chain fatty acid in the culture medium,
transmits this chemical signal to the yeast mating-pathway (mustard), which relays it to a synthetic
transcription factor (STF, orange). The STF activates transcription of GFP. The image for b was
reprinted with permission of the American Chemical Society (ACS). The original file can be found
at https://doi.org/10.1021/sb500365m. Further permissions regarding this image should be directed
to the ACS
Significantly, the mating factor-response GPCRs activate a signal transduction

pathway that leads to activation of mating-factor-responsive genes. Thus, it seems
logical that one could plug human GPCRs into this pathway to induce transcription
of a reporter. Unfortunately, researchers have found that most medically relevant
human GPCRs require significant optimization as to expression level, membrane
transport, and coupling to the downstream transduction machinery in order to work
usefully in yeast (reviewed by (Shaw et al. 2019)). However, progress is being
made through approaches such as genome engineering to remove interfering path-
ways (Shaw et al. 2019) and humanization of the yeast membrane to promote
proper folding, processing, and allosteric behaviors (Routledge et al. 2016). The
result of all this work is that yeast expressing human GPCRs are becoming strong
systems for biotechnological and medical applications (Lengger and Jensen 2020).
One question that must be addressed for extracellular analyte receptors is how
the presence of the bound analyte is conveyed from the extracellular part of the
receptor across the membrane to the intracellular part. The typical answer is that
the information crosses the membrane in the form of a conformation change. While
that is a reasonable answer for GPCRs (which cross the membrane seven times,
providing a 3-D structure by which to convey allosteric change), the mechanism
of this information transfer is less clear for proteins like receptor kinases, which
generally cross the membrane only one time (Fig. 2). One plausible explanation is
that binding of the receptor to analyte induces dimerization, and it is the dimeriza-
tion event that turns on downstream signalling (Nakamura et al. 2016). Thus, this
dimerization response could be considered the first step in the transduction pro-
cess. This point suggests that regulation of the level of expression of the receptor
itself can be very important for functionality of a sensing system: if the level is
too low or too high, performance of a system that relies on regulated dimerization
could be severely compromised.
5 Transduction and Amplification Pathways
5.1 Typical Transduction Pathways Used by Whole-Cell

Yeast-Based Biosensors
As discussed above, while receptors can either be endogenous or exogenous,

the transduction process typically occurs via cellular machinery: the transcrip-
tion/translation system (as exemplified by the copper or tetracycline response
systems), signaling machinery (as seen with GPCR-mediated responses), or
metabolism (e.g., sensors for nutrients such as phosphate). We won’t repeat these
discussions, except to stress that in the case of imported detection or response sys-
tems, it is essential to identify all necessary components, ensure they are present,
and determine that these components interact properly with the cellular transduc-
tion machinery. In addition, it is important to remember that many endogenous
biological information processing elements integrate inputs from multiple sources.
For example, metal-response promoters can sometimes respond to other metals
or even other stresses, meaning that responses may not be as specific as expected.
For example, the copper-responsive promoter CUP1 is induced not only by copper
(through Ace1p) (Jeyaprakash et al. 1991) but also heat shock, glucose starva-
tion, and oxidation stress (through Hsf1p) (Tamai et al. 1994). A related issue is
that the biosensor elements themselves may have potentially confounding effects
caused either by simple overexpression (e.g., through selfish recruitment of cellu-
lar machinery) or protein-specific toxicity (which can cause off-target cell stress
responses that are aggravated by overexpression). Thus, the strength of the pro-
moters and/or types of plasmids can affect the overall success of the biosensor.
Finally, cellular sensing pathways often include mechanisms for adaptation and
desensitization, similar to those experienced by human eyes upon exposure to
light. Engineering out these additional inputs/influences can be helpful or even
necessary (Versele et al. 2001; Mukherjee et al. 2015; Hilger et al. 2018; Shaw
et al. 2019).
5.2 Other Potentially Useful Transduction Pathways
While most extracellular reporters interface with endogenous signal transduction

systems, other possibilities exist, and they may provide inspiration for synthetic
biologists designing new types of whole-cell biosensors. One intriguing example
is provided by the Notch pathway of animal development, where a transcription
factor is released upon ligand binding by an induced proteolytic cleavage (Hori
et al. 2013). This and related systems offer the possibility of utilizing tension (not
just conformational change) as part of the transduction process. Harnessing the
Notch system would likely be challenging because of the number of components
involved and the need to introduce them to yeast, but this type of approach could
potentially be useful for connecting an extracellular sensor to a transcription-based
transduction system.
5.3 Amplification
A key element common to most of the transduction systems discussed above is

that the signal is amplified as part of the transduction process. For example, a
single analyte binding to a single DNA-binding receptor can result in production of
many mRNA molecules and many protein reporter proteins. Similarly, endogenous
signal transduction pathways typically incorporate amplification at multiple steps.
For example, activation of an initial receptor enzyme activates multiple additional
enzymes, creating a “signaling cascade” (Nelson and Cox 2017). Amplification can
also occur through cell division or the use of a population of yeast as the detecting
unit instead of single cells (e.g., (Ong et al. 2021)). Amplification at some point
in the detection process is important to high-sensitivity sensing because without it
there would be a 1:1 correspondence between analyte-binding and reporter signal,
meaning that only high-concentration analytes could be detected. Amplification
could occur either during the biological part of the transduction process or during
readout (e.g., as part of a fluorescence detection process). However, if the goal is to
harness the biosensor for a technology-limited environment, it can be advantageous
to utilize biological amplification.
The need for amplification for high-sensitivity sensing tasks reduces the util-
ity of engineered molecules such as aptamers for whole-cell sensing applications.
Briefly, aptamers are short single-stranded DNA or RNA molecules that can be
selected from a library to bind specifically to a target (Adachi and Nakamura
2019; Wang et al. 2020). These and similar tools (e.g., phage display antibodies
(Peltomaa et al. 2019)) are attractive as sensing tools because in theory one can
use selection (perhaps as optimized by directed evolution) to select molecules that
bind specifically to essentially anything. However, the challenge to using these
molecules for whole-cell biosensing applications is figuring out how to couple
the molecule to endogenous transduction/amplification systems or design novel
systems with sensitivity sufficient to detect exogenous analytes.
For example, one response system commonly used in conjunction with aptamers
is FRET (Fluorescence/Förster Resonance Energy Transfer). With FRET-based
aptamer-sensing molecules, the probes are placed such that FRET increases (or
decreases) upon binding to analyte (reviewed by (Yoon et al. 2012)). FRET
reporters can indeed be very effective. However, the ~ 1:1 ratio between detec-
tion (binding event) and response means that the concentration of analytes needs
to be relatively high for this method to be effective, or that the detection system
needs to be very sensitive. Thus, unless one can design a way to amplify the signal
from the aptamer (or similar molecule) and do so in a way that takes advantage
of the cellular environment, it might be more effective to stick with a non-cellular
context for using these tools to detect extracellular analytes if the goal is to do so
in a technology-limited environment.
6 Response and Readout
The biological part of the transduction process of a whole-cell biosensor ends

at the response, which often consists of production or activation of a reporter, a
biomolecule or process that directly or indirectly creates a signal that is detectable
(through readout) by the outside world (Table 2).
6.1 Color-Based Reporters
One early reporter for DNA-binding intracellular receptors was beta-galactosidase,

a cytosolic protein from bacteria that cleaves an exogenous chemical called X-
gal to release a dark blue product. This reporter is functional in the context of
whole-cell biosensors (see e.g., (Weaver et al. 2015)), but has several drawbacks,
especially for applications in technology-limited environments. One major concern
is the need for X-gal itself, a rather expensive and unstable compound. Another
is that X-gal does not cross the cell membrane, which means that cell lysis (e.g.,
by liquid nitrogen) is required to enable the enzyme to access the X-gal sub-
strate (Möckli and Auerbach 2004; Weaver et al. 2015). An additional drawback
is the time required for signal development: any reporter based on transcrip-
tion/translation is likely to need several hours to accumulate sufficient amounts
of the reporter protein. This beta-galactosidase reporter system needs several addi-
tional hours to develop the color after the reporter protein is expressed (Möckli
and Auerbach 2004; Weaver et al. 2015).
A variety of researchers have tried to enable improved color-producing reporters
by using other combinations of enzymes and substrates or by secreting the reporter
enzyme out of the cell so that the color-producing substrate can be provided in
the exterior environment. The recent development of technologies for producing
betalains (a family of colored compounds including beet-red betanin) in yeast
is promising (Grewal et al. 2018). Regardless, many approaches for colorimetric
biosensors still rely on the lacZ reporter system (Martin-Yken 2020). Due to the
Table 2 Examples of reporting methodologies for yeast biosensors

Type of Examples Advantages Disadvantages Selected examples
response from this review
process
Color-based X-gal, betalains, Readout visible Expensive and Möckli and
lycopene to the naked eye unstable substrate Auerbach (2004),
for reaction; cell Weaver et al,
lysis must occur (2015),Ostrov
for X-gal; color (2017), Grewal
development can et al. (2018), Chen
take a long time et al. (2018),
(~hours-days) Martin-Yken
(2020)
Light-based GFP, YFP, RFP Relatively quick Requires specific Leskinen et al.
Luciferase (~hours); filters/light (2003), Allard
noninvasive wavelengths (this (2008), Adeniran
readout; may be helped et al. (2015),
sensitive; with advances in Borse et al.
fluorescent portable (2017), Miller
proteins do not fluorescence et al. (2020a),
require substrate readers); Martin-Yken
luciferase requires (2020)
an expensive
substrate
Olfactory Isoamyl-acetate Readout only Difficult to Miller et al.
using human quantify (2020b)
nose; minimal
training required
Probe-based Amperomentry Quick (~hours or Requires Renneberg et al.
measurement (current), faster); immobilization (2004), Seo et al.
potentiometry noninvasive and specialized (2009), Yudina
(pH) readout; sensitive equipment et al. (2015),
Yudina et al.
(2015)
Metabolism Cell growth/death No Less specific than Hung et al. (2018),
bioengineering other typical Shepherd et al.
required reporting systems; (2021), Trentman
systems based on et al. (2021)
nutrient depletion
or colony
formation can take
several days for
final readout
Hybrid Cell growth Readout visible Systems based on Lehmann et al.
process following to the naked eye colony formation (2000)
analyte-induced can take several
expression of days for final
required enzyme readout
lower protein expression levels and the longer replication times of yeast compared
to other potential whole-cell biosensors (e.g., E. coli), signals produced by these
color-based systems are relatively weak and can take a long time (e.g., >24 h) to
develop (Chen et al. 2018).
6.2 Light-Based Reporters
Because of these challenges with color-based systems, a popular set of reporters

for transcription/translation-based transduction processes is GFP (green fluores-
cent protein). GFP and similar polypeptides emit fluorescent light in response to
excitation. Readout of the reporter can be assessed quickly, non-invasively, and
without additional manipulation simply by exposing cells to the appropriate exci-
tation wavelength and assessing the light level at the expected emission wavelength
(Lei et al. 2006). A potential drawback of these systems is that filters and lights
of specific wavelengths are generally needed to assess the level of fluorescence.
However, recent work has shown that inexpensive, portable fluorescence readers
have made the use of fluorescence as a reporter much cheaper and has allowed
for data collection in the field (Borse et al. 2017; Miller et al. 2020a). Decreasing
background noise/interference and enhancing the fluorescence signal can improve
fluorescence detection.
Another popular reporter is luciferase, which produces light when provided with
the substrate luciferin. A disadvantage of luciferase is that the luciferin substrate
is both somewhat expensive and has a limited shelf-life (Leskinen et al. 2003).
However, because the substrate is dark, there is a very low background, meaning
that readout of luciferase levels is 10–1000 × more sensitive than a comparable
assay with GFP (Allard 2008).
6.3 Olfactory Reporters
The reporters discussed thus far are light-based: they produce light or
color during the readout process. A more recently developed reporter for
transcription/translation-based transduction processes in yeast is an enzyme that
produces a scent: “scentsor” assays involve the induction of a reporter enzyme
that produces banana smell (Miller et al. 2020b). Aromatic compounds and their
properties have been paired with yeast for years through bioengineering and fer-
mentation (Van Wyk et al. 2018), making diverse olfactory reporters in whole-cell
yeast-based biosensors a realistic possibility. The benefit of these assays is that the
readout requires no equipment other than the human nose and minimal training
or resources outside of the biosensor itself. However, it is difficult to quantify a
scent, limiting scentsor-based assays to yes/no assessments. Despite these chal-
lenges, the ease of use suggests that olfactory-based reporter systems could be
ideal for technology-limited environments (Miller et al. 2020b).
6.4 Other Potentially Useful Reporters
As noted above, cell growth and/or metabolism can be used as reporters for biosen-
sors used to detect nutrients (e.g., (Shepherd et al. 2021; Trentman et al. 2021)) or
toxins (Hung et al. 2018).
In addition, while the readouts of the reporters above follow logically from
the activities of the reporter proteins themselves, it is possible to have situations
where the responses, reporters, and readouts are less directly connected and/or
have additional layers. For example, Lehmann et al. (2000) engineered a yeast
strain in which a lactose-metabolizing enzyme (LacZ) was placed under control
of a copper-responsive promoter. Because yeast cannot otherwise metabolize lac-
tose, this created cells that require copper to grow on lactose. Thus, with this
strain, one can use growth on media lacking a carbon source other than lactose
to detect the presence of copper. In this case, LacZ is a reporter, and induction of
LacZ is one level of response, but cell growth resulting from the copper-induced
ability to metabolize lactose is another level of response. The overall readout for
detection of copper would be whatever method is convenient to assess cell growth
and/or metabolism; Lehmann and colleagues actually used amperometry to mea-
sure oxygen consumption. Using a similar strain in which LacZ was induced by
copper, Tag et al. (2007) measured changes in lactose concentration in response to
heavy metals. This lactose-based method works in combination with flow injection
analysis to allow for semi-continuous measurements and to increase the sensor’s
capability as an in-line sensor for heavy metal waste produced by manufacturers.
7 Whole-Cell Biosensor Housing and Reading Devices
The text thus far focuses on the characteristics of the yeast cells used for whole-
cell biosensor work. To make a useful whole-cell biosensor-based device, one
generally needs to design a type of "housing" to hold the cells, expose them to
a potential analyte, and develop the signal. Also needed is a reading device to
interpret (recognize and/or quantify) the signal, though in some cases the senses
of the biosensor-users themselves (e.g., eyes to detect color, nose to detect smell)
can provide this functionality. An additional issue is that one also needs a way to
distribute the biosensor cells. Because this review focuses on whole-cell biosen-
sors for use in technology-limited settings, we concentrate here on reading and
housing devices that require minimal skills or access to specialized equipment for
use; such devices are generally based on large populations of cells. However, it is
important to recognize that yeast-based whole-cell biosensors have a long history
of use in laboratory settings (see e.g., (Routledge and Sumpter 1996)), and appli-
cations based on small populations (as in microtiter plates) or individual cells (as
in microfluidic devices) have been proliferating. We discuss some microtiter plate-
based approaches below, but for a deeper discussion of lab-based yeast whole-cell
biosensor-devices, see (Adeniran et al. 2015; Jarque et al. 2016a; Martin-Yken
2020).
First, it is important to clarify what we mean by "low-tech, field-friendly" hous-

ing devices. Such devices should be able to be distributed and employed at the
point of use without sterile technique and preferably without micro-pipettes. While
the end use of such sensor devices may have few requirements, it is important to
emphasize that the manufacturing of these devices generally will require a labo-
ratory setting. Similarly, low-tech field-friendly reading devices should be small,
inexpensive, require minimal training, and have minimal power requirements (i.e.,
preferably they would require no power or be battery-operable).
Given these restrictions, three basic categories of low-tech devices for hous-
ing whole-cell yeast-based biosensors become particularly apparent: bio-paper
analytical devices (bioPADs), microtiter plates, and plastic culture tubes. In a
yeast bioPAD, biosensor yeast cells are immobilized in a hydrogel on paper, and
are exposed to the potential analyte by soaking the PAD in an aqueous solu-
tion containing the potential analyte. In present implementations of yeast PADs,
this solution also contains some yeast nutrients as well as antibiotics to reduce
the growth of bacteria (Weaver et al. 2015; Miller et al. 2020a). However, one
could imagine lateral flow versions where nutrients, antibiotics, and any additional
reagents necessary for function of the reporter (e.g., color-generating substrates)
are placed on the PAD below the yeast spot.
Advantages to bioPADs include transportability (they are small and light), ease
of use, and stability (bioPADs have been shown to be shelf-stable for > 1 year
at −20 °C, > six months at 4 °C, and at least 56 days at 37 °C) (Weaver et al.
2015; Miller et al. 2020a). However, one challenge thus far has been reading them.
An initially published color-based reporter (LacZ) is not field-friendly because it
requires that the yeast be lysed with liquid nitrogen to release the LacZ enzyme
and subsequently “developed” by exposing the bioPADs to a solution containing
the color-developing substrate X-gal (Weaver et al. 2015). If a suitably intense
and rapidly developing color-based reporter could be developed, then a cell phone
might be an ideal reader (see e.g., (Ballard et al. 2020)). More recently, we have
developed a bioPAD that utilizes a fluorescent reporter (Miller et al. 2020a). For
reading this bioPAD, we utilized a small and inexpensive home-built reader device,
which is collapsible and light for ease of transport (Miller et al. 2020a). Others
have worked to develop cell-phone-based fluorescence readers, suggesting this will
be an option in the future (Wei et al. 2013; Vashist et al. 2014; Berg et al. 2015).
Another type of housing device that should be adaptable to work in a field envi-
ronment is the microtiter plate. Microtiter plate-based whole-cell biosensor assays
have been long-established in laboratory environments (see e.g., (Cevenini et al.
2018; Miller et al. 2020a)). As presently described, most microtiter plate assays
require sterile technique, incubating shakers, and use of micro-pipettes. However,
we have developed an assay where yeast can be grown in microtiter plates without
shaking, at room temperature (Shepherd et al. 2021; Trentman et al. 2021). Read-
ing these microtiter plates is then the remaining challenge. Remarkably, a number
of authors have worked to develop cell-phone based microtiter plate readers (Berg
et al. 2015; Cevenini et al. 2018; Wang et al. 2018), meaning that microtiter-based
assays may become suitable for technology-limited settings. In the interim, many
relatively modest laboratories have access to standard plate-reading devices. One

question is how yeast would be distributed across the microtiter plate. If access to
micropipettes is lacking, dry yeast could perhaps be preloaded.
For cases where microtiter plates are problematic, another possibility is sim-
ply growing the yeast in capped plastic centrifuge tubes; for this approach, tubes
might be distributed after preloading them with freeze-dried yeast and media (more
about yeast long-term storage below). For any particular assay developed under
more standard lab conditions, controls would have to be performed to ascertain
if/how growing in a capped tube at room temperature would affect the biosensor
performance. However, based on our experience, yeast-based biosensors can be
remarkably resilient to changes in aeration and/or temperature: the main effect is
an alteration to the time needed for signal development. While housing biosen-
sors in pre-loaded plastic tubes is not currently common practice, one that fvcould
imagine that pre-loading plastic tubes for whole-cell yeast-based biosensors could
increase their utility in the field or use in LMICs where materials may be scarce.
Two examples of assays that could benefit from such a process would be the
“Scentsor” (Miller et al. 2020b) and the phosphorus centrifuge tube assay (Shep-
herd et al. 2021; Trentman et al. 2021). While neither system relies on complex
signal reading devices (they use scent and optical density respectively) they do
rely on culturing the yeast with their analyte of interest. Through the use of pre-
loaded tubes containing the components necessary for the assays to function, these
biosensors could be made available for use in community testing sites or other
technology-limited settings by only requiring the addition of the sample of interest.
8 Long-Term Storage
Whole-cell yeast-based biosensors must be stored in a way that the biosensors can
be transported and used at a later time. When biosensor yeast are used in paper-
based assays, yeast cultures are typically stored using a sugar-based hydrogel to
protect the yeast until the biosensor is ready for use (Fine et al. 2006; Weaver
et al. 2015; Jarque et al. 2016b; Miller et al. 2020a). As noted above, bioPADs
constructed with yeast in hydrogel have been shown to be shelf-stable for > 1 year
at -20 °C, > six months at 4 °C, and at least 56 days at 37 °C (Weaver et al. 2015;
Miller et al. 2020a). Storage of yeast spotted onto paper-based "dipstick" biosen-
sors in argon has also been reported (Ostrov et al. 2017). For long-term storage,
drying methods such as lyophilization (freeze-drying) or storage in hydrogel have
been effective. Depending on when the biosensors will need to be used, freeze-
dried yeast can be stored for 2 months at 4 °C or 10 months at -18 °C and still
retain normal activity and sensitivity in assays (Jarque et al. 2016b). Immobiliza-
tion in polyvinyl alcohol hydrogel particles increased yeast storage time to 1 year
(Herkommerová et al. 2018). More high-tech treatments may also have utility in
enhancing long-term storage. For example, the application of different coatings
(e.g. polyelectrolytes, biomolecules, metal nanoparticles, or oxides) has been found
to improve reproducibility and cell sensitivity, viability, and stability as compared
to bare cells (Bittner et al. 2015; Dai et al. 2018). In particular, bio-silica sol-gels
can act as protective shells against environmental factors. These gels have even
been found to protect cells from exposure to heavy metals and UV radiation for
up to 28 days (Ponamoreva et al. 2015).
9 Conclusions
Whole-cell yeast-based biosensors are affordable and transportable devices that

can detect analytes of interest in an environment with limited resources. These
biosensors excel at detecting biologically relevant analytes, including pharma-
ceuticals, toxins, and chemically undefined or complex mixtures ranging from
environmental mutagens to bioavailable phosphorous. They can be tailored to
detect a desired analyte using a range of possible readout mechanisms by har-
nessing the large existing (and continuously expanding) toolbox of modular
parts assembled from across the biological world. Recent work on whole-cell
yeast-based biosensors has been focused on expanding the applications of the
biosensors and improving the sensors in terms of signal response, sensitivity, and
reporter/readout mechanisms. The largest roadblock to utilization of whole-cell
yeast-based biosensors is the time associated with the transduction and response
processes; harnessing other natural biological processes (e.g., using signal trans-
duction pathways instead of transcription/translation) or creating new processes
through synthetic biology should provide significant improvements in analyte
detection time. Ideally, improved transduction/response processes would be paired
with a readout mechanism that requires minimal additional technology (e.g., color-
changing reporters requiring only the user’s eyes for analyte detection) so that the
target analyte could be detected quickly in a field setting. Despite these challenges,
yeast cell hardiness and biosensor modularity suggests these obstacles can be
overcome, affirming the utility of whole-cell yeast-based biosensors as an analyte
detection tool in the lab and field.
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2780
Applications of Yeast Synthetic Biology
Yeast Synthetic Biology Approaches
for the Production of Valuable
Polyphenolic Compounds
Daniela Gomes, João Rainha, Ligia R. Rodrigues,

and Joana L. Rodrigues
Abstract
Polyphenols are secondary metabolites isolated from plants that are known
for their biological and therapeutical properties. However, the extraction from
plants renders low yields, besides being expensive and not environmentally
friendly. Therefore, the use of heterologous microorganisms, so-called micro-
bial chassis, became an interesting alternative approach to produce polyphenols.
With the advances in the metabolic engineering and synthetic biology fields, the
development of such microbial chassis able to produce these compounds with
higher yields and productivities became easier. Several yeast species, such as
Saccharomyces cerevisiae, Yarrowia lipolytica, and Pichia pastoris, have already
been engineered to produce some polyphenols. However, there is still a long
way to go before these compounds can be produced by heterologous organisms
at an industrial scale. In this chapter, we review the recent advances in the pro-
duction of polyphenolic compounds using yeasts as heterologous hosts, as well
as several synthetic biology approaches used to improve such production.
1 Introduction
Polyphenolic compounds or polyphenols are valuable compounds that are naturally

present in plants. These secondary metabolites have several recognized health ben-
efits due to their antioxidant, anti-inflammatory, anticancer, antiviral, and wound
healing properties (Hussain et al. 2016; Panda et al. 2017; Park 2015; Rodrigues
et al. 2015c; Tsao 2010). However, polyphenols are present in very low amounts in
plants. For this reason, their isolation from plants besides being difficult and expen-
sive is not enough to support their increasing demand (Krivoruchko and Nielsen
D. Gomes · J. Rainha · L. R. Rodrigues · J. L. Rodrigues (B)

Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal
https://doi.org/10.1007/978-3-030-89680-5_5
120 D. Gomes et al.
2015; Rodrigues et al. 2015c). The production of polyphenolic compounds using

microorganisms as heterologous chassis could be an interesting alternative method
to produce them in a fast, cheap, and more environmentally friendly way (Rainha
et al. 2020a). To achieve the production of polyphenols in microorganisms, it is
necessary to design and construct the biosynthetic pathways responsible for their
production. This step is challenging since it requires the introduction of several
heterologous genes into the chassis. Moreover, these genes should be efficiently
expressed and produce functional enzymes (Braga et al. 2018, 2019). With the
exploitation of metabolic engineering and synthetic biology tools, the construction
of complete biosynthetic pathways in microorganisms became easier. These tools
have also been used to improve the host chassis toward an increase in the produc-
tion of the desired compounds. The heterologous production of plant polyphenolic
compounds was mostly explored using Saccharomyces cerevisiae as yeast chas-
sis (Rainha et al. 2020a). Nevertheless, in the last years, other yeasts such as
Pichia pastoris and Yarrowia lipolytica have been also explored as alternatives to
produce relevant polyphenolic compounds. In the following subchapters, the bio-
logical relevance and the biosynthetic pathways responsible for the production of
different polyphenolic compounds are described. Moreover, several examples of
heterologous production of plant polyphenols in yeasts will be explored.
2 Polyphenolic Compounds
Polyphenolic compounds are naturally produced in plants and interfere in several

physiological processes, namely in plant growth and development, seed germi-
nation, and response to biotic and abiotic stresses (Sharma et al. 2019; Tanase
et al. 2019). Polyphenolic compounds have been widely used in the pharmaceuti-
cal industry due to their relevant biological activities. Polyphenols have also been
widely used in the food industry as food additives and in active food packaging to
improve food preservation and stability (de Araújo et al. 2020; Martillanes et al.
2017). Moreover, these compounds have also other commercial applications, such
as in the production of cosmetics, fertilizers, paints, surfactants, among others (de
Araújo et al. 2020). Due to these potential applications, the global market size for
polyphenols is expected to reach USD 2.26 billion by 2027 with a CAGR of 7.2%
(Proficient Market 2020).
Phenolic compounds are composed of one aromatic ring containing one or more
hydroxyl groups. These compounds are divided into two subclasses: hydroxyben-
zoic acids and hydroxycinnamic acids. Hydroxycinnamic acids are the precursors
of polyphenolic compounds. These compounds are constituted by multiple phe-
nol building blocks. Due to their large diversity, polyphenols are also organized
in different subclasses (Fig. 1). The largest subclass of polyphenols is flavonoids.
However, polyphenols can also be divided into stilbenoids, curcuminoids, lignans,
polyphenolic amides, and coumarins (Singla et al. 2019).
Yeast Synthetic Biology Approaches for the Production of Valuable … 121
Fig. 1 Different classes and examples of polyphenolic compounds
3 Biological Activities
Polyphenolic compounds are widely distributed in nature, namely in plants, fruits,

and vegetable species. Moreover, more than 8000 different structures are known
(Tsao 2010). In the last years, polyphenols have been widely studied as biolog-
ically active compounds and have shown health benefits in the prevention and
treatment of several diseases due to their recognized antioxidant, anticancer, antivi-
ral, anti-inflammatory, and wound healing properties (Braga et al. 2018; Cassidy
et al. 2020; Dayem et al. 2016; Rodrigues et al. 2015c; Sobhani et al. 2020; Van
de Velde et al. 2019).
3.1 Antioxidant
Polyphenols are recognized as natural antioxidant molecules due to their ability

to scavenge, inhibit, and neutralize reactive species. The antioxidant properties
of these compounds are affected by the number of hydroxyl groups and their
arrangement in the aromatic ring. For example, the flavonoids 3 -hydroxygenistein,
3 -hydroxyldaidzein, 6-hydroxyldaidzein, and 8-hydroxyldaidzein were found to
be more potent antioxidants than quercetin and ascorbic acid due to the presence
of one more hydroxyl group (Rüfer and Kulling 2006). Moreover, polyphenols are
also able to activate antioxidant enzymes and to perform the inhibition of enzymes
involved in the oxidation process (Hussain et al. 2016).
The application of several polyphenolic compounds in the treatment of diseases
caused by oxidative stress has been widely studied (Huang et al. 2018; Panda et al.
2017; Rashmi et al. 2017; Sökmen and Akram Khan 2016). The protective effect
of naringenin in Alzheimer’s disease was studied. Ghofrani et al. (2015) showed
122 D. Gomes et al.
that naringenin pretreatment improves the memory and learning capacities in Aβ-
injected rat model. These beneficial effects were achieved due to the decrease
in lipid peroxidation and apoptosis in the hippocampus. Moreover, curcumin was
found to protect biomembranes by scavenging oxygen free radicals like hydroxyl
radicals and superoxide anions responsible for initiation of lipid peroxidation in
liver and kidneys of diabetic db/db mice. These results showed that curcumin could
be used to prevent oxidative stress in diabetes patients (Soto-Urquieta et al. 2014).
3.2 Anticancer
Cancer is considered one of the most frequent causes of death worldwide, and
it is considered a health problem due to its high incidence in society. In 2018,
the World Health Organization estimated that among 18.1 million cases of cancer,
9.6 million cases would result in death (World Health Organization 2018).
To reduce cancer mortality, the discovery of new therapeutic compounds
became a priority. Polyphenols have been reported as potential therapeutic com-
pounds for several types of cancer. These compounds can interfere in cell
proliferation, tumor initiation and proliferation, metastasis, angiogenesis, and
apoptosis (Niedzwiecki et al. 2016). These effects are attributed to the ability of
polyphenols to interact with transcription factors, signaling molecules, growth and
apoptotic regulators, and adhesion molecules (Avtanski and Poretsky 2018; Khan
et al. 2020; Park 2015). For example, it was shown that curcumin could decrease
the Warburg effect in different cancer cell lines. The Warburg effect is character-
ized by the high productions of lactate and high glucose uptake by tumor cells
even when oxygen is available. The reduction of the Warburg effect was achieved
due to the ability of curcumin to downregulate pyruvate kinase M2 expression
by regulating mammalian target of rapamycin (mTOR)/ hypoxia-inducible factor
1–α (HIF1α) pathway. Additionally, it was also demonstrated that the viability of
cancer cells was reduced after curcumin treatment (Siddiqui et al. 2018). Eriodyc-
tiol also showed anticancer effect against human lung cancer cell line A549. This
effect was characterized by downregulating the expression of the apoptotic protein
Bcl-2 and upregulating the expression of Bax protein. Consequently, apoptosis
was induced. Additionally, phosphatidylinositol 3 kinase (PI3K)/protein kinase B
(AKT)/mTOR signaling pathway was also inhibited. All these modifications in the
protein expression have contributed to the inhibition of the cancer cell’s growth
(Zhang et al. 2020). Another polyphenolic compound that has been widely stud-
ied as an anticancer agent is quercetin. This flavonoid showed activity against
several types of cancer cell lines. For example, Hashemzaei et al. (2017) have
studied the effect of quercetin treatment in nine different tumor cell lines. In all
the tested tumor cell lines, it was concluded that quercetin induces apoptosis.
Additionally, the effect of quercetin in vivo was also assessed in mice bear-
ing estrogen receptor-positive breast cancer MCF-7 and colon carcinoma CT-26
tumors. After the treatment with quercetin, a significant reduction in the tumor
volume was found. The authors concluded that quercetin has the potential to be
used as an anticancer agent due to its in vitro and in vivo effects. The anticancer
properties of resveratrol have been also studied. For example, Zeng et al. (2017)
studied the effect of resveratrol in human colon cancer cells. The authors have
found that resveratrol inhibits the proliferation and induces the apoptosis of these
cells. Moreover, it was also reported that resveratrol upregulates the activity of
bone morphogenetic protein 7 (BMP7). BMP7 is considered an effective tumor
suppressor that interacts with the PI3K/AKT signaling pathway. This signaling
pathway was inactivated by resveratrol, and its inactivation was potentiated by
BMP7, thus suggesting that resveratrol could be applied in the treatment of colon
cancer. In addition to these studies, there are many more that demonstrate the abil-
ity of polyphenols to act on several cancer types (Abbaszadeh et al. 2019). For this
reason, these compounds have a lot of potentials to be used as anticancer drugs.
3.3 Antiviral
Beyond the antioxidant and anticancer activities, polyphenols have also been
reported as natural compounds with antiviral properties due to the presence of
hydroxyl and ester groups (Kamboj et al. 2012). Several studies have reported the
antiviral activity of polyphenols against a large variety of virus, such as herpes
simplex virus (HSV), dengue virus (DENV), zika virus, human immunodeficiency
virus (HIV), severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2),
among others (Ali and Banerjea 2016; Chen et al. 2012; Jasso-Miranda et al.
2019; Lee et al. 2017; Paraiso et al. 2020; Vázquez-Calvo et al. 2017). For exam-
ple, the in vitro effect of a mixture of polyphenols extracted from almond skin in
HSV type I was tested. The treatment with 0.4 mg/mL of the mixture contain-
ing catechin, naringenin-7-O-glucoside, kaempferol-3-O-glucoside, epicatechin,
isorhamnetin-3-O-rutinoside, and isorhamnetin-3-O-glucoside led to a reduction
in the expression of viral proteins ICP0, UL42, and Us11. Moreover, the viral
DNA accumulation was lower compared to the control (infected cells without any
treatment). The results showed that the mixture of polyphenols tested is efficient in
the treatment of HSV type I infected cells (Musarra-Pizzo et al. 2019). Quercetin
has also shown activity against HSV type I. In this study, Raw 264.7 cells were
infected with HSV type I in the presence of quercetin at different concentrations.
Quercetin reduced the expression of HSV-1 glycoprotein D that is essential for
viral entry into the host cells and ICP0 that is expressed in the HSV-1 replica-
tion cycle. The effect on the viral proteins responsible for the virus replication
was also studied. The levels of expression of ICP0, UL13, and UL52 were signifi-
cantly reduced in cells treated with quercetin. With these results, it was possible to
conclude that quercetin affects the virus entrance in the host cells and its further
replication (Lee et al. 2017). Another compound with reported antiviral proper-
ties is curcumin. For example, this compound has shown activity against HIV by
the degradation of the Tat protein that is responsible for viral replication. More-
over, the treatment of infected cells with this compound resulted in significant
inhibition of virus production (Ali and Banerjea 2016). Polyphenols, like fisetin
124 D. Gomes et al.
and quercetin, were also studied as efficient antiviral compounds in DENV infec-
tion. Fisetin and quercetin were found to inhibit DENV-2 and DENV-3 infections.
Both compounds exhibited an effect in the inflammatory response by decreas-
ing the production and secretion of tumor necrosis factor-alpha (TNF-α) that is
related to severe DENV infections (Jasso-Miranda et al. 2019). Recently, polyphe-
nols have also shown potential activity against SARS-CoV2 (Paraiso et al. 2020).
It was found that angiotensin-converting enzyme-2 (ACE2) cellular receptor is
downregulated during SARS-Cov2 infection, and this downregulation could be
the reason for the pathogenesis and progression of SARS-CoV2. Horne and Vohl
(2020) have suggested that resveratrol has the potential to control the pathogenesis
of SARS-Cov2 by upregulating the ACE2 cellular receptor.
3.4 Anti-Inflammatory and Wound Healing Agents
Inflammation is a natural response of the body to the presence of pathogens

or non-infectious factors like irradiation, toxic compounds, damaged cells, and
among others. This inflammatory response is characterized by the production
and secretion of pro-inflammatory cytokines and microbial products. During the
inflammatory response, inflammatory cytokines, like TNF-α, interleukin (IL) 1β
and 6, are synthesized and secreted and their interaction with different receptors
leads to the activation of signaling pathways (Chen et al. 2018).
Polyphenols have been reported as natural compounds with the ability to mod-
ulate inflammatory responses. These compounds can interfere in the regulation of
pro-inflammatory gene expression and the synthesis of pro-inflammatory cytokines
(Yahfoufi et al. 2018). For example, genistein showed an ability to suppress the
skin inflammatory response induced by psoriasis. This flavonoid was able to
decrease the production of inflammatory cytokines, such as TNF-α, IL-1β, IL-
6, IL-17, and IL-23, in skin mouse and human keratinocyte HaCaT cells with
induced psoriasis-like inflammation. Moreover, genistein also inhibited STAT3 and
nuclear factor kappa-β (NF-kB) phosphorylation. Consequently, it was concluded
that genistein reduced the inflammatory response in psoriasis-like models (Wang
et al. 2019). Naringenin has also shown anti-inflammatory activity in murine mod-
els of induced liver injury. After the treatment with naringenin, the authors have
found a decrease in the production of TNF-α and IL-6 in the macrophages and
T-cells. Moreover, the NF-kB activation was reduced. For this reason, it was con-
cluded that naringenin could be useful to attenuate the inflammatory response in
acute inflammatory diseases (Jin et al. 2017). Scopoletin has also shown a capac-
ity to decrease the inflammatory response in cerulein-induced acute pancreatitis
(AP) and associated lung injury in mice. This coumarin was found to downregu-
late NF-kB as well as TNF-α, IL-1β, IL33, and monocyte chemoattractant protein
1 (MCP-1). The downregulation of the NF-kB pathway and the production of
inflammatory cytokines had a protective effect against inflammation (Leema and
Tamizhselvi 2018).
Polyphenols have also wound healing properties. The wound healing process
involves three different phases that are the inflammatory phase, proliferative phase,
and remodeling phase (Ibrahim et al. 2018). For example, curcumin has been found
to accelerate the wound closure in mice with excisional wound healing. This effect
is related to a reduction in the expression of TNF-α and matrix metallopeptidase
9, which are up-regulated in the presence of wounds, by inhibiting the NF-kB
signaling pathway. Moreover, this compound was also able to increase cell pro-
liferation and collagen synthesis (Yen et al. 2018). Quercetin has shown positive
effects on the wound healing process in pressure ulcer lesions. This compound was
also found to accelerate wound closure. Moreover, the production of TNF-α and
IL-1β was also significantly reduced through suppression of the MAPK signaling
pathway (Yin et al. 2018).
4 Biosynthetic Pathway
The biosynthetic pathway responsible for polyphenols production in plants is

complex given that several different enzymes are involved. The polyphenols
biosynthesis includes the shikimate pathway and phenylpropanoid pathway.
Shikimate pathway is responsible for the production of chorismate through suc-
cessive enzymatic reactions (Fig. 2). This pathway uses two molecules derived
from the primary metabolism as starter substrates: Erythrose-4-phosphate (E4P)
and phosphoenol pyruvate (PEP) (Zabalza et al. 2017). These molecules are
condensed to produce 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP)
through the action of the DAHP synthase (DAHPS) (Munack et al. 2016). DAHP is
converted into 3-dehydroquinate (DHQ) by DHQ synthase (DHQS). Then, DHQ is
converted into shikimate through the successive action of the bifunctional enzyme
DHQ dehydratase/shikimate dehydrogenase (DHQD/SDH) (Ding et al. 2007). In
the next step, shikimate is phosphorylated by shikimate kinase (SK) originat-
ing shikimate 3-phosphate (S3P). Through the action of 5-enolpyruvylshikimate
3-phosphate (EPSP) synthase (EPSPS), S3P, and other molecules of PEP are
condensed and one molecule of EPSP is produced. Afterward, EPSP is dephospho-
rylated by chorismate synthase (CS) to produce chorismate (Marchiosi et al. 2020).
Chorismate is the main precursor of the aromatic amino acids L-phenylalanine,
L-tyrosine, and L-tryptophan. Polyphenols are only derived from L-phenylalanine
and L-tyrosine, and these two amino acids can be obtained from chorismate by two
alternative routes. The first step is common in both routes and consists of the pro-
duction of prephenate from chorismate by chorismate mutase (CM) (Parthasarathy
et al. 2018). To produce L-phenylalanine, prephenate could be converted into
phenylpyruvate or arogenate by prephenate dehydratase (PDT) or prephenate
aminotransferase (PAT), respectively. Then, phenylpyruvate and arogenate are con-
verted into L-phenylalanine by the action of aromatic amino acid aminotransferase
(AAT) or arogenate dehydratase (ADT), respectively. Prephenate can also be con-
verted into p-hydroxyphenylpyruvate by the action of prephenate dehydrogenase
(PDH). Afterward, L-tyrosine could be produced from p-hydroxyphenylpyruvate
126 D. Gomes et al.
Fig. 2 Schematic representation of the shikimate pathway involved in the synthesis of the
aromatic amino acids L-tyrosine and L-phenylalanine. AAT, aromatic amino acid aminotrans-
ferase; ADH, arogenate dehydrogenase; ADT, arogenate dehydratase; CM, chorismate mutase;
CS, chorismate synthase; DAHP, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate; DAHPS,
3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase; DHQ, 3-dehydroquinate; DHQD,
3-dehydroquinate dehydratase; DHQS, 3-dehydroquinate synthase; E4P, erythrose-4-phosphate;
EPSP, 5-enolpyruvylshikimate 3-phosphate; EPSPS, 5-enolpyruvylshikimate 3-phosphate syn-
thase; PAT, prephenate aminotransferase; PDH, prephenate dehydrogenase; PDT, prephenate dehy-
dratase; PEP, phosphoenol pyruvate; S3P, shikimate 3-phosphate; SDH, shikimate dehydrogenase;
SK, shikimate kinase
and arogenate by the action of aromatic amino acid aminotransferase (AAT) and
arogenate dehydrogenase (ADH), respectively (Tzin and Galili 2010).
L-tyrosine and L-phenylalanine are converted into the hydroxycinnamic acids
through the phenylpropanoid pathway (Fig. 3). When L-phenylalanine is used as a
substrate, the first enzyme of this biosynthetic pathway is phenylalanine ammonia-
lyase (PAL). This enzyme performs the deamination of L-phenylalanine to produce
cinnamic acid. Afterward, cinnamic acid is hydroxylated by cinnamic acid 4-
hydroxylase (C4H). In this step, p-coumaric acid is produced (Fraser and Chapple
2011). Alternatively, p-coumaric acid could be produced in a single enzymatic
step from L-tyrosine. This alternative route uses tyrosine ammonia lyase (TAL) to
perform the deamination of L-tyrosine (Watts et al. 2006).
Subsequently, p-coumaric acid could be used to produce other hydroxycin-
namic acids. By the action of 4-coumarate 3-hydroxylase (C3H), p-coumaric acid
is converted into caffeic acid. Moreover, caffeic acid could be converted into
ferulic acid by caffeic acid 3-O-methyltransferase (COMT). These hydroxycin-
namic acids could be activated to the correspondent coenzyme A (CoA) esters by
Fig. 3 Schematic representation of the biosynthetic pathway responsible for the production of
polyphenols (curcuminoids, resveratrol, and naringenin chalcone) using aromatic amino acids as
substrates. The enzymes involved in the phenylpropanoid pathway are phenylalanine ammonia-
lyase (PAL), tyrosine ammonia-lyase (TAL), cinnamic acid 4-hydroxylase (C4H), 4-coumarate
3-hydroxylase (C3H), caffeic acid 3-O-methyltransferase (COMT), and 4-coumarate-CoA lig-
ase (4CL). p-coumaroyl 5-O-shikimate 3’-hydroxylase (CS3’H), p-coumaroyl shikimate trans-
ferase (CST), and caffeoyl-CoA 3-O methyltransferase (CCoAOMT) catalyze the conversion
of p-coumaric acid into other hydroxycinnamic acids. The Type III polyketide synthase (PKS)
enzymes responsible for curcuminoids, stilbenoids, and flavonoids scaffold production, which are
underlined, are diketide-CoA synthase (DCS), curcumin synthase (CURS), curcuminoids synthase
(CUS), chalcone synthase (CHS), and stilbene synthase (STS)
4-coumarate-CoA ligase (4CL). Moreover, p-coumaroyl-CoA could also be con-

verted into caffeoyl-CoA by p-coumaroyl 5-O-shikimate 3’-hydroxylase (CS3’H)
or p-coumaroyl shikimate transferase (CST). Subsequently, caffeoyl-CoA could be
converted into feruloyl-CoA by caffeoyl-CoA 3-O methyltransferase (CCoAOMT).
After the CoA esters biosynthesis, the pathway deviates toward the formation
of the different polyphenols. The enzymes involved in these steps are different,
depending on the type of polyphenol that is produced. Generally, these steps
include the action of Type III polyketide synthase (PKS) enzymes (Fig. 3). Type
III PKSs are involved in the condensation of the CoA esters (starter units) with
malonyl-CoA (extender unit) leading to the polyphenol scaffold (Yu et al. 2012).
For example, stilbene synthase (STS) is responsible for the condensation of three
molecules of malonyl-CoA with p-coumaroyl-CoA. By the action of this enzyme,
it is obtained resveratrol which is considered the stilbene backbone (Parage et al.
2012). Chalcone synthase (CHS) is the Type III PKS involved in the forma-
tion of the flavonoid scaffold. This enzyme also catalyzes the condensation of
128 D. Gomes et al.
three molecules of malonyl-CoA with p-coumaroyl-CoA leading to the forma-

tion of naringenin chalcone (Liou et al. 2018). Other examples of Type III PKSs
involved in the production of polyphenols are diketide-CoA synthase (DCS),
curcumin synthase (CURS), and curcuminoids synthase (CUS). These enzymes
are responsible for the production of curcuminoids. DCS is responsible for the
condensation of one molecule of malonyl-CoA with feruloyl-CoA or p-coumaroyl-
CoA forming a diketide intermediate. CURS is responsible for the formation of
curcuminoids by condensation of the diketide intermediates (second extender sub-
strates) with another molecule of CoA ester (second starter substrates) (Couto
et al. 2017; Katsuyama et al. 2009; Rodrigues et al. 2017a, 2015a; Rodrigues
et al. 2020). Additionally, CUS could also catalyze the formation of curcum-
inoids in a unique manner. This enzyme can catalyze the two steps that are
performed by DCS and CURS (Katsuyama et al. 2010). Afterwards, the polyphe-
nol scaffold can be modified by the action of several enzymes to produce a large
variety of polyphenols. These modifications include hydroxylation, glycosylation,
methylation, prenylation, among others (Yu et al. 2012).
5 Yeasts as Valuable Chassis
Polyphenols, like other plant secondary metabolites, are produced and accumu-
lated in very low amounts in plants. Due to the low amounts produced in plants,
the extraction of polyphenols from their natural resources is very difficult and the
yields are considered low (Krivoruchko and Nielsen 2015; Rainha et al. 2020a;
Rodrigues et al. 2015c; Rodrigues and Rodrigues 2020). Moreover, the amounts
of these compounds are affected by climatic and seasonal variations. The chemi-
cal synthesis could be an alternative to produce some polyphenols. However, some
of these compounds have complex structures being hard to chemically synthesize.
Additionally, chemical synthesis involves the use of toxic compounds and expen-
sive substrates. For this reason, the process is considered non-environmentally
friendly and expensive (Liu et al. 2017). Due to their potential applications and to
try to satisfy their industrial demand, there has been an increased interest in the
heterologous production of polyphenols.
The heterologous production in plants has the advantage of only requiring the
introduction of one or two genes of the biosynthetic pathway (Rodrigues et al.
2015c). The other genes from the pathway, such as the ones responsible for the
phenylpropanoid pathway, are already present in the plant. However, heterologous
production in plants has also disadvantages. One of these disadvantages is the
long period that the plant takes to grow. The growth is also dependent on the
climacteric conditions and fertile land occupation. Additionally, the production
yields are usually very low and variable due to cellular heterogeneity. Beyond
this, the downstream purification process is more difficult since several similar
compounds are also produced in plants (Ebrahimi and Mokhtari 2017; Wilson and
Roberts 2012).
The use of microorganisms as chassis has emerged as an alternative solution to

heterologously produce several compounds of interest. Heterologous production
has several advantages comparing to plant and chemical synthesis. Microorgan-
isms have rapid growth cycles and, consequently, it allows short production times
compared to plants. Moreover, they can grow in inexpensive substrates. For this
reason, heterologous production is considered a more efficient and inexpensive
method (Krivoruchko and Nielsen 2015). As with plants, microorganisms have
also the capacity to produce the required precursors involved in the synthesis of
plant natural compounds, namely aromatic amino acids and malonyl-CoA (Milke
et al. 2018). Nevertheless, the reconstruction of large biosynthetic pathways in
microorganisms is challenging since it requires the introduction of several genes
of the pathway in the chassis, their efficient expression, and the production of
functional enzymes (Braga et al. 2019).
The heterologous production became easier with all the advances achieved
in the metabolic engineering and synthetic biology fields. Within bacteria,
Escherichia coli has been the most used to produce polyphenolic compounds. In
the case of yeasts, S. cerevisiae has been the most widely explored. These microor-
ganisms have been widely used since they are easy to grow and manipulate and
well-characterized. Moreover, there are available several genetic tools to geneti-
cally modify them (Krivoruchko and Nielsen 2015; Rainha et al. 2020b; Rodrigues
et al. 2017b; Rodrigues and Rodrigues 2017). Comparing to E. coli, S. cerevisiae
has several advantages to produce plant compounds. S. cerevisiae, unlike E. coli,
can perform post-translational modifications, and it contains intracellular com-
partments resembling the ones present in plant cells such as the endomembrane
system. This characteristic allows the functional expression of genes derived from
plants, namely the eukaryotic cytochrome P450 enzymes that are involved in the
synthesis of polyphenolic compounds (C4H and C3H). Moreover, this microorgan-
ism is generally recognized as safe (GRAS) facilitating its use in the production of
nutritional and pharmaceutical compounds (Xu et al. 2020). However, other yeasts
like P. pastoris and Y. lipolytica have been also explored for the production of
polyphenols. P. pastoris has also several characteristics that make it a good host
for heterologous production of relevant compounds. Like S. cerevisiae, P. pastoris
is also easy to grow in a simple and low-cost medium. Moreover, it is known that
this microorganism can produce proteins with high yields, perform the appropri-
ate folding, and secret them to the extracellular medium. P. pastoris is also able
to perform post-translational modifications (Karbalaei et al. 2020). Y. lipolytica is
also considered a good chassis for the heterologous production of polyphenols.
This oleaginous yeast has a high metabolic flux of the tricarboxylic acid (TCA)
cycle being able to produce high amounts of acetyl-CoA and malonyl-CoA that
are essential precursors in the synthesis of polyphenols. Y. lipolytica is also able to
grow on inexpensive substrates like renewable feedstocks and wastes making the
industrial production process more economic and environmentally friendly. This
microorganism also contains intracellular compartments facilitating the expression
of plant-derived genes and it is considered food-grade safe (Gu et al. 2020; Xu
130 D. Gomes et al.
et al. 2020). Due to these unique features, S. cerevisiae, Y. lipolytica, and P. pas-
toris were exploited as a microbial chassis for the heterologous production of
several compounds, including polyphenols.
6 Case Studies: Production of Hydroxycinnamic Acids

and Polyphenolic Compounds in Yeasts
To achieve the industrial production of polyphenols in yeasts, it is essential to

optimize several steps such as improve the precursor and extender substrates
availability, improve the expression of the enzymes involved in the biosynthetic
pathway, decrease or inhibit the expression of enzymes involved in competing
pathways, among others (Chen et al. 2020). The emergence of synthetic biology
and metabolic engineering tools made this process easier. Within the subclasses of
polyphenolic compounds, the production of flavonoids and stilbenoids using yeasts
as heterologous hosts has been the most explored. Additionally, there are some
reports of heterologous production of coumarins, curcuminoids, and polypheno-
lic amides. The production of lignans was the only one that was never reported
in any yeast strain. The highest production titers of hydroxycinnamic acids and
polyphenols using S. cerevisiae, Y. lipolytica, and P. pastoris as heterologous hosts
are summarized in Table 1.
6.1 Heterologous Production of Hydroxycinnamic Acids
Hydroxycinnamic acids belong to the group of the phenolic compounds. These

compounds could be found in several vegetables, fruits, and beverages such as
coffee and tea (Abramovič 2015). p-Coumaric acid, caffeic acid, and ferulic acid
are the hydroxycinnamic acids involved in the synthesis of polyphenols, and they
have a C6-C3 structure (Fig. 4). These compounds have also associated strong
antioxidant properties as well as anti-inflammatory, anticancer and antimicrobial
properties, among others (Rodrigues et al. 2015a; Taofiq et al. 2017).
The development of yeast chassis with the ability to produce industrial sig-
nificant amounts of polyphenolic compounds became a priority. The production
of significant amounts of hydroxycinnamic acids, especially p-coumaric acid, is
important since these compounds are the main precursors of polyphenols. In the
last years, several attempts were performed to construct engineered strains with
the ability to produce relevant amounts of hydroxycinnamic acids. S. cerevisiae
and Y. lipolytica were explored to produce hydroxycinnamic acids, especially p-
coumaric acid since this compound is the main precursor of all the subclasses
of polyphenols. Until now, to the best of our knowledge, there are no reports of
hydroxycinnamic acid production in P. pastoris.
In S. cerevisiae, the construction of one strain able to produce p-coumaric
acid from glucose was reported. Several metabolic engineering approaches were
explored to prevent the pathway deviation and to improve the flux toward amino
Table 1 Highest values reported of heterologous production of hydroxycinnamic acids and polyphenols in Saccharomyces cerevisiae, Yarrowia lipolytica, and
Pichia pastoris. The strain, genes, modifications in the host chassis, substrate, and production titers obtained are presented
Class Compound Strain Genesa Modifications in the Substrate Production (mg/L) References
chassis
Hydroxycinnamic p-Coumaric acid S. cerevisiae IMX AtPAL and Construction of Glucose (20 g/L) 12,500 Liu et al. (2019a)
acids AtC4H aromatic amino acids
overproducing strain
Caffeic acid S. cerevisiae BY4741 RtTAL, PaHpaB, – Tyrosine (0.5 g/L) 289.4 Liu et al.
and SeHpaC (2019b)
p-Coumaric acid Y. lipolytica W29 RtTAL Construction of Glucose (40 g/L) 595.3 Gu et al. (2020)
aromatic amino acids
Ferulic acid Y. lipolytica P01g Tf AXE – Corncob biomass (2%)b 77 Huang et al.
(2011)
(continued)
Yeast Synthetic Biology Approaches for the Production of Valuable …
131
132
Table 1 (continued)
chassis
Flavonoids Naringenin S. cerevisiae BY4741 FjTAL, At4CL, Construction of Glucose (20 g/L) 220 Lyu et al. (2019)
HaCHS, and aromatic amino acids
PhCHI overproducing strain
Kaempferol S. cerevisiae BY4741 FjTAL, At4CL, Construction of Glucose (20 g/L) 168.1 Du et al. (2020)
AtCHS, AtCHI, aromatic amino acids
NtF3H, and and malonyl-CoA
AtFLS overproducing strain
Quercetin FjTAL, At4CL, 154.2
AtCHS, AtCHI,
NtF3H, PhF3’H,
and AtFLS
Myrcetin FjTAL, At4CL, 145
AtCHS, AtCHI,
NtF3H, PhF3’H,
SlF3 5’H, and
AtFLS
Delphinidin FjTAL, At4CL, 26.1
AtCHS, AtCHI,
NtF3H, PhF3’H,
SlF3 5’H,
AaDFR, and
GeANS
Pelargonidin FjTAL, At4CL, 33.3
AtCHS, AtCHI,
NtF3H, AaDFR,
and GeANS
Cyanidin FjTAL, At4CL, 31.7
AtCHS, AtCHI,
NtF3H, PhF3’H,
AaDFR, and
GeANS
(continued)
D. Gomes et al.
Table 1 (continued)
chassis
Flavonoids Naringenin Y. lipolytica Po1f SeTAL, Nt4CL, Construction of Glucose (80 g/L) 898 Palmer et al.
and HsCHS aromatic amino acids (2020)
and malonyl-CoA
Eriodictyol Y. lipolytica Po1f RtTAL, Pc4CL, Construction of a Glucose (40 g/L) 134.2 Lv et al. (2019b)
PhCHS, MsCHI, strain with improved
GhF3’H, and chorismate and
CrCPR malonyl-CoA
Taxifolin RtTAL, Pc4CL, biosynthesis 110.5
PhCHS, MsCHI,
GhF3’H, CrCPR,
and SlF3H
Liquiritigenin Y. lipolytica (ATCC201249) ZmPAL, PcC4H, – Glucose (20 g/L) + 62.4 Akram et al.
Pc4CL, PhCHS, p-Coumaric acid (2020)
MsCHR, and (100 mg/L)
MsCHI
8-hydroxydaidzein P. pastoris X-33 AoCYP57B3 and – Daidzein (25.4 mg/L) 0.58 Chang et al.
3 -hydroxydaidzein BmBM3R 0.23 (2013)
6-hydroxydaidzein 9.1
3’-hydroxygenistein P. pastoris X-33 AoCYP57B3 and Strain modified by Genistein (135 mg/L) 20.3 Wang et al.
ScCPR treatment with (2016)
hydrogen peroxide
to induce oxidative
stress and,
consequently, select
the most resistant
strains
(continued)
133
134
Table 1 (continued)
chassis
Stilbenoids Resveratrol S. cerevisiae AtPAL, AtC4H, Construction of Glucose (88 g/L) 800 Li et al. (2016)
CEN.PK102-5B At4CL, and aromatic amino acids
VvVST and malonyl-CoA
Pterostilbene AtPAL, AtC4H, overproducing strain 34.9
At4CL, VvVST,
and VvROMT
Pinostilbene AtPAL, AtC4H, 5.52
At4CL, VvVST,
and SbROMT
Resveratrol Y. lipolytica W29 FjTAL, At4CL, Construction of Glucose (40 g/L) 12,400 Sáez-Sáez et al.
and VvSTS aromatic amino acids (2020)
and malonyl-CoA
(continued)
D. Gomes et al.
Table 1 (continued)
chassis
Coumarins Scopoletin S. cerevisiae BY4742 Pc4CL, AtF6 H1, – Lignin hydrolysate 4.79 Zhao et al.
PaHpaB, and containing p-coumaric (2020)
EnHpaC acid (2.3 g/L) and ferulic
acid (0.5 g/L)
Curcuminoids Bisdemethoxycurcumin Y. lipolytica Po1f Nt4CL and Construction of p-Coumaric acid 0.17 Palmer et al.
OsCUS aromatic amino acids (0.33 g/L) (2020)
and malonyl-CoA
Polyphenolic p-Coumaroyl-3-hydroxyanthranilic S. cerevisiae CENPK113-5d Nt4CL and – p-Coumaric acid 120 Moglia et al.
amides acid ccHCT (462 mg/L) and (2015)
3-Hydroxyanthranilic
acid (77 mg/L)
Caffeoyl-3-hydroxyanthranilic acid Caffeic acid (540 mg/L) 22
and 3-Hydroxyanthranilic
acid (77 mg/L)
a Aa—Anthurium andraeanum, At—Arabidopsis thaliana, Ao—Aspergillus oryzae, Bm—Bacillus megaterium, Cc—Cynara cardunculus, Cr—Catharanthus roseus, En—Enterobacteriaceae, Fj—Flavobac-
terium johnsoniaeu, Ge—Gerbera specie, Gh—Gerbera hybrid, Ha—Hypericum androsaemum, Hs—Huperiza serrata, Ms—Mendicago sativa, Nt—Nicotiana tabacum, Os—Oryza sativa, Pa—Pseudomonas
aeruginosa, Pc—Petroselinum crispum, Ph—Populus hybrid, Rc—Rhodobacter capsulatus, Rt—Rhodosporidium toruloides, Sb—Sorghum bicolor, Sc—Saccharomyces cerevisiae, Se—Salmonella enterica,
Sl—Solanum lycopersicum, Tf—Thermobifida fusca NTU22, Vv—Vitis vinifera, Zm—Zea mays; 4CL—4-coumarate-CoA ligase, ANS—anthocyanidin synthase, AXE—thermostable esterase, BM3R—reductase
domain of the CYP102A1 gene, C3H—4-coumarate 3-hydroxylase, C4H— cinnamic acid 4-hydroxylase, CHR—chalcone reductase, CHI—chalcone isomerase, CHS—chalcone synthase, CPR- cytochrome
P450 reductase, CUS—curcuminoid synthase, CYP57B3—cytochrome P450 monooxygenase, DFR—dihydroflavonol 4-reductase, F3H—flavanone 3-hydroxylase, F3’H—flavonoid 3’-hydroxylase, F3´5´H—
flavonoid 3’,5’-hydroxylase, F6’H1—feruloyl-CoA 6’-hydroxylase, FLS—flavonol synthase, FSI—flavone synthase I, HpaB and HpaC—4-hydroxyphenylacetate 3-hydroxylase, PAL— phenylalanine ammonia
lyase, ROMT—resveratrol O-methyltransferase, STS—stilbene synthase, TAL—tyrosine ammonia lyase, VST—resveratrol synthase; b Corncob biomass (2%) contains feruloyl-polysaccharide that is AXE
substrate
135
136 D. Gomes et al.
Fig. 4 Structure of the hydroxycinnamic compounds p-coumaric acid, caffeic acid, and ferulic
acid
acids since they are present in limited amounts in the cells. The constructed strain
holds a knockout in the pyruvate decarboxylase (PDC) genes, PDC5 and Aro10.
These genes are responsible for the conversion of the precursor of phenylalanine,
that is, phenylpyruvate, into phenylacetaldehyde contributing to the pathway devi-
ation. Moreover, DAHP synthase and chorismate mutase genes were replaced by a
feedback-insensitive version to prevent the inhibition of aromatic amino acids syn-
thesis since these genes are strongly affected by the amounts of aromatic amino
acids produced. Moreover, the authors have overexpressed a shikimate kinase from
E. coli since this could be a limiting step in the production of higher amounts of
p-coumaric acid. TAL from Flavobacterium johnsoniae was also expressed in this
engineered strain that was able to produce almost 2000 mg/L of p-coumaric acid in
fed-batch fermentation (Rodriguez et al. 2015). Later, the same group studied the
influence of the production of p-coumaric acid in the metabolism of S. cerevisiae-
producing strains to construct more efficient and robust strains (Rodriguez et al.
2017a). After transcriptomic and metabolomic analysis, the authors have found
that genes involved in the sugars and amino acids transport were downregulated
in the producer strain. When these downregulated genes were knocked out, it was
found that the production of p-coumaric acid was improved. The highest produc-
tion of p-coumaric acid (2400 mg/L) was obtained in the S. cerevisiae CEN.PK
strain that holds the deletion of the tyrosine and tryptophan amino acid transporter
1 (TAT1), which is probably involved in the transport of tyrosine to the outside
of the cells being less available for p-coumaric acid production (Rodriguez et al.
2017a). More recently, Liu et al. (2019a) have also constructed a S. cerevisiae strain
able to produce higher amounts of p-coumaric acid. In this strain, PAL and C4H
genes from Arabidopsis thaliana were expressed to produce p-coumaric acid. To
increase the cytochrome P450 activity, cytochrome B5 (CYB5) from S. cerevisiae
and cytochrome P450 reductase (CPR1) from A. thaliana were overexpressed.
Moreover, the strain was also modified to overexpress a feedback-insensitive ver-
sion of the DAHP synthase and chorismate mutase genes. The TAL gene from F.
johnsoniae was also expressed, and the production of p-coumaric acid was verified
using both tyrosine and phenylalanine branches. Prephenate dehydrogenase (PDH)
genes from different microorganisms were also tested, and it was found that PDH
from Medicago truncatula significantly improves the shikimate pathway flux and,
consequently, the production of p-coumaric acid. The production of E4P was also
improved by expressing a heterologous phosphoketolase pathway in conjugation
with the expression of the gene that encodes glycerol-1-phosphatase. To prevent

the pathway deviation and formation of by-products, PDC5 and Aro10 were also
deleted. Additionally, the expression levels of several genes were also optimized by
promoter replacement. After all these modifications, the yeast chassis was able to
produce 12,500 mg/L of p-coumaric acid from glucose in a fed-batch fermentation
experiment. Currently, this was the highest production of p-coumaric acid reported
and this strain could be considered an excellent starting strain for the production
of polyphenolic compounds with high titers and yields (Liu et al. 2019a).
The heterologous production of caffeic acid was also achieved in S. cerevisiae.
To produce caffeic acid using tyrosine as substrate, a strain holding the TAL
gene from Rhodosporidium toruloides, 4-hydroxyphenylacetate 3-monooxygenase
(HpaB), and NADPH-flavin oxidoreductase (HpaC) genes derived from Pseu-
domonas aeruginosa and Salmonella enterica, respectively, was constructed. HpaB
and HpaC can catalyze the transformation of p-coumaric acid into caffeic acid.
This strain was able to produce 289.4 mg/L of caffeic acid (Liu et al. 2019b).
Y. lipolytica was also explored as a heterologous host to produce p-coumaric
acid. Gu et al. (2020) reported the construction of a strain able to produce p-
coumaric acid from glucose. The platform strain has modifications toward an
increase in the aromatic amino acids biosynthesis, namely the substitution of the
native DAHP synthase for a feedback-insensitive version of the gene. In order
to increase the flux toward the shikimate pathway, transketolase gene was overex-
pressed in this strain to convert fructose-6-phosphate into E4P, and pyruvate kinase
gene was deleted to prevent PEP consumption. Moreover, TAL gene was expressed
in this strain and 593.5 mg/L of p-coumaric acid was produced (Gu et al. 2020).
The production of ferulic acid was also obtained in Y. lipolytica by expressing an
esterase gene from Thermobifida fusca. This esterase can release the ferulic acid
that is linked to lignin and other polysaccharides in the cell wall of plants. In this
study, the esterase was able to convert corncob biomass residue, that is a ligno-
cellulose residue, into 77 mg/L of ferulic acid (Huang et al. 2011). However, the
production of ferulic acid from tyrosine or glucose was not explored yet.
6.2 Heterologous Production of Flavonoids
Within polyphenolic compounds, flavonoids are the class with more variety of
structures. These compounds are responsible for organoleptic properties and for the
pigments that confer color to vegetables, fruits, and herbs. Due to their medical and
industrial applications, the market value of these compounds is estimated to reach
USD 1.06 billion by 2025 (Grand View Research 2016). The flavonoid skeleton
is characterized by two benzene rings that are connected by one pyrene ring that
contains an oxygen molecule (Fig. 5). Depending on the differences in the struc-
ture of these compounds, flavonoids could be grouped into different subclasses.
These subclasses are flavonols, flavones, flavanones, flavonols, isoflavonoids, and
anthocyanins (Panche et al. 2016).
138 D. Gomes et al.
Fig. 5 Basic structure of flavonoids compounds. A and B correspond to two benzene rings and C
corresponds to a pyrene ring containing an oxygen molecule
In the last years, several research efforts have been made to construct yeast cell
factories with the ability to produce flavonoids with high titers and yields. S. cere-
visiae has been the most explored yeast to produce these compounds. Several S.
cerevisiae strains were constructed to produce flavonoids using p-coumaric acid or
amino acids as precursors (Jiang et al. 2005; Lyu et al. 2017; Trantas et al. 2009;
Yan et al. 2005). However, these substrates are more expensive, and the industrial
production process was unfeasible (Santos et al. 2011). For this reason, de novo
biosynthesis of these compounds from cheap carbon sources became a priority.
Koopman et al. (2012) have constructed a S. cerevisiae strain able to produce narin-
genin from glucose. This strain harbors the genes responsible for the naringenin
biosynthetic pathway (PAL1, C4H, CPR1, 4CL3, CHS3, and chalcone isomerase
(CHI1)) from A. thaliana. The production of naringenin was evaluated in shake
flasks fermentations, and it was found that low amounts of naringenin were pro-
duced (1.47 mg/L). The authors have modified the S. cerevisiae strain to construct a
new yeast chassis able to overproduce amino acids and, consequently, optimize the
production yields and titers. In this strain, the DAHP synthase gene was replaced
for a feedback-insensitive version of the gene. PDC gene was also deleted to pre-
vent the pathway deviation. Additionally, the number of copies of CHS gene was
also increased to improve its catalytic efficiency. To improve the production of
p-coumaric acid, TAL from R. capsulatus was also expressed. These modifications
have resulted in an improvement of naringenin production reaching 54.5 mg/L in
shake flask cultures and 108.9 mg/L in aerated, pH-controlled batch reactors from
20 g/L of glucose (Koopman et al. 2012). Rodriguez et al. (2017b) have also con-
structed S. cerevisiae strains able to produce several flavonoids using glucose as a
substrate. In this work, the genes responsible for the biosynthesis of naringenin,
liquiritigenin, kaempferol, resokaempferol, quercetin, and fisetin were integrated
into the yeast genome. These strains contained modifications to improve the flux
toward amino acids, namely the knockout of the PDC5 and Aro10 genes and the
overexpression of DAHP synthase and chorismate mutase. The highest productions
obtained in this study were 26.57 mg/L of kaempferol and 20.38 mg/L of quercetin
(Rodriguez et al. 2017b). Duan et al. (2017) also constructed a S. cerevisiae strain
to produce kaempferol de novo. Since malonyl-CoA low availability represents
a limiting step in the high-level production of polyphenols, the authors overex-
pressed the endogenous genes responsible for the synthesis of malonyl-CoA from
ethanol to increase its availability. Using a fed-batch fermentation strategy, the pro-
duction of kaempferol has reached 66.29 mg/L. More recently, Lyu et al. (2019)
also optimized a S. cerevisiae strain to improve the flux toward amino acids and
prevent the pathway deviation. As performed by other research groups, the authors
constructed a strain with a knockout in the PDC5 and Aro10 genes. Additionally,
the genes responsible for the biosynthetic pathway were overexpressed and the
promotors were replaced by the glucose-inducible strong promoter pGAL1. This
strain was able to produce 86 mg/L of kaempferol. Moreover, this strain was also
able to produce 220 mg/L of naringenin when Tween 80, that is a surfactant that
could increase the enzymatic activity and cell biomass growth, was added to the
fermentation medium. This is the highest de novo production of naringenin using
S. cerevisiae as chassis reported so far (Lyu et al. 2019).
The biosynthetic pathway responsible for the synthesis of anthocyanins has
been firstly engineered in S. cerevisiae by Eichenberger et al. (2018). In this study,
the genes responsible for the biosynthetic pathway were integrated into a single
copy in the yeast genome. The constructed strains were able to produce 0.85 mg/L
of pelargonidin-3-O-glucosidase, 1.55 mg/L of cyanidin-3-O-glucosidase and
1.86 mg/L of delphinidin-3-O-glucosidase (Eichenberger et al. 2018). Afterward,
a co-culture strategy was designed by Du et al. (2020) to produce naringenin,
kaempferol, quercetin, myricetin, delphinidin, pelargonidin, and cyanidin. In this
co-culture strategy, one strain harbors the biosynthetic pathway responsible for the
production of naringenin, and the other strains harbor the other genes responsi-
ble for the production of the three flavonols and the three anthocyanidins from
naringenin. The first strain contains several modifications to improve the pro-
duction of aromatic amino acids and malonyl-CoA and to prevent the deviation
of the pathway for competing pathways. The first strain was able to produce
144.1 mg/L of naringenin. The co-culture of both strains resulted in the pro-
duction of 168.1 mg/L of kaempferol, 154.2 mg/L of quercetin, 145 mg/L of
myrcetin, 26.1 mg/L delphinidin, 33.3 mg/L of pelargonidin, and 31.7 mg/L of
cyanidin. This study demonstrated the importance of using co-culture strategies to
reduce the metabolic burden caused by the presence of large biosynthetic pathways
(Du et al. 2020; Rodrigues et al. 2020). Additionally, these studies strengthened
the relevance of improving the synthesis of aromatic amino acids and the exten-
der substrates availability to significantly increase the production of the desired
compounds.
Although Y. lipolytica has been less used than S. cerevisiae, it has been recently
explored as a yeast chassis for the production of flavonoids. Lv et al. (2019a) have
constructed the biosynthetic pathways responsible for the production of naringenin,
eriodyctiol, and taxifolin. The authors have developed a method for integration
of the genes responsible for the biosynthetic pathway in multiple copies in ran-
dom sites of the Y. lipolytica genome by combining 26 s ribosomal DNA (rDNA)
multi-copy integration with Cre-loxP system. For the integration into the yeast
genome, this methodology uses the sequence of the repetitive 26 s rDNA sites of
Y. lipolytica genome as regions of homology for the cassette integration. Beyond
the genes of the biosynthetic pathway, the cassette also contains the selection
140 D. Gomes et al.
marker URA3 with loxP flanking sequences. After integration in the genome and
selection of the transformants, Cre recombinase is expressed and recognizes the
loxP sequences and, consequently, the selection marker is removed. The devel-
opment of this method of integration was important because this microorganism
has few auxotrophic markers available. Moreover, the use of plasmids can lead to
genomic instability as well as copy number variations. After integrating the genes
responsible for the biosynthetic pathways, the authors have evaluated the abil-
ity of the constructed strains to produce naringenin, eriodyctiol, and taxifolin in
Yeast Extract–Peptone–Dextrose (YPD)-rich medium. The best performing strains
were able to produce 71.2 mg/L, 54.2 mg/L, and 48.1 mg/L of naringenin, eri-
odyctiol, and taxifolin, respectively (Lv et al. 2019a). Later, the same research
group has performed several steps to optimize the production of these compounds.
The authors have optimized the copy number of the CHS and CPR genes that
are responsible for limiting steps in the production of these flavonoids. Moreover,
Lv et al. (2019b) also enhanced the availability of the chorismate and malonyl-
CoA precursors by overexpressing the genes responsible for the synthesis of both
compounds. Moreover, fermentation parameters such as pH and C/N ratio were
also optimized. In the best conditions, the optimized strains were able to produce
252.4 mg/L, 134.2 mg/L, and 110.5 mg/L of naringenin, eriodictyol, and taxi-
folin, respectively. Comparing with the previous study, these optimizations led to
3.5-fold, 2.5-fold, and 2.3-fold improvement in the production of naringenin, eri-
odictyol, and taxifolin, respectively (Lv et al. 2019b). More recently, a mixture
of glucose and xylose was used as a substrate to produce naringenin (Wei et al.
2020). Due to the need to produce flavonoids in an inexpensive way, the search for
low-cost substrates, such as agricultural residues, has become a priority to reduce
the production costs of these compounds at an industrial scale (Gudiña et al. 2020).
Since xylose is one of the sugars that is present in a higher amount in agricultural
residues, Wei et al. (2020) engineered the Y. lipolytica strain toward the use of
xylose as a substrate to produce naringenin since the wild-type strain is not able
to metabolize xylose. This strain was constructed by integration into the genome
of a xylose-inducible module for activation of the expression of the genes respon-
sible for xylose utilization. By activating the expression of these genes, xylose is
metabolized and originates E4P which is a precursor of the shikimate pathway.
Moreover, the genes responsible for the naringenin biosynthetic pathway were
also integrated into the genome by Clustered Regularly Interspaced Short Palin-
dromic Repeats—associated Cas9 endonuclease (CRISPR-Cas9). The production
of naringenin was evaluated in the strain that only contains the naringenin biosyn-
thetic pathway and in the strain that contains the naringenin biosynthetic pathway
and the xylose-inducible utilization module. The first strain was able to produce
239.1 mg/L of naringenin in YPD fermentation medium. The strain that also con-
tains the xylose-inducible utilization module was able to produce 715.3 mg/L of
naringenin in YPD fermentation medium containing 60 g/L of xylose. This study
showed that the xylose utilization pathway is a good alternative to be explored to
increase the precursor availability and, consequently, improve the production of
the desired polyphenolic compound. Moreover, this study opens doors to explore
the production of polyphenols using different bio-wastes as low-cost substrates. In

the same year, Palmer et al. (2020) also engineered a Y. lipolytica strain toward
the production of naringenin. The genes responsible for the biosynthetic pathway
were codon-optimized for Y. lipolytica and integrated into random sites of the
yeast genome. The constructed strain was also modified to increase the production
of naringenin. The modifications performed in this strain include the replacement
of DAHP synthase gene for a feedback-insensitive version, expression of the per-
oxisome biogenesis factor gene (PEX10) involved in the peroxisome synthesis and
improves the formation of acetyl-CoA, and integration of another copy of the CHS
gene. This strain was able to produce 898 mg/L of naringenin from 80 g/L of glu-
cose, being the highest production of naringenin reported so far in any yeast, thus
demonstrating the high potential of using Y. lipolytica as heterologous host to pro-
duce polyphenols. Moreover, Y. lipolytica was also recently engineered toward de
novo production of liquiritigenin. Different genes from diverse sources were tested
to evaluate the best combination to produce higher amounts of liquiritigenin. The
gene combination that results in the best production of liquiritigenin was PAL
from Zea mays, C4H and 4CL from Petroselinum crispum, CHS from Pterolophia
hybrida, and CHR and CHI from Medicago sativa. CHR and CHI were fused
to increase the production of liquiritigenin. Additionally, the TEF promoter that
initially regulated CHI expression was replaced by the lipase promoter. All these
modifications allowed the improvement of the production of liquiritigenin reaching
8.2 mg/L from glucose. Moreover, feeding p-coumaric acid led to the production
of 62.4 mg/L (Akram et al. 2020).
P. pastoris has been less explored than the other yeasts in the heterologous pro-
duction of polyphenols. The yeast was used to produce hydroxylated isoflavones.
However, this production was achieved by bioconversion of the non-hydroxylated
compound (Chang et al. 2013; Ding et al. 2015; Wang et al. 2016). Chang et al.
(2013) reported the production of 8-hydroxydaidzein, 3 -hydroxydaidzein, and 6-
hydroxydaidzein from daidzein. In this study, the authors constructed a fusion
gene by combining the gene encoding CYP57B3, which is responsible for the
hydroxylation of genistein, with the reductase domain of the CYP102A1 gene
(BM3R) from Bacillus megaterium. The fusion gene was inserted into the P.
pastoris genome using an integrative vector. The conversion of daidzein by the
constructed strain was evaluated and 0.58 mg/L, 0.23 mg/L, and 9.1 mg/L of
8-hydroxydaidzein, 3 -hydroxydaidzein, and 6-hydroxydaidzein were produced,
respectively (Chang et al. 2013). Moreover, the production of 3’-hydroxygenistein
from genistein was also achieved in P. pastoris through the expression of a
cytochrome P450 hydroxylase (CYP57B3) from Aspergillus oryzae fused with
a cytochrome P450 oxidoreductase gene (ScCPR) from S. cerevisiae. The pro-
duction of 3’-hydroxygenistein was evaluated in this strain, and it was verified
that 3.5 mg/L of 3’-hydroxygenistein were produced (Ding et al. 2015). Later,
Wang et al. (2016) have obtained mutants of the strain constructed by Ding
et al. (2015) upon treatment with periodic hydrogen peroxide. This treatment
was used to induce oxidative stress. Afterward, the most resistant strains were
selected since it was hypothesized that these strains produced higher amounts of
142 D. Gomes et al.
3’-hydroxygenistein that has recognized antioxidant properties. The best mutant

could produce 20.3 mg/L of 3’-hydroxygenistein (Wang et al. 2016). These are
the only studies that report heterologous production of flavonoids in P. pastoris.
6.3 Heterologous Production of Stilbenoids
Stilbenoids are polyphenolic compounds with limited distribution in plant species.

These compounds are mostly found in berries and grapes, and they are involved
in the plant’s response against several stress factors including pathogens (Rivière
et al. 2012). Due to their interesting biological activities, resveratrol has been the
most extensively studied and explored stilbenoid (Shen et al. 2013). The global
market size of resveratrol was estimated to be USD 65 million in 2020, and it is
expected to reach USD 106 million by 2026 (Market Watch 2020). Stilbenoids are
composed of a skeleton with a C6-C2-C6 structure (Fig. 6). However, this skeleton
could be decorated to produce several stilbenoids by hydroxylation, methylation,
glycosylation, among others (Pecyna et al. 2020).
The first reports of stilbenoids heterologous production in S. cerevisiae used
p-coumaric acid as a substrate to obtain resveratrol (Becker et al. 2003; Beek-
wilder et al. 2006; Shin et al. 2011; Sydor et al. 2010; Wang et al. 2011; Wang
and Yu 2012). The highest production of resveratrol using p-coumaric acid as sub-
strate was achieved by Sydor et al. (2010). In this study, an industrial Brazilian
S. cerevisiae strain, that was isolated from a sugar cane plantation, carrying the
plasmids containing 4CL1 from A. thaliana and STS from Vitis vinifera showed a
higher ability to produce resveratrol compared to the well-described S. cerevisiae
CEN.PK2-1C strain. This industrial strain was able to produce 391 mg/L of resver-
atrol from 2.46 g/L of p-coumaric acid in a rich fermentation medium. However,
other studies showed the application of interesting metabolic engineering, as well
as synthetic biology approaches to improve production titers and yields. For exam-
ple, Wang et al. (2011) have codon-optimized a TAL gene since the native gene
from Rhodobacter sphaeroides did not demonstrate in vivo activity in S. cerevisiae.
By using a codon-optimized gene, the probability of occurring translation errors
or the termination of the translation is lower. Moreover, codon-optimized genes
are usually highly expressed. Also, the authors have also expressed arabinose-H
Fig. 6 Basic structure of stilbenoids

+ transport protein (AraE). This protein is involved in arabinose transport. How-

ever, it was verified that resveratrol could also be transported when its intracellular
concentration reaches the threshold. For this reason, it was hypothesized that AraE
probably increases the lipid membrane permeability for the diffusion of resveratrol.
These modifications resulted in a 2.44-fold increase in the production of resvera-
trol (2.3 mg/L) (Wang et al. 2011). The same research group also constructed one
synthetic scaffold between 4CL1 and STS to improve the resveratrol production.
The scaffold interacts with the enzymes by small ligand peptides and improved the
substrate flux between both heterologous enzymes. Compared to the non-scaffold
strategy, the production of resveratrol increased 5-fold reaching 14.4 mg/L (Wang
and Yu 2012). Moreover, resveratrol was also produced from tyrosine by Shin et al.
(2012). In this study, PAL, C4H, 4CL1, and STS were expressed in S. cerevisiae
W303-1A. In order to increase the extender substrate availability, the promoter of
the native ACC gene was replaced by pGAL1. This modification resulted in a 1.3-
fold improvement in the resveratrol production reaching 5.8 mg/L from 2.17 g/L
of tyrosine (Shin et al. 2012). Later, de novo production of resveratrol in S. cere-
visiae was achieved by Li et al. (2015) and Li et al. (2016). First, it was evaluated
the influence of different isoforms of the 4CL gene from A. thaliana in the resver-
atrol production (Li et al. 2015). The authors have found that the expression of
At4CL1 resulted in higher productions of resveratrol comparing with At4CL2,
thus being possible to conclude that the choice of the enzymes is a step that can
greatly influence the production titers and yields. The construction of aromatic
amino acids overproducing strain was also evaluated. This strain was constructed
by overexpressing feedback-insensitive versions of the DAHP synthase and cho-
rismate mutase genes. This modification resulted in a 1.8-fold improvement in
the production of resveratrol. The authors have also overexpressed a version of
the ACC gene with a double mutation into the serine residues 659 and 1157 to
increase malonyl-CoA availability. These mutations prevent the ACC phosphory-
lation and, consequently, its inactivation by the action of sucrose non-fermenting
protein 1. The overexpression of the ACC mutant version has resulted in a 1.3-fold
improvement in the production of resveratrol. In order to increase the expression
levels and, consequently, the production of resveratrol, the genes responsible for
the resveratrol biosynthetic pathway were integrated into multiple copies in the Ty
repetitive sites that are present in the yeast genome using an integrative plasmid.
The determination of the copy number was performed by qPCR. The maximum
number of integrations found in the selected transformants was eight copies. The
strain that holds the eight copies of the genes responsible for the biosynthetic
pathway was able to produce 36-fold more resveratrol than the strains holding
only one copy of the genes. In fed-batch fermentation, this strain was able to pro-
duce 531.4 mg/L of resveratrol from 40 g/L of glucose (Li et al. 2015). Later, the
same research group optimized the resveratrol production to 800 mg/L from glu-
cose in fed-batch fermentation. This improvement was obtained by changing the
heterologous pathway for phenylalanine utilization by expressing PAL and C4H
genes instead of TAL. CPR from A. thaliana and CYB5 from S. cerevisiae were
overexpressed toward an improvement in the cytochrome P450 activity. Moreover,
144 D. Gomes et al.
the flux toward the aromatic amino acids production was improved by replacing
DAHP synthase and chorismate mutase by feedback-resistant versions of these
genes. Malonyl-CoA synthesis was also improved by expressing the acetyl-CoA
synthase from Salmonella enterica and the mutated version of the ACC gene.
Beyond these modifications, the pathway deviation was also prevented by delet-
ing the PDC gene. In this study, two stilbenoids that are derived from resveratrol
were also produced. By expressing resveratrol O-methyltransferase (ROMT) from
Sorghum bicolor, the constructed strain produced 5.52 mg/L of pinostilbene. By
expressing ROMT from V. vinifera, the strain produced 34.92 mg/L of pterostil-
bene (Li et al. 2016). Pterostilbene was also previously produced using p-coumaric
acid as a substrate in S. cerevisiae expressing 4CL, STS, and ROMT (Wang et al.
2015a). However, the production was even lower (2.2 mg/L) than the one reported
by Li et al. (2016).
The heterologous production of resveratrol was also achieved using Y. lipolytica
as microbial chassis. Huang et al. (2006) reported for the first time the production
of resveratrol in Y. lipolytica. The strain expressing PAL, C4H, 4CL, and STS
was able to produce 1.46 mg/L of resveratrol from 36.2 mg/L of tyrosine. More
recently, there have been many advances to produce larger amounts of resveratrol
in Y. lipolytica. Beyond the construction of the p-coumaric acid biosynthetic path-
way, Gu et al. (2020) also constructed the pathway responsible for the resveratrol
production. The authors expressed 4CL and STS in the strain previously con-
structed to produce p-coumaric acid. Although this strain has several modifications
to improve the flux toward the synthesis of aromatic amino acids and the shikimate
pathway, only 12.67 mg/L of resveratrol were produced from 40 g/L of glucose.
Palmer et al. (2020) have also reported the construction of a Y. lipolytica strain
able to de novo produce resveratrol. This strain also contains modifications to
improve the synthesis of malonyl-CoA and amino acids, and it was able to produce
48.7 mg/L of resveratrol from 20 g/L of glucose and 32.8 mg/L of p-coumaric acid.
Afterward, the heterologous production of resveratrol in Y. lipolytica was optimized
by Sáez-Sáez et al. (2020). In this study, TAL from Flavobacterium johnsoniae,
4CL from A. thaliana and STS from V. vinifera were successfully integrated into
the Y. lipolytica genome using integrative plasmids. DAHP synthase and choris-
mate mutase were replaced by feedback-insensitive versions. Moreover, PCD5 and
Aro10 were also deleted to prevent the pathway deviation. Two mutations were
introduced in the ACC gene to prevent the phosphorylation and inactivation of
malonyl-CoA synthesis. This strain was able to produce 85 mg/L of resveratrol.
In order to improve the resveratrol titer, five copies of the genes responsible for
the biosynthetic pathway were introduced using integrative plasmids in the pre-
viously constructed strain reaching the production of 409 mg/L of resveratrol. In
fed-batch fermentation, this strain was able to produce 12.400 mg/L of resvera-
trol from glucose. This is the highest production of resveratrol reported so far in
any microorganism (Sáez-Sáez et al. 2020). More recently, He et al. (2020) also
constructed a strain for de novo production of resveratrol. The authors studied
the production using the tyrosine or the phenylalanine routes or the conjugation
of both routes. The biosynthetic pathways were firstly integrated into a single
copy in the Y. lipolytica genome. Afterward, one extra copy of the biosynthetic
pathways was also integrated to increase the metabolic flux toward the produc-
tion of resveratrol. When glucose was used as a substrate, the strain carrying two
copies of the tyrosine route was the best resveratrol producer (85 mg/L). Addi-
tionally, the performance of the three strains was also tested using glycerol as an
alternative substrate. In these experiments, the strain carrying two copies of both
tyrosine and phenylalanine routes showed the best results of resveratrol production.
In bioreactor fermentation, this strain was able to produce 430 mg/L of resveratrol
from 100 g/L of glycerol without any supplementation of amino acids (He et al.
2020). All these studies show the great potential to use Y. lipolytica as a chassis to
industrially produce resveratrol and other resveratrol-derived compounds.
6.4 Heterologous Production of Coumarins
Coumarins are polyphenolic compounds that are largely found in many plant
species being fundamentally present in essential oils and fruits (Jain and Joshi
2012). These compounds act in the defense mechanism of plants against stress
factors such as fungal infections (Stringlis et al. 2019). The core structure of
coumarins is composed of one benzene ring and one α-pyrone ring (Fig. 7). These
compounds are divided into two main classes that are simple coumarins and com-
plex coumarins. In the group of complex coumarins, the compounds are classified
as furanocoumarins, pyranocoumarins, phenylcoumarins, and bicoumarins (Matos
et al. 2015).
Relatively to the flavonoids and stilbenoids, the heterologous production of
coumarins has been less explored. Simple coumarins, such as umbelliferone,
scopoletin, and esculetin, were already produced in E. coli (Lin et al. 2013; Yang
et al. 2015; Zhao et al. 2019). However, the use of yeasts as microbial chassis
to produce these compounds practically has not been explored. Until now, only
scopoletin was already produced in S. cerevisiae (Zhao et al. 2020). The genes
4CL from Petroselinum crispum and feruloyl-CoA 6 -hydroxylase (F6 H1) from
A. thaliana responsible for the synthesis of scopoletin from ferulic acid were
expressed into S. cerevisiae BY4742. This strain was able to produce 0.364 mg/L
of scopoletin. Since these results of production were very low, the authors fused
4CL and F6’H1 with different linkers. The best production result was obtained
when the genes were fused with the linker (GGGGS)4 , resulting in a 3-fold
improvement in the production of scopoletin. After integrating the fusion gene
Fig. 7 Basic structure of coumarins

146 D. Gomes et al.
into the S. cerevisiae genome, 3.42 mg/L of scopoletin were produced using fer-
ulic acid as substrate. The authors also tested the production of scopoletin using
p-coumaric acid as substrate. HpaB from P. aeruginosa, HpaC from Enterobacte-
riaceae and COMT from A. thaliana were integrated into the genome of the strain
carrying the fusion gene 4CL-F6’H1. This strain was able to produce 4.98 mg/L of
scopoletin using p-coumaric acid as substrate. Due to the interest of using alterna-
tive substrates to produce compounds of interest, the production was also evaluated
using lignin hydrolysate as substrate (Gudiña et al. 2020). Since ferulic acid and
p-coumaric acid are monomers of lignin, this strain produced 4.79 mg/L of scopo-
letin (Zhao et al. 2020). As far as we know, this is the only report of heterologous
production of coumarins in yeasts. Although the production titers are lower com-
pared with the productions obtained in E. coli, we believe that this production could
be successfully improved and reach higher levels. Moreover, the construction of the
biosynthetic pathway to produce the other simple and complex coumarins should
also be explored using S. cerevisiae and other yeasts as chassis.
6.5 Heterologous Production of Curcuminoids
Curcuminoids are natural polyphenolic compounds that can be found in the rhi-
zome of the plant Curcuma longa which is widely known as turmeric. The
rhizome of turmeric is mainly composed of curcumin, demethoxycurcumin, and
bisdemethoxycurcumin. These compounds have been largely used as a food addi-
tive, as well as therapeutic agents due to their several recognized therapeutical
activities (Amalraj et al. 2017). For this reason, the market value of curcumin,
which is present in higher amounts in turmeric and exhibits the most potent bio-
logical activities, is expected to reach USD 151.9 million by 2027 (Hewlings and
Kalman 2017; Grand View Research 2020). The core structure of curcuminoids
is composed of a C6-C7-C6 structure being classified as diarylheptanoids (Fig. 8)
(Rodrigues et al. 2015c).
Fig. 8 Structure of the three curcuminoids compounds: curcumin, demethoxycurcumin, and bis-
demethoxycurcumin
The heterologous production of curcuminoids has been widely explored in E.

coli. These compounds were already produced using hydroxycinnamic acids and
tyrosine as starter substrates (Couto et al. 2017; Fang et al. 2018; Katsuyama
et al. 2008; 2010; Kim et al. 2017; Rodrigues et al. 2017a, 2015a, 2020; Wang
et al. 2013, 2015b). However, to the best of our knowledge, the construction of
the biosynthetic pathway responsible for the curcuminoids production was not
achieved until now in S. cerevisiae and P. pastoris. The only report of the produc-
tion of curcuminoids in yeast species was the production of bisdemethoxycurcumin
using Y. lipolytica as yeast chassis (Palmer et al. 2020). In this study, the authors
constructed a Y. lipolytica strain expressing 4CL gene from Nicotiana tabacum
and a codon-optimized version of CUS gene from Oryza sativa. PEX10 and ACC1
genes were overexpressed toward an improvement in the flux toward malonyl-CoA
biosynthesis. This strain was able to produce 0.17 mg/L of bisdemethoxycurcumin
from 2 mM of p-coumaric acid (Palmer et al. 2020). Although this production
value is lower than the productions reported in E. coli, we believe that this study
can be a starting point for producing larger amounts of these compounds in Y.
lipolytica and also in other yeasts. The construction of the whole biosynthetic
pathway to produce curcuminoids from glucose as well as the modification of the
constructed strains using synthetic biology and metabolic engineering strategies
could be an interesting approach to produce these compounds with a high yield
and purity.
6.6 Heterologous Production of Polyphenolic Amides
Polyphenolic amides are polyphenolic compounds that could be found in foods

such as peppers and oats (Tsao 2010). Avenanthramide, which belongs to the
polyphenolic amides group, is present in oats and is involved in the plants’
response to the infection with pathogens (Meydani 2009). The structure of these
compounds consists of an anthranilic acid and a hydroxycinnamic acid linked by
an amide bond (Fig. 9). More than 40 different structures are known for avenan-
thramide compounds due to the presence of radical substituent groups in the
structure (Perrelli et al. 2018).
Fig. 9 Basic structure of avenanthramides

148 D. Gomes et al.
As occurred with coumarins and curcuminoids, the heterologous production of

polyphenolic amides using yeasts as chassis has been also less explored. There is
only one report of the heterologous production of two avenanthramide-derived
compounds in S. cerevisiae. 4CL from N. tabacum and hydroxycinnamoyl-
CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) from Cynara car-
dunculus were successfully expressed in S. cerevisiae for the conversion of the
hydroxycinnamic acids into CoA esters and the CoA esters into the respective
amide. In this study, 120 mg/L of p-coumaroyl-3-hydroxyanthranilic (avenan-
thramide I) were produced from 462 mg/L of p-coumaric acid and 77 mg/L of
3-hydroxyanthranilic acid. Moreover, 22 mg/L of caffeoyl-3-hydroxyanthranilic
acid (avenanthramide II) was produced from 540 mg/L of caffeic acid and 77 mg/L
of 3-hydroxyanthranilic acid (Moglia et al. 2015). This study only tested the
production from the hydroxycinnamic acids. The next step could be the construc-
tion of the complete biosynthetic pathway for the de novo production of these
compounds.
7 Conclusions
Polyphenolic compounds have been recognized as promising compounds to be

used in the treatment of several diseases. Due to the increasing demand for these
compounds by the pharmaceutical and nutraceutical industry, the development of
methodologies to industrially produce them became a priority. In the last years,
several research efforts have been made toward the production of polyphenolic
compounds with high yields and titers using microorganisms. The use of microor-
ganisms, namely yeast species, became an attractive solution since the process is
faster and more economic and environmentally friendly. Regarding flavonoids, the
heterologous production of naringenin has been the most explored. The maximal
production of this compound was 898 mg/L in Y. lipolytica (Palmer et al. 2020).
Within stilbenoids, the production of resveratrol using yeasts as chassis has been
the most studied. This compound was already produced in a significant amount
in Y. lipolytica (12.5 g/L) (Sáez-Sáez et al. 2020). Although these reports are a
good starting point, there is still a long way to go to produce these compounds
at an industrial scale. Nevertheless, it was possible to conclude that Y. lipolyt-
ica is potentially an excellent heterologous host for the industrial production of
these compounds due to the higher yields reported. Moreover, the production of
coumarins, curcuminoids, and polyphenolic amides remains much less explored.
In this case, exploiting different enzyme combinations performs a step-by-step
optimization, as well as constructing the complete biosynthetic pathway for the de
novo production of these compounds are important steps to improve and achieve
relevant productions of these polyphenols. The main bottlenecks in the produc-
tion of polyphenols using microbial cell factories are the substrates and precursors
availability and the deviation of these compounds for competing pathways. The
use of metabolic engineering and synthetic biology approaches are useful to over-
come these issues and, consequently, to construct more robust and adapted strains.
In summary, we believe that the production of polyphenols could reach industrial

levels after performing several optimizations in the yeast chassis and also in the
operational methods of the fermentation process.
Acknowledgements This study was supported by the Portuguese Foundation for Science and
Technology (FCT) under the scope of the strategic funding of UIDB/BIO/04469/2020 unit and
BioTecNorte operation (NORTE-01-0145- FEDER-000004) funded by the European Regional
Development Fund (ERDF) under the scope of Norte2020—North Portugal Regional Pro-
gram. DG and JR are recipients of a fellowship supported by a doctoral advanced training
(SFRH/BD/04433/2020 and SFRH/BD/138325/2018, respectively) funded by FCT.
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Yeast Synthetic Biology
for Production of Artemisinin
as an Antimalarial Drug
Arman Beyraghdar Kashkooli, Karim Farmanpour-Kalalagh,

and Alireza Babaei
Abstract
Artemisinin, a sesquiterpene endoperoxide lactone derived from Artemisia
annua, is highly effective against malaria parasite Plasmodium falciparum.
Artemisinin and its derivatives (collectively termed artemisinins) demonstrate
additional anticancer, anti-inflammation, antiviral, and anti-SARS-CoV-2 activ-
ity. Because of the expensive medicine of artemisinins and the low content
of artemisinin biosynthesis in native host, researchers have been endeav-
ored to produce artemisinin via alternative approaches. Previous attempts give
attention to increase artemisinin biosynthesis via common plant breeding tech-
niques and on engineering cultivated plants for wide production. However,
current trends are focusing on the bioengineering of artemisinin biosynthetic
pathway in heterologous expression platforms. To date, in planta artemisinin
production in two model plants, tobacco (Nicotiana benthamiana) and moss
(Physcomitrella patens), has been reported successfully. Nevertheless, to meet
the large-scale demands, de novo reconstituting of artemisinin biosynthetic
pathway in heterologous microorganisms such as Escherichia coli and Sac-
charomyces cerevisiae has had documentary achievements. Since there are
challenges for the expression of cytochrome P450s (CYPs) from the eukary-
otic origin (such as CYP71AV1) in E. coli, S. cerevisiae has been proposed as a
heterologous host for production of artemisinin precursors such as amorphadi-
ene, artemisinic acid, and dihydro artemisinic acid. Moreover, the presence of
the MVA biosynthetic pathway for production of universal sesquiterpenes pre-
cursor, farnesyl pyrophosphate (FPP), and relative compatibility of S. cerevisiae
cell environment for the expression of plant-origin genes (enzymes) have made
this microorganism as a favorable platform for artemisinins production.
A. Beyraghdar Kashkooli (B) · K. Farmanpour-Kalalagh · A. Babaei

Department of Horticultural Science, Faculty of Agriculture, Tarbiat Modares University,
14115-365, Tehran, Iran
https://doi.org/10.1007/978-3-030-89680-5_6
158 A. Beyraghdar Kashkooli et al.
1 Introduction
Plants have developed the capacity to make a considerable diversity of special-

ized (secondary) metabolites. These metabolites have different classifications and
groups which are produced via specific biosynthetic pathways. Terpenoids, con-
taining the 5 carbon backbone in their structure, are the largest class of secondary
metabolites. One of the most widely studied groups in this class is sesquiterpene
lactones (SLs). SLs have different types and each has various applications in differ-
ent industries. Artemisinin is an SL that is exclusively found in the plant Artemisia
annua. Artemisinin, artemisinin derivatives (collectively termed as artemisinins),
and artemisinin-based combination therapies (ACTs) are the first-line antimalar-
ial drugs recommended by World Health Organization (Lin and Pakrasi 2018). In
addition, artemisinins are effective in the treatment of various cancers, inflamma-
tions, viruses (Efferth 2018), and very recently in the fight against SARS-CoV-2
(Krishna et al. 2021; Nair et al. 2021; Uckun et al. 2021). Due to the low produc-
tion of artemisinin in A. annua, classical methods such as nutrient manipulations,
biotic effectors, enhancing glandular trichomes through plant breeding methods,
and application of plant growth regulators have been done to increase artemisinin
production. However, obviously, these attempts and the use of classical methods
did not meet global artemisinin demand. Recent studies indicated that artemisinin
production can be enhanced via plant manipulation techniques as well as the appli-
cation of metabolic engineering methods of microorganisms. Plant biotechnology
techniques including tissue culture (native host regeneration, callusgensis, etc.),
hairy root and gall induction by Agrobacterium rhizogenes and A. tumefaciens,
respectively, ploidy induction, adventitious roots induction, suspension culture, use
of bioreactors, metabolic engineering in native and in planta heterologous host(s)
have had significant success in artemisinin production. On the other hand, de novo
reconstitution of artemisinin biosynthetic pathway in heterologous microorganisms
such as E. coli and S. cerevisiae (baker’s or brewer’s yeast) increased artemisinin
precursor production on large scale. In this chapter, we reviewed artemisinin
production advances via S. cerevisiae using metabolic engineering and synthetic
biology techniques.
2 Importance of Terpenoids Production
Terpenoids (isoprenoids) as the oldest and most diverse group of natural molecules
represent the first instance of natural bioactive compounds (Pateraki et al. 2015).
Most terpenes are isolated from plants which play an important role in some plant’s
metabolism (Davis and Croteau 2000). In plants, terpenes are involved in the
biosynthesis of the phytohormones gibberellic acid, abscisic acid, and cytokinins
and serve as precursors of steroid hormones. They also act as biosynthetic precur-
sors for carotenoid pigments and phytol side chains in chlorophyll and components
of electron-carrying coenzymes such as quinone, ubiquinone, and plastoquinone.
The terpenes–environment interactions are more important as a chemical defense
Yeast Synthetic Biology for Production of Artemisinin … 159
system against herbivores and pathogens, but they also have other roles such as
absorption of pollinators and allopathic properties. Apart from their potential roles
in plants, bioactive terpenoids have raised considerable interest as pharmaceuticals,
nutraceuticals, and flavors and fragrances (Pateraki et al. 2015).
Structurally, terpenoids are various polymers created from the fundamental C5
block, the isoprene unit. They are organized and grouped based on the number of
isoprene units. Therefore, hemiterpenes, monoterpenes, sesquiterpenes, triterpenes,
diterpenes, and carotenoids contain 5, 10, 15, 20, 30, and 40 isoprene units, respec-
tively. The long-chain polymers possess thousands of units and comprise natural
products such as rubber. Regardless of their diversity, all terpenoids are biosynthe-
sized from the universal precursors dimethylallyl pyrophosphate (DMAPP) and
isopentenyl pyrophosphate (IPP, isopentenyl diphosphate, or IDP). This occurs
via two different pathways, the methyl erythritol 4-phosphate (MEP; mevalonate-
independent, or non-mevalonate) pathway and the mevalonate pathway (MVA)
(Capell and Christou 2004).
3 Terpenoids Biosynthetic Pathway in Plants
Biosynthesis of terpenes can be divided into four stages. The first step involves
the production of isopentenyl diphosphate (IPP) and its isomer, dimethylal-
lyl pyrophosphate (DMAPP), which is carried out by isopentenyl diphosphate
isomerase (IPP isomerase). In the second stage, IPP and DMAPP are struc-
turally elongated to form more complex isoprenoid backbones called geranyl
pyrophosphate (GPP C10), farnesyl pyrophosphate (FPP, C15), geranylgeranyl
pyrophosphate (GGPP, C20). The third stage involves the conversion of GPP,
FPP, and GGPP to the corresponding terpene groups producing monoterpenes,
sesquiterpenes, and diterpenes, respectively. The final step in the biosynthesis of
terpenes occurs in the cytosol (e.g., cyclization, hydroxylation, and carboxylation)
which produces derivatives of the basic terpene groups (Davis and Croteau 2000;
Moses et al. 2013).
In general, the major pathway for the biosynthesis of monoterpenes is within
plastids. Monoterpenes can also be metabolically engineered to be produced in the
cytosol, although these pathways can also produce intermediates (Bouwmeester
2006). In plants, two distinct but interacting pathways have been identified for IPP
biosynthesis: the mevalonate (MVA) or cytosolic pathway, the methylerythritol
4-phosphate (MEP) pathway or the plastid pathway.
3.1 MEP (Methylerythritol 4-Phosphate) Pathway Versus

the MVA (Mevalonate) Pathway
In MEP, pyruvate is first combined with glyceraldehyde-3-phosphate (G3P). After

a series of reactions, 1-deoxy-d-xylulose-5-phosphate (DXP) is formed. This com-
pound is then converted to methylerythritol-4-phosphate, and after a series of other
steps, it is also converted to isopentyl diphosphate (IPP) (Moses et al. 2013).
The MEP pathway also referred to as the 1- deoxy-D-xylulose 5-phosphate

(DXP) contains seven enzymatic steps and starts with the condensation of D-
glyceraldehyde 3-phosphate (GAP) and pyruvate to generate 1-deoxy-D-xylulose
5-phosphate (DXP), which then undergoes isomerization/reduction with the forma-
tion of MEP. Five sequential steps are essential to shift MEP to IPP and DMAPP
(Fig. 1). The MEP pathway depends on early metabolism for the provision of pyru-
vate and GAP, with the subsequent resulting from both glycolysis and the pentose
phosphate pathway (PPP). Both IPP and DMAPP are substrates for short-chain
prenyltransferases (PTs), which create prenyl diphosphate precursors, geranyl
diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate
(GGPP), for a numerous group of terpene synthases/cyclases (Dudareva et al.
2013; Moses et al. 2013; Vranova et al. 2013).
Fig. 1 MEP (methylerythritol 4-phosphate) and MVA (mevalonate) biosynthetic pathway in

plants
In parallel to the MEP pathway, the mevalonate pathway (MVA) is one of the
most important cellular metabolic pathways in all eukaryotes and many bacte-
ria. This pathway aims to produce dimethylallyl diphosphate (DMAPP) as well
as isopentenyl diphosphate (IPP), both of which act as a basis for molecular
biosynthesis in a variety of pathways, including the synthesis of terpenoids, cell
membrane preservation and regeneration, and many other cellular hormones. In the
MVA pathway, two molecules of acetyl-CoA first combine to form acetoacetyl-
CoA. Then, by adding another molecule of acetyl-CoA to acetoacetyl-CoA, and
then by performing a series of other steps, mevalonate is obtained. Mevalonate
is then converted to IPP under the influence of other steps and enzymes, and
IPP is converted to DMAPP under the influence of isopentenyl diphosphate iso-
merase. IPP is elongated by the enzyme prenyltransferase, and geranyl diphosphate
is produced by the enzyme geranyl diphosphate synthase, which is also called
the precursor (monoterpene synthases) produces different types of monoterpenes
(Vranova et al. 2012; Moses et al. 2013) (Fig. 1).
3.2 Sesquiterpene Lactones
Sesquiterpene lactones (SLs), a subgroup of the sesquiterpenoids, contain over

4000 various known structures. SLs are mostly colorless, bitter, and often aro-
matic constituents of plant essential oils found mostly in some plant species
from Asteraceae. Their biological activities such as anticancer, antimalarial, anti-
migraine, antioxidant, antibacterial, antifungal, anti-inflammation, antiviral, and
anti-SARS-CoV-2 have generated attention for medical uses. SLs are classified
into six bicyclic or tricyclic subclasses including guaianolides, pseudoguaiano-
lides, xanthanolides, eremophilanolides, eudesmanolides, and germacranolides.
The subsequent modification of SLs is followed by double-bond reductases and
glycosyltransferases (reviewed in Beyraghdar Kashkooli et al. 2018).
Sesquiterpenoids are biosynthesized from FPP, via the catalytic activity of ter-
pene synthases (in this case, sesquiterpene synthases (STPS)). Molecular genetics
and phytochemistry studies indicated that the biosynthesis of STLs in plants cre-
ated a multiplex network of metabolic routes that compete for general precursors.
Furthermore, it is also exclusively regulated by numerous temporal, spatial, and
environmental factors. Some SLs biosynthetic pathways including costunolide,
parthenolide, kauniolide (Liu et al. 2005), and artemisinin production have been
fully characterized. On the other hand, a number of transcription factors that
manage the transcription of biosynthetic genes have been characterized, which
specified a great comprehension of how these STLs are biosynthesized. This infor-
mation has led to a high contribution in characterization understanding of the other
SL biosynthetic pathways in plants. More precisely, the regulation is specific for
each SL biosynthetic pathway. Thus, it is imperative to identify the various factors
that regulate and control the upstream (MVA and) and SLs-specific downstream
biosynthetic pathway in every SL-producing plant (Perassolo et al. 2018).
3.3 Artemisinin Biosynthetic Pathway in Artemisia annua
It is important to deeply understand the key biosynthetic pathway routes and genes
involved in artemisinin biosynthesis in order to be able to enhance artemisinin
yield in the natural artemisinin producing plant, A. annua, as well as other heterol-
ogous host platforms. artemisinin is a sesquiterpene lactone, containing 15 carbon
atoms with lipophilic properties (Lv et al. 2017). Both artemisinin and arteannuin
B are produced via a biosynthetic pathway in the glandular trichomes (GTs) in
leaves and oval of the A. annua (Wang et al. 2016). However, some reports have
shown that the highest GTs densities are detected on flowers, buds, and young
leaves of this plant (Olofsson et al. 2011). The biosynthetic pathway of GTs occurs
via the terpenoids biosynthetic pathway. Isoprene units of terpenoids are obtained
from two pathways: the MVA pathway, which occurs in the cytosol, MEP pathway
in the plastids (Dewick 2009; Vranová et al. 2013). The artemisinin biosynthetic
pathway starts with the conversion of 5-carbon units of IPP and MAPP units to FPP
(Fuentes et al. 2016). A study on the artemisinin biosynthetic pathway shows that
the first and key step in biosynthesis of this compound is the conversion of FPP to
amorpha 4, 11-diene (amorphadiene), which is catalyzed by a well-known terpene
cyclase, the amorphadiene synthase (ADS) (Bertea et al. 2005; Mercke et al. 2000).
The cytochrome P450 hydroxylase (CYP71AV1) (Teoh et al. 2006) then converts
amorphadiene to artemisinic alcohol. CYP71AV1 also oxidizes artemisinic alcohol
to artemisinic aldehyde and artemisinic acid, respectively. Artemisinic aldehyde
reductase (DBR2) and aldehyde dehydrogenase 1 (ALDH1) (Zhang et al. 2008),
as the branching point, converts artemisinic aldehyde to dihydroartemisinic alde-
hyde (Bertea et al. 2005; Schramek et al. 2010). Bifurcation points of artemisinic
aldehyde biosynthetic pathway can synthesize artemisinic acid by CYP71AV1
and ALDH1. Also, due to the non-enzymatic reaction, dihydroartemisinic acid
and artemisinic acid are converted to artemisinin and arteannuin B, respectively
(Brown 2010; Han et al. 2014; Ikram and Simonsen 2017). ALDH1 then pro-
duces dihydroartemisinic acid (DHAA) from dihydroartemisinic aldehyde. DHAA
is considered as a precursor to the artemisinin (bio)synthesis (Tang et al. 2014),
and finally its conversion to artemisinin is considered as a non-enzymatic reaction
which occurs under the influence of UV light and oxygen (Bertea et al. 2005;
Ikram et al. 2019; Schramek et al. 2010; Teoh et al. 2006) (Fig. 2).
Studies show that all genes responsible for biosynthesis of artemisinin are
fully identified (Ikram et al. 2019). Identification of the genes involved in the
biosynthetic pathway of artemisinin offers an opportunity to increase artemisinin
production and subtle reduction in its production costs through plant breeding
and/or the engineering of other putative hosts (Zhang et al. 2008).
Fig. 2 Artemisinin biosynthetic pathway in Artemisia annua L
4 Application of Artemisinins in Medicine
4.1 Antimalarial
Malaria has been a worldwide outbreak health threat since old times. Scientific and
hospital reports show that many patients die every year due to malaria infections.
Artemisinins and artemisinin-based combination therapies (ACTs) are frontline
antimalarial agents accepted for their potency and low toxicity (Yang et al. 2020).
In the early 1970s, a group of Chinese scientists led by Professor Tu Youyou
(winner of Nobel prize in physiology or medicine 2015) succeeded in obtaining
the effective results of A. annua plant extracts on malaria (Liu and Liu 2016;
Su and Miller 2015). By testing the natural extract from the A. annua, Professor
Tu found that the compound could have 100% inhibitory effect on malaria para-
sites in vivo experiments (Tu 2011). In the late 1970s, artemisinin was effectively
recognized as an anti-malarial drug (Klayman 1985; Tu 2011; Xie et al. 2016).
So far, several mechanisms of action such as interfering with the heme detoxifica-
tion pathway, creating protein damage and inhibiting parasite proteasome function,
inducing the alkylation of PfTCTP and inhibiting PfATPase, and membrane depo-
larizing of mitochondria via ROS production have been reported for artemisinin to
fight malaria parasites (Plasmodium falciparum and Plasmodium vivax).
4.2 Anti-inflammation
The anti-inflammatory activity of artemisinins have been attributed to the regulat-

ing of pro-inflammatory cytokines expression, inhibition of nuclear factor-kappa
B (NF-κB), toll-like receptors, Syk tyrosine kinase, phospholipase Cγ, PI3K/Akt,
MAPK, STAT-1/3/5, Sp1, Nrf2/ARE signaling pathways, matrix metallopro-
teinases (MMPs), vascular endothelial growth factor (VEGF), promoting cell cycle
arrest, driving reactive oxygen species (ROS) production, and inducing Bak or
Bax-dependent or independent apoptosis (Ho et al. 2014; Cheong et al. 2020).
Artemisinin prevents the secretion and the mRNA amount of TNF-α, interleukin
(IL)-1ß, and IL-6 in a dose-dependent manner in PMA-induced THP-1 human
monocytes. It also inhibits the phosphorylation of IKKα/ß, phosphorylation and
degradation of IκBα, and the nuclear translocation of the NF-κB p65 subunit
(Wang et al. 2011).
4.3 Anticancer
Beyond the anti-malarial activity of artemisinins, they have also been proven to
contain anticancer effects, indicating cytotoxic effects in combating numerous
cancer types. These activities look to be done by artemisinin-induced modifica-
tions in several signaling pathways, interfering with various hallmarks of cancer at
the same time. Considerable successes have been obtained to distinguish these
pathways and to display their anticancer mechanisms of action (Wong et al.
2017). Recent investigations have documented the strong anticancer activity of
artemisinins (Slezakova and Ruda-Kucerova 2017). The main mechanisms of
action are reported for artemisinin in renal (Yu et al. 2019), breast (Cao et al.
2019; Li et al. 2019), ovarian (Liang et al. 2019), prostate (Willoughby et al.
2009), colon (Riganti et al. 2009), cervical (Mondal and Chatterji 2015), ishikawa
endometrial (Tran et al. 2014), neuroblastoma (Zhu et al. 2014), nasopharyngeal
(Wu et al. 2011), gastric (Zhang et al. 2014), leukemia (Ohgami et al. 2010),
lung (Li et al. 2018), fibrosarcoma tumors (Singh and Lai 2004), for artesunate
in cancer stem cells (Subedi et al. 2016), prostate (Nunes et al. 2017), head and
neck (Roh et al. 2017), cervical (Thanaketpaisarn et al. 2011), colorectal (Chen
et al. 2017), bladder (Zhao et al. 2020; Zhou et al. 2020), leukemia (Zhou et al.
2007), skin (Jiang et al. 2012), liver (Yin et al. 2020), laryngeal (Singh and Verma
2002), ovarian (McDowell et al. 2021), glioblastoma (Berdelle et al. 2011), rhab-
domyosarcoma (Beccafico et al. 2015), breast (Pirali et al. 2020), endometrial (Yin
et al. 2021), lung (Ma et al. 2011), colon (Hao et al. 2020), non-small cell lung
(Wang et al. 2020), pancreatic (Wang et al. 2019), gastric (Zhang et al. 2015),
beta-cell lymphoma (Våtsveen et al. 2018), and esophageal (Fei et al. 2018), for
dihydroartemisinin in colorectal (Yao et al. 2018), cervical (Disbrow et al. 2005),
esophageal (Li et al. 2018a, b), breast (Mao et al. 2013), hepatocellular (Wu et al.
2019), ovarian (Liu et al. 2018), cholangiocarcinoma and hepatocarcinoma (Chai-
jaroenkul et al. 2011), pancreatic (Chen et al. 2009), non-small-cell lung (Jiang
et al. 2016), for artemisone in melanoma, breast, colon, and pancreatic (Gravett
et al. 2011; Dwivedi et al. 2015), and for artemether in gastric (Alcântara et al.
2013) cancer cells.
4.4 Antiviral and Anti-SARS-CoV-2
Antiviral activity of artemisinins against DNA viruses of the Hepadnaviridae and

Herpesviridae families such as Epstein-Barr virus, cytomegaloviruses, herpes sim-
plex viruses 1 and 2, hepatitis B virus, and human herpesvirus 6 have been reported
based on in vitro and in vivo studies. The confirmation is weaker for papilloma
and polyomaviruses. Also, low or no prevention in vitro activity has been docu-
mented for RNA viruses such as influenza virus, human immunodeficiency viruses
1 and 2, and hepatitis C virus. (Efferth 2018). As mentioned above, artemisinins
with anti-inflammatory properties contain wide-spectrum antiviral activity. There-
fore, the inhibition of interleukins such as interleukin-6 (IL-6) plays a key role in
combating SARS-CoV-2 (Krishna et al. 2021). Also, it is reported that the anti-
SARS-CoV-2 activity of artemisinins has been attributed to its potency to prevent
spike-protein-mediated and TGF-β-dependent primary phases in the inflammation
process as well as its capability to destroy the post-entry intracellular events of the
SARS-CoV-2 inflammation cycle needed for viral replication (Uckun et al. 2021).
5 Yeast Synthetic Biology for Artemisinin Precursors

Production
Extensive application of the beneficial secondary metabolites has been hindered

by little yield in vivo and the high expense of chemical synthesis in vitro. New
production techniques are essential to provide the ever-growing requirements for
these valuable metabolites. Previous attempts give attention to raising production
via common breeding techniques, with low positive results, and on engineering
cultivated plants for wide production in bioreactors. However, recent studies are
focusing on bioengineering for these special biochemical routes (Arsenault et al.
2008). Also, the use of recombinant DNA technology in reconstituting metabolic
pathways can increase the biosynthesis of metabolites by modification in the net-
work rates and distribution (Farid et al. 2019). On the other hand, because of the
economic importance of plant metabolites and the increased demand, researchers
are looking for methods to produce extraordinary amounts of these compounds.
Synthetic biology (SynBio) is a fascinating field in modern science, which is grow-
ing rapidly. There is no definite and accepted SynBio definition yet, but in general,
SynBio is a new interdisciplinary field that includes the application of engineer-
ing in biology. Furthermore, recent advances in SynBio in microorganisms and
metabolic engineering of compounds have made it possible to produce molecules
that were previously impossible to be made heterologously (Paddon and Keasling
2014). The possibility of creating heterogeneous pathways in different microor-
ganisms has led to an increase in the production of shrinking molecules (Tsuruta
et al. 2009).
Due to the low performance of artemisinin biosynthesis, synthetic artemisinin
biology started to eliminate these problems using various approaches. As previ-
ously mentioned, artemisinin biosynthesis pathway requires 11 different enzymatic
steps which the pathway starts with the acetyl-CoA precursor. Also, newly rec-
ognized genes, such as cytochrome P450 and its related reductase, have been
indicated to catalyze several phases in the artemisinin biochemical network. This
has the promising results to create a semi-synthetic strategy to production that is
both feasible and profitable. Artemisinin precursors de novo biosynthesis in engi-
neered yeast is about many orders of the content above field-grown A. annua plant
(Arsenault et al. 2008). Because cytochromes P450 enzymes (CYPs) from eukary-
otic origin (such as CYP71AV1) cannot be perfectly expressed in E. coli, yeast was
selected as a heterologous host for artemisinin precursor production (Paddon and
Keasling 2014). In addition, this platform is more methodically and technically
strong and less sensitive to phage contamination (Kong et al. 2013; Krivoruchko
and Nielsen 2015; Perassolo et al., 2018). Attempts to raise flux by engineered net-
works are growing in yeast via combinations of engineering precursors networks
and downstream improving gene expression (Arsenault et al. 2008).
Yeast is a single-celled fungi and one of the best-characterized eukaryotic
organisms. Many cost-effective and beneficial varieties of yeast are available.
Yeasts contain considerable functions in the fermentation of many food prod-
ucts such as bread and cheese. (Tamang and Fleet 2009). S. cerevisiae is one
of the most studied eukaryotic platform organisms. Availability of a high den-
sity of elucidated information regarding its physiology, genetics, and biochemistry
offers a significant improvement in the industrial application of this microorgan-
ism. The yeast model gives numerous similar advantages as prokaryotic platforms
performed for terpene hydrocarbon biosynthesis, which also supplies the biosyn-
thetic tools necessary for the appropriate functional expression of the downstream
modifying enzymes such as cytochrome P450 hydroxylases (Kong et al. 2013). S.
cerevisiae has been extensively used in biotechnology due to its generally regarded
as safe (GRAS) is proper for large-scale performance. As a eukaryotic platform,
cell and molecular biology of S. cerevisiae have been investigated comprehensively
with many genetic engineering existing techniques. Additionally, S. cerevisiae dis-
plays high tolerance against inappropriate industrial conditions (Hong and Nielsen
2012). Thus, S. cerevisiae has been introduced as a model microorganism for syn-
thetic biology (Lian et al. 2018). Therefore, de novo production of the artemisinin
via metabolic engineering and SynBio in S. cerevisiae has relied on the com-
plete knowledge of this organism. In this regard, an effective effort to produce
semi-synthetic artemisinin was made in collaboration with the University of Cali-
fornia, Amyris, and OneWorld Health, with the primary goal of host engineering
to produce high content of artemisinin precursors that could be converted to the
artemisinin by chemical processes (Paddon and Keasling 2014). Remarkably, it
is noteworthy to imply that Amyris company is an industrial claimant for the
production of artemisinic acid in the S. cerevisiae platform (Tang et al. 2014).
The efficient application of SynBio for the semi-synthetic industrial production
of artemisinin was reported in S. cerevisiae (Paddon et al. 2013), as one of the
prominent points of synthetic artemisinin biology (Immethun et al. 2013). Here,
we review and compartmentalize the de novo reconstitution and production of
components involved in the artemisinin biosynthetic pathway in S. cerevisiae.
5.1 Amorphadiene Production
In order to increase amorphadiene, a volatile precursor of artemisinin, success

reports have been documented. An amorphadiene synthase (ADS), a sesquiterpene
synthase, can catalyze the cyclization of farnesyl pyrophosphate (FPP) to amor-
phadiene biosynthesis. The genomic ADS contains a complex structure containing
six introns and seven exons and is classified as class III terpene synthase (Li et al.
2006). It is believed that transcription of ADS possesses tissue and cell specificity
(Alejos-Gonzalez et al. 2011). ADS is detected at a bifurcation point of terpene
metabolism, and it is regarded as an essential enzyme in the artemisinin biosyn-
thesis. The little amount of the volatile amorphadiene in the A. annua and the high
ADS activity was suggested to be potent confirmation that amorphadiene is an
intermediate in artemisinin biosynthesis (Kong et al. 2013). FPP is a usual inter-
mediate metabolite of various compounds in the MVA pathway of yeast (Baadhe
et al. 2013). Therefore, the pivotal plan is to improve FPP biosynthesis and to
regulate it toward the amorphadiene synthesis network instead of other compet-
ing pathways like sterol synthesis. Several genes in the biosynthesis of FPP in
S. cerevisiae including HMGR and ERG20 have been cloned successfully (Wang
et al. 2013). Enhancing the expression of HMGR and ERG20 can boost the titer
of FPP (Han et al. 2008; Ram et al. 2010; Peng et al. 2011). The expression of
the ADS gene plus GAL1 promoter in the BY4742 strain produces a low titer
of amorphadiene (Ro et al. 2006). It is described that carbon flux extends toward
IPP when the N-terminal regulatory region of HMGR is removed (Donald et al.
1997). In the WHT strain of S. cerevisiae, the expression of tHMGR, ADS, and
ERG20 via GAPDH1, GAPDH1, and ADH1 promoters, respectively, enhances the
amorphadiene production in yeast (Kong et al. 2007).
Because of some competing networks commencing with the universal FPP
precursor, the overexpression of ERG20 and tHMGR genes may not exclusively
enhance the carbon flux toward the biosynthesis of amorphadiene (Paddon et al.
2013). Squalene synthase (SQS), encoded by EGR9, which is responsible for the
biosynthesis of squalene in the full process is the main competing branch point
(Paradise et al. 2008). Results of other studies indicated that the FPP conversion
to amorphadiene by expression of heterologous amorphadiene synthase (ADS) has
been successful in yeast. First, ERG9 promoter of S. cerevisiae was replaced with
repressible methionine (MET3) promoter via bipartite gene fusion method. Then,
to dominate the reduction of the intermediate FPP by competitive networks in S.
cerevisiae, fusion protein technique was used, and FPP synthase of yeast has been
combined with ADS of A. annua where amorphadiene production was increased
in yeast strains YCF-002 (11.2 mg/l) and YCF-005 (25.02 mg/l) compared with
the control YCF-AD (5.5 mg/l) strain, respectively (Baadhe et al. 2013).
Totally, as mentioned above, since the expression of ADS alone leads to low
levels of amorphadiene, some modifications were created in the MVA pathway
intending to enhance FPP formation such as coupling the overexpression of two
copies of tHMGR, the overexpression of the gene encoding FPS (ERG20), the
overexpression of UPC2–1, which is an activated allele of UPC2 (a common tran-

scription factor which influences the expression of several enzymes from the MVA
pathway), and the downregulation of ERG9 by a methionine repressible promoter
(PMET3) amorphadiene production. (Perassolo et al. 2018).
5.2 Artemisinic Acid Production
To introduce an industrially potent yeast strain, several biological engineering

attempts have been used to notably increase artemisinic acid production in the
engineered microorganism (Kong et al. 2013). Regarding the cost and possibil-
ity in industry, S. cerevisiae is distinguished as a good model for artemisinic acid
synthesis (reviewed in Li et al. 2016). To increase the biosynthesis of artemisinic
acid in S. cerevisiae cells, some plans have been established, such as the over-
expression of basic genes, and the repression (knock-down) of competing for
biosynthetic pathway networks (Paddon and Keasling 2014). All these common
approaches are mostly focused on enhancing carbon flux toward heterologous
biosynthetic pathways in microorganism cells. It has been shown that the down-
regulation or upregulation of crucial genes can lead to malfunction of metabolism
and consequently, the cell growth could be prevented by the accumulation of toxic
intermediates (Sun et al. 2014).
The first heterologous artemisinic acid production in S. cerevisiae was reported
by Ro et al., in 2006. In the engineered S. cerevisiae, production of artemisinic acid
increased to 100 mg L−1 when all A. annua-derived genes (ADS, CYP71AV1, and
CPR) were expressed on a single platform. The transgenic S. cerevisiae synthesized
high artemisinic acid compared to relative native host biomass and in a shorter
time. The effective conversion of amorphadiene to artemisinic acid by S. cerevisiae
expressing the ER-bound CYP71AV1 convinced enough scientists to suggest it as
an optimal model for expression of genes encoding membrane-bound enzymes.
In the last part of the pathway, although the complete biosynthetic pathway
of artemisinin has been discovered, the final step, the conversion of dihy-
droartemisinic acid to artemisinin, which occurs non-enzymatically must be
accomplished via chemical processes (Li et al. 2016) (Fig. 3).
6 Production of Artemisinin in planta Versus Application

of Yeast SynBio
6.1 Metabolic Engineering of Artemisinin in Artemisia annua
The unstable yield and production of artemisinin is a major problem in the

global need for this compound (Judd et al. 2019). So that, the highest amount
of artemisinin was reported 2 % based on plant dry weight (Zhang et al. 2008).
Metabolic engineering is considered a promising biotechnological technique and
as a new method to improve the production of ARTs in native and heterologous
Fig. 3 De novo reconstitution of artemisinin biosynthetic pathway in Saccharomyces

cerevisiae. ERG10, acetyl-CoA C-acetyltransferase; ERG13, hydroxymethylglutaryl-CoA
synthase; ERG12, mevalonate kinase; tHMGR, truncated 3-hydroxy-3-methylglutaryl-CoA
reductase; ERG8, phosphomevalonate kinase; ERG19, diphosphomevalonate decar-
boxylase; IDI1, isopentenyl-diphosphate delta-isomerase; ERG20, farnesyl diphosphate
synthase/dimethylallyltranstransferase; ERG9, squalene synthase; BTS1, geranylgeranyl
diphosphate synthase
hosts (Covello 2008; Ikram et al. 2019; Ma et al. 2015; Xie et al. 2016). Many
efforts have been made to increase the production of artemisinin during various
experiments. However, there are still lots of potential routes to increase the pro-
duction of this valuable compound (Lv et al. 2017). To overcome the unsustainable
production and low-yield of artemisinin in A. annua, attempts led to positive results
in biological systems as native and heterologous expression systems (Paddon et al.
2013). One of the main approaches pursued in metabolic engineering is the sus-
tainable production of artemisinin to benefit underdeveloped and more vulnerable
communities (Ikram et al. 2017).
Insertion of Agrobacterium’s rol genes is considered an effective and efficient
method for increasing the secondary metabolites in plants (Bulgakov 2008). Dil-
shad et al. (2015) indicated that transformation via rol B and rol C genes show high
levels of gene expression and artemisinin production (9 and 4 times more than a
non-transgenic plant, respectively). Also, the expression of the ipt gene transmitted
by A. tumefaciens increases the content of artemisinin (30–70%) (Sa et al. 2001).
As mentioned earlier, ADS, Cytochrome P450 (CYP71AV1), double-bond
reductase 2 (DBR2), and aldehyde dehydrogenase 1 (ALDH1) catalyze the dif-
ferent stages of artemisinin biosynthesis pathway. One of the strategies used in
artemisinin metabolic engineering is to increase the expression of genes involved
in the biosynthetic pathway. 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR)
gene is known to be one of the most important factors in MVA pathway (Farhi
et al. 2013). Different studies have shown that high expression of the HMGR
increases artemisinin production (Aquil et al. 2009; Ma et al. 2017; Nafis et al.
2011). Aquil et al. (2009) by transferring the HMGR gene using the Agrobac-
terium-mediated gene transformation, succeeded in producing transgenic lines
with a 22.5% increase in artemisinin content compared to the non-transgenic A.
annua. Squalene synthase (SQS), as another potent enzyme in artemisinin biosyn-
thesis, converts the FPP to squalene production, thereby reduces the strength of
the biosynthetic pathway toward the artemisinin production. Studies have shown
that suppressing this enzyme can increase artemisinin biosynthesis (Paradise et al.
2008; Yang et al. 2008; Zhang et al. 2009). Zhang et al. (2009), by suppressing
the expression of SQS gene using RNA interference (RNAi) technique, indicated
that the amount of artemisinin in transgenic A. annua increased significantly to
31.4 mg on a dry weight scale. Also, with overexpression of genes involved in
the biosynthetic pathway, the amount of artemisinin increases significantly to 3.6-
fold higher than control in transgenic plants, which includes FPS, CYP71AV1, and
CPR, among the most effective genes in biosynthesis of artemisinin (Chen et al.
2013). Furthermore, in separate studies, the amount of artemisinin in transgenic
plants was 38% higher than normal plants, and it was observed that the overex-
pression of the farnesyl pyrophosphate synthase (FPS) in A. annua significantly
increased compared to the wild-type (Banyai et al. 2010; Han et al. 2006). It
has also been reported that overexpression of the chimeric farnesyl diphosphate
synthase in native plant strengthens the biosynthetic pathway of sesquiterpenes,
leading to increased artemisinin biosynthesis in some lines (up to threefold) (Chen
et al. 2000). In another study Kiani et al. (2016), with overexpression of rol ABC
genes, also in diploid plants and HMGR, FPS, ADS, and Aldh1 and upregulation of
ADS in tetraploid plants of A. annua increased artemisinin level (Lin et al. 2011).
Moreover, transcription factors (TFs) known as regulators of the location and time
of secondary metabolites production (Yang et al. 2012), are one of the modules
that can be used in the regulation of special metabolites production (Verpoorte and
Memelink 2002). Identification of TFs involved in the regulation of artemisinin
metabolism is important (Jiang et al. 2016; Shen et al. 2016). AP2/ERF, bHLH,
MYB, and WRKY are members of various TFs in artemisinin (Lv et al. 2017) that
influence the artemisinin biosynthesis in A. annua.
6.2 In Planta Artemisinin Production
To date, in planta artemisinin production in two model plants, tobacco (Nicotiana

benthamiana) and moss (Physcomitrella patens), have been reported successfully.
N. benthamiana is considered as one of the most widely used platforms for the pro-
duction of various secondary metabolites. In addition to the possible disadvantages,
the ability to change the pathway at tissue and intracellular levels, the possibility
of rapid and transient expression, and no need for external primary sources are
the main dominant features of N. benthamiana for the production of secondary
metabolites (Carqueijeiro et al. 2020). Wang et al (2016), in the study of the role
of AaLTPs (Lipid Transfer Protein 3) and AaPDRs (Pleiotropic Drug Resistance

2) from A. annua using transient expression in N. benthamiana leaves to increase
the accumulation of dihydro artemisinic acid in the apoplastic space, indicated
that the AaLTP3 and AaPDR2 prevent dihydroartemisinic acid reflux from the
apoplast to the cell and strengthen the pathway. Recent reports demonstrated that
transient expression is the most practical and successful method for the production
of medicinal compounds, especially in this model plant (Reed et al. 2017).
Interestingly, in addition to N. benthamiana, P. patens, which is known as a
vascular plant with an appropriate growth rate, is used as a model plant in applied
biotechnology research (Büttner-Mainik et al. 2011; Ikram et al. 2015; Reski et al.
2015; Simonsen et al. 2009). Due to the complete sequencing of the moss genome
and its haploid life cycle compared to other green hosts, it has become an attractive
and efficient industrial production system for the production of secondary metabo-
lites. On the other hand, studies have shown that P. patens have the potential to
produce large-scale artemisinin (Ikram et al. 2017) (Fig. 4). So far, it has been pos-
sible to produce several recombinant drug proteins and other valuable molecules
Fig. 4 Highest reported artemisinin/artemisinin precursor production via bioengineering of

artemisinin biosynthetic pathway in native and heterologous hosts
in the moss system (Anterola et al. 2009; Pan et al. 2015; Reski et al. 2015; Zhan
et al. 2014). Ikram et al. (2017) by engineering and inserting 5 genes involved
in the biosynthetic pathway of artemisinin into P. patens, produced 0.21 mg/g of
dry weight artemisinin after three days. Various reports indicate that the insertion
of genes involved in the production of artemisinin has been successful using a
constitutive expression in P. patens.
7 Conclusions
Given the fact that artemisinins, in addition to treating malaria, also play a key
role in fighting other diseases, meeting global demands which has been a chal-
lenge for researchers and industry. Since the artemisinin levels in Artemisia annua
plant is very low, its large-scale planting does not meet global needs. To this end,
biotechnological tools have been able to eliminate these concerns to a large extent.
In this regard, biosynthetic pathway engineering in the main host has been suc-
cessfully performed. For further production, the engineering of the artemisinin
biosynthetic pathway in two model plants (tobacco and moss) has led to signifi-
cant successes. But for much more production, the reconstitution of the artemisinin
biosynthetic pathway in microorganisms has been able to meet global demand so
that the baker’s yeast with a series of special mechanisms and potentials is more
recommended for this purpose.
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2014.3323
Yeast Synthetic Biology
for the Production of Terpenoids
Derived from Traditional Chinese
Medicinal Plants
Yongjun Wei
Abstract
Traditional Chinese medicine has been widely used to cure diverse diseases in
China. Modern medicinal chemistry and pharmaceutical research help to iden-
tify active natural products that functioned during disease treatment. Among
them, terpenoids are one of the largest natural bioactive products in traditional
Chinese medicine. Traditionally, these active terpenoids are extracted from tra-
ditional Chinese medicinal plants whose yield of the active terpenoids derived
from the plants is low and the quality is not stable. The yield cannot sat-
isfy the increasing terpenoid demand. Therefore, another sustainable supply
of active terpenoids is of great interest. The development of synthetic biol-
ogy enables yeasts to be ideal microbial cell factories for plant-derived natural
product production. In the past few years, several typical active terpenoids
derived from traditional Chinese medicinal plants have been produced in yeasts,
including artemisinic acid, rare ginsenosides, rare licorice triterpenoids, and
other value-added terpenoids. In this chapter, the terpenoid biosynthetic path-
way and current synthetic biology strategies for the production of some typical
terpenoids using yeasts are summarized. Moreover, future synthetic biology
strategies for efficient terpenoid production are discussed.
Y. Wei (B)
Key Laboratory of Advanced Drug Preparation Technologies, Ministry of Education, School of
Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, Henan Province, China
Laboratory of Synthetic Biology, Zhengzhou University, Zhengzhou 450001, Henan Province,
China
https://doi.org/10.1007/978-3-030-89680-5_7
182 Y. Wei
1 Introduction of Terpenoids and Their Production

from Traditional Chinese Medicinal Plants
Terpenoids, also known as isoprenoids, represent a highly diverse group of natural

products with wide use in cosmetics, pharmaceuticals, food, and other industries
(Pichersky and Raguso 2018). The terpenoids are the most abundant plant natural
products and have diverse structures (Zhou and Pichersky 2020). There are more
than 55,000 different kinds of terpenoids derived from plants. The terpenoids are
composed of the basic unit of isoprene and can be classified based on the iso-
prene numbers (Paramasivan and Mutturi 2017). Usually, the terpenoids can be
divided into hemiterpenoids, such as isoprene; monoterpenoids, such as menthol,
menthone, camphor, and eucalyptol; sesquiterpenoids, such as artemisinin (Qing-
haosu青蒿素), humulene, caryophyllene, and zingiberene; diterpenoids, such as
triptolide, oridonin, and taxol; triterpenoids, such as ginsenosides, glycyrrhetinic
acid; and tetraterpenoids, such as lycopene, zeaxanthin, and astaxanthin (Jiang
et al. 2016).
Traditional Chinese herbs harbor diverse terpenoids, and many of them
show medicinal characteristics (Guan et al. 2020). The terpenoids have a wide
array of clinical or other pharmacological properties, including antitumor, anti-
inflammatory, antiviral, antimalarial effects, preventing and treating cardiovascular
diseases, and other activities towards diverse metabolic diseases (Jaeger and Cuny
2016). The most famous terpenoids in the world might be artemisinin, which helps
to cure malarial disease and saves many lives (Tu 2016). The medicinal plants har-
bor abundant terpenoids (Jaeger and Cuny 2016). With the development of modern
pharmaceutical sciences, the demand for terpenoids increased (Paramasivan and
Mutturi 2017). Traditionally, the terpenoids are extracted from plants, especially
from herbs (Bergman et al. 2019). The herb plants need to grow for months or even
years before they can be used for active terpenoid extraction, such as Panax Gin-
seng for five to seven years, Glycyrrhiza uralensis for several years, and Artemisia
annua for several months (Fig. 1). Moreover, the terpenoids in plants are low;
for example, the artemisinin composition in the biomass of Artemisia annua is
0.01–2% (Namuli et al. 2018); the rare ginsenoside compositions in Panax plants
are less than 0.1% (Hwang et al. 2014). Besides, the plant growth and terpenoid
content in the plant are affected by climate, soil types, and other environmental
factors (Fig. 1) (Wei et al. 2017a). Chemical synthesis of these structurally com-
plex terpenoids suffers from the high cost and low yield (Jansen and Shenvi 2014).
Therefore, another sustainable supply of terpenoids is of great interest (Moser and
Pichler 2019).
Synthetic biology approaches had been applied to produce value-added ter-
penoids, which provide a bright way to supply enough terpenoids for future
commercial use (Bian et al. 2017; Liu et al. 2019b). The most commonly used
model microorganism, Saccharomyces cerevisiae, grows rapidly on low-cost sub-
strates and is robust to different adverse fermentation conditions (Wei et al. 2017b).
Additionally, S. cerevisiae is easy for genetic manipulation, which has been widely
used for the biosynthesis of novel non-native natural products (Liu et al. 2019b).
Yeast Synthetic Biology for the Production of Terpenoids … 183
Fig. 1 Comparison of plant production of terpenoids and yeast synthetic biology for the pro-
duction of terpenoids. The traditional Chinese herbal plants often grow months to years, and
production of terpenoids requires plant biomass collection, extraction, and purity. The omics tech-
nologies and available database can help to identify key terpenoid biosynthetic genes, and these
genes can be used for building yeast cell factories. It takes several days to ferment. Compared with
plants, the native natural products in yeasts are few and simple, and it is easy to extract targeted
bioactive terpenoids from yeast biomass
Other non-model yeasts, such as Yarrowia lipolytica and Rhodosporidium toru-

loides, had been applied in the biosynthesis of terpene and other natural products
(Ma et al. 2020; Wang et al. 2020a; Yaegashi et al. 2017). The yeasts are consid-
ered to be ideal hosts for terpenoid biosynthesis, and they were easier to express
some terpenoid biosynthetic genes than Escherichia coli, such as cytochrome
P450s in the terpenoid biosynthetic pathway (Liu et al. 2020). Engineering yeasts
for terpenoid production needs to build terpenoid precursor chassis cells, iden-
tify terpenoid biosynthetic pathways, and understand their metabolic networks in
plants and yeasts (Zu et al. 2020). After the introduction of heterologous terpenoid
biosynthetic genes in yeast terpenoid biosynthetic genes precursor chassis cell,
strengthening the terpenoid biosynthetic pathway, downregulating competing path-
ways, alleviating the toxic of the targeted terpenoids, enhancing cofactor supply,
and other strategies used to rewire the whole metabolism network for terpenoid
production should be implemented to enable high titer, rate, and yield of targeted
terpenoid production in yeasts (Nielsen and Keasling 2016; Liu et al. 2019b).
In this chapter, current knowledge of the terpenoid biosynthetic pathways in
plants and yeasts, the omics technologies used for the discovering of key ter-
penoid biosynthetic enzymes, and engineering strategies for high-level terpenoid
production in yeasts are summarized and discussed.
184 Y. Wei
2 Terpenoid Biosynthesis Pathways in Nature
The carbon skeleton and building block for terpenoids is isopentenyl pyrophos-
phate (IPP), and the precursor substrates for IPP formation are acetyl-CoA,
pyruvate, and glycerol-3-phosphate (Wang et al. 2019d). Two distinct pathways,
mevalonate (MVA) and 2-C-methyl-D-Erythritol-4-phosphate (MEP) pathways,
can be used for the production of IPP in nature. Normally, the MVA pathway for
IPP production is mainly reacted in the cytoplasm of the higher eukaryotes; while
in plastids of plant cells, protists, and most microorganisms, the MEP pathway is
used to synthesize IPP (Fig. 2). The intermediates of MVA and MEP pathways
have exchanges and interactions in plants (Liao et al. 2016).
For the MVA pathway, the acetyl-CoA is catalyzed by acetyl-CoA C-
acetyltransferase (AACT) to form acetoacetyl-CoA. The acetoacetyl-CoA is
further catalyzed by 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) to form
3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). The HMG-CoA is used to gen-
erate MVA catalyzed by the HMG reductase (HMGR). Under multi-step cat-
alytic reactions of MVA kinase and phosphomevalonate kinase, mevalonate
diphosphate (MVAPP) is synthesized. The MVAPP is further decarboxylated
to form IPP by mevalonate diphosphate decarboxylase. The IPP is used for
the synthesis of dimethylallyl pyrophosphate (DMAPP) under the catalysis
of isopentenyl-pyrophosphate delta isomerase (IDI) (Fig. 2) (Vranová et al.
2013). For the MEP pathway, the first step is the formation of D-xylulose-5-
phosphate (DXP) catalyzed by DXP synthase, with pyruvate and glyceraldehyde-
3-phosphate (G3P) as substrates. The second step is the reduction of DXP
to MEP catalyzed by DXP reductoisomerase (Kim and Keasling 2001). With
the help of multi-step enzymatic reactions, MEP is further catalyzed to a
series of products of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME), 4-
diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-MEP), 2-C-methyl-
D-erythritol 2,4-cyclopyrophosphate (MECPP), and 1-hydroxy-2-methyl-2-(E)-
butenyl 4-pyrophosphate (HMBPP) (Liu et al. 2014). The HMBPP is transformed
to IPP and DMAPP by 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase
(ispH) (Fig. 2).
The terpenoids are synthesized with the common five-carbon substrate of iso-
prenoid (Fig. 2) (Vranová et al. 2012). After the production of IPP and DMAPP,
the geranyl pyrophosphate synthase (GPPS) uses IPP and DMAPP to synthesize
geranyl diphosphate (GPP), and GPP is the precursor for monoterpenoid biosyn-
thesis (Nagegowda and Gupta 2020). Farnesyl pyrophosphate synthase uses IPP
and GPP to synthesize farnesyl diphosphate (FPP), and FPP is the precursor for
sesquiterpenoid and triterpenoids biosynthesis. GPPS uses IPP and FPP to syn-
thesize geranylgeranyl pyrophosphate (GGPP), and GGPP is the precursor for
diterpenoid and tetraterpenoids biosynthesis (Nagegowda and Gupta 2020). In
order to synthesize the final terpenoids, identification of the core structure mod-
ification enzymes, such as the terpene synthases, P450 and their redox partners
of NADPH-cytochrome P450 reductases (CPRs), and glycosyltransferases, are
essential (Wang et al. 2018).
186 Y. Wei
Fig. 2 Terpenoid biosynthetic pathways, MVA pathway and MEP pathway, are described.
G3P, glyceraldehyde-3-phosphate; DXP, 1-deoxy-D-xylulose 5-phosphate; MEP, 2-C-methyl-
D-erythritol 4-phosphate; CDP-ME, 4-diphosphocytidyl-2-C-methyl-D-erythritol; CDP-MEP,
4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate; MECPP, 2-C-methyl-D-erythritol 2,4-
cyclopyrophosphate; HMBPP, 1-hydroxy-2-methyl-2-(E)-butenyl 4-pyrophosphate; HMG-CoA,
3-hydroxy-3-methylglutaryl-CoA; MVA, mevalonic acid; MVAP, mevalonate phosphate; MVAPP,
mevalonate diphosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate;
GPP, geranyl diphosphate; FPP, farnesyl diphosphate; GGPP, Geranylgeranyl pyrophosphate.
DXS, D-xylulose-5-phosphate synthase; DXR, D-xylulose-5-phosphate reductoisomerase;
ispD, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; ispE, 4-diphosphocytidyl-2-
C-methyl-D-erythritol kinase; ispF, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; ispG,
(E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase; ispH, 4-hydroxy-3-methylbut-2-en-1-yl
diphosphate reductase; ipk, isopentenyl phosphate kinase; AACT, acetyl-CoA C-acetyltransferase;
HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA
reductase; MK, mevalonate kinase; PMK, phosphomevalonate kinase; MDC, mevalonate
diphosphate decarboxylase; IDI, isopentenyl-pyrophosphate delta isomerase; IDI, isopentenyl-
pyrophosphate delta isomerase (IDI); GPPS, Geranyl pyrophosphate synthase; FPPS, Farnesyl
pyrophosphate synthase; GGPPS, Geranylgeranyl-PP synthetase
3 Identification of Key Genes Involved in Terpenoid

Production from Traditional Chinese Medicinal Plants
The next-generation sequencing technologies have generated huge amounts of

genomic and transcriptomic data in public databases (Leebens-Mack et al. 2019).
The sequencing price decreases, and many labs can sequence multiple tissues of
different plants and microorganisms. Assembly of the sequences data and anno-
tation of them can identify the primary metabolic pathways and most of the
known secondary metabolic pathways in the sequencing species (Kumar et al.
2016). However, plants normally have several similar enzymes for one biochem-
ical reaction. Especially, for secondary metabolism, many similar/high-identity
enzymes contribute to the diverse metabolites in plants (Yang et al. 2020a). Multi-
ple omics data can help to identify key genes in terpenoids biosynthetic pathway.
The genome and tissue-specific expression data of Canabis sativa help to charac-
terize its complete terpene synthase gene family (Allen et al. 2019). The terpene
synthases genes of Lathyrus odoratus flowers were identified with the help of L.
odoratus transcriptome data (Bao et al. 2020). Even so, identifying the right mod-
ified enzymes for the production of diverse terpenoids is challenging. Therefore,
developing some feasible strategies for key gene identification is necessary (Nett
et al. 2020).
Many different kinds of annotation and screening strategies have been devel-
oped for genomic and transcriptomic data analyses (Nett et al. 2020; Srinivasan
and Smolke 2020), and some of them have been used for key gene discovery
(Scossa et al. 2018; Han et al. 2016). Triterpenoid saponins are the main bioac-
tive components of licorice. The final step for the production of triterpenoid
glycyrrhizin is to attach two glucuronic acids to the glycyrrhetinic acids. The gly-
cosylation reactions normally occur in the cytosol, and the 3-o-glucuronosylation
is believed to be a cytoplasmic glycosyltransferase. Recently, a cellulose synthase-

like enzyme, an endoplasmic reticulum-membrane localization enzyme, can glu-
curonidate of specialized plant metabolites of triterpenoid saponins (Jozwiak et al.
2020). Nearly at the same time, a cellulose synthase-derived enzyme was identi-
fied to catalyze the 3-o-glucuronosylation in glycyrrhizin biosynthesis using gene
co-expression analyses (Chung et al. 2020). All these two studies used the tran-
scriptomic data of different plant tissues to identify the key genes for terpene
biosynthesis, suggesting multi-omics data provide one efficient way for key gene
identification.
Phylogenetic analyses, sequence similarity networks, homology-based model
prediction, and other bioinformatics-aided strategies have been applied for key
gene identification (Wang et al. 2020b). Key genes from the same plant genera
normally have high identities with each other, and it provides the opportunities to
use sequence similarity network and phylogenetic analyses to identify key genes in
terpene biosynthesis. In order to identify the key glycosyltransferase genes for the
production of triterpenoid saponin ginsenosides, all the potential glycosyltrans-
ferase genes were assembled from a Panax cDNA and EST database using the
microbial OTU classification strategy (Wei et al. 2015b; Liang et al. 2019; Mai
et al. 2020). The classified glycosyltransferase genes were further aligned, and the
phylogenetic tree was built (Yan et al. 2014; Wang et al. 2015). Based on the
phylogenetic analysis results, some glycosyltransferase genes were selected for
microbial characterization, and the glycosyltransferases used for the ginsenosides
of CK, Rh2, and Rg3 were identified (Yan et al. 2014; Wang et al. 2015). The
sequence similarity network has been used to classify terpene synthase-like pro-
teins. With the help of hidden Markov models, some putative terpene synthase
genes from Marchanita polymorpha were identified (Kumar et al. 2016). Recently,
a streamlined computational workflow was provided to explore natural product
biosynthetic gene clusters, and it had potential application in terpene biosynthesis
gene identification in the future (Navarro-Muñoz et al. 2020).
4 Engineering Yeasts for the Production of Terpenoid

Precursors
Yeasts, including the model organisms of S. cerevisiae and Y. lipolytica, are attrac-
tive choices for microbial terpene production (Nielsen and Keasling 2016; Arnesen
et al. 2020). In order to produce terpene at a high titer, rate, and yield (TRY),
the terpenoid precursor biosynthesis in the yeasts were enhanced (Nielsen and
Keasling 2016). The common metabolic engineering and newly developed syn-
thetic biology strategies had been applied to overcome precursor limitation in yeast
terpenoid precursor biosynthesis (Farhi et al. 2011; Zu et al. 2020). The yeasts are
not the natural hosts for terpenoid production, and rewiring cellular metabolism
and directing of metabolic flux for terpenoid precursor biosynthesis is necessary
(Worland et al. 2020; Bian et al. 2017). The yeasts only have the MVA pathway
188 Y. Wei
for terpenoid biosynthesis. Based on the MVA pathway in yeast (Fig. 3), overex-
pression of some genes in the MVA pathway and optimization of the terpenoid
precursor biosynthetic pathway by introducing exogenous efficient genes are the
normal strategies (Paramasivan and Mutturi 2017). In fact, diverse genes have been
selected for terpenoid precursor production (Fig. 3).
The rate-limiting enzymes in the MVA pathway are often selected for improving
yeast terpenoid precursor synthesis. The HMGR is the first rate-limiting enzyme
Fig. 3 Engineering targets for the production of terpenoids in S. cerevisiae. HMG-CoA, 3-

hydroxy-3-methylglutaryl-CoA; MVA, mevalonic acid; MVAP, mevalonate phosphate; MVAPP,
mevalonate diphosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate;
GPP, geranyl diphosphate; FPP, farnesyl diphosphate; GGPP, Geranylgeranyl pyrophosphate
in the MVA pathway. In order to enhance HMGR activity, the tHMG1 (encod-
ing the catalytic domains of yeast HMGR) is overexpressed in yeast, and the
metabolic fluxes in the MVA pathway are increased and the final production of
terpenoids is improved (Fig. 3) (Wu et al. 2018). The overexpression of tHMG1
often leads to the improvement of terpenoid production, such as the limonene pro-
duction using engineered S. cerevisiae (Amiri et al. 2016; Wu et al. 2018). HMG2
is the isoenzyme of HMG1 and is the dominant HMGR isoform under a low oxy-
gen environment (Mantzouridou and Tsimidou 2010). Overexpression of HMG2
variant (K6R) and IDI1 can improve monoterpene production levels (Ignea et al.
2011, 2012). In fact, overexpression of tHMG1, IDI1, and MAF1 increased geranyl
acetate production in S. cerevisiae (Wu et al. 2018).
The ERG20 and ERG9 are two of the most essential enzymes for terpenoid
precursor production (Fig. 3). Engineering ERG20 significantly improved the pro-
duction of sclareol and other terpenoids (Ignea et al. 2015), and overexpression of
fusion genes of ERG20 and BTS1 resulted in the increment of terpenoid production
(Dai et al. 2012). Recently, the overexpression of ERG20 and its mutant mERG20
(F96C mutation in ERG20, which might function as a geranylgeranyl diphosphate
synthase) significantly improved geranylgeraniol content of S. cerevisiae (Dong
et al. 2020). Dynamic control of ERG20 expression level might improve terpenoid
precursor production in yeast. The dynamic control of ERG20 combined with min-
imized endogenous downstream metabolism improved geraniol production (Zhao
et al. 2017); moreover, dynamic control of the expression of ERG20 and ERG9
improved diterpenoid casbene production in S. cerevisiae (Callari et al. 2018).
Downregulation of ERG9 expression level leads to the high-level production of
FPP (Asadollahi et al. 2008), and the common strategy is replacing native ERG9
promoter with HXT1 or other promoters (Scalcinati et al. 2012). Besides, the intro-
duction of exogenous rate-limiting genes of the MVA pathway in yeast could also
improve terpenoid production, such as PaGGPPS and SaGGPPS (Cao et al. 2020).
The lipid production would affect the terpenoid precursor of squalene by storage
of squalene in the lipid body, and co-overexpression of tHMG1 and DGA1 coding
for diacylglycerol acyltransferase led to over 250-fold higher squalene accumula-
tion than a control strain (Wei et al. 2018). The transcriptional activators regulated
terpenoid precursor synthesis in yeasts (Zhang et al. 2017; Davies et al. 2005). The
overexpression of the transcription factor of UPC2 led to the increase of terpenoid
production (Shianna et al. 2001), and other transcription factors, such as Rox1 and
Mot3, repressed ergosterol biosynthesis in yeasts (Montañés et al. 2011).
5 Production of Artemisinin Precursor of Artemisinic Acid

Derived from Artemisia Annua in Yeasts
The extraction of Artemisia annua had been used as an antimalarial drug in tradi-
tional Chinese medicine. In the 1970s, Youyou Tu discovered the active component
artemisinin in the plant extraction of Artemisia annua. Since then, artemisinin was
used as a potent antimalarial drug (Jung et al. 2004). Recent studies suggested
190 Y. Wei
that artemisinin and its derivatives had immune-modulation effects and antitumor
activities (Yao et al. 2016; Kiani et al. 2020). Artemisinin is a sesquiterpene lac-
tone endoperoxide, and it is mainly obtained by extracting from the leaves and
other biomass of A. annua (Efferth 2017). The A. annua needs to grow several
months before harvest, and the artemisinin component in wild A. annua is low (Cai
et al. 2017). Moreover, planting A. annua occupies large amounts of fields, and the
artemisinin component is affected by the climate. Therefore, artemisinin supply is
uncertain and unstable, and the price fluctuates. The production of artemisinin
using microorganisms is of great interest, for it might produce large amounts of
artemisinin with limited space and short time.
Keasling’s group developed bacterial chassis for terpenoid production, and the
obtained E. coli strains are capable of synthesizing diverse terpenoid precursors
(Martin et al. 2003, 2001). The cytochrome P450 is not easy to express in E.
coli, and S. cerevisiae was selected for the production of high-level artemisinin.
The FPP biosynthetic pathway was engineered to increase FPP production and
decrease sterol production by overexpression of tHMGR, UPC2-1, and ERGs, and
downregulation of ERG9. The amorphadiene synthase genes from A. annua was
introduced to S. cerevisiae, in order to construct one strain with amorphadiene
producing ability. Moreover, a novel cytochrome P450 (CYP71AV1) was identified
to have the ability to perform three-step oxidation which can convert amorphadiene
to artemisinic acid. The expression of the CYP71AV1 and its redox partner from
A. annua led to the production of artemisinic acid (Ro et al. 2006). As artemisinic
acid can be easily oxidized to artemisinin using a chemical process, the production
of artemisinic acid is a good proof of concept that using yeasts as microbial cell
factories can synthesize plant natural products. The engineered yeast strains can
produce artemisinic acid in a short time (4–5 days). Compared with the fact that
planting A. annua needs months or years, these synthetic biology strategies save
time and labor for valuable natural product synthesis (Fig. 1).
Further, overexpressing every gene in the MVA pathway doubled artemisinic
acid production; however, the amporph-4,11-diene production is tenfold higher
than artemisinic acid (Westfall et al. 2012). Therefore, they developed one opti-
mized process to convert amporph-4,11-diene to dihydroartemisinic acid. The
dihydroartemisinic acid can be further transformed to artemisinin (Westfall et al.
2012). In order to produce affordable artemisinic acid using yeasts, the artemisinic
acid biosynthetic pathway was optimized by using proper promoters for genes and
introducing efficient artemisinic acid biosynthetic genes. The fermentation titers
of artemisinic acid reached as high as 25 g/L and a low-cost chemical process for
the conversion of artemisinic acid to artemisinin was established, which provided
a second semi-synthetic artemisinin source independent of botanical production
(Paddon et al. 2013). The success of artemisinic acid production using yeasts paves
the way for industrial-scale production of artemisinin derived from the traditional
Chinese medicinal plant.
6 Production of Rare Ginsenosides Derived from Panax

Plants in Yeasts
Ginsenosides are the main pharmacological components in Panax ginseng C. A.

meyer which is one of the most famous traditional Chinese medicinal plants in East
Asia. The ginsenosides are triterpenoids, and they are mainly distributed in Panax
genus (Pace et al. 2015). The ginsenosides can be classified into two main types,
dammarane-type and oleanate-type, and most ginsenosides are dammarane-type
terpenoids (Shin et al. 2015). The ginsenosides have diverse biological activities,
such as antitumor, anti-inflammatory, antioxidation, and antiaging. However, the
ginsenoside compositions in Panax plant are lower, normally less than 4% (Schlag
and McIntosh 2006). Moreover, the main functional ginsenosides are rare in natu-
ral Panax plants (Piao et al. 2020). Compound K (CK), a triterpenoid compound
that has potential anti-inflammation, anti-diabetes and other biological activities, is
only detected in animal or human blood (Yang et al. 2020b). Though CK is clas-
sified as ginsenoside and is proved to be transformed from natural ginsenosides,
none CK was detected in Panax plants (Han et al. 2020). It takes five to seven years
to obtain ginsenosides from Panax plants (Schlag and McIntosh 2006). Besides,
the structure of ginsenosides is complex, and large-scale de novo chemical synthe-
sis of ginsenosides is impossible until now. Microbial production of ginsenosides
using synthetic biology strategies was applied (Chu et al. 2020).
The core skeletons of ginsenosides are protopanaxadiol (PPD) and protopanax-
atriol (PPT). The precursors of PPD and PPT were synthesized via the MVA
pathway in yeasts. The wild-type yeasts can synthesize 2,3-oxidosqualene. In
order to produce ginsenosides in yeast, other heterologous genes need to be intro-
duced to the yeasts. The compound of 2,3-oxidosqualene can be further converted
to dammarenediol-II with Dammarenediol-II synthase (Fig. 4) (Han et al. 2006;
Tansakul et al. 2006). The Cyt P450 enzyme of CYP716A47 was identified to be
responsible for the conversion of dammarenediol-II to PPD (Han et al. 2011), and
one P450 enzyme from Arabidopsis thaliana had the same function (Wang et al.
2015). Subsequently, the Cyt P450 enzyme of CYP716A53v2 can synthesize PPT
with PPD as a substrate (Fig. 4) (Zhao et al. 2019).
Diverse UDP-glycosyltransferases (UGTs) are responsible for the glycosyla-
tion of PPD, PPT, and their glycosylation products (Fig. 4). The UGTPg1 was
identified to glycosylate PPD and produced CK in yeasts. There are two possi-
ble pathways for CK production. One pathway is from the dammarenediol-II to
PPD, and then to CK; the other pathway is from dammarenediol-II to 20S-O-β-
(D-glucosyl)-dammarenediol II, and then to CK (Yan et al. 2014). Besides these
catalysis abilities, UGTPg1 can also convert Rg3 to Rd, and Rh2 to F2, showing
UGTPg1 might be responsible for the production of several different ginseno-
sides (Yan et al. 2014; Wei et al. 2015a). Moreover, several other Panax UGTs
with high amino acid identities (>84%) indicated divergent functions, and they
can convert PPT and PPT-type compounds to diverse ginsenosides (Wei et al.
2015a). The investigation of UGT94 family genes in Panax plants indicated that
an unprecedented group of UGT94s with high amino acid identity had diverse
192 Y. Wei
Fig. 4 Engineering for the production of ginsenosides in S. cerevisiae. GPP, geranyl diphosphate;
FPP, farnesyl diphosphate
catalyzing activities in Panax plants (Yang et al. 2020a). These UGT94 enzymes
were responsible for the synthesis of diverse ginsenosides (Yang et al. 2020a).
Moreover, the UGT diversity in Panax plant was investigated, and two UGTs of
UGTPg45 and UGTPg29 for ginsenosides Rh2 and Rg3 biosynthesis were identi-
fied (Wang et al. 2015). The production of Rh2 and Rg3 using synthetic biology
strategies was applied in engineered yeasts (Wang et al. 2015). A semi-rationally
designed UGT51 derived from yeast was also demonstrated to have the ability to
synthesize Rh2, showing the synthetic biology strategies had great potential for
terpenoid production in yeast (Zhuang et al. 2017). The Rh2 titer increased to
2.25 g/L via expressing multiple copies of UGTPg45 in a PPD-producing chas-
sis, which provides the possible large-scale production of ginsenosides in yeasts
(Wang et al. 2019c). Moreover, the non-conventional yeast, Y. lipolytica, had been
engineered for CK production, and the final CK titer reached to 161.8 mg/L via
fed-batch fermentation in a 5 L fermenter (Li et al. 2019a).
7 Production of Licorice Triterpenoid Derived

from Glycyrrhiza Plants
Glycyrrhiza plants, including Glycyrrhiza uralensis Fisch., Glycyrrhiza flata Bat.

and Glycyrrhiza glabra L., are widely used herbal plants in traditional Chinese
medicine (Asl and Hosseinzadeh 2008). One of the main bioactive components
of Glycyrrhiza plants is glycyrrhetinic acid (GA) (Li et al. 2014; Zhang and Ye
2009). GA has anti-inflammatory, antitumor, immunomodulatory, and other bio-
logical activities (Kowalska and Kalinowska-Lis 2019; Roohbakhsh et al. 2016).
GA can be generated by the hydrolysis of glycyrrhizic acid. Glycyrrhizic acid is
extracted from the roots of the licorice plants, and is one kind of triterpenoids.
Glycyrrhizic acid is mainly used as a sweetening and flavoring agent for bever-
ages, candies, chewing gum, and bitter drugs (Pastorino et al. 2018). The wild
and cultivated Glycyrrhiza plants cannot satisfy the growing market demand for
GA and glycyrrhizic acid. Moreover, the extraction of Glycyrrhizic acid from Gly-
cyrrhiza plants pollutes the environment. For GA structure is complex, chemical
synthesis yield is low and the cost is high (Wang et al. 2019a). Therefore, another
sustainable supply of GA is necessary (Guan et al. 2020).
Compared with the ginsenosides biosynthetic pathway, the GA biosynthetic
pathway is different. The precursors were synthesized with the MVA pathway,
but the 2,3-oxidosqualene is converted to β-amyrin by β-amyrin synthase. The β-
amyrin is oxidized to 11-oxo-β-amyrin with a licorice β-amyrin 11-oxidase which
is a cytochrome P450 enzyme (Seki et al. 2008). Another licorice cytochrome
P450 enzyme of CYP72A154 catalyzed three sequential oxidation steps at C-30
of 11-oxo-β-amyrin, and GA is obtained (Seki et al. 2011). In the catalysis reac-
tion, CPRs provide electrons to the cytochrome P450s (Zhu et al. 2018). The UGTs
(GuGT14 and UGT73P12) are responsible for the biosynthesis of glycyrrhizic acid
(Nomura et al. 2019; Chen et al. 2019). In order to produce high-level GA in
yeasts, the MVA pathway was strengthened and an acetyl-CoA competing path-
way was disrupted. The final β-amyrin production reached 279 mg/L (Liu et al.
2019a). By introducing efficient β-amyrin synthase and P450 enzymes and opti-
mizing metabolic flux to GA synthesis, the final 11-oxo-β-amyrin and GA titer in
shake flask are 80 mg/L and 8.78 mg/L, respectively (Wang et al. 2019a).
8 Production of Other Terpenoids Derived

from Traditional Chinese Medicinal Plants in Yeasts
Besides the sesquiterpenes and triterpenes, many monoterpenoids and diterpenoids

have antitumor and other activities (Huang et al. 2012). The synthetic biology
and metabolic engineering strategies had been used for the yeast biosynthesis of
monoterpenoids and diterpenoids (Table 1) (Gao et al. 2020; Vickers et al. 2017).
The Nepeta cataria is widely used in traditional Chinese medicine, and the main
bioactive components are the monoterpenoids of menthone and pulegone (Gao
et al. 2020). As the yeast Erg20p can function as the GPPS and FPPS, which
limited monoterpenoid production by catalyzing GPP to FPP. In order to over-
come this limitation, the GPP function of Erg20p was strengthened, and this led
to a significant increase in monoterpenoid titers. By further engineering Erg20p to
reduce its FPPS activity and fusion of the engineered ERG20 genes with the ter-
pene synthase, the yield of the obtained engineered strain increased 340-fold over
the starting strain (Ignea et al. 2014). The native terpene precursor GPP is tightly
coupled with yeast sterol biosynthesis which is essential for yeast survival; there-
fore, engineering the native pathway is unable to direct the GPP metabolic flux
to FPP. Ignea et al. established a synthetic orthogonal monoterpenoid biosynthetic
pathway in S. cerevisiae. They identified the selectivity mechanism of monoterpene
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Table 1 Some selected terpenoids derived from traditional Chinese medicinal plants which have been synthesized by yeasts
Terpenoid name Traditional Chinese Yeast chassis Titer Terpenoid type References
medicinal plant source
Geraniol Diverse plants Saccharomyces cerevisiae 1.68 g/L Monoterpenoid (Jiang et al. 2017)
strain CEN.PK2-1C
α-Terpineol Pine and cypress plants Saccharomyces cerevisiae 21.88 mg/L Monoterpenoid (Zhang et al. 2019)
strain LCB08
Limonene Citrus fruits Saccharomyces cerevisiae 166 mg/L Monoterpenoid (Ignea et al. 2019)
strain BY4741
Miltiradiene Salvia miltiorrhiza Bge Saccharomyces cerevisiae 3.5 g/L Diterpenoid (Hu et al. 2020)
strain S288C
Triptolide Tripterygium wilfordii Saccharomyces cerevisiae 30.5 μg/g yeast Diterpenoid (Tu et al. 2020)
strain BY-HZ16 biomass
(-)-β-elemene Curcuma wenyujin Saccharomyces cerevisiae 190.7 mg/L Sesquiterpenoid (Hu et al. 2017)
strain SCIGS22a
Artemisinin Artemisia annua L Saccharomyces cerevisiae 25 g/L Sesquiterpenoid (Paddon et al. 2013)
strain Y1284
Glycyrrhetinic acid Glycyrrhize glabra L Saccharomyces cerevisiae 8.78 mg/L Triterpenoid (Wang et al. 2019a)
strain Y7
Ginsenoside compound K Panax plants Saccharomyces cerevisiae 1.7 g/L Triterpenoid (Nan et al. 2020)
(CK) strain (WLN-3)
Ginsenoside F1 Panax plants Saccharomyces cerevisiae 450. 5 mg/L Triterpenoid (Wang et al. 2019b)
strain (BY-F1)
Ginsenoside Rh2 Panax plants Saccharomyces cerevisiae 2.25 g/L Triterpenoid (Wang et al. 2019c)
strain ZWDRH2-10
(continued)
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Table 1 (continued)
Terpenoid name Traditional Chinese Yeast chassis Titer Terpenoid type References
medicinal plant source
Ginsenoside Rh1 Panax plants Saccharomyces cerevisiae 92.8 mg/L Triterpenoid (Wei et al. 2015a)
strain ZW-Rh1-20
Ginsenoside Rg3 Panax plants Saccharomyces cerevisiae 3.49 μmol/g dry cell Triterpenoid (Wang et al. 2015)
strain D20RG1 weight
Protopanaxadiol (PPD) Panax plants Saccharomyces cerevisiae 152.37 mg/L Triterpenoid (Gao et al. 2018)
strain GW10
Yeast Synthetic Biology for the Production of Terpenoids …
195
196 Y. Wei
synthases and engineered enzymes to accept neryl diphosphate as the substrate. By

combining the engineered monoterpene synthase and dynamic control of FPP and
neryl diphosphate biosynthesis, the production of monoterpenes using the orthog-
onal monoterpenoid biosynthetic pathway increased sevenfold over using yeast
canonical pathway (Ignea et al. 2019).
Some traditional Chinese medicinal plants can produce bioactive diterpenoid
components, such as triptolide from Tripterygium wilfordii and oridonin from
Isodon rubescens (Song et al. 2019; Cheng et al. 2019). The triptolide has anti-
inflammatory, antitumor, and other biological activities, but its composition in the
biomass of T. wilfordii is very low (from 0.0001 to 0.002%) (Zeng et al. 2013;
Zhou et al. 2012). Integration analysis with the genomic, transcriptomic, and
metabolomic data of T. wilfordii recovered the cytochrome P450 (CYP728B70)
for triptolide intermediate of dehydroabietic acid. Introduction of CYP728B70 to
engineered yeast achieved the production of diterpene alcohols and acids, which
paves the way for triptolide biosynthesis in yeasts (Tu et al. 2020). The key genes
of oridonin biosynthetic pathway are unclear (Pelot et al. 2017). Though paclitaxel
(taxol) is not extracted from traditional Chinese medicinal plants, it is one of the
most famous plant diterpenoids used as an effective antitumor drug (Gallego-Jara
et al. 2020). The paclitaxel is mainly extracted from the bark of several Taxus
species, and most Taxus species are used for taxol production which threatens the
survival of these old trees (Expósito et al. 2009). The biosynthesis of paclitaxel has
been realized in Taxus baccata suspension cultures and Nicotiana benthamiana, but
its yield is low (Li et al. 2019b; Malik et al. 2011). Production of paclitaxel in yeast
is still challenging, many essential enzymes need to be identified (Gallego-Jara
et al. 2020). Diterpene biosynthesis involves the oxidation steps which requires
identifying cytochrome P450 enzymes, and the total biosynthesis of diterpenes in
yeast is difficult (Hu et al. 2020).
The multi-omics data can be collected from the available database or obtained
by sequencing different fresh tissues of the plant at different growth phases or
areas. By comparing the multi-omics data and constructing a network between
different omics data, the genes in the terpenoid biosynthetic pathway can be iden-
tified. Using structural biology and protein engineering strategies, the activities of
key enzymes would be improved and adapted to the yeast hosts. These key genes
can be further used to redesign the heterologous pathway in engineered yeast,
and the redesigned pathway can be applied to build yeast strains with enhanced
terpenoid production. The synthetic biology and metabolic engineering strategies
could direct yeast metabolic flux to the biosynthesis of terpenoids. Finally, yeast
strains with a high titer, rate, and yield production of terpenoids can be obtained.
Based on the proper scale-up fermentation strategies and bioprocess optimization,
large-scale production of terpenoids in yeasts can be achieved (Fig. 1).
9 Conclusion and Perspective
The omics-based technologies can help to identify key genes easily from omics
data of the traditional Chinese medicinal plants, and these key genes can be
used for building yeast cell factories. Engineering for efficient yeast terpenoid
precursor-producing chassis, introducing heterogenous terpenoid synthetic path-
ways, increasing key gene expression level, directing metabolic flux for terpenoid
synthesis by downregulating competing pathway, enhancing cofactor supply for
MVA synthesis, engineering sub-organelle for terpenoid production, and other fre-
quently used strategies would significantly improve titer, rate, and yield of yeast
terpenoid production.
With the development of genomic-scale models and high-throughput automated
screening, engineered yeasts for efficient terpenoid production would be acceler-
ated. The optimized fermentation and bioprocess could decrease the cost of yeast
terpenoids. Besides, to produce affordable or commercial terpenoids, developing
smart biomanufacturing with a low-cost, intelligent, and continuous process will
lay the foundation for industrial-scale yeast production of valuable terpenoids. The
production of many valuable terpenoids derived from traditional Chinese medicinal
plants will be achieved in the near future.
Acknowledgements This work was supported by the National Natural Science Foundation of
China (No. 31800079).
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10.1016/j.copbio.2020.07.005
Application of Yeast Synthetic
Biology for the Production of Citrus
Flavors
Karim Farmanpour-Kalalagh, Arman Beyraghdar Kashkooli,

and Alireza Babaei
Abstract
Plants have the potential to produce an extensive variety of sesquiterpenoid
compounds being used as flavor, fragrance, and medicine. Among the sesquiter-
penoids of the citrus family, (+)-valencene and (+)-nootkatone are considered
the most valuable compounds used in various industries. Due to the low yield of
these compounds in their native hosts, commercial (+)-nootkatone is produced
from (+)-valencene by oxidation, either chemically or enzymatically. Further-
more, chemical synthesis is complex, toxic, and harmful to the environment.
Therefore, more attention should be paid to the environment-friendly and safe
methods for (+)-nootkatone synthesis. Recently, however, significant progress
has been achieved on enzymatic oxidation of (+)-valencene to (+)-nootkatone
(or to its precursor nootkatol) in the biotransformation/bioconversion process.
Nevertheless, ideally de novo (+)-nootkatone could also be produced from
(+)-valencene via biotechnological approaches in heterologous hosts using the
enzymes involved in its biosynthetic pathway. Among some of the organisms
used for the production of (+)-valencene and (+)-nootkatone, the yeast platform
has the highest potential for the production of these two valuable products. De
novo reconstitution of related-biosynthetic pathways in Saccharomyces cere-
visiae, Yarrowia lipolytica, and Pichia pastoris has led to high production of
these compounds, expressing hope to meet the needs of various industries on
large scales which will be described in the following chapter.
Keywords
(+)-valencene • (+)-nootkatone • Synthetic biology • Saccharomyces

cerevisiae • Yarrowia lipolytica • Pichia pastoris
K. Farmanpour-Kalalagh · A. Beyraghdar Kashkooli (B) · A. Babaei

Department of Horticultural Science, Faculty of Agriculture, Tarbiat Modares University, P.O.
Box 14115-365, Tehran, Iran
https://doi.org/10.1007/978-3-030-89680-5_8
208 K. Farmanpour-Kalalagh et al.
1 Introduction
Plants produce a vast diversity of secondary metabolites including terpenes (also

termed as isoprenoids) that possess physiological and ecological properties (Chap-
pell 2004). Low-molecular-weight terpenoids mostly exist as volatiles in different
parts of plants (Lücker et al. 2004). Terpenoids include the largest group of sec-
ondary metabolites; lots of them show biological functions and are applied as
pharma and nutraceutical ingredients and compounds used in food, flavor, fra-
grance, and cosmetics. The grouping of terpenoids is originated from the number
of C5-subunits (isoprene) in their backbone (e.g., hemiterpenoids (C5), monoter-
penoids (C10), sesquiterpenoids (C15), diterpenoids (C20), sesterterpenoids (C25),
triterpenoids (C30), and tetraterpenoids (carotenoids) (C40)). The main hydrocar-
bon skeletons constructed from isoprene units are mainly altered by enzymes such
as cytochromes P450s, glycosyltransferases, hydrogenases, and methyltransferase.
The amount of terpenoids in natural origins is commonly low and extraction may
result in by-products (Asmund Arnesen et al. 2020). Among the plant-derived
terpenoids, sesquiterpenes which are classified as a large group of natural prod-
ucts show diverse properties for agriculture, food, pharmacology, and cosmetics
industry (Beyraghdar Kashkooli 2018; Troost et al. 2019). (+)-Valencene and (+)-
nootkatone are high-value ingredients for flavor, fragrance, and pharmaceutical
industry (Cankar et al. 2015; Milhim et al. 2019). Because of valencene’s fruity fla-
vor, it is industrially applied as an additive in food and drinks. In addition to using
as an additive, (+)-valencene can be oxidized to (+)-nootkatone. (+)-Nootkatone
(oxidation product (+)-valencene which is found in grapefruit and heartwood of
the Nootka cypress) is also a considerable added-value sesquiterpene with slightly
bitter taste and low odor threshold (Girhard et al. 2009; Frohwitter et al. 2014).
Since raw plant-derived material of nootkatone is insufficient on the global mar-
ket, semisynthetic (+)-nootkatone is derived from (+)-valencene in commercial
approaches. Chemical oxidation of (+)-valencene to (+)-nootkatone requires exten-
sive use of tert-butyl chromate, which is a carcinogenic material. On the other
hand, non-carcinogenic tert-butyl peracetate or tert-butyl hydroperoxide can be
replaced which are extremely corrosive and are flammable (Cankar et al. 2011).
As mentioned above, both (+)-valencene and (+)-nootkatone are valuable com-
pounds in the flavor and fragrance industry, but the low yield in nature and
high cost of extraction restricts their application (Chen et al. 2019). Addition-
ally, the quality, value, and availability of these two compounds are very much
related to seasonal variations and the harvest time (Beekwilder et al. 2014).
On the other hand, since commercial nootkatone is presently produced from
valencene by oxidation, either by a chemical process or enzymatically, meeting the
global demands seems difficult. Hence, increasing the production of (+)-valencene
and (+)-nootkatone from (+)-valencene via bioengineering of their biosynthetic
pathway in heterologous hosts is critical. In this chapter, we review all possi-
ble techniques for improving (+)-valencene and (+)-nootkatone production using
chemical and enzymatical processes. In particular, we highlight heterologous
Application of Yeast Synthetic Biology for the Production … 209
advances in the production of these compounds using microorganism platforms,

specifically yeast-based ones and their application flavor and fragrance industry.
2 Natural Origin(s) of Valencene and Nootkatone
The bicyclic sesquiterpene (+)-valencene, a natural sesquiterpene, is a constituent

of the essential oils of different members of the citrus genus, including Valen-
cia or sweet orange (Citrus × sinensis) as well as in grapevine (Vitis vinifera L.)
and in small amounts from rhizomes of Cyperus rotundus (Tsoyi et al. 2011; Nam
et al. 2016), Chinese bayberry (Myrica rubra) (Fujita et al. 2014; Langhasova et al.
2014; Ambrož et al. 2015), Welensali (Croton flavens) (Sylvestre et al. 2006), man-
darins cultivars (Merle et al. 2004), dehydrated lime [Citrus aurantifolia (Christm.)
Swingle] (Ramesh Yadav et al. 2004), Egyptian Eucalyptus camaldulensis var. bre-
virostris (El-Ghorab et al. 2002), heartwood of the Nootka cypress (also known as
the Alaska yellow cedar, native to the Pacific Northwest coast of North America)
(Callitropsis nootkatensis), and Leyland cypress (Cupressoparis leylandii) which
is an inter-genetic hybrid of Nootka cypress (Chamaecyparis nootkatensis) and
Monterey cypress (Cupressus macrocarpa) (Liu 2009).
(+)-Nootkatone is an important oxidized sesquiterpene for the flavor and fra-
grance industry. It has a characteristic grapefruit-like flavor and a low odor
threshold. Natural (+)-nootkatone can be extracted in small amounts from flavedo
of grapefruit (Citrus × paradisi), pomelo (Citrus grandis) (Tocmo et al. 2020),
vetiver (or khus) grass (Chrysopogon zizanioides) (Mao et al. 2006; Filippi et al.
2013), rhizome of Cyperus rotundus (Tsoyi et al. 2011; Jaiswal et al. 2014),
Alpiniae Oxyphyllae Fructus (Wang et al. 2018), and fruits of Alpinia oxyphylla
Miquel (Xie et al. 2009) and in high amounts from heartwood of the Nootka
cypress (Callitropsis nootkatensis) (Beekwilder et al. 2014) (Fig. 1.).
Fig. 1 Oxidation of valencene to nootkatone under different treatments/conditions via

Cytochrome P450s (CYPs) or through chemical conversion
3 Yields of Valencene and Nootkatone in Native Hosts
Valencene is the main sesquiterpene in orange peel oil, and its titer has been
commonly applied to specify the oil’s commercial degree. To characterize the
contribution of valencene to the aroma quality of a marketable orange oil by using
multidimensional GC–O/GC–MS, thirty-seven aroma-active compounds have been
identified in orange oil with valencene concentration between 54 and 68 μg/g
(Elston et al. 2005). In addition to orange oil, valencene is reported in Chinese
bayberry (Myrica rubra) with a relative abundance of 0.27–4.25% (Fujita et al.
2014), 5.97% (Langhasova et al. 2014), and 6% (Ambrož et al. 2015), 1.24% in
Welensali (Croton flavens) (Sylvestre et al. 2006), 0.01–0.4% in mandarins culti-
vars (Merle et al. 2004), 0.1 (μL)/100 g in dehydrated lime [Citrus aurantifolia
(Christm.) Swingle] (Ramesh Yadav et al. 2004), 0.44% in Egyptian Eucalyptus
camaldulensis var. brevirostris (El-Ghorab et al. 2002).
Analyzing and comparison of essential oil components extracted from the heart-
woods of Leyland cypress, Alaska yellow cedar, and Monterey cypress via steam
distillation and solvent extraction by Liu (2009) indicated that the eighteen com-
pounds are found both in Alaska cedar and Leyland cypress although there are
only eight compounds found in Monterey cypress from steam distillation (6 and
12 h) of their heartwoods. The GC–MS analysis of volatile constituents from two
fractions of steam distillation and solvent extraction, respectively, indicates that
the main components in Alaska cedar, Monterey cypress, and Leyland cypress, are
carvacrol, nootkatene, nootkatol, nootkatone, and nootkatin, which display about
60–90% of the whole capacity of the essential oil. These ingredients are from three
diverse families which are p-Cymene, eremophilane, and tropolone (Fig. 2a and b).
The evaluation of volatile constituents from Alaska cedar lumber and fresh wood
exhibits that in parallel with the maturing and aging of the wood, the quantity of
nootkatol and epi-nootkatol decreased, whereas the content of nootkatene increases
significantly, which indirectly shows the conversion of nootkatol to nootkatene in
the lumber boosted via the increasing of acidity of the heartwood (Fig. 2c).
Many compounds are found by solvent extraction when detected with the
GC–MS. Because of the low titer of some components, only some of them are
identified. There are twelve compounds in Alaska cedar, eleven in Leyland cypress,
and only seven from Monterey cypress. Excluding two compounds, valencene-11,
12- diol and an unknown tropolone, all the others are detected in the steam distil-
lation fractions. The lacking of valencene-11, 12- diol and the unknown tropolone
are probably modified during steam distillation due to higher temperature (Fig. 2d).
Nootkatone detection in other plants such as vetiver grass oil by applying com-
prehensive two-dimensional gas chromatography techniques (GC × GC-FID/MS)
showed that the amount of this sesquiterpene is between 0.30 and 0.60 g/100 g oil
among different varieties.
Fig. 2 a The components of the first 6 h distillation fraction from Alaska yellow cedar, Leyland
cypress, and Monterey cypress; b The components of the 6-12 h distillation fraction from Alaska
yellow cedar, Leyland cypress, and Monterey cypress; c The components of the 6 h distillation
fraction from Alaska yellow cedar fresh wood and lumber; d The components of the 24 h solvent
extraction from Alaska yellow cedar, Leyland cypress and, Monterey cypress analyzed by GC–MS.
(Adapted from Liu 2009)
4 Importance and Application of Valencene

and Nootkatone
4.1 Flavor and Fragrance Industry
Valencene is considered an important ingredient in consumer priority among

a wide range of products, such as food, flavor, fragrance, beverage, personal,
and home care products. Because of nootkatone’s strong citrusy, sweet juicy
notes, peely, woody, and grapefruit-like aroma characteristics as an oxidized
sesquiterpene from valencene, it is involved in the formulation of citrus and
grapefruit-flavored beverages. In fragrances, it is applied in the production of cit-
rusy and dry components for men’s perfumes. (+)-Nootkatone contains a ϕ value
of 2.7 × 1011 , while (-)-nootkatone value estimated a ϕ 3.6 × 108 with a slightly
fresh, sour, green, organoleptic profile. Nootkatone is obtainable from various mar-
kets with diverse concentrations. This compound can be added in grapefruit drinks
at a level of 2–6 ppm and can be used along with other citrus oils in fragrance for-
mulations (such as bergamot oil, bitter orange oil, etc.,) to increase an interesting
citrus note (Zviely 2009).
4.2 Therapeutic Benefits
Anti-cancer: The results of various studies show the anti-cancer activity of

valencene and nootkatone. Essential oil from Myrica rubra leaves containing
valencene plus seven dominant compounds prevents cancer cell increasing and
induces apoptosis in some human intestinal lines. The anti-proliferative activity of
extracted M. rubra essential oil (MEO) has been tested in human colon and ileoce-
cal adenocarcinoma cell lines and results show that the MEO remarkably prevents
cell proliferation in a concentration-dependent manner in all cell lines. In cancer
cells, MEO creates apoptosis and leads to a considerable development of activities
of the initiator as well as effector caspases (Langhasova et al. 2014).
The anti-proliferative effect of nootkatone on lung and bladder cancer cell
lines at different concentrations has been investigated. Nootkatone showed a
strong anti-proliferative effect on the A549 lung cancer cell line (Šunjerga
2019). In another successful study, nootkatone prevents v-Ki-ras2 Kirsten rat
sarcoma (RAS) viral oncogene homolog (KRAS) downstream pathway and weak-
ens non-small-cell lung cancer A549 cells to Adriamycin. Nootkatone activated
AMP-activated protein kinase (AMPK) by liver kinase B1 (LKB1)-independent
and calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2)-dependent
pathways causes prevention of cell growth and development and induction of G1
cell limiting (Hung et al. 2019).
Efficacy of chemotherapy drug: Evaluation of the valencene, β-caryophyllene,
β-caryophyllene oxide, α-humulene, and trans-nerolidol effects as essential com-
pounds of the volatile constituents from Myrica rubra leaves against cancer cells
and on the potency and toxicity of the anticancer drug doxorubicin in CaCo-2 can-
cer cells (an immortalized cell line of human colorectal adenocarcinoma cells) and
in primary culture of rat hepatocytes display substantial antiproliferative activity
in various intestinal cancer cell lines, with CaCo-2 cells as the most susceptible
line. Results indicate that the valencene prevents the proliferation of CaCo-2 can-
cer cells and does not have an impact on the viability of hepatocytes. Furthermore,
valencene can also strengthen the pro-oxidative activity of doxorubicin in CaCo-2
cells (Ambrož et al. 2015).
Anti-Alzheimer: Analyzing the therapeutic neuroprotective activity of nootka-
tone on an Alzheimer’s disease mouse model caused by intracerebroventricular
injection of lipopolysaccharide displays that nootkatone (10 mg/kg) has an effec-
tive efficiency in behavior trials including Y-maze and Morris water maze tests.
The outcomes of the histopathological and immunohistochemical test exhibit that
lipopolysaccharide leads to degeneration of neurons and initiation of microglia
especially in the hippocampus and nootkatone (10 mg/kg) reverse these modifi-
cations. Enzyme-linked immunosorbent evaluation and western blot results also
indicate that the model group enhances the expression of IL-1β, IL-6, TNF-α,
NLRP3, and NF-κB p65, particularly in the hippocampus compared to the control
group, and nootkatone (10 mg/kg) drops the high-level expression of inflammatory
cytokines (Wang et al. 2018).
Anti-bacterial: Nootkatone displays anti-bacterial properties toward gram-

positive bacteria such as Corynebacterium diphtheriae (most effective against
this bacteria), Enterococcus faecalis, Listeria monocytogenes, Bacillus cereus, and
Staphylococcus aureus. Nevertheless, no bactericidal activity has been reported
against gram-negative bacteria. Additionally, high-concentration nootkatone shows
anti-bacterial properties against gram-positive bacilli. These findings propound that
the nootkatone may create a bactericidal property via attacking to cell wall or a
specific metabolite. In addition, nootkatone can prevent the formation of biofilms
by Staphylococcus aureus even at low concentrations (0.25 mM) (Yamaguchi
2019).
Skin protection: Electrophysiological assays in the evaluation of the antagonis-
tic properties of C. rotundus extract and their compositions on TRPV1 and ORAI1
channels indicate that valencene extracted from the hexane fraction significantly
prevents capsaicin-induced TRPV1 and ORAI1 flows at 90 μM (69 ± 15% and
97 ± 2% at −60 and −120 mV, respectively). Also, valencene contains a preventive
effect on cytoplasmic Ca2+ amounts and concentrations toward ORAI1 activation
(85 ± 2% at 50 μM). In addition, valencene concentration-dependently reduces
the melanin quantity after UVB irradiation in murine B16F10 melanoma cells via
82.66 ± 2.14% at 15 μg/mL (Nam et al. 2016).
Insect repellent: Nootkatone can be applied as an insecticide or insect repellent,
according to a decision from the US Environmental Protection Agency (EPA) in
August 2020. Registration and introduction of a compound with industrial applica-
tion make the process easier for producers to improve nootkatone-based products
(Waltz 2020).
5 Nootkatone and Valencene Biosynthetic Pathway

in Native Hosts
Terpenoids, also termed isoprenoids, are known as a wide and highly diverse group
of natural products. They are regarded as one of the largest groups of secondary
metabolites with various biological functions and valuable properties for diverse
industrial applications (Kallscheuer et al. 2019). Classification of terpenoids is
based on the number of carbon atoms forming the main skeleton. The general
divisions of them are hemiterpenoids (C5), monoterpenoids (C10), sesquiter-
penoids (C15), diterpenoids (C20), sesterterpenoids (C25), triterpenoids (C30), and
tetraterpenoids (also known as carotenoids; C40) (Ashour et al. 2010; Beyraghdar
Kashkooli et al. 2018). The central hydrocarbon backbone created from isoprene
units is regularly changed via enzymes like cytochromes P450, hydrogenases,
methyltransferases, and glycosyltransferases. Totally, terpenoids content in natu-
ral sources is typically low and extraction may result in by-products (Asmund
Arnesen et al. 2020). Biosynthetically, all terpenoids are biosynthesized from
the isoprene C5 backbones isopentenyl pyrophosphate (IPP) and dimethylallyl
pyrophosphate (DMAPP), which are produced via the 2-C-methyl-D-erythritol
4-phosphate (MEP), also called 1-deoxy-D-xylulose 5-phosphate (DXP), and
mevalonate (MVA) pathway. The MEP pathway, which commences from pyruvate
and glyceraldehyde-3-phosphate (GAP), is commonly found in plastids of bacte-
ria, cyanobacteria, green algae, and plants (Frank and Groll 2017). The cytosolic
MVA pathway mainly exists in eukaryotes (mammals, plants, and fungi), but also
archaea and a few bacteria. In the cytosol, sesquiterpenes are biosynthesized from
isoprenoid precursors by the MVA pathway. The MVA pathway starts from acetyl-
CoA and leads to the formation of isopentenyl diphosphate (IPP) and dimethyl
allyl diphosphate (DMAPP). Then, farnesyl diphosphate synthase (FPS) accumu-
lates DMAPP and IPP into farnesyl diphosphate (FPP). Terpene synthases (TPSs)
use FPP to create terpene hydrocarbons, which can be further altered via oxi-
dation into a diverse range of terpenoids (Chappell 2002). TPSs are classified
based on their substrate characteristics. Notable advances have been achieved dur-
ing the past decade in the biochemical reactions catalyzed by TPSs detail (Chappell
2004). Sesquiterpene synthases use the 15-carbon substrate farnesyl diphosphate
(FPP) (Chappell 2004). Sharon-Asa et al. (2003) have isolated and characterized a
sesquiterpene synthase (Cstps1) from Citrus that is presenting a new insight into
the biochemical process of this enzyme, and bioengineering of (sesqui)terpenoids
in native and heterologous hosts. Only a few functional (+)-valencene synthases
were reported in previous studies including VvVal from Vitis vinifera (Lücker
et al. 2004), Cstps1 from Citrus sinensis (Sharon-Asa et al. 2003), GFTpsD
from Citrus × paradisi, and CnVS from Nootka cypress (Callitropsis nootkatensis)
(Beekwilder et al. 2014).
The biosynthesis of (+)-valencene, as a precursor of (+)-nootkatone, is done
through the MVA pathway. IPP is frequently condensed via prenyl transferases to
create Farnesyl pyrophosphate (FPP). FPP is converted to (+)-valencene by the
germacrenyl carbocation. So that the formation of internal bond and deprotonating
is catalyzed by valencene synthase (Figs. 3 and 4). Not much information has been
published about the enzymatic steps of converting (+)-valencene to (+)-nootkatone.
It has been propounded that the biosynthetic pathway continues by a regioselec-
tive allylic hydroxylation of (+)-valencene to form nootkatol and then oxidized
to (+)-nootkatone. Both phases can be accelerated by the catalytic function of a
single multifunctional hydroxylase/oxidase or via a consecutive enzyme-mediated
reaction. But the reason has not been determined yet why (+)-valencene accumu-
lates in the peel of Valencia orange throughout fruit maturation while is further
oxidized to (+)-nootkatone in grapefruit (Fraatz et al. 2009a).
6 Methods for Production of Valencene and Nootkatone
6.1 Extraction from Native Hosts
Valencene: In the extraction of valencene from citrus fruits, such as Valencia

orange (Citrus × sinensis) peel, the following is estimated to have the required
volume of the raw material: Approximately, 5 kg of active ingredient and oil is
extracted from each ton of Valencia oranges. It is noteworthy to mention that the
Fig. 3 Valencene and Nootkatone biosynthetic pathway in four major producing plants
Fig. 4 Overview of yeast synbio for production of valencene and nootkatone. ERG10, acetyl-
CoA C-acetyltransferase; ERG13, hydroxymethylglutaryl-CoA synthase; ERG12, mevalonate
kinase; tHMGR, truncated 3-hydroxy-3-methylglutaryl-CoA reductase; ERG8, phosphomeval-
onate kinase; ERG19, diphosphomevalonate decarboxylase; IDI1, isopentenyl diphosphate delta-
isomerase; ERG20, farnesyl diphosphate synthase/dimethylallyltranstransferase; VS: valencene
synthase, Cyp450: cytochrome P450, valencene oxidase
valencene concentration in citrus fruits is low (0.2%–0.6% w:w), and availabil-

ity, quality, and price depends on seasonal variations and the harvesting process
(Beekwilder et al. 2014). In other words, from every 2.5 million kg of oranges,
1 kg of valencene is obtained (Asmund Arnesen et al. 2020). The general process
is that the peels of fruits, which are wastes from the juice industry, enter the oil
extraction process after washing and reducing moisture. The oil is extracted using a
cold-press followed by several stages of refining. After extracting Valencia orange
peel oil, which contains an average of 0.2% valencene, it must be processed with a
solvent such as methanol to separate valencene from other components. After this
stage, the valencene concentration will reach almost 30%. Subsequently, fractional
distillation should be used for final purification. Then, valencene can be harvested
in different purities according to the grades available in the market. The final goal,
however, is to reach 98% valencene crystals after fractional distillation and using
a crystallizer.
Nootkatone: Extraction of nootkatone from plant tissues is always costly and
dependent on agricultural fluctuations. At least 400 tons of grapefruit (Citrus ×
paradisi) are needed to produce 1 kg of nootkatone. Therefore, the sustainable
supply of raw materials for the industry is one of the biggest challenges in the pro-
duction of nootkatone. Like valencene extraction methods, nootkatone is extracted
from grapefruit peel oil. The concentration of nootkatone is then elevated by liq-
uid–liquid extraction (LLE; also known as solvent extraction and partitioning) and
then distilled to high purity.
6.2 Chemical Synthesis
The chemical synthetic production of (+)-nootkatone from the (+)-valencene has

been conducted using the oxidizing agents known to be toxic to the environ-
ment, such as tert-butyl peracetate or tert-butyl hydroperoxide along with metal
catalysts supported on silica (Wilson and Shaw 1978; Salvador and Clark 2002).
Hence, obtained (+)-nootkatone cannot be considered as a “natural” compound
and does not meet the high demand for this compound in the markets. In conclu-
sion, because of toxic heavy metals or peroxides, highly flammable and/or strong
oxidants, chemical synthetic nootkatone methods are neither safe nor environment-
friendly. Therefore, more consideration is being paid to environment-friendly and
unharmed techniques for (+)-nootkatone production (Wriessnegger et al. 2014).
6.3 Biotransformation (or Bioconversion)
Valencene biotransformation into nootkatol and nootkatone is believed to be cat-

alyzed via the cytochrome P450 enzymes from both eukaryotic and prokaryotic
(micro) organisms. Some (micro)organisms contain enzymes from the cytochrome
P450 monooxygenase family, of which many were significantly proven for adding
an oxygen atom into allyl groups and are the main candidates for biotransforma-
tion of (+)-valencene into (+)-nootkatone. However, finding regioselective P450
enzymes relevant to industrial demands is still a challenge in this regard (Gavira
et al. 2013). A two-step enzymatic transformation of (+)-valencene has been sug-
gested by a regioselective allylic hydroxylation of the 2-position of (+)-valencene
to nootkatol and then by the oxidation to (+)-nootkatone. There are two hypotheses
in this case. The first is that both reactions can be enhanced by a multifunctional
cytochrome P450 enzyme. Second, the oxidative activity of cytochrome P450 leads
to the production of nootkatol, followed by an alcohol dehydrogenase activity
to produce (+)-nootkatone. Some enzymes originated from plants and microor-
ganisms catalyzing the production of either nootkatol and/or (+)-nootkatone from
(+)-valencene have been successfully identified (Cankar et al. 2014).
Cytochrome P450cam and P450BM-3 : (+)-valencene oxidation of the wild-type
and mutants of P450cam from Pseudomonas putida, and of P450BM-3 from Bacil-
lus megaterium, have been studied as a promising way towards (+)-nootkatone
production. Wild-type P450cam does not oxidize (+)-valencene but the mutants
produce (+)-trans-nootkatol and (+)-nootkatone as the major products. Ignoring
the non-selective properties, wild-type P450BM-3 and its mutant forms contain
dominant activities compared with the P450cam . Among the many compounds,
cis-(+)-valencene-1, 10-epoxide, cis- and trans-(+)-nootkatol, (+)-nootkatone, (+)-
nootkatone-13S, 14-epoxide, and trans-(+)-nootkaton-9-ol were characterized from
whole-cell reactions. The selectivity patterns express that (+)-valencene contains
a single binding direction in P450cam but several directions in P450BM-3 (Sowden
et al. 2005).
CYP from ascomycete Chaetomium globosum: Oxidizing the exogenous (+)-
valencene to nootkatone via the stereoselective production of alpha-nootkatol
can be applied in submerged cultures of the ascomycete Chaetomium globosum.
Inhibition tests propose that the insertion of the first oxygen atoms catalysis
by cytochrome P450 monooxygenase. Hence in addition to nootkatone, non-
volatile oxidation products along with flavor-active and inactive compounds can be
identified. (+)-valencene, valencene-11, 12-epoxide, alpha-nootkatol, and nootka-
tone accumulated mostly inside the C. globosum cells. On the other hand,
nootkatone-11, 12-epoxide is only in the culture medium. Therefore, active trans-
porting of compounds into the extracellular sections is done during (+)-valencene
detoxification (Kaspera et al. 2005).
CYP from algae and fungi: Studies indicate that the cytochrome P450s have
an important role in (+)-valencene biotransformation by the green algae Chlorella
species and fungi such as Mucor species, Botryosphaeria dothidea, and Botry-
odiplodia theobromae to yield nootkatone in high quantity (Furusawa et al.
2005).
CYP from Gynostemma pentaphyllum: It has been known that the suspension
cultures of Gynostemma pentaphyllum can transform valencene into nootkatone
as the major product. Also, biotransformation of valencene by Caragana cham-
lagu and Hibiscus cannabinus cultured plant cells show largely the same results
(Sakamaki et al. 2005).
Premnaspirodiene oxygenase CYP71D55: Probing (+)-valencene oxidizing

activity of cytochrome P450 enzymes from the CYP71 family shows that the
premnaspirodiene oxygenase CYP71D55 from Hyoscyamus muticus contains cat-
alytic oxidizing activity in conversion of (+)-valencene firstly to nootkatol in vitro
(Takahashi et al. 2007).
Fungal dioxygenase from Pleurotus sapidus: A selective and strong allylic
oxidation of the (+)-valencene to (+)-nootkatone can be obtained with lyophiliza-
tion of the basidiomycete Pleurotus sapidus. Therefore, a novel oxygenase from P.
sapidus can transform valencene to nootkatone. (Fraatz et al. 2009b).
CYP109B1 from Bacillus subtilis: It has been known that the cytochrome
CYP109B1 from Bacillus subtilis can catalyze the oxidation of (+)-valencene
to nootkatol and (+)- nootkatone. On the other hand, when (+)-valencene is
bio-oxidized in vivo, a number of unfavorable multi-oxygenated by-products are
produced. But as mentioned in Girhard et al. (2009) study, there are some tech-
niques in the production of nootkatol and (+)-nootkatone up to 97% of the total
product.
Dioxygenase of Pleurotus sapidus: A dioxygenase from Pleurotus sapidus con-
verts (+)-valencene to (+)-nootkatone regio-specifically by a stereo-specific allylic
hydroperoxidation. The isolation and characterizing of two allylic (+)-valencene-
derived hydroperoxides along with homology data from amino acid sequencing
expresses a lipoxygenase-like type of enzyme (Krügener et al. 2010).
CYP71AV8 from Cichorium intybus roots: A new P450 cDNA has been found
in a chicory root EST library. Co-expression of the enzyme with a valencene
synthase in yeast led to the formation of trans-nootkatol, cis-nootkatol, and
(+)-nootkatone (Cankar et al. 2011).
Valencene dioxygenase from Pleurotus sapidus: Valencene dioxygenase from
the edible basidiomycete Pleurotus sapidus transforms the (+)-valencene to the
(+)-nootkatone and to nootkatol through intermediate hydroperoxides (Zelena et al.
2012).
CYP71D51v2 from tobacco and a P450 reductase from Arabidopsis: Recom-
binant yeast with P450 reductase from Arabidopsis and CYP71D51v2 from
tobacco has been studied for optimizing the bioconversion mechanism. The bio-
conversion process produces β-nootkatol and nootkatone, with low efficiency
which is reduced upon increasing the substrate concentration. Toxicity and
harmfulness of the products for yeast platforms at concentrations higher than
100 mg/L, gathering of β-nootkatol in yeast endomembranes, and prevention of the
CYP71D51v2 hydroxylation reaction by the obtained products are considered as
the main reasons for low bioconversion yield. In addition, it has been distinguished
that the generation of nootkatone from β-nootkatol is not a P450-dependent reac-
tion. According to these achievements, researchers suggest new strategies for the
performance of a P450-based bioconversion mechanism (Gavira et al. 2013).
CYP706M1 from Alaska yellow cedar: After co-expression of intended
cytochrome P450s from Alaska yellow cedar in yeast with a valencene syn-
thase, a C. nootkatensis valencene oxidase (CnVO) has been found to generate
trans-nootkatol and (+)-nootkatone. Generation of (+)-nootkatone was observed
at 144 ± 10 μg/L yeast culture. CnVO is one of the CYP706 family members of
cytochrome P450 oxidases (Cankar et al. 2014).
CYP from Botryodiplodia theobromae 1368, Yarrowia lipolytica 2.2ab, and
Phanerochaete chrysosporium: It has been observed that Botryodiplodia theobro-
mae 1368, Yarrowia lipolytica 2.2ab, and Phanerochaete chrysosporium oxidize
(+)-valencene to (+)-nootkatone, with concentrations of 231.7 ± 2.1, 216.9 ± 5.8,
and 100.8 ± 2.6 mg/l (+)-nootkatone, respectively. Various biotransformation
experiments (organic, aqueous, and biphasic) have been also tested which led to
the same production levels of nootkatone (Palmerín-Carreño et al. 2015).
Peroxidase combined with a laccase from Funalia trogii: an incorporated
platform of a dye-decolorizing peroxidase (Ftr-DyP) and a laccase isolated from
the basidiomycete Funalia trogii transformed the (+)-valencene totally to the
(+)-nootkatone, with a high concentration of 1100 mg/L (Kolwek et al. 2018).
6.4 Application of Synthetic Biology Using Yeast Platforms
Industrially, many (sesqui) terpenes have applications as a pharmaceutical fla-

vor, fragrance, etc. (Cankar et al. 2015). Inserting related-gene(s), overexpres-
sion/knockdown of the enzyme(s) in the metabolic biosynthetic pathway, and use
of diverse subcellular sections for production have led to high levels of (sesqui)
terpenes (reviewed in Scholtmeijer et al. 2014). Microorganisms are used because
of the variety of naturally existing strains, high metabolic diversity, and high pro-
duction of related metabolite(s). On the other hand, production in microorganisms
is a reliable technique to supply (sesqui) terpenoids for industrial applications.
These processes, however, require the targeted transfer of the related biocatalytic
pathways in an appropriate heterologous microorganism (Troost et al. 2019). Some
yeast species are the most common microorganisms applied for the heterologous
production of secondary metabolites. Here, we review the heterologous production
of citrus flavors in three different yeast platforms as an attractive alternative for
high production of (+)-valencene and (+)-nootkatone.
6.4.1 Saccharomyces cerevisiae

Saccharomyces cerevisiae, also known as baker’s yeast, is one of the most common
platforms used in the heterologous production of secondary metabolites. Studying,
designing, and constructing S. cerevisiae cell factories for high-level production
of citrus flavors is more appealing due to the short growth cycle and low costs
(Chen et al. 2019). So far, successful studies have been performed on the pro-
duction of citrus flavors in S. cerevisiae via bioengineering of related-pathway.
After co-expression of candidate cytochrome P450 enzymes from Alaska yellow
cedar with a valencene synthase in S. cerevisiae, a C. nootkatensis valencene oxi-
dase (CnVO) has been identified to produce trans-nootkatol and (+)-nootkatone
(144 ± 10 μg/L) (Cankar et al. 2014). Also, overexpression of CnVS in S. cere-
visiae strain WAT11 only has produced 1.36 mg/L of (+)-valencene (Beekwilder
et al. 2014). Whereas with overexpressing of (+)-valencene synthase GFTpsD and
downregulating the squalene synthase, (+)-valencene is produced up to 3 mg/L

(Asadollahi et al. 2008).
For the production of trans-nootkatol from (+)-valencene, two protocols (1):
converting externally added (+)-valencene with resting cells or (2): cultivating
engineered self-sufficient production strains) have been established and opti-
mized with recombinant S. cerevisiae co-expressing CYP Hyoscyamus muticus
premnaspirodiene oxygenase (HPO) and the cytochrome P450 reductase from Ara-
bidopsis thaliana (CPR) to hydroxylate the C2 atom of (+)-valencene. The phase
transfer process displays a substantial problem of whole-cell biotransformation
of hydrophobic substrates. To solve the problem, Emmerstorfer et al. (2015) have
designed an efficient S. cerevisiae strain containing the ability to produce valencene
intracellularly. Furthermore, the self-sufficient production of trans-nootkatol has
been carried out in biphasic systems via n-dodecane for catching the synthesized
compounds to prevention of toxicity or inhibitory effects of high concentrations of
compounds on yeasts (Emmerstorfer-Augustin and Pichler 2016).
In another successful study in order to overproduce valencene, S. cerevisiae
was used as the suitable host for cell factory construction. In this regard, some
bioengineering strategies were accomplished in the MVA biosynthetic pathway.
First of all, a renewable CRISPR/Cas9 system was designed to obtain mul-
tiple genome editing to enhance the FPP pool toward the valencene synthesis
via the introduction of Cre/loxP. Furthermore, the main genes of the FPP biosyn-
thetic pathway in the MVA pathway were overexpressed in the yeast genome to
increase the metabolic flux to precursor FPP. To continue, the expression cassette
of valencene synthase was developed by designing a promoter–terminator library,
with PHXT7- VS-TTPI1 containing high performance in valencene production.
(It is important to note that the identification of suitable expression cassettes
containing promoter and terminator is critical for the improvement of valencene
production). Finally, after fed-batch fermentation in a 3 L bioreactor, the valencene
yield titer increased to 539.3 mg/L (Chen et al. 2019).
More recently, (+)-valencene was produced in high quantities (217.95 mg/L) in
S. cerevisiae via overexpressing Callitropsis nootkatensis (+)-valencene synthase
(CnVS) along with the expression of farnesyl diphosphate synthase (ERG20) as
a fused protein, overexpression of tHMG1, and downregulating the squalene syn-
thase enzyme (ERG9). Afterward, (+)-valencene oxidation by overexpressing the
Hyoscyamus muticus premnaspirodiene oxygenase (HPO) variant (V482I/A484I)
and cytochrome P450 reductase (ATR1) from Arabidopsis thaliana led to (+)-
valencene oxidation to β-nootkatol and only low amounts of (+)-nootkatone
(9.66 mg/L). Oxidation of β-nootkatol to (+)-nootkatone demonstrated that the
short-chain dehydrogenase/reductase (SDR) superfamily dehydrogenases ZSD1 of
Zingiber zerumbet and ABA2 of Citrus sinensis were effective in such an oxida-
tion process. Lastly, overexpression of ZSD1 and ABA2 increased (+)-nootkatone
yield to 59.78 mg/L and 53.48 mg/L, respectively (Meng et al. 2020).
6.4.2 Yarrowia lipolytica

Y. lipolytica is an ascomycetous yeast that is known as a safe microorganism. Y.
lipolytica is part of the oleaginous yeast group, with cells containing more than
20% fat content (Nicaud 2012). Due to the endogenous MVA pathway, it is the
potential platform for (sesqui) terpenoid production. Also as an oleaginous yeast,
Y. lipolytica can utilize both hydrophilic and hydrophobic substrates as carbon
sources to produce valuable compounds (Guo et al. 2018).
In the first heterologous production of (+)-nootkatone in Y. lipolytica, Guo et al.,
(2018) constructed (+)-nootkatone biosynthetic pathway in Y. lipolytica ATCC
201,249. Co-expressing (+)-valencene synthase (CnVS), codon-optimized (+)-
nootkatone synthase opCYP706M1, and codon-optimized NADPH-cytochrome
P450 reductase opAtCPR1 produced (+)-nootkatone with initial quantities of
45.6 g/L. Fusion of opCYP706M1 and opt46AtCPR1 (opAtCPR1 with 46 amino
acids truncated at N-terminal) enhanced (+)-valencene transformation performance
to (+)-nootkatone and (+)-nootkatone production raised to 312.2 g/L. In the next
step, overexpression of tHMG1 and FPP synthase (ERG20) enhanced the (+)-
nootkatone production. Then, the last engineered strain led to the (+)-nootkatone
production with a titer of 978.2 g/L. Also in another study, overexpression of
HMG1, ERG12, ACL1, SeACS, IDI, ERG20, and CnVS along with SQS knock-
down in Y. lipolytica strain ST9204 has led to 113.9 ± 6.6 mg/L (+)-valencene
(Asmund Arnesen et al. 2020).
6.4.3 Pichia pastoris

P. pastoris is classified as a species of methylotrophic yeast. A comparative study
including different yeast species and Escherichia coli has shown that the methy-
lotrophic yeast P. pastoris is an appropriate platform for the functional expression
of membrane-associated cytochrome P450 enzymes. The advantages of P. pas-
toris over S. cerevisiae include the capability to grow in high densities in simple
media along with containing the potently regulated alcohol oxidase (AOX1) pro-
moter (reviewed in Wriessnegger et al. 2014). Bearing these in mind, the first
heterologous production of (+)-nootkatone in P. pastoris was done by Wriessneg-
ger et al. (2014). In this study, P. pastoris was first used as a whole-cell biocatalyst
for the production of (+)-nootkatone from (+)-valencene. Therefore, a strain co-
expressing the premnaspirodiene oxygenase of Hyoscyamus muticus (HPO) and
the Arabidopsis thaliana cytochrome P450 reductase (CPR) that hydroxylated
extracellularly added (+)-valencene was generated. Then, phase transfer issues
of (+)-valencene resolved via intracellular production of (+)-valencene by co-
expression of valencene synthase from Callitropsis nootkatensis. Production of
trans-nootkatol was performed via biphasic cultivations of P. pastoris; then oxi-
dation process to (+)-nootkatone production was catalyzed by an intrinsic P.
pastoris activity. Further, overexpression of a P. pastoris alcohol dehydrogenase
and tHmg1p increased the (+)-nootkatone yield to 208 mg/L.
7 Conclusion
The flavor and fragrance industry has always been an attractive and rich industry
in the world. Today, meeting the needs of the people and the market is one of the
main challenges of these industries. (+)-Valencene and (+)-nootkatone are known
as natural flavors and aromas and are produced in low amounts in their native
hosts which requires a lot of raw material. On the other hand, although chem-
ical synthesis of these compounds has been successful, due to the side effects
and the fact of not being categorized as natural, this method is not very much
accepted and popular. Significant success has also been achieved in the biotrans-
formation/bioconversion method. In order to meet the market demand on a large
scale, high amounts of these compounds have been obtained via the heterolo-
gous production of (+)-valencene and (+)-nootkatone in microorganisms, including
yeasts S. cerevisiae, Y. lipolytica, and P. pastoris.
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Synthesis of Polyols and Organic
Acids by Wild-Type and Metabolically
Engineered Yarrowia lipolytica Strains
Chong Li, Weichao Lin, Khai Lun Ong, Jinhua Mou,

Carol Sze Ki Lin, and Patrick Fickers
Abstract
In the yeast Yarrowia lipolytica, sugar polyols and organic acids are derived
from central metabolism, namely the citrate cycle and the pentose phosphate
pathway. Although these metabolites have numerous applications in agro-food,
chemical, and pharmaceutical industries, the main challenge is to gain produc-
tivity to obtain processes that are economically viable. Y. lipolytica is known
for its ability to use industrial wastes or raw materials as feedstock and to grow
at high cell density in large-scale bioreactor. Recent advances in metabolic
engineering and synthetic biology allowed the development of efficient Y.
lipolytica-based cell factories to bioconvert these feedstocks into added-value
metabolites. This book chapter will focus on current knowledge on the synthesis
of the most important polyols and organic acids in Y. lipolytica.
C. Li · W. Lin
Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Shenzhen Key
Laboratory of Agricultural Synthetic Biology, Genome Analysis Laboratory of the Ministry of
Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of
Agricultural Sciences, Shenzhen, China
W. Lin
School of Life Sciences, Henan University, Kaifeng 47500, China
K. L. Ong · J. Mou · C. S. K. Lin
School of Energy and Environment, City University of Hong Kong, Kowloon Tong, Hong Kong
P. Fickers (B)
Microbial Process and Interaction, TERRA Teaching and Research Centre, University of
Liege – Gembloux Agro-Bio Tech, Gembloux, Belgium
https://doi.org/10.1007/978-3-030-89680-5_9
228 C. Li et al.
1 Introduction
Several polyols and organic acids have been included in the top 12 platform
chemical list of the US Department of Energy (Werpy and Petersen 2004). This
highlights their importance in nowadays industries. Historically, most of these
compounds have been produced industrially by chemical synthesis in a fossil
fuel-dependent fashion. For instance, succinic acid (SA) could be produced by
the catalytic hydrogenation of maleic anhydride (Ong et al. 2020) while erythri-
tol, four-carbon sugar alcohol, could be obtained from dialdehyde starch in the
presence of nickel as a catalyst at high temperatures (Carly and Fickers 2018).
In microorganisms, these compounds are also intermediates of biochemical
pathways, such as SA that is involved in the Krebs cycle, or cell metabolites
such as erythritol that is produced by certain osmotolerant yeasts in response to
osmotic stress (Carly and Fickers 2018). Actually, there is an increasing demand
from global drivers for greener and more sustainable processes to be developed in
the context of circular economy. With the advent of molecular technologies such
as metabolic engineering and synthetic biology, industrial microorganisms could
now be engineered to bioconvert organic and industrial wastes into value-added
chemicals using the so-called biorefinery concept. In this book chapter, we will
focus on the non-conventional yeast Yarrowia lipolytica as a cell factory and detail
recent advances made in strain and process engineering to produce polyols and
organic acid of biotechnological interest.
2 Yarrowia lipolytica
Y. lipolytica is an Hemiascomycetes yeast isolated from dairy products, dry

sausages, lipid-rich or alkane-containing matrix (polluted soil and sewage) or
hypersaline environments such as seawater (Nicaud 2012). This yeast has specific
metabolisms for alkanes and triglycerides catabolism (Fickers et al. 2005). Also,
it presents other metabolic features such as the ability to sustain high osmotic
pressure owing to the synthesis of a specific metabolite, namely erythritol, or
the ability to synthesize and secrete enzymes (lipase, protease) with high effi-
ciency (up to several g/L). Interest in Y. lipolytica started in the mid-60s for the
production of single-cell proteins (SCP) from alkanes and later the yeast was
a research focus for heterologous protein production. It is also considered as a
model organism to study dimorphism as Y. lipolytica could grow either as yeast-
like cell and pseudo-filamentous forms (Barth and Gaillardin 1996). Y. lipolytica
has gained a GRAS status (Generally Recognized As Safe) that is of interest for
the development of food-related applications (Groenewald et al. 2014). The com-
plete annotated genome from strain E150 was released in 2004 (Dujon et al. 2004)
and the genome of several Yarrowia strains are now publicly available (http://gryc.
inra.fr/). Numerous molecular tools have been also developed such as efficient pro-
moters and vectors for fine-tuned gene expression (Shabbir Hussain et al. 2016;
Synthesis of Polyols and Organic Acids by Wild-Type … 229
Sassi et al. 2016; Trassaert et al. 2017; Park et al. 2019), DNA assembly meth-
ods (Wong et al. 2017; Vandermies et al. 2017; Celińska et al. 2017; Larroude
et al. 2019), gene disruption systems (Fickers et al. 2003), and genome editing
tools including CRISPR/Cas9 (Larroude et al. 2020). All these technologies have
been recently reviewed (Abdel-Mawgoud et al. 2018; Larroude et al. 2018; Bilal
et al. 2020). Efficient strategies have been also developed to cultivate Y. lipolytica
in bioreactors with the aim to maximize recombinant protein or metabolite pro-
duction titers (Do et al. 2019; Vandermies and Fickers 2019). Also, review papers
related to Y. lipolytica physiology have been published (Barth and Gaillardin 1996;
Fickers et al. 2005; Nicaud 2012).
3 Organic Acid
Y. lipolytica is considered as a model organism to study the production of organic

acids from different carbon sources including glycerol, glucose but also waste
carbon sources (waste cooking oil, pineapple waste, olive-mill wastewater, or rape-
seed oil, Table 1). Organic acids such as citric acid, iso-citric acid, succinic acid,
itaconic acid, and α-ketoglutaric acid which are all intermediates of Krebs cycle
will be discussed in this section. The main production data for these organic acids
are summarized in Table 1.
3.1 Citric Acid
Citric acid (CA) is a tricarboxylic organic acid that plays a central role in the
metabolism of all aerobic organisms. It is an intermediate of the Krebs cycle
(Fig. 1). Based on its non-toxic, pH buffering, and chelating properties, CA has
become an important industrial product. Applications for CA include the food,
cosmetic, and pharmaceutical industries, where it is used as a flavoring agent,
antioxidant, preservative, or pH buffering system. About 70% of the global total
CA production is used by the food industry, 20% by detergent, and the remaining
10% by the chemical and pharmaceutical industries (Carsanba et al. 2019). The
global CA production exceeded 2 million tons in 2018. The market is projected
to reach a volume of nearly 3 million tons by 2024, with an expected Compound
Annual Growth Rate (CAGR) of 4% between 2019 and 2024 (Fickers et al. 2020;
Ciriminna et al. 2017). Historically, CA was isolated from citrus fruits, but this
method was displaced by fermentation of sugars using the filamentous fungus
Aspergillus niger. In bench-top bioreactors, a production titer of 130 kg/m3 could
be obtained after five to eight days of batch cultivation (Ciriminna et al. 2017).
Because of its high productivity, A. niger dominated the market for CA production.
However, the processes developed for CA production from molasses are multi-
stage, are not environmentally friendly, and are being limited by the raw material
sources used (Morgunov et al. 2013).
230 C. Li et al.
Table 1 Some examples of organic acid produced in Y. lipolytica

Y. lipolytica strain Genetic Medium Organic acid, References
modification production
(g/L)
AJD-pADUTGut1/2 GUT1OE , Glycerol 93, CA Mironczuk et al.
GUT2OE (2016)
SWJ-1b acl1, Inulin 84, CA Forster et al.
ICL OE (2007b)
Cooking oil 80, CA Papanikolaou
et al. (2008)
W29 YHM2OE , Glucose 97, CA Liu et al. (2013)
AMPD OE
NG40/UV7 MT Glycerol-containing 122.2, CA Carsanba et al.
wastes (2019)
NCIM 3589 WT Pineapple waste 202.4, CA Wojtatowicz
et al. (1991)
A101-1.22 MT Glycerol 124.2, CA Yuzbasheva et al.
(2019)
A101-1.44 MT Glucose 100, CA Morgunov et al.
(2013)
K57 WT Glucose 72.1, CA Rymowicz et al.
(2010)
VKM Y-2723 WT Rapeseed oil 70.6, ICA Forster et al.
(2007a)
Ethanol 109.6, ICA Kamzolova et al.
(2016)
704-UV4-A/NG50 MT Rapeseed oil 86, ICA Forster et al.
(2007a)
Strain 20 ACO1OE Rapeseed oil 72.6, ICA Kamzolova et al.
(2018)
PSA3.0 EO Glucose 76.8, SA Tan et al. (2013)
PSA02004 WT Sugarcane bagasse 33.2, SA Yang et al.
hydrolysates (2017)
Fruit and vegetable 140.6, SA Li et al. (2018a)
waste
H222-AZ2 Ylshd5OE Glycerol 25, SA Bondarenko
et al. (2016)
PGC01003 Ylshd5 YPD medium 65.7, SA Jost et al. (2014)
Food waste 87.9, SA Jost et al. (2014)
hydrolysate
Glycerol 160, SA Yuzbashev et al.
(2010)
(continued)
Table 1 (continued)
Y. lipolytica strain Genetic Medium Organic acid, References
modification production
(g/L)
PGC202 Ylshd5, ach; Glycerol 110.7, SA Cui et al. (2017)
PCKEO,
SCS2EO
Mix food waste 31.7, SA Li et al. (2016)
PO1f-CAD CADEO Glucose 0.033, IA Blazeck et al.
(2015)
PCM-ACO-MFS MTT EO , Glucose 22, IA Zhao et al.
CADEO (2019)
H355 WT N-alkanes 195, KG Otten et al.
(2015)
H355A IDH1EO , Raw glycerol 186, KG Chin et al.
PYC1EO (2015)
VKM Y -2412 WT Ethanol 172, KG Chernyavskayaá
et al. (2000)
Rapeseed oil 102.5, KG Yin et al. (2012)
RoPYC2 RoPYC2EO Glycerol 62.5, KG Kamzolova et al.
(2012)
Abbreviations: OE: gene overexpression, WT: wild-type, MT: mutant strain (obtained by chem-
ical and/or UV mutagenesis), ME: evolved strain, CA: citric acid, ICA: iso-citric acid, SA: suc-
cinic acid, IA: itaconic acid, KG: ketoglutarate. GUT1: glycerokinase, GUT2: glyceraldehyde-3-
phosphate dehydrogenase, ACL1: ATP citrate lyase, ICL: isocitrate lyase, YHM2: mitochondrial
citrate carrier, YIAMPD: adenosine monophosphate deaminase, ACO1: aconitase, Ylshd5: succi-
nate dehydrogenase subunit 5, ACH: acetyl-coenzyme A hydrolase, PCK: phosphoenolpyruvate
carboxykinase, SCS2: succinyl CoA synthetase, CAD:cis-aconitate decarboxylase, MTT: mito-
chondrial cis-aconitate transporter, IDH1: isocitrate dehydrogenase, PYC1: pyruvate carboxylase,
RoPYC2: pyruvate carboxylase
In a study by Kamzolova and Morgunov, 43 different wild yeast isolates were

evaluated for CA production (Kamzolova and Morgunov 2017). Among these, Y.
lipolytica was found as the best CA producer on minimal medium with titer and
yield of 85 g/L and 0.70 g/g, respectively (Kamzolova and Morgunov 2017). Based
on this special ability and other biochemical characteristics, Y. lipolytica has been
recognized as a potential host to produce Krebs cycle intermediates, including
CA. In addition, cultivation conditions such as pH 4.5–5.0, temperature of 28 °C,
and glycerol concentration in culture medium from 20 to 80 g/L were found the
optimal values for CA production (Morgunov et al. 2013). The main drawback
of using Y. lipolytica to produce CA is the co-production of iso-CA (ICA) in high
amounts (Cavallo et al. 2017). However, several authors have reported that nitrogen
limitation during yeast growth could alleviate this disadvantage. Indeed, nitrogen
starvation activates adenosine monophosphate (AMP) deaminase (Beopoulos et al.
2009a; b). This leads to a decrease in the mitochondrial AMP concentration that
inhibits the iso-citrate dehydrogenase. This finally blocks the Krebs cycle at the
232 C. Li et al.
Fig. 1 Overview of the principal metabolic pathways for organic acid synthesis in Y. lipolytica.
GK: glycerokinase; GDH: Glyceraldehyde-3-phosphate dehydrogenase; PCK: Phosphoenolpyru-
vate carboxykinase; PYC: pyruvate carboxylase; PDC: pyruvate decarboxylase; ACS: acetyl-
coenzyme A synthetase; ACH: acetyl-coenzyme A hydrolase; CIT: citrate synthetase; ACO: aconi-
tase; IDH: isocitrate dehydrogenase; KGDH: αketoglutarate dehydrogenase; SCS: succinyl CoA
synthetase; SDH: succinate dehydrogenase; FUM: fumarase; ACL: ATP citrate lyase; ICL: isoci-
trate lyase; MLS: malate synthase; PEP: phosphoenol pyruvate; CAD: cis-aconitate decarboxylase
ICA stage allowing accumulation of early metabolites in the mitochondria, such

as CA (Fig. 1) (Beopoulos et al. 2009a; b). Compared to other limiting factors,
nitrogen starvation yielded to high accumulation of CA. Indeed, upon limitation
of nitrogen, phosphorus, or sulfur, CA could accumulate in the culture medium up
to 18.0 g/L, 16.0 g/L, and 16.8 g/L, respectively (Morgunov et al. 2013).
Low pH value is considered as a limiting factor for CA production by Y. lipoly-
tica wild-type strains (Tomaszewska et al. 2014). However, the engineered strain
AJD pADUTGut1/2 was obtained by overexpression of GUT1 and GUT2 genes
encoding, respectively, glycerol kinase (YALI0F00384g) and glycerol-3-phosphate
dehydrogenase (YALI0B02948g) were able to produce CA efficiently from glycerol
at pH 3 (Mironczuk et al. 2016). Initially, the aim of these modifications was to
improve erythritol production (see below), which was indeed achieved when strain
AJD pADUTGut1/2 was grown under high osmotic pressure (Mironczuk et al.
2016). However, in the absence of osmotic stress, CA becomes the main metabo-
lite produced during culture. Moreover, it was shown that overexpression GUT1
alone significantly increases glycerol uptake rate, whereas in the strain overex-
pressing GUT2 gene, higher quantities of CA were obtained. This suggested that
GUT2 could be crucial for CA production (Mironczuk et al. 2016). With strain
Y. lipolytica AJD pADUTGut1/2, CA titer of 63.9 g/L was obtained at pH 3.0
which corresponds to a 14.5-fold increase as compared to the wild-type strain
A101 under the same experimental conditions. On glycerol-based medium, a CA
titer of 93 g/L was obtained at pH 6.0 (Mironczuk et al. 2016). The purification
of glycerol is an expensive process, leading to CA production process unprof-
itable. As an economical alternative, crude glycerol, which is a direct byproduct
of biodiesel production or saponification could be used. However, the utilization
of such substrates could be limited by their high content of impurities such as
salts, sodium hydroxide, methanol, or other organic compounds, which could be
toxic for yeasts or hinder the production process. Despite these facts, Y. lipolytica
can easily grow on such substrates and was found to produce CA with titer of
76 g/L at pH 3 (Rzechonek et al. 2018a). Overexpression of gene ICL1 encod-
ing iso-citrate lyase led to a decrease in ICA production from 10–12 to 3–6%
(Forster et al. 2007b). However, the overexpression of ICL1 did not influence the
total production of CA and ICA. When ICL1 overexpression was coupled with the
disruption of ACL1 gene (YALI0E34793g) encoding ATP citrate lyase and over-
expression of the K. marxianus INU1 gene encoding inulinase, CA and ICA titers
of 84.0 g/L and 1.8 g/L were obtained, respectively, from inulin (100 g/L) after
214 h of culture in a 2L scale bioreactor (Liu et al. 2013). Besides this, overex-
pression of genes encoding mitochondrial citrate carrier (YHM2, YALI0B10736g)
and adenosine monophosphate deaminase (AMDP, YALI0E11495g) in Y. lipolyt-
ica wild-type strain W29, a CA titer with productivity and yield from glucose of
97.1 g/L, 0.93 g/(L h) and 0.5 g/g were obtained, respectively, during culture in
bioreactor (Yuzbasheva et al. 2019).
The CA production by different Y. lipolytica wild-type strains has been also
reported. With an acetate-negative mutant strain Y. lipolytica A101-1.22 grown
on glycerol-containing waste from the biodiesel industry, a CA titer of 124.2 g/L
was obtained in repeated batch culture (Rymowicz et al. 2010). From a screening
study, strain K57 was found able to produce CA with titer of 72.1 g/L and yield
of 0.77 g/g on glucose-based medium during batch culture (Carsanba et al. 2019).
Industrial wastes have been also considered as possible carbon substrates for
CA production (Wojtatowicz et al. 1991; Imandi et al. 2008; Papanikolaou et al.
2008; Morgunov et al. 2013; Liu et al. 2014; Morgunov and Kamzolova 2015).
Using mutant strain Y. lipolytica NG40/UV7 and glycerol-containing wastes, CA
production was found to increase by 40.6% (122.2 g/L) as compared to that
obtained with pure glycerol (Morgunov et al. 2013; Morgunov and Kamzolova
2015). Pineapple wastes were also used successfully with a production titer of
202 g/kg of waste in optimized conditions (namely, 0.34% yeast extract, 70.71%
moisture content of the substrate, 0.64% KH2 PO4, and 0.69% Na2 HPO4 ) (Imandi
et al. 2008). Olive-mill wastewater (OMW) blended with glucose was used also
for CA production in nitrogen-limited conditions. The titer obtained was 28.9 g/L
(Papanikolaou et al. 2008). With Y. lipolytica SWJ-1b grown in the presence of
waste cooking oil (80 g/L), a CA titer of 31.7 g/L was obtained within 336 h
of culture in a 10 L bioreactor (Liu et al. 2014). The highest CA concentration,
234 C. Li et al.
100 g/L, was obtained by the mutant strain A-101–1.14 following the shortest
growth (24 h) and production (80 h) phases from potato starch (Wojtatowicz et al.
1991).
3.2 Iso-citric Acid
Iso-citric acid (ICA) exists in the form of four stereoisomers, of which only the
threo-Ds-form is an intermediate of Krebs cycle (Kamzolova et al. 2016, 2018).
A promising application of ICA is sports medicine. ICA has a marked energetic
and anti-hypoxic effect and can be used as a physiological stimulant of sports-
men undergoing intensive long-term physical training (Kamzolova et al. 2016).
Recently, ICA has been tested as a natural prophylactic and therapeutic agent. It
has been shown efficient for the treatment of iron-deficiency anemia and in the
resorption of blood clots (Morgunov et al. 2019).
At present, ICA is produced via chemical synthesis. However, this results in a
racemic mixture of stereoisomers that cannot be separated by chemical methods.
ICA can be found also in the leaves and stems of some plants from the Cras-
sulaceae family or in fruits such as blackberry and blackcurrant. However, the
purification of ICA from plant extracts or fruit juices that contain a wide range of
organic acids and other components is a complicated and very expensive techno-
logical process. Therefore, the development of biotechnological methods for ICA
production is extremely important. At present, the most promising method for ICA
production is considered to be microbiological synthesis (Kamzolova et al. 2016;
Laptev et al. 2017).
It is known that Y. lipolytica accumulates ICA and CA in the culture medium
when cell growth is nutrients limited. Moreover, the ICA/CA ratio greatly depends
on the carbon source used for cell growth. For example, wild-type strains of Y.
lipolytica secrete mainly CA and about 8–16% ICA on carbohydrates or glycerol
while approximately 50–65% CA and 35–50% ICA on substrates like alkanes,
triglycerides, ethanol, or acetate. When using ethanol as a carbon source, an ICA
proportion of 35–67% was found depending on the cultivation conditions (Kam-
zolova et al. 2016; Holz et al. 2009). Some authors have conducted several studies
to increase the ICA production titer. The recombinant Y. lipolytica H222-S4 strain
deleted for ICL1 gene, when grown on glycerol or glucose, showed only a smaller
enhancement (by 2–5%) of ICA /CA ratio. With Y. lipolytica H222-S4 T5 that
overexpress ICL1 gene, the relative ICA content in the medium was as low as
5–7% of the total acids (CA and ICA) (Forster et al. 2007a, b).
Y. lipolytica VKM Y-2723 and its mutant derivative 704-UV4-A/NG50 were
selected from 60 yeast strains for their ability for ICA production. Under optimal
culture conditions (i.e., iron concentration of 1.2 mg/L, temperature of 29 °C, pH
6.0, pO2 50–55% of saturation, and 30 mM itaconic acid), Y. lipolytica VKM
Y-2373 produced 70.6 g/L ICA and 22.4 g/L CA with an ICA/CA ratio of
1:0.32 when grown on rapeseed oil. In similar conditions, a titer of 86 g/L ICA
and 20 g/L CA with an ICA/CA ratio of 1:0.23 was obtained with Y. lipolyt-
ica 704-UV4-A/NG50 (Kamzolova et al. 2013). Under the above optimal culture
conditions, Y. lipolytica VKM Y-2723 produced 90.5 g/L ICA with a yield of
0.77 g/g (Kamzolova et al. 2018). Also, catalytic activities of enzymes such as
alcohol dehydrogenase, citrate synthase, aconitate hydratase, iso-citrate dehydro-
genase, and iso-citrate lyase but not aldehyde dehydrogenase were found higher
in a repeated-batch cultivation of 748 h than in batch cultivation. Therefore, under
optimal repeated-batch culture using ethanol as substrate, Y. lipolytica strain VKM
Y-2723 produced 109.6 g/L ICA with a production rate of 1.35 g/L/h (Morgunov
et al. 2019). Besides this, overexpression of ACO1 (YALI0D09361g) encoding
aconitase allowed to increase ICA titer (Laptev et al. 2017; Holz et al. 2009). For
the wild-type strain H222 and H222-S4 grown on sunflower oil, the ICA propor-
tion ranged between 35 and 49%, whereas it increased up to 71% with the ACO1
multi-copy transformant T1 without any modification in the total organic acid titer
(both CA and ICA). However, ICA production was only moderately increased from
8–12% up to 13–17% with carbon sources such as glucose, sucrose, and glycerol
(Holz et al. 2009). When ACO1 was expressed in multicopy, ICA and CA titers
were, respectively, of 72.6 g/L and 29.0 g/L with an ICA/CA ratio of 2.5:1 during
culture in 10L bioreactor in a rapeseed oil-based medium (Laptev et al. 2017).
3.3 Succinic Acid
As an almost ubiquitous metabolite in many organisms, succinic acid (SA) is an

intermediate of the Krebs cycle. Due to its numerous potential applications, SA
has been recognized as one of the most important and high value-added bio-based
building block chemicals by the US Department of Energy (DOE) (Beauprez et al.
2010; Ahn et al. 2016). In 2004 and 2010, the US DOE reported SA as one of
the five most promising bio-based platform chemicals. SA market worldwide is
forecasted to grow at a compound annual growth rate (CAGR) of 15.7% between
2020 and 2026 (Ahn et al. 2016; Tan et al. 2014, 2020a). Among all the plat-
form chemicals, SA is currently used as a surfactant, ion chelator, additive in
agricultural and food, and pharmaceutical industries. It can be used as a precur-
sor to synthesize γ-butyrolactone, 1,4-butanedioic acid, tetrahydrofuran, and other
value-added chemicals (Ahn et al. 2016; Tan et al. 2014; McKinlay et al. 2007).
Currently, succinate is produced mainly from petroleum-derived maleic anhydride.
However, due to near future shortages of petroleum resources, severe environmen-
tal concerns related to chemical synthesis processes and to reach an economical
bio-based production of SA, extensive research works have been focusing on the
development of efficient microbial strains by metabolic engineering as well as
optimized fermentation and downstream processes (Ahn et al. 2016; Tan et al.
2013). Besides, bio-based SA production technologies can reduce greenhouse gas
emissions by 50% and energy demand by 30–40% as compared to the chemical
production process (Tan et al. 2014).
236 C. Li et al.
By contrast to prokaryotes, some yeasts, such as Y. lipolytica, are highly tol-

erant to low pH, rendering them attractive as an industrial host for SA synthesis
(Cui et al. 2017). A Y. lipolytica evolved strain, named PSA3.0, which can pro-
duce SA at low pH using glucose as substrate was selected from a long-lasting
culture in in situ fibrous bed bioreactor (isFBB) (Li et al. 2018a). Strain PSA3.0
produced SA with a titer of 18.4 g/L and yield of 0.23 g/g at pH 3.0. These val-
ues are, respectively, 4.8 and 4.6-fold higher than those obtained with the parental
strain PSA02004 at pH 3.0. Using fed-batch culture in bioreactor, a SA titer of
76.8 g/L was obtained, which is the highest value ever achieved from a glucose-
based medium at low pH (Li et al. 2018a). Furthermore, Y. lipolytica PSA02004
produces 33.2 g/L SA with a yield of 0.58 g/g and productivity of 0.33 g/L/h
from sugarcane bagasse hydrolysates. Using glucose-rich fruit and vegetable waste
(FVW) hydrolysates, SA titer of 140.6 g/L with a productivity of 0.69 g/L/h has
been obtained (Li et al. 2018b; Ong et al. 2019). Under optimal conditions of
repeated-batch fermentation, SA titer of 55.3 g/L and the maximal productivity
2.6 g/L/h was reached with Y. lipolytica strain VKPM Y3753 (Bondarenko et al.
2016).
An increase in SA titer was also achieved by replacing the native promoter of
the succinate dehydrogenase by strong promoters (Cui et al. 2017; Yuzbashev et al.
2010; Jost et al. 2014; Gao et al. 2016; Li et al. 2016, 2017; Yang et al. 2017).
Under oxygen limitation, Y. lipolytica strain H222-AZ2 obtained by exchange of
the native promoter of the succinate dehydrogenase subunit 2 encoding gene by
the inducible promoter POT1, a SA productivity of 0.152 g/L/h and titer of 25 g/L
after 165 h of culture in glycerol medium were obtained (Jost et al. 2014). With
Y. lipolytica strain PGC01003, deleted for Ylsdh5 genes encoding succinate dehy-
drogenase subunit 5, SA titer of 43 g/L was obtained during batch culture in 2.5
L bioreactor in medium containing crude glycerol. With a fed-batch strategy, the
strain produced 160 g/L SA (Gao et al. 2016). After 21 days of evolution to enable
the strain PGC01003 to catabolize glucose, the evolved strain produced 65.7 and
87.9 g/L SA using YPD medium and food waste hydrolysate, respectively (Yang
et al. 2017).
Enhanced SA produced was also achieved by genetic modifications of the
strains in succinate dehydrogenase or fermentation technology innovation. For
example, when the native promoter of SDH2 was replaced by an inducible pro-
moter of POT1, SA titer at 25 g/L with productivity at 0.15 g/L/h was obtained
by Y. lipolytica H222-AZ2 from glycerol under oxygen limitation (Jost et al.
2014) After inactivation of SDH5 that encodes succinate dehydrogenase subunit
5, SA titer at 43 g/L was obtained by batch fermentation from crude glycerol
By PGC01003, and a much higher titer was achieved at 160 g/L SA via fed-
batch fermentation strategy (Gao et al. 2016). Based on this, an in-situ fibrous bed
bioreactor (isFBB) which could improve the initial cell density was developed,
and SA titer at 198.2 g/L with average productivity of 1.46 g/L/h was achieved by
PGC01003 via fed-batch strategy (Li et al. 2016). This value was further improved
to 209.7 g/L when the immobilization matrix in isFBB was replaced by the more
porous material (e.g., sugarcane bagasse), which is the highest value ever reported
(Li et al. 2017). However, it was reported that the accumulation of acetate was
a major factor that impeded the fermentation at low pH, resulting in an obvious
increase in downstream process cost (Cui et al. 2017). In order to solve this prob-
lem, the gene encoding the acetyl-coenzyme A hydrolase (ACH1) was deleted,
and PCK from S. cerevisiae encodes phosphoenolpyruvate carboxykinase together
with SCS2 encoding endogenous succinyl CoA synthetase were overexpressed in
PGC01003 to achieve the strain PGC202. As result, SA titer at 110.7 g/L with
a yield of 0.53 g/g and productivity of 0.8 g/L/h was obtained by this strain via
fed-batch fermentation with pH without control (final pH at 3.4) (Cui et al. 2017).
Nevertheless, all the metabolic evolution of Y. lipolytica for SA production has
led to a partial or total loss of its ability to grow in glucose-based medium, which
limits its industrial application (Yang et al. 2017). In this case, a strategy termed
metabolic engineering was applied by Yang et al. to obtain a glucose-consuming
Y. lipolytica (PSA02004) after a 21-day repeated fermentation of PGC01003 (Yang
et al. 2017). As a result, the evolved strain PSA02004 could produce SA at
65.7 g/L and 87.9 g/L from the YPD medium and food waste hydrolysate at pH
6.0, respectively. Interestingly, with the help of isFBB, the pH for the cultivation
of PSA02004 could be decreased to a level lower than 3.0 gradually by metabolic
evolution, and the evolved strain named Y. lipolytica PSA3.0 that could produce
SA with a titer of 19.3 g/L, productivity of 0.52 g/L/h, and yield of 0.29 g/g at
pH 3.0 from YPD was achieved (Li et al. 2018a). The enzyme activity analysis
demonstrated that the pathway from pyruvate to acetate was partially blocked in
Y. lipolytica PSA3.0 after the evolution, which is beneficial to cell growth and SA
production at low pH.
However, as for PGC01003, the accumulation of acetate is a limiting factor
for further improvement of SA production as SA recovery request a pH adjust-
ment leading to an increase of downstream process cost (Cui et al. 2017). To
solve this issue, a new recombinant strain, named PGC202, was deleted for gene
ACH1 (YALI0E30965g) encoding the acetyl-coenzyme A hydrolase and overex-
pressing PCK gene from Saccharomyces cerevisiae encoding phosphoenolpyruvate
carboxykinase together with gene SCS2 encoding endogenous succinyl CoA syn-
thetase was constructed. It allowed improving the SA production process through
the elimination of acetic acid overflow and by-products formation. Indeed, SA titer
of 110.7 g/L with a maximum yield from glycerol of 0.53 g/g and a productivity of
0.8 g/L/h were obtained during fed-batch fermentation of strain PGC202 (Cui et al.
2017). Furthermore, the strain produces 31.7 g/L SA with a yield of 0.52 g/g and
productivity of 0.60 g/L/h in isFBB fermentation when using glucose-containing
MFWs hydrolysate as the carbon source supplemented with 3% of tryptone (Li
et al. 2019).
3.4 Itaconic Acid
Itaconic acid (IA) is naturally produced by several Aspergillus species (Blazeck

et al. 2015). The industrially versatile usability of IA and its derivatives are
238 C. Li et al.
reflected in the wide range of applications such as in plastics, styrene-butadiene

rubber, synthetic latex, super-absorbent polymers, unsaturated polyester resins, and
detergents. The field of application of these products is widespread and ranges
from paint, lacquer, paper industries, hygiene, and medical products as well as
in the construction sector (Kuenz and Krull 2018). Thus, IA was recognized as
one of 12 value-added chemicals from biomass by the US DOE in 2004 (Blazeck
et al. 2015). IA market is estimated at US$28.4 million in the year 2020 in the
USA while the world market is expected to reach $129.3 million by 2027 due to
increasing demand for bio-based chemicals (2020b). To date, two strategies have
been developed to produce IA, a chemical method based on the pyrolysis of citric
acid and a biosynthesis process that relies on the decarboxylation of cis-aconitic
acid by microorganisms (Kuenz and Krull 2018; Zhao et al. 2019).
Although current industrial production of IA is based on A. terreus fermentation
allowing titer higher than 80 g/L, A. terreus suffers from poor growth in optimal
media used for IA production and is negatively affected by shear stress in the
bioreactor, thus precluding fermentations under conventional conditions (Blazeck
et al. 2015). Moreover, a complex and expensive downstream process has been
developed at an industrial scale (Kuenz and Krull 2018; Okamoto et al. 2014;
Bellasio et al. 2015; Chin et al. 2015; Otten et al. 2015). In order to address
these concerns, Y. lipolytica was considered as an alternative IA production host
as this organism can natively grow at low pH and sustains a high flux toward
the citric acid cycle (Blazeck et al. 2015). There are currently two reports on
engineered Y. lipolytica strains used to produce IA. In the study of Blazeck et al.,
the native cis-aconitate decarboxylase (CAD) gene was overexpressed, and the
resulting strain produced IA with a titer of 33 mg/L (Blazeck et al. 2015). Further
strain optimizations of the metabolic pathway, enzyme localization, and media
optimization strategies enabled IA titer of 4.6 g/L during culture in bioreactors,
representing a 140-fold improvement. A recombinant strain which overexpresses
genes encoding the mitochondrial cis-aconitate transporter MTT and CAD allowed
an IA titer of 22.0 g/L in optimized conditions (Zhao et al. 2019). This represents
a 60-fold improvement over the initial titer (0.36 g/L).
3.5 α-ketoglutaric Acid
α-ketoglutaric acid (α-KG), an important dicarboxylic acid, is an intermediate of

the Krebs cycle. It is also involved in amino acid metabolism and has an important
role in the regulation of the balance between carbon and nitrogen metabolism in
many microorganisms. α-KG is widely applied in the industrial scope, e.g., as
a building block for the chemical synthesis of heterocycles, dietary supplement,
component of infusion solutions, and wound healing compounds (Otto et al. 2011;
Yovkova et al. 2013). Currently, α-KG is synthesized by chemical processes or
biosynthesis at an industrial scale. The main chemical routes utilized succinic acid
and oxalic acid diethyl esters or relied on the oxidation of glyoxylic acid with
sodium glutamate using copper as a catalyst. The drawbacks of these chemical
routes are a lack of selectivity, a low yield, high risk from manipulation of harsh
chemicals, and the generation of environmental hazards. These drawbacks sharply
increased the downstream process cost and restricted the utilization of α-KG in
food, medicine, and cosmetics applications.
Y. lipolytica is unable to synthesize the pyrimidine structure of the thiamine
molecule. Thiamine is the cofactor of α-ketoglutarate dehydrogenase (KGDH)
which is a key enzyme in the metabolism of α-KG; thus, the limitation of thi-
amine availability can reduce the activity of α-ketoglutarate dehydrogenase of the
Krebs cycle. Under conditions of thiamine deficiency, the conversion of α-KG in
the Krebs cycle is inhibited, which leads to its accumulation in the fermentation
broth (Chernyavskayaá et al. 2000; Kamzolova et al. 2012; Yin et al. 2012).
Currently, different strains producing α-KG from different carbon sources have
been reported. Y. lipolytica H355 allows α-KG titer of 195 g/L with a productivity
of 1.3 g/L/h in the medium containing a mixture of n-alkanes (Guo et al. 2016b).
Under optimal conditions, Y. lipolytica VKM Y-2412 produced 172 g/L of α-KG
with a yield of 0.70 g/g and 102.5 g/L α-KG with the yield of 0.95 g/g from
ethanol and rapeseed oil, respectively (Kamzolova et al. 2012; Kamzolova and
Morgunov 2013). However, in order to overcome the disadvantages of low yield
and accumulation of byproducts, several approaches including metabolic engi-
neering strategies and different fermentation configurations have been investigated
(Guo et al. 2016a). Y. lipolytica H355A that overexpress the iso-citrate dehydroge-
nase (IDH1) and pyruvate carboxylase (PYC1) genes can produce 186 g/L α-KG
from raw glycerol. This represents a 19% increased production as compared to
the control strain H355. Y. lipolytica-RoPYC2 which overexpress RoPYC2 gene
encoding pyruvate carboxylase produced α-KG with a titer of 62.5 g/L with only
13.5 g/L pyruvate (PA). As compared to the parental strain, α-KG production in
strain RoPYC2 increased by 35.3% while PA production is reduced by 69.8%
(Yovkova et al. 2013; Yin et al. 2012).
4 Polyols
In some biological systems, polyols are obtained by the reduction of their keto or
aldo groups into hydroxyl groups (Rice et al. 2019). As polyols, Y. lipolytica syn-
thesize mainly mannitol (MAN), which is suggested to provide cofactor NADPH
for fatty acid synthesis and erythritol (EOL). The latter being produced in response
to osmotic stress. Derivatives of erythritol, namely erythrulose (EOSE) and threitol
(TOL), are also of industrial interest and will be also discussed below (Table 2).
4.1 Mannitol
MAN is a six-carbon alcohol involved in stress tolerance in microorganisms

notably in the scavenging of reactive oxygen species (ROS) (Zhang et al. 2018;
Sekova et al. 2019). A possible role of MAN in fatty acid metabolism has been
240 C. Li et al.
Table 2 Some examples of polyols produced in Y. lipolytica

Y. lipolytica Genetic modification Medium Production, References
strain polyol
AIG GUT1OE (YALI0F00384g) Crude 11 g/L, MAN Rakicka et al.
glycerol (2017a, b)
Molasse
AMM EROE (YALI0F18590g) Glycerol 44 g/L, EOL Janek et al. (2017)
HCY118 ER10OE (YALI0D07634g), Glucose 196 g/L, EOL Cheng et al.
ER25OE (YALI0C13508g), (2018)
ZWF1EO (YALI0E22694g),
GND1EO (YALI0B15598)
Po1d TKL1OE (YALI0E06479g) Glycerol 43 g/L, EOL Carly et al.
(2017a, b)
MK1 TKL1OE (YALI0E06479g) Glycerol 51 g/l, EOL Mironczuk et al.
(2017)
MK1 TKL1OE (YALI0E06479g), Glycerol 46 g/l, EOL Mironczuk et al.
TAL OE (YALI0F15587g) (2017)
Po1d GUT1OE (YALI0F00384g), Glycerol 78 g/l, EOL Carly et al.
TKL1OE (YALI0E06479g) (2017a, b)
M53 snf1 (YALI0D02101g) Peanuts 185 g/kg, EOL Li et al. (2019)
cake
Wratislavia K1 WT Glycerol 180 g/L, EOL Rakicka-Pustułka
et al. (2020)
RIY210 EYD1OE (YALI0F01650g), EOL 0.12 g/gDCW, Carly et al.
eyk1 (YALI0F01606g) EOSE (2017a, b)
CGMCC7326 Ss-XDH OEcn Glucose 112 g/L, TOL Chi et al. (2019)
Abbreviations: MAN: mannitol, EOL: erythritol, EOSE: erythrulose, TOL: threitol, GUT1:
glycerokinase, ER: erythrose reductase, ZWF1: glucose-6P dehydrogenase, GND1: 6-
phosphogluconate dehydrogenase, TKL1: transketolase, TAL: transaldolase, SNF1: regulator
of lipid accumulation, EYD1: erythritol dehydrogenase, EYK1: erythritol kinase, XDH: xylitol
dehydrogenase
also reported (Dulermo et al. 2015). MAN production has been investigated for
several Y. lipolytica strains and different experimental conditions. For cells grown
in shake-flasks in the presence of crude glycerol, mannitol was co-produced with
EOL with titers ranging from 2.6 to 14.9 g/L. During glycerol fed-batch bioreactor
culture, strains A-15 and A UV’1 synthetized MAN with a titer of, respectively,
41.4 and 38.1 g/L, corresponding to the productivity of 0.29 and 0.28 g/(L h)
(Tomaszewska et al. 2012). With a derivative of strain AIB overexpressing GUT1
gene encoding glycerol kinase grown in a batch bioreactor on a mixture of molasse
and crude glycerol, maximal MAN titer of 11 g/L was obtained (Rakicka et al.
2017a). Similarly, using a mixture of crude glycerol and olive oil mill wastewater,
the MAN titer reached 13 g/L within 140 h with a yield from glycerol of 0.21 g/g
(Sarris et al. 2019).
4.2 Erythritol
Erythritol (EOL) is a four-carbon polyol. Its industrial interest relies on its sweet-
ening property as it has the texture and taste of table sugar. EOL has also
other interesting properties regarding its application. It is not metabolized by the
human body, and thus, it is calories-free and does not modify glycemia (Carly
and Fickers 2018). It could be synthesized by several osmotolerant microorgan-
isms, including Y. liploytica and Candida magnoliae, as an osmoprotectant. In
Y. lipolytica, EOL derives from erythrose-4P, an intermediate of the pentose phos-
phate pathway (PPP) (Fig. 2). The latter is dephosphorylated by an erythrose-4P
phosphatase (E4PP) before being reduced by an erythrose reductase (ER) into
erythritol. In this yeast, genes coding for erythrose reductase have been character-
ized (namely, YALI0F18590g, YALI0D07634g, and YALI0C13508g) (Janek et al.
2017; Cheng et al. 2018). The complete EOL catabolic pathway has been also
Fig. 2 Overview of the principal metabolic pathways for polyols and derivatives synthesis in Y.
lipolytica. DHAP: dihydroxyacetone-P; E4PP: Erythrose 4P phosphatase; ER: erythrose reduc-
tase; EDH: erythritol dehydrogenase; EK: erythritol kinase; E1PI: erythrulose-1P isomerase;
E4PI: erythrulose-4P isomerase; FBA: fructose-bisphosphate aldolase; F6P-P: Fructose-6P phos-
phatase; GK: glycerol kinase; G3PDH: glycerol-3P dehydrogenase; GPI: glucose-6P isomerase;
G6PDH: glucose-6P dehydrogenase; HK: hexokinase; MDH: mannitol dehydrogenase; PFK:
phosphofructokinase; PGDH: phopshogluconate dehydrogenase; RPE: Ribulose-P3 epimerase;
R5PI: ribose-5P isomerase; TK: transketolase; TIM: triose isomerase; TAL: transaldolase; 6PGL:
6-phosphonogluconolactonase, Ss-XDH: xylitol dehydrogenase from Scheffersomyces stipites.
Pathway in italics is heterologous
242 C. Li et al.
reported recently (Niang et al. 2020). It is first converted into erythrulose by an ery-
thritol dehydrogenase (EYD1, YALI0F01650g) and subsequently phosphorylated
into L-erythrulose-1P by an erythrulose kinase (EYK1, YALI0F01606g). Then, L-
erythrulose-1P is isomerized into D-erythrulose-4P by an erythrulose-1P isomerase
(EYL1, YALI0F01584g). Finally, erythrose-4P is generated by the activity of an
erythrulose-4P isomerase (EYL2, YALI0F01628g).
Several review papers focusing on EOL have been published, and only the
most striking information will be reported below (Carly and Fickers 2018; Reg-
nat et al. 2018; Rzechonek et al. 2018b). An efficient strategy for EOL synthesis
consisted of constitutively expressing genes encoding erythrose reductase (ER).
Overexpression of ER gene YALI0F18590g in Y. lipolytica strain AMM led to
a 20% increase in EOL titer as compared to the parental strain (44.4 g/L)
(Janek et al. 2017). This corresponded to the productivity of 0.77 g/(L h) and
a yield from glycerol of 0.44 g/g. More recently, two additional ERs have been
characterized (YALI0D07634g and YALI0C13508g) (Cheng et al. 2018). Upon
overexpression of these three ERs under the control of the strong constitutive
promoter hp4d, an EOL titer of 178 g/L was obtained from an initial glucose
concentration of 300 g/L within 84 h. This corresponded to productivity and
yield of 2.1 g/(L h) and 0.59 g/g, respectively. As ERs are NADPH-dependent
enzymes, the authors engineered the co-factor metabolism by overexpressing genes
YALI0B15598g and YALI0E22694g encoding 6-phosphogluconate dehydrogenase
(GND1) and glucose-6P dehydrogenase (ZWF1), respectively. The correspond-
ing enzymes are known to generate NADPH from NADP+ . The resulting strain,
HCY108, produced EOL with a titer of 190 g/L within 80 h of culture with produc-
tivity and yield from the glucose of 2.4 g/(L h) and 0.63 g/g, respectively (Cheng
et al. 2018). Other genes from the PPP pathway have been also overexpressed
to increase EOL titer. Overexpression of gene YALI0E06479g encoding transketo-
lase (TKL1) in strains Po1d and MK1 yielded a 19% (43 g/L) and 51% (51 g/L)
increased EOL titer, respectively. This corresponded to a productivity of 0.04
and 0.05 g/(gDCW h) (Carly et al. 2017b; Mirończuk et al. 2017). By coexpres-
sion of genes YALI0E06479g (TKL1) and YALI0F15587g encoding transaldolase
(TAL1), an increase in EOL titer and productivity of 45% and 46% was obtained,
respectively (46.7 g/L and 0.5 g/(L h) (Mirończuk et al. 2017). Gene involved
in carbon source catabolism were also overexpressed to feed the PPP pathway
with precursors (i.e., fructose-6P and glyceraldehyde-3P). Overexpression of genes
YALI0F00384g (GUT1) and YALI0B02948g (GUT2) encoding, respectively, glyc-
erol kinase (GK) and glycerol-3-P dehydrogenase (G3P-DH) allowed an EOL titer
of 78 g/L from an initial glycerol concentration of 100 g/L within 72 h in a 5-L
bioreactor (Mirończuk et al. 2016). Besides this, overexpression of genes GUT1
and TKL1 in a strain disrupted for gene EYK1 allowed an erythritol titer, produc-
tivity, and yield from glycerol of 80 g/L, 1.03 g/(L h), and 0.53 g/g, respectively
(Carly et al. 2017b). By overexpressing invertase encoding gene SUC2 from Sac-
charomyces cerevisiae and native GUT1 gene, an EOL titer of 100 g/L with a
productivity and yield of 1.1 g/(L h) and 0.67 g/g, respectively, were obtained
from a mixture of raw industrial molasses (60 g/L) and crude glycerol (100 g/L)
(Rakicka et al. 2017b). Disruption of gene SNF1 (YALI0D02101g) coding for a

regulator of lipid accumulation allowed an EOL production of 185.4 mg/g in
solid-state fermentation (SSF) using peanut press cake mixed with 40% sesame
meal and 10% waste cooking oil as substrate (Li et al. 2019). EOL production
in large-scale bioreactor was recently reported. Using raw glycerol feeding, strain
Y. lipolytica Wratislavia K1 produced EOL with titer and yield from glycerol of
180 g/L and 0.53 g/g, respectively, after 144 h of cultivation in 500 L bioreactor
(Rakicka-Pustułka et al. 2020). With metabolically engineered Y. lipolytica strain
HCY118, a production titer of 196 g/L with productivity and yield from glucose of
2.51 g/L h and 0.65 g/g, respectively, were obtained in a 30 m3 bioreactor within
78 h (Wang et al. 2020).
4.3 Erythrulose
As mentioned already, erythrulose (EOSE) is an intermediate of the erythritol

catabolic pathway. It has many applications in cosmetics as a sunless tanning agent
and in chemistry as a precursor of several drugs such as anticancer (Bengamide
E), antifungal (Tanikolide), substitute β-lactam, cytokine modulator (Cytoxazone),
or cholesterol-lowering drugs (Crestor, Zetia) (Carly et al. 2018). In a Y. lipolyt-
ica Δeyk1 derivative (RIY210), the conversion of EOSE into L-erythrulose-1P is
impaired. Therefore, such a mutant strain can convert EOL into EOSE. The con-
version rate could be increased by the overexpression of EYD1 encoding erythritol
dehydrogenase in a eyk1 genetic background. With such a strategy, an efficient
fed-batch bioreactor process was developed to convert EOL into EOSE with a con-
version rate and yield of 0.116 g/g DCW·h and 0.64 g/g, respectively (Carly et al.
2017a).
4.4 Threitol
Threitol (TOL) is a diastereoisomer of erythritol that is produced naturally as an

antifreeze molecule by the fungus Armillaria mellea as well as the Alaskan bee-
tle Upis ceramboides. TOL has application as a precursor for the synthesis of
anticancer drugs (treosulfan and threitol ceramide). It is also a constitutive ele-
ment of oxygen-sensitive pigments incorporated in a smart plastic film used for
food packaging (Chi et al. 2019). Recently, xylitol dehydrogenase (Ss-XDH) from
Scheffersomyces stipites CBS6054 was found able to oxidize EOL into EOSE irre-
versibly and then reduce EOSE into threitol (Chi et al. 2019). By overexpression
of the corresponding gene in the Y. lipolytica strain CGMCC7326, a good producer
of EOL from glucose, a TOL production titer of 112 g/L with a yield from the
glucose of 0.37 g/g has been reported (Chi et al. 2019).
244 C. Li et al.
5 Conclusion and Prospects
Due to its potential for organic acids and polyols production and good tolerance to
low pH, Y. lipolytica has gradually become a cell factory for their production. How-
ever, several key steps allowing to increase strain production titer and productivity
remain to be identified. Process operations are still not optimal, especially on a
large scale. Further investigations must be made to obtain cost-effective processes
for most of the metabolites described herein.
Acknowledgements The authors would like to thank Andrew Zicler for his contribution in editing
Figs. 1 and 2.
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Recent Advances in Synthetic Biology
Applications of Pichia Species
Wan Sun, Yimeng Zuo, Zhanyi Yao, Jucan Gao, Zengyi Shao,
and Jiazhang Lian
Abstract
The rapid development of metabolic engineering and synthetic biology has
recently attracted great attention to exploring many non-model microbial
species. Pichia pastoris and Pichia kudriavzevii are the two representative
Pichia species, which possess uniquely desired biochemical, metabolic, and
physiological features favoring large-scale industrial production. This review
begins with illustrating synthetic biology parts and tools that have been devel-
oped for manipulating these two species, followed by summarizing their
applications in biomanufacturing as hosts to produce recombinant proteins, bulk
W. Sun, Y. Zuo, Z. Yao and J. Gao contributed equally to this work.
W. Sun · Z. Shao (B)

Interdepartmental Microbiology Program, Iowa State University, Ames, IA, USA
W. Sun · Z. Yao · Z. Shao
NSF Engineering Research Center for Biorenewable Chemicals, Iowa State University, Ames, IA,
USA
W. Sun · Z. Shao
The Ames Laboratory, Ames, IA, USA
Y. Zuo · J. Gao · J. Lian (B)
College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, Zhejiang, China
Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University, Hangzhou
Zhejiang, China
Z. Yao · Z. Shao
Department of Chemical and Biological Engineering, Iowa State University, Ames, IA, USA
Z. Shao
4140 Biorenewables Research Laboratory, Iowa State University, Ames, IA, USA
https://doi.org/10.1007/978-3-030-89680-5_10
252 W. Sun et al.
chemicals, and natural products, and concludes on the challenges and potential
strategies for advancing them for broader biotechnological applications.
1 Introduction
With an increasing demand for fuels and petrochemicals, in addition to the growing
food shortage and global warming, researchers have been working on engineer-
ing microbes to generate desirable products (Choi et al. 2015; Curran and Alper
2012). Model organisms such as Escherichia coli and Saccharomyces cerevisiae
have been studied extensively and engineered as cell factories for the production
of value-added chemicals and proteins (Pontrelli et al. 2018; Lian et al. 2018).
With the rapid development of next-generation sequencing and synthetic biology
tools, non-conventional organisms with unique traits (e.g., thermotolerance (Varela
et al. 2017), stress tolerance (Abdel-Mawgoud et al. 2018), special feedstock uti-
lization (Agbogbo and Coward-Kelly 2008; Gong et al. 2015; Yaegashi et al. 2017;
Yaguchi et al. 2017), and high protein secretion capacity (Li et al. 2007)) have been
explored as well-suited hosts for industrial processes.
The most renowned Pichia species is Pichia pastoris, a methylotrophic yeast
that can utilize methanol as the sole carbon source. It can grow to a very high
cell density (>100 g/L dry cell weight) (Wang et al. 2012). The medium for
growing P. pastoris is simple and inexpensive (containing only methanol or glyc-
erol, biotin, salts, and trace elements), making it ideal for large-scale industrial
production (Cereghino et al. 2002). In addition, P. pastoris can express heterol-
ogous proteins intracellularly or extracellularly at high levels whilst minimizing
secretion of endogenous proteins. It possesses the machinery required for proper
post-translational modifications (e.g., glycosylation, disulfide bond formation, and
proteolytic processing) (Cereghino and Cregg 2000). As a “generally regarded as
safe” (GRAS) yeast, it has been engineered to produce industrial enzymes and
chemicals (Zhu 2019), therapeutic agents such as vaccines (Wang et al. 2016) and
drugs (Yu et al. 2007), and protein-based polymers (Werten et al. 2019). In recent
years, it has also been engineered to produce a leghemoglobin protein (LegH) from
soy, to create a meat-like flavor in plant-based meat products (Fraser et al. 2018).
The well-annotated genome sequence of P. pastoris (Love et al. 2016; Sturm-
berger et al. 2016) and a few genome-scale metabolic models are also available
for engineering purposes (Ye et al. 2017; Saitua et al. 2017; Torres 2019).
This review change it to summarizes the current synthetic biology parts and
tools available for P. pastoris, including promoters, terminators, plasmids, genome-
editing tools, and signal peptides, followed by a discussion on the engineering
efforts carried out to improve protein secretion and various applications of this
yeast. In addition, we have also highlighted Pichia kudriavzevii, another Pichia
species whose acid tolerance is attractive in the production of value-added organic
acids such as succinic acid (Xiao et al. 2014), D-lactic acid (Park et al. 2018),
and itaconic acid (Sun 2020). This review concludes with future perspectives in
establishing Pichia species for biotechnological applications.
Recent Advances in Synthetic Biology Applications … 253
2 Synthetic Biology Parts and Tools
2.1 Promoters and Terminators
The strength and tunability of promoters are crucial for efficient production (Porro
et al. 2005). Strong promoters are usually preferred because of the usually low
expression levels of heterologous genes, whereas tunable promoters are desirable
when multiple heterologous genes are involved in complex pathways. Commonly
used promoters include inducible and constitutive promoters. The former allows
genes of interest to be switched on or off at different stages through induction
or repression via transcription factors. In practice, applying an inducible promoter
can increase the cell density of cultures first and then initiate heterologous protein
production. Such a separation of biomass accumulation and protein production
provides a great advantage if the accumulated intermediates or products are toxic
to the cells (Cereghino and Cregg 2000; Ahmad et al. 2014; Macauley-Patrick
et al. 2005). However, using an inducible promoter requires an extra step during
cultivation (e.g., carbon source swapping or compound supplementation), which
incurs an additional cost for large-scale industrial production. In contrast, strong
and steady expression of genes of interest mediated by constitutive promoters con-
tributes to decreasing the operational cost while enhancing the yield, which is a
commonly adopted strategy if the constitutive expression does not negatively affect
cell growth (Vogl and Glieder 2013).
Inducible promoters are usually identified from unique biochemical pathways,
and constitutive promoters generally originate from housekeeping genes. The two
most commonly used promoters in P. pastoris are the inducible PAOX1 and the
constitutive PGAP (Rajamanickam et al. 2017). PAOX1 is the promoter of the alcohol
oxidase gene AOX1, which is induced by the inexpensive carbon source, methanol.
When methanol is used as a carbon source, the methanol-induced alcohol oxidase
expression can reach 30% of the total soluble protein content (Cregg et al. 1993). It
is one of the most effective promoters that have been found for protein expression
in P. pastoris (Cregg et al. 1989; Jahic et al. 2006; Koutz et al. 1989). Heterologous
protein synthesis at levels of approximately 20 g/L was achieved two decades ago
(Hasslacher et al. 1997; Werten et al. 1999). Extensive efforts have been made to
elucidate the regulatory mechanisms of PAOX1 . The determination of its cis-acting
regulatory sequence elements has allowed researchers to engineer the promoter
by means of deletion and duplication of putative transcription factor-binding sites,
yielding a library of variants with strengths ranging from 6 to 160% of the wild-
type PAOX1 (Hartner 2008). A dozen transcription factors have been identified to
be involved in the induction of PAOX1 . This work provided various PAOX1 variants
with tunable activities by combining cis-acting elements with the basal promoter
identified based on deletion analysis.
PGAP is the promoter of the glycolytic glyceraldehyde 3-phosphate dehydro-
genase gene GAP. It remains constitutively expressed, although its activity varies
when different carbon sources are used (Waterham et al. 1997). Similar to the
engineering strategy applied to PGAP , a library of PGAP variants was constructed
254 W. Sun et al.
using mutagenesis, in which the activity varied from 0.6% to 1960% of the wild-
type PGAP (Qin et al. 2011). Several transcription factors have been examined and
suggested to play roles in the regulation of PGAP . Most of the available P. pastoris
promoters are summarized in two review articles, with the strengths benchmarked
to those of PAOX1 and PGAP (Vogl and Glieder 2013; Turkanoglu Ozcelik et al.
2019). These promoters have been much less extensively studied but are highly
desired for controlling multi-gene pathways. Most of their cis-acting regulatory
sequences and mechanisms remain unclear. Beyond the ones in the two lists, there
are also a strong native promoter (PCAT1 ) from the catalase gene in P. pastoris and
a strong heterogeneous one (PMOX ) originating from the methanol oxidase gene in
Hansenula polymorpha. The former is induced by methanol with the PCAT1 variant
P4 even stronger than PAOX1 (Nong et al. 2020); the latter was completely inac-
tivated in the presence of xylose and sorbitol but showed strong activities in the
glucose, glycerol, and methanol feeds (Mombeni 2020).
In conjunction to a promoter, a terminator (tt) also plays a critical role in reg-
ulating the expression level, mainly by influencing the mRNA stability and the
subsequent translation process (Shalgi et al. 2005). However, compared with pro-
moter engineering, much less attention has been focused on terminators. In S.
cerevisiae, hundreds of terminators have been examined and characterized, which
have been demonstrated to regulate protein expression levels over a broad range,
whereas in P. pastoris, only a few recent studies have investigated the impact of
terminators.
In P. pastoris, both endogenous and heterogeneous terminators have been used
for heterologous expression. The commonly used endogenous terminators are
mostly from either the methanol utilization pathway (i.e., AOX1tt) or housekeep-
ing genes (i.e., GAPtt), whereas heterogeneous terminators are from other yeasts
such as S. cerevisiae (e.g., CYC1tt from cytochrome C isoform 1, PRM9tt from
pheromone-regulated membrane protein 9, and VPS13tt from vacuolar protein
sorting-associated protein 13), H. polymorpha (e.g., MOXtt from methanol oxi-
dase), and Kluyveromyces lactis (e.g., LAC4tt from beta-galactosidase). To date,
the impact of different terminators on heterologous expression has been primarily
examined by evaluating the expression level under the control of PAOX1 and PGAP .
Vogl et al. examined 20 terminators, of which 15 originated from the endoge-
nous methanol assimilation pathway and the remaining five from S. cerevisiae.
These terminators provided comparable expression levels of green fluorescent pro-
tein (GFP) under the control of PAOX1 , with the lowest active terminator still
reaching 57% of the highest active terminator (Vogl et al. 2016). Prielhofer et al.
focused more on the terminators from highly expressed endogenous genes, includ-
ing many ribosomal terminators. Using CYC1tt from S. cerevisiae as a reference,
all ten terminators yielded similar GFP levels under the control of PGAP (Priel-
hofer et al. 2017). Interestingly, in both studies, the heterogeneous terminators of
genes of interest offered comparable and sometimes even higher activities than the
endogenous ones, indicating that the terminators isolated from other yeasts could
be effectively recognized in P. pastoris. Recently, Ito et al. created a catalog of 72
terminators, including 28 endogenous terminators, 41 heterogeneous terminators
from S. cerevisiae, and three strong synthetic terminators developed originally for
S. cerevisiae. Under the control of PGAP , these terminators resulted in a 17-fold
degree of tunability in P. pastoris (Ito et al. 2020). In these studies, AOX1tt seemed
to yield the highest activity, regardless of the promoter being used (Karbalaei et al.
2020; Weninger et al. 2016).
A recent study using Candida antarctica lipase B (CALB) as a reporter pro-
tein showed that the activities of terminators are closely associated with those
of promoters (Ramakrishnan et al. 2020). Ten terminators from the endogenous
methanol utilization pathway, glycolysis, tricarboxylic acid (TCA) cycle, and other
housekeeping genes and five terminators from S. cerevisiae were compared. Their
activities were estimated by evaluating the corresponding CALB activity under the
control of PAOX1 and PGAP . Compared to AOX1tt, three terminators led to lower
lipase activities when paired with PAOX1 but higher activities when paired with
PGAP , which suggested that the performance of terminators is not insulated from
promoter influences and may also be subjected to regulatory mechanisms as seen
in promoter studies. However, the mechanism by which individual terminators
enhance expression along with different promoters remains unclear in P. pastoris.
In addition, the terminator of dihydroxyacetone synthase (DHAStt) provided a
slightly higher CALB expression level than AOX1tt under the control of PAOX1 , but
nearly threefold higher activity under the control of PGAP . Therefore, DHAStt can
potentially serve as a strong terminator when seeking high heterologous expression
in P. pastoris. In general, terminators play a critical role in protein expression, but
more studies are needed to elucidate terminator-mediated regulatory mechanisms.
2.2 Episomal Plasmids and Integration Plasmids
Most of the protein expression and metabolic engineering tasks in P. pastoris were
achieved through genome integration, many of which targeted the AOX1 gene via
homologous recombination (HR) (Cereghino and Cregg 2000). However, unlike S.
cerevisiae, non-homologous end joining (NHEJ) is the dominant mechanism for
repairing chromosomal double-stranded breaks (DSBs) in P. pastoris. Transfor-
mants with targeted integration have to be identified using a laborious screening
process because of the uncontrollable random integration events, including large-
scale relocation of an integration locus, off-target integration potentially affecting
cell growth, and even co-integration of the DNA elements originating from the
F-plasmid and the genome of the E. coli host used to prepare the shuttle plasmid
(Schwarzhans et al. 2016).
An alternative strategy is to use replicative plasmids, which yield higher trans-
formation efficiency and are easier to screen, although stability is sometimes an
issue in this case (Lee et al. 2005). An autonomously replicating sequence (ARS)
is a key element in replicative plasmids. The first P. pastoris-specific ARS, desig-
nated PARS1, was identified over 35 years ago and enabled the use of replicative
plasmids with high transformation efficiency (Cregg et al. 1985). Over the past
decade, other elements have been discovered that can serve as alternative ARSs
256 W. Sun et al.
in P. pastoris, such as a 452 bp panARS identified from K. lactis and a 1442 bp

mitochondrial DNA fragment from P. pastoris itself (Liachko and Dunham 2014;
Schwarzhans et al. 2017). Unfortunately, plasmids using these ARSs demonstrate
poor stability during mitotic segregation, which is fatal for industrial applica-
tions. Recent studies suggest that this inherent instability is caused by the lack
of a centromere (CEN), another genomic element that guides stable segregation of
chromosomes, and therefore, can be used to improve plasmid stability during cell
division (Cao et al. 2017a).
CENs are DNA sequences recognized by kinetochore complexes, which sub-
sequently interact with spindle microtubules and enable equal partitioning of
chromosomes to the two dividing cells during mitosis and meiosis. In S. cere-
visiae, a 125 bp CEN and an ARS have been widely applied as a combination
in all low-copy episomal plasmids. Like ARS, a CEN is also species-specific,
and recent studies have identified four putative CENs corresponding to the four
P. pastoris chromosomes (Sturmberger et al. 2016; Coughlan et al. 2016). In a
more recent study, a new autonomously replicating plasmid was constructed, har-
boring an entire putative centromeric region from chromosome 2 (Cen2). The
plasmid can be replicated and stably distributed in P. pastoris. Within this Cen2, a
~111 bp sequence was found to enable autonomous replication, which can serve
as a new ARS (Nakamura, et al. 2018). Another study confirmed that the entire
CENs from chromosomes 1 and 4 (Cen1 and Cen4) could confer replicative sta-
bility to plasmids (Piva 2020) although Cen4 did not support a high number of
transformants in a separate study (Nakamura, et al. 2018). Although these new
plasmids exhibit relatively high stability and have the potential to expedite cloning
and high-throughput screening, harboring the entire Cen sequence may lead to
other undesired outcomes. First, this kind of plasmid can only be maintained at
low copy numbers due to their chromosome-like segregation mechanism, in con-
trast to those plasmids using PARS1. Moreover, the sizes of the plasmids will be
much larger since all Cen sequences are above 6 kb in length, which does not ben-
efit transformation, especially when large pathways are cloned. Lastly, having the
entire centromeric sequence may cause unexpected genomic integration or DNA
exchange with chromosomes, which increases the difficulty of screening.
Therefore, genome integration appears to be an alternative for heterologous
gene and pathway expression before the functioning mechanisms of ARS and CEN
are clearly elucidated and the sequences are optimized in P. pastoris. Genome inte-
gration is generally achieved via a single crossover or double crossover. A single
crossover requires that the circular vector contains a sequence identical to the tar-
get locus in the P. pastoris genome. After transformation, the linearized vector,
including genes of interest, a selectable marker, and backbone, will be inserted
into the target site, leaving two copies of the target site flanking the inserted vec-
tor. The PAOX1 region and the auxotrophic gene HIS4 have been widely selected
as the target sites for single crossover-mediated integration, with an efficiency
of 50–80% (Cereghino and Cregg 2000). However, this kind of integration will
introduce elements beyond the expression cassette, such as the elements respon-
sible for the replication of the shuttle vector in E. coli. In addition, a second
single crossover may occur again on the genome between two identical target
sequences, especially when the selection pressure is removed, which results in a
loss of integrated expression cassette. To achieve a double crossover, a selection
marker-containing expression cassette flanked by two homologous arms to the tar-
get site is usually transformed, resulting in the direct replacement of the genomic
sequence located between the two homologous sequences by the expression cas-
sette. Flanking the desired genes of interest and a selectable marker with the 5’
and 3’ AOX1 sequences leads to the disruption of AOX1, thereby changing the phe-
notypic substrate utilization of P. pastoris. Lastly, the selection markers commonly
used for screening include auxotrophic genes such as HIS4, URA3, and ADE1, as
well as the genes encoding resistance to zeocin and G418 sulfate (Daly and Hearn
2005; Papakonstantinou et al. 2009).
2.3 Genome Editing and Integration Loci
In addition to integration vectors or fragments, the Clustered Regularly Interspaced

Short Palindromic Repeats (CRISPR) technology has become a revolutionary tool
for achieving genomic integration in P. pastoris because Cas9, in principle can
target any accessible locus containing a 2–6 bp protospacer adjacent motif with
the help of a guide RNA (gRNA) molecule. However, it has been reported that
a Cas9/gRNA complex may lead to toxic effects due to off-targeting and there-
fore, the expression level of Cas9 needs to be at an appropriate level to yield a
desired efficiency (Weninger et al. 2016). For example, using a weaker promoter
for Cas9 expression can lead to higher transformation efficiencies and growth rates
(Prielhofer et al. 2017). Cas9 cleaves a genome and introduces a DSB that can
be repaired by the NHEJ machinery when donor DNA is not provided, result-
ing in insertion and deletion (indel) mutations at the target locus. In P. pastoris,
the inherently strong NHEJ activity enables highly efficient multi-locus disruption
or deletion with co-transformation of different gRNAs. For example, Weninger
et al. successfully mutated GUT1 and AOX1 simultaneously by expressing two
gRNAs on a CRISPR/Cas9 plasmid. By testing different combinations of gRNAs,
the highest double-editing efficiency of 69% was obtained (Weninger et al. 2016).
However, the active NHEJ machinery is a hurdle for achieving precise locus-
specific integrations via HR. When a donor DNA is provided, HR must compete
with the predominant NHEJ to repair the DSB, and under the selection pressure,
the marker can be randomly integrated via NHEJ. The HR activity in the wild-
type P. pastoris is naturally much lower than that of NHEJ, yielding many false
positives (Li et al. 2007). Therefore, it is much more difficult to achieve precise
integration in P. pastoris than in other yeast species with HR dominancy, such as
S. cerevisiae. This limitation can be overcome by deleting the KU70 and KU80
genes encoding the two proteins comprising a heterodimer threading onto the bro-
ken DNA ends. In a recent study, it was reported that upon knock-out of KU70,
258 W. Sun et al.
NHEJ was repressed to a great extent, thereby heightening the role of HR in repair-
ing DSB repair and raising the target integration efficiency to approximately 100%
(Weninger et al. 2016, 2018).
In addition to repressing NHEJ, future studies should focus on enhancing HR
performance to increase editing efficiency when using CRISPR/Cas9 in P. pastoris.
For example, the overexpression of RAD family recombinases can be considered.
In S. cerevisiae, overexpression of Rad51 has been shown to elevate the integra-
tion correctness and overexpression of an engineered Rad51 variant, which has a
higher affinity to recombinase Rad54, thus significantly increasing the targeting
efficiency in S. cerevisiae (Liu et al. 2004). Furthermore, expressing Rad52 from
S. cerevisiae in Yarrowia lipolytica has been shown to increase the targeting effi-
ciency from 15 to 95% (Ji 2020). When KU70 was disrupted, certain chemicals,
such as hydroxyurea, were applied to synchronize Y. lipolytica cells to the S-phase
of the cell cycle, which is when HR has the highest activity (Jang 2018). Similar
strategies can be used to enhance the HR performance in P. pastoris.
Theoretically, any locus on a genome can serve as a potential target site for
integration, except for those related to essential genes. However, several additional
features directly affect the integration efficiency. First, a higher accessibility ren-
ders a higher chance for the Cas9/gRNA complex, and later, the donor DNA to
form a complex with the genome during the cutting and repair process, which is
especially important for the integration of large pathways. AOX1, DAS1, DAS2,
and GUT1 have been widely targeted owing to their easy accessibility (Pena et al.
2018). In addition, the nano-environment surrounding the integration locus is key
to determining the expression level and dynamics of the integrated gene. Although
a high expression level does not necessarily lead to high production, it is usu-
ally preferred by the rate-limiting step for the synthesis of a product through a
multi-step pathway.
Increasing the copy number of a gene of interest has been found to be an
efficient way to increase heterologous protein production. Accordingly, ribosomal
RNA (rRNA)-encoding loci, namely ribosomal DNA (rDNA), have been popu-
lated because of their highly repeated sequences, where several loci can be targeted
simultaneously using a single plasmid containing one gRNA design (Marx et al.
2009). In P. pastoris, each repeat consists of 25S, 5.8S, and 18S rRNA genes
arranged identically in a head-to-tail tandem array and a non-transcribed spacer
(NTS) is located between two rDNA repeats. The sequences and locations of
these rDNA repeats on the chromosomes in many yeasts have been specified for
decades. For example, Wang et al. successfully integrated ten copies of the resver-
atrol biosynthetic pathway consisting of genes from Herpetosiphon aurantiacus,
Arabidopsis thaliana, and Vitis vinifera, onto the NTS regions of O. polymor-
pha simultaneously via CRISPR/Cas9, without using a selection marker (Wang
et al. 2018). In Y. lipolytica, Luu et al. obtained an integrant with eight copies
of the genes encoding capsid proteins originating from red-spotted grouper ner-
vous necrosis virus at the 26S rRNA loci through HR (Luu et al. 2017). In P.
pastoris, high copy-number integration of human serum albumin and human super-
oxide dismutase was achieved by targeting the NTS region of the rDNA locus
and repeated selection on increasing the concentration of zeocine™ (Marx et al.

2009). In addition to expressing proteins as products, this strategy has also been
applied to enhance the production of D-lactic acid in P. pastoris by integrating
the D-lactate dehydrogenase gene (D-LDH) into the rDNA locus, followed by
copy number amplification enabled by gradually increased antibiotic concentration
(Yamada et al. 2019).
2.4 Commonly Used Strains
Strains Y-11430 (CBS-7435), GS115, and X-33 are the most commonly used P.
pastoris strains. Y-11430 is a wild-type strain that was originally isolated from
California Black oak and deposited in the United States Department of Agri-
culture Culture Collection (USDA-NRRL). It is renowned because of its robust
growth rate and high activity in the methanol utilization pathway. GS115 is a his-
tidine auxotroph obtained by mutagenizing Y-11430 with nitrosoguanidine. It has
become popular for its ease of integration and screening while using HIS4 as a
selectable marker (Cregg et al. 1985; Schutter et al. 2009). X-33 is the revertant
of GS115 created by the complementation of HIS4, and zeocin or blasticidin can
still be used to select X-33 transformants carrying the antibiotic resistance gene
(Higgins et al. 1998). X-33 shares a few mutations with GS115 in genes encod-
ing cell wall biosynthesis, which enhance the secretion of membrane-associated
proteins and result in a higher transformation efficiency than other species with
thick cell walls, making X-33 a popular commercial strain for heterologous protein
expression (Brady et al. 2020).
2.5 Signal Peptides for Mediating Protein Secretion
Protein secretion is a complex process that involves multiple steps to generate a

mature active protein. A signal peptide is a short sequence that is usually located
at the N-terminus of a nascent polypeptide and directs a protein into the secre-
tory pathway (Owji et al. 2018). The most commonly used signal peptide for
recombinant protein expression in P. pastoris is α-mating factor (α-MF) prepro
peptide originating from S. cerevisiae (Fig. 1). The α-MF prepropeptide consists
of a 19-amino acid presignal sequence and a 66-amino acid pro-sequence. The
pre-peptide typically has three domains: a positively charged N-terminal region, a
central hydrophobic region, and a polar C-terminal region. There are three main
steps in processing an α-MF secretion signal. First, the presignal is cleaved by
signal peptidases in the endoplasmic reticulum (ER). Kex2 endopeptidase in the
Golgi then cleaves the pro-leader sequence at the dibasic KR site, and finally,
Ste13 protein removes the EA repeats (Julius et al. 1984; Brake et al. 1984). α-MF
has been used in P. pastoris to produce recombinant proteins such as endoglu-
canase III from Trichoderma harzianum (Generoso et al. 2012), human P53 protein
260 W. Sun et al.
Fig. 1 General organization of the α-mating factor (α-MF) secretion signal originating from S.
cerevisiae and applied in mediating protein secretion in P. pastoris. The pre-sequence consists of
three parts and is cleaved by signal peptides in ER. The pro-sequence is cleaved at KR site by
endoprotease Kex2 and the two EA repeats are removed by dipeptidyl aminopeptidase Ste13 in
the Golgi
(Abdelmoula-Souissi et al. 2013), and manganese superoxide dismutase (PoMn-

SOD) from Pleurotus ostreatus (Yin et al. 2014), etc. Many recombinant proteins
have been successfully expressed in P. pastoris with their native signal peptides.
For example, the activity of alkaline protease from Aspergillus oryzae with its
native signal peptide is 1.5-fold higher than that with the α-MF secretion signal
peptide (Guo and Ma 2008); the activity of the laccase from white-rot fungus Poly-
porus grammocephalus TR16 with its native signal peptide is threefold higher than
that with the α-MF secretion signal peptide (Huang et al. 2011).
To enhance the efficiency of the α-MF signal sequence, various approaches
such as codon optimization (Xiong et al. 2005; Ahn et al. 2016), error-prone
PCR mutagenesis (Rakestraw et al. 2009), deletion mutagenesis (Aggarwal and
Mishra 2020; Chahal et al. 2017; Lin-Cereghino et al. 2013), and synthetic sig-
nal peptides (Aza et al. 2021; Obst et al. 2017) have been applied. Upon codon
optimization of α-MF, the phytase yield increased approximately sevenfold and
CALB production increased by 132–295% compared to the version prior to codon
optimization (Xiong et al. 2005; Ahn et al. 2016). Through error-prone PCR and
library screening, one α-MF mutant could increase the secretion of a single-chain
antibody 4m5.3 up to 16-fold, compared to the wild type. This improvement has
also been found in the production of other single-chain antibody fragments and
two structurally unrelated proteins, interleukin-2 (IL-2) and horseradish peroxi-
dase (HRP) (Rakestraw et al. 2009). A recent study in S. cerevisiae resulted in
an optimized α-MF named αOPT with four mutations (Aα9D, Aα20T, Lα42S, and
Dα83E), through a bottom-up (i.e., iterations of directed evolution on the native
α-MF) and top-down strategy (i.e., examining the evolved signal peptide, namely
α9H2 leader, and removing potential deleterious or neutral mutations) (Aza et al.
2021). The obtained αOPT could increase the secretion of two laccases, PK2 and
ApL, approximately 14- and 26-fold compared to α-MF, respectively. Combina-
torial saturation mutagenesis at positions 86 and 87 of the αOPT leader could
further enhance laccase secretion. It is appealing to apply this αOPT to protein
expression in P. pastoris. In addition, deletion of amino acids 57–70 in the pro-
peptide of α-MF enhanced the HRP activity by more than 50% and CALB activity
approximately onefold compared to the wild-type α-MF signal sequence (Lin-

Cereghino et al. 2013). In a separate study, this strategy led to an increased titer
of granulocyte colony-stimulating factor (G-CSF) to 39.4 ± 1.4 mg/L (Aggarwal
and Mishra 2020). Structural studies suggested that a specific orientation between
both the N- and C-termini of α-MF pro-peptide is required to interact with secre-
tion machinery and therefore facilitate protein secretion. Mutations generated near
these termini usually impact secretion negatively, and changes within the interior of
the pro-peptide could benefit secretion if these mutations can stabilize the N- and
C-termini (Chahal et al. 2017). By combining established leader sequences and α-
MF with deletions, Obst et al. designed several synthetic secretion signal peptides
and characterized them with a red fluorescent protein (RFP) and yeast-enhanced
green fluorescent protein (yEGFP) as reporters under different promoters. How-
ever, although these synthetic hybrid peptides yielded a more than tenfold variation
in secretion efficiency, all except αMF_no_EAEA with certain promoters were less
efficient than α-MF (Obst et al. 2017). The fusion of the S. cerevisiae Ost1 signal
sequence and α-MF pro-region with two mutations could enhance the secretion
of far-red fluorescent protein E2-Crimson by 20-fold and lipase BTL2 by tenfold
(Barrero et al. 2018).
In addition to modifications in α-MF, putative secretory signal peptides can
be determined by in silico analysis and further confirmed by experiments. Using
five computer programs, SignalP4.1, Phobius, WolfPsort0.2, ProP1.0, and NetNG-
lyc1.0, Massahi and Calik were able to identify eight signal peptides from the
sequences of 56 endogenous and exogenous proteins that had higher D-scores
than that of S. cerevisiae α-MF (Massahi and Çalık 2015). Among the eight signal
peptides, five with D-scores higher than 0.8 (SP13, SP23, SP24, SP26, and SP34)
were selected for investigation of their efficiency in secreting recombinant human
growth hormone. SP23 had the highest secretion efficiency, reaching 70%–80% of
the efficiency of α-MF (Massahi and Çalık 2016). There are also eight commer-
cially available signal peptides (The PichiaPink™ Secretion Signal Set) for protein
expression in P. pastoris. Table 1 summarizes the major signal peptides reported
in the literature.
2.6 Co-expression of Chaperones to Facilitate Protein Folding
Secretory proteins enter the ER by translocation in an unfolded state and then

undergo chaperone-assisted folding for maturation into their native conformation.
Only properly folded proteins are exported from the ER to the Golgi apparatus for
further modifications before delivery to intra- or extracellular destinations (Idiris
et al. 2010). The folding of secretory proteins is error-prone. When unfolded pro-
teins accumulate in the ER, unfolded protein responses are triggered to decrease
the amount of newly unfolded proteins from entering the ER and increasing ER
folding capacity. If the ER is overburdened by misfolded proteins, cell apoptosis
occurs (Yu et al. 2015; Hetz et al. 2020). Misfolded proteins can also be trans-
ported from the ER to the cytosol for ubiquitination and subsequently degraded by
262 W. Sun et al.
Table 1 Signal peptides used for extracellular protein secretion in P. pastoris

Signal peptide Origin Target protein References
α-MF S. cerevisiae α-mating factor Endoglucanase III (300 mg Generoso
L−1 ) et al. (2012)
W1 Synthetic signal peptide β-galactosidase (440 mg Cao et al.
L−1 ) (2017b)
nsB Candida antarctica lipase B Lipase A (220 U mL−1 ) Vadhana et al.
and lipase B (383 U mL−1 ) (2013)
PHA-E Phaseolus vulgaris agglutinin Snowdrop lectin (GNA) Raemaekers
E and GFP et al. (1999)
PHOI P. pastoris acid phosphatase 1 Rabies virus glycoprotein Ben Azoun
(RABV-G) et al. (2016)
SUC2 S. cerevisiae sucrose invertase Recombinant human Kuwae et al.
antithrombin (rAT, 324 mg (2005)
L−1 )
HFBII Trichoderma reesei class II Endoglucanase (EG27I, Su et al.
hydrophobin HFBII 47.7 mg L−1 ) (2017)
nsB-AP2 Candida antarctica lipase B + Leech hyaluronidase Kang et al.
amphipathic peptide (LHAase, 1.24 × 106 U (2016)
mL−1 )
SP23 P. pastoris protein disulfide Human growth hormone Massahi and
isomerase (rhGH, 56 mg L−1 ) Çalık (2016)
SPIgG1 Murine IgG1 Anti-HIV antibody Aw et al.
(VRC01, 3.05 μg mL−1 ) (2017)
HSA P. pastoris human serum Human lysozyme (hLM) Xiong and
albumin Chen (2008)
cSIG Chicken lysozyme signal Classical swine fever virus Li (2020)
peptide glycoprotein E2
SP killer protein S. cerevisiae killer protein Phytase Helian et al.
(2020)
SP invertase S. cerevisiae invertase Phytase Helian, et al.
(2020)
SP α-amylase A. niger α-amylase Phytase Helian, et al.
(2020)
SP Inulinase Kluyveromyces maxianus Phytase Helian, et al.
Inulinase (2020)
SP Lysozyme Gallus gallus lysozyme Phytase Helian, et al.
(2020)
Scw P. pastoris Scw11p EGFP, CALB Liang et al.
(2013)
Dse P. pastoris Dse4p EGFP, CALB Liang et al.
(2013)
(continued)
Table 1 (continued)
Signal peptide Origin Target protein References
Exg P. pastoris Exg1p EGFP, CALB Liang et al.
(2013)
MF4I Codon-modified Sc α-mating Phytase (6.1 g L−1 ) Xiong et al.
factor (2005)
HFBI Trichoderma reesei class II β-galactosidase Cao et al.
hydrophobin HFBI (2017b)
Apre S. cerevisiae, preregion of β-galactosidase Cao et al.
α-factor (2017b)
SP FAEC Talaromyces stipitatus feruloyl FAEC (297 mg L−1 ) Crepin et al.
esterase (FAEC) (2003)
SP Fae-1 Neurospora crassa Fae-1 FAEC (260 mg L−1 ) Crepin et al.
feruloyl esterase (2003)
SP Bovine Bovine β-casein Xylanase He et al.
β-casein (2,755.126 IU mL−1 ) (2012)
Pptox Viral K28 preprotoxin GFP Eiden-Plach
et al. (2004)
SP pGKL pGKL killer toxin Mouse α-amylase Kato et al.
(~240 mg L−1 ) (2001)
SP DDDK PMT1-gene-inactivated P. Porcine carboxypeptidase Govindappa
pastoris DDDK protein B (CPB) and Erythrina et al. (2014)
trypsin inhibitor (ETI)
Modified signal Mouse salivary α-amylase Glucoamylase (GA, Liu et al.
peptide (MSP) (S8L) + pro-region of S. 12.619 IU mL−1 ) (2005)
cerevisiae α-MF
proteasome. This process is called ER-associated degradation (ERAD) (Römisch

2005). To enhance recombinant protein production in P. pastoris, endogenous or
exogenous chaperones can be overexpressed to facilitate proper protein folding and
secretion (Shen et al. 2012; Navone et al. 2021; Damasceno et al. 2007; Sallada
et al. 2019; Jariyachawalid et al. 2012; Summpunn et al. 2018). There are two fam-
ilies of chaperones: molecular chaperones and chaperonins. Molecular chaperones
bind to a short segment of substrate proteins and chaperonins form barrel-shaped
folding chambers to sequester all or part of the unfolded proteins for proper folding
(Evstigneeva et al. 2001).
Protein disulfide isomerase (Pdi) is a commonly used chaperone that is present
in the ER lumen. It catalyzes both the formation and isomerization of disulfide
bonds (i.e., changing an incorrectly bonded protein to a correct disulfide-bonded
protein) and helps with the correct protein folding (Wilkinson and Gilbert 2004).
Upon co-expression of Pdi with an IL-1 receptor antagonist and human serum
albumin fusion protein (IH) that contains 18 disulfide bonds, there was a signifi-
cant increase in the yield of IH, as compared to that from the strain expressing only
the IH protein at a high copy (Shen et al. 2012). Another study with E. coli AppA
264 W. Sun et al.
phytase that contains an extra non-consecutive disulfide bond showed that co-
expression of Pdi increased the phytase ApV1 thermostability, and consequently,
the production by ~12-fold compared to the expression of ApV1 alone (Navone
et al. 2021). Immunoglobulin binding protein (BiP) is another abundant chaper-
one protein that resides in the ER. Belonging to the heat shock protein Hsp70
family, it facilitates protein folding and plays an important role in the ERAD path-
way. Co-expression of BiP with an A33 single-chain antibody fragment (A33scFv)
in P. pastoris increased the ER folding capacity and resulted in an approximately
threefold increase in A33scFv secretion (Damasceno et al. 2007). Co-expression of
the chaperon gene KAR2 with different copies of the gene encoding hydrophobin
(HFBI) also resulted in increased HFBI secretion. The highest HFBI secretion with
3-copy HFBI was 22 ± 1.6-fold higher than that of the strain overexpressing only
single-copy HFBI (Sallada et al. 2019).
Apart from molecular chaperones, chaperonins have also been engineered
to facilitate protein production in P. pastoris. D-phenylglycine aminotransferase
(D-PhgAT) from Pseudomonas stutzeri ST-201 is an intracellular protein that is dif-
ficult to express in the soluble active form. Jariyachawalid et al. overexpressed this
enzyme in P. pastoris and found that most of the D-PhgAT protein was insoluble.
By co-expressing E. coli chaperonins GroEL-GroES intracellularly with D-PhgAT,
a considerable amount of soluble D-PhgAT was produced, and the activity also
increased significantly. Compared to the D-PhgAT gene expressed alone, a 14,400-
fold higher volumetric activity was achieved when ten copies of chaperonins were
co-expressed (Jariyachawalid et al. 2012). In another study, GroEL-GroES resid-
ing in the ER was co-expressed with extracellular bacterial phytase or intracellular
D-PhgAT in P. pastoris. The volumetric activity of extracellular phytase was 1.5–
2.3-fold higher than that of phytase expression alone. However, the majority of
the D-PhgAT protein was inactive and found in the insoluble protein fraction
(Summpunn et al. 2018). These results suggested that the GroEL-GroES chaperone
could potentially enhance the production of functional proteins in P. pastoris when
they are present within the same compartment. Some of the major chaperones
overexpressed in P. pastoris are summarized in Table 2.
2.7 Cell Surface Display
Cell surface display is a promising method for engineering functional proteins to

be expressed on the cell surface through fusing with an anchor protein. Appli-
cations include, but are not limited to: whole-cell biocatalysts, bioadsorption and
bioremediation, biosensor design, vaccine and antibody development, epitope map-
ping, library screening, protein engineering (Gai and Wittrup 2007; Kuroda and
Ueda 2011; Tanaka et al. 2012; Ueda 2016; Andreu and Olmo 2018). The anchor
protein can be fused with a target protein either at either the N-terminus or at the C-
terminus (Tanaka et al. 2012). Both the fusion order and the linker between a target
protein and an anchor protein can affect the display efficiency and functional prop-
erties (Ueda 2016). Commonly used anchor proteins in P. pastoris are Aga1 (Wang
Table 2 Commonly used chaperones to increase recombinant protein production in P. pastoris

Chaperone Target protein References
Glutathione peroxidase 1 RABV-G Ben Azoun et al. (2016)
(Gpx1)
Protein disulfide Isomerase HFBI, lipase, rhG-CSF, and Sallada et al. (2019), Sha et al.
(Pdi1p) recombinant Bombyx mori (2013), Zhang et al. (2006), Li
acetylcholinesterase 2 (2021)
(rBmAChE2)
ER-oxidoreductin 1 protein HFBI Sallada et al. (2019)
(Ero1p)
CNE1p, FAD1p 9 -tetrahydrocannabinolic Zirpel et al. (2018)
acid synthase (THCAS)
Bmh2, Sso2, Ssa4, VHb Rhizopus oryzae lipase (ROL) Jiao et al. (2018)
Sse1, Bfr2, Cup5, Kin2 Fab fragment of a monoclonal Gasser et al. (2007)
antibody fragment (2F5mAb)
Immunoglobulin binding A33scFv, HFBI Damasceno et al. (2007), Sallada
protein (BiP/Kar2p) et al. (2019)
Ydj1p, Ssa1p, Sec63p Lipase Zhang et al. 2006)
HAC1 Chitinase, lipase Jiao et al. (2018), Gasser et al.
(2007), Song et al. (2020)
Sis1 Porcine growth hormone Deng et al. (2020)
(pGH)
GroEL-GroES D-phenylglycine Jariyachawalid et al. (2012),
aminotransferase (D-PhgAT) Summpunn et al. (2018)
et al. 2007; Su et al. 2010a; Dong 2013), Sed1 (Su et al. 2010b; Li et al. 2015a),
Tip1 (Jo et al. 2011), Aga2 (Jacobs et al. 2008), and Flo1 (Jiang et al. 2007), all
of which are from S. cerevisiae, as well as, Pir1 (Khasa et al. 2011; Yang et al.
2017) and Pir2 (Khasa et al. 2011) from P. pastoris. In another study, 13 endoge-
nous glycosylphosphatidylinositol-modified cell wall proteins were identified upon
screening the genome of P. pastoris GS115 (Zhang et al. 2013), three of which
were chosen as anchor proteins for displaying CALB (Wang et al. 2017). These
three anchors (i.e., GCW21, GCW51, and GCW61) have also been applied to dis-
play bacterial PETase on the surface of P. pastoris, to degrade highly crystallized
polyethylene terephthalate (PET). The turnover rate of the whole-cell biocata-
lyst displaying PETase was approximately 36-fold higher than that of the purified
PETase (Chen 2020). Another anchor protein identified in P. pastoris is Flo9. The
displayed lipase B with Flo9 showed higher thermostability at 45 °C and stability
in organic solvents (Moura 2015).
P. pastoris X-33 has also been engineered to assemble protein complexes
such as minicellulosomes on the cell surface (Ou and Cao 2014). The truncated
CipA, which contains a cellulose-binding module and two cohesin modules from
Clostridium acetobutylicum, was fused to the C-terminus of the anchor flocculation
266 W. Sun et al.
protein Flo1 from S. cerevisiae, whereas a Nasutitermes takasagoensis endoglu-

canase (NtEG) was fused with the dockerin. Fusion proteins were expressed
separately in two P. pastoris X-33 strains, which were co-cultured for minicellulo-
some assembly. The surface displayed CipA and assembly of cohesin and dockerin
were confirmed using immunofluorescence and western blotting. The hydrolysis
efficiencies of NtEG for carboxymethyl cellulose (CMC), microcrystal cellulose
(Avicel), and filter paper were enhanced by 1.4-fold, 2.0-fold, and 3.2-fold, respec-
tively, when compared to free NtEG. Another study conducted by Dong et al.
utilized an ultra-high-affinity IM7/CL7 protein pair for minicellulosome assem-
bly (Dong et al. 2020). IM7 (including one, two, or three units) was fused to the
N-terminus of the anchor protein SED1 from S. cerevisiae and expressed in P. pas-
toris. An endoglucanase (EG), an exoglucanase (CBH), a β-glucosidase (BGL),
and a carbohydrate-binding module (CBM) from Thermobifida fusca, each fused
with an N-terminal CL7 tag, were expressed individually in E. coli. The secreted
proteins from E. coli cultures were assembled and displayed on the P. pastoris cell
surface in vitro. The display system with two or three IM7 showed comparable or
even higher efficiency for the hydrolysis of Avicel, phosphoric acid-swollen cel-
lulose (PASC) and CMC, compared to free cellulases. The ethanol titer reached
5.1 g/L when three IM7 units were engaged in CMC fermentation.
In a recent study, Silva et al. displayed specific immunogenic epitopes of ZIKV
envelope, NS1 protein, and both on the surface of P. pastoris GS115 by fusing
these epitopes at the N-terminus of a partial Agα1 (C-terminal portion, nucleotides
970–1950) (Silva et al. 2021). The ability of the recombinant yeast to stimulate
immune cells was evaluated in vitro using mouse immunological cells isolated
from the spleen. P. pastoris displaying EnvNS1 epitopes showed better efficacy
in producing IL-6, IL-10, and tumor necrosis factor-alpha (TNF-α) cytokines and
an increase in lymphocytes CD4+ , CD8+ , and CD16+ , similar to ZIKV. These
epitopes will be beneficial for the development of vaccines against ZIKV infection.
3 Applications of P. pastoris in Biomanufacturing
After more than 20 years of development, P. pastoris has become one of the most
popular protein expression systems that is widely used in protein preparation,
structural analysis, and functional characterization. As a GRAS microorganism
approved by the United States Food and Drug Administration, thousands of pro-
teins, including medicinal proteins (i.e., insulin, human serum albumin, hepatitis B
surface antigen, and epidermal growth factor (Weinacker et al. 2013)) and indus-
trial enzymes (i.e., mannanase, phytase, xylanase, and lipase (Rabert et al. 2013))
have been successfully expressed in P. pastoris. In addition, due to the develop-
ment of pathway assembly and genome editing tools, a growing interest has been
seen in establishing P. pastoris as a microbial cell factory to produce chemicals
and natural products.
3.1 Recombinant Proteins
Recombinant proteins can be produced using bacterial, yeast, mold, insect, plant,
and mammalian expression systems. P. pastoris is particularly attractive for the
large-scale production of recombinant proteins. It naturally contains several highly
expressed genes that encode methanol assimilation and dissimilation pathway
enzymes, enabling growth with methanol as the sole carbon and energy source
(Wegner and Harder 1987). Thus, high-level expression of target proteins can
be readily achieved using these methanol-inducible promoters. When compared
with the plant and mammalian expression systems, the P. pastoris expression sys-
tem offers the advantages of low cost, fast growth, high cell density fermentation
(HCDF), and consequently high expression levels. In contrast to prokaryotes, the
biggest advantage of P. pastoris is the capability of post-translational modifica-
tions (e.g., O- and N-glycosylation and disulfide bond formation. When compared
to S. cerevisiae, P. pastoris poses little concerns regarding over-glycosylation, and
the secretory expression level of recombinant proteins is much higher (Karbalaei
et al. 2020). Therefore, P. pastoris has been widely used to produce therapeutic
glycoproteins. Moreover, P. pastoris can efficiently secrete the target proteins into
the fermentation broth, making the downstream separation and purification pro-
cess simpler, which is a paramount variable in designing viable industrial-scale
processes.
3.1.1 Medicinal Proteins

Recombinant proteins represent a growing market in medical biotechnology. Many
approved biopharmaceuticals are protein-based, such as monoclonal antibodies,
growth factors, blood factors, hormones, interleukins, anticoagulants, interfer-
ons, and vaccines. Some of the representative medicinal proteins produced by P.
pastoris are listed in Table 3.
Vaccines represent the largest class of recombinant medicinal proteins produced
by P. pastoris. Vaccines can be divided into three types: inactivated vaccines, live
attenuated vaccines, and recombinant subunit vaccines (Gasser et al. 2006). Recent
studies have found that P. pastoris is preferred for producing recombinant subunit
vaccines compared to other expression systems (Gasser et al. 2007). Compared
to the non-glycosylated antigen, the mannose-glycosylated antigen produced by
P. pastoris has enhanced antigen presentation and T cell activation. Enterovirus
71 (EV71) is the main pathogen that causes hand-foot-mouth disease in children.
The establishment of a microbial system for large-scale and safe production of
the EV71 vaccine has value in medicinal applications. Yang et al. cloned P1 and
3C genes of EV71 and established a microbial system for efficient production of
recombinant EV71-VLP (virus-like particle) in P. pastoris. Expression levels of
as high as 270 mg/L EV71-VLPs antigen have been achieved (Yang et al. 2020).
Another example is related to cervical cancer, the fourth most common cancer
that threatens the health of women worldwide. Although there is already a market-
oriented vaccine, its high price limits its wide application. Recently, Sanchooli
et al. inserted the cross-neutralizing epitope of L2-HPV-16 into L1-HPV-16 to form
268 W. Sun et al.
Table 3 Representative medicinal proteins expressed in P. pastoris

Product Strain Vector Titer Application References
(mg/L)
Bovine IFN-α GS115 pPIC9K 200 Prevention and Tu et al. (2016)
therapy of viral
diseases
hPAB-β GS115 pPIC9K 241 Antibacterial Chen et al. (2011)
peptide
Bovine KM71H pJ902 3500 Transferrin and Iglesias-Figueroa,
lactoferrin antibacterial et al. (2016)
protein
Human GS115 pPICZα 11 Antitumor Chan et al. (2018)
topoisomerase I SMD1168H
Insulin SuperMan5 pPICZα 5 Diabetes Baeshen et al.
(2016)
Human gastric X-33 pGAPZα 7 Diseases of the Sams et al. (2017)
lipase digestive system
Rabies virus GS115 pHIL-S1 1.23 Rabies vaccine Ben Azoun et al.
glycoprotein (2016)
L1-L2 proteins GS115 pBLHIS-IX 23.61 Cervical cancer Bredell et al.
of HPV virus (2018)
type 16
Human KM71H pPICZαB 130 Pharmacological Luley-Goedl et al.
sialyltransferase uses (2016)
Human X-33 pPICZαA 600 Treatment of Vallet-Courbin
antiplatelet scFv atherosclerosis (2017)
antibody
IL-1β GS115 pPICZαA 250 Proinflammatory Li et al. (2016a)
SMD1168 cytokine
X-33
IL-3 X-33 pPICZαA 2230 Multipotent Dagar and Khasa
hematopoietic (2018)
cytokine
IL-11 GS115 pPINKαHC 300 Thrombopoietic Yu et al. (2018)
growth factor
IL-15 X-33 pPICZαA 75 Differentiation Sun et al. (2016)
and proliferation
of T, B, and NK
cells
an L1/L2-HPV-16 chimeric fragment, which was cloned into the pPICZA plasmid
for heterologous expression. The chimeric protein could be positively detected by
both L1-HPV-16 and L2-HPV-16 antibodies (Sanchooli et al. 2018). Meanwhile,
when Bredell et al. expressed the HPV-16L1/L2 chimeric protein (VLP) in P. pas-
toris KM71 (MutS ) or GS115 (Mut+ ) under a constant dissolved oxygen level
(DO stat) fed-batch culture supplemented with methanol, they achieved a titer of
23.61 mg/L for the chimeric protein (Bredell et al. 2018).
Although a panel of medicinal proteins has been expressed in P. pastoris, cor-
rect folding of target proteins is a major concern. Due to the lack of sufficient
chaperone factors, a considerable number of recombinant proteins cannot fold into
their correct configurations. To overcome this limitation and improve the expres-
sion level, rational design and reverse engineering strategies can be adopted to
improve the protein folding microenvironment. As mentioned above, genes related
to protein folding in the ER, such as BiP (an Hsp70 chaperone), can help secretory
proteins fold correctly. The secretion of A33scFv fragment was increased approx-
imately threefold upon overexpression of the chaperone protein BiP in P. pastoris
(Damasceno et al. 2007). Overexpression of Pdi (responsible for the formation of
disulfide bonds) in P. pastoris can increase the expression of the antibody protein
2F5Fab by 2-fold (Gasser et al. 2006). Zhang et al. tested three factors related
to protein transport from S. cerevisiae (Sec63p, Ydj1p, and Ssa1p), whose overex-
pression increased the expression of GCSF by 2.8-, 3.6-, and 6.8-fold, respectively,
in P. pastoris. Therefore, finding suitable protein mates remains a major challenge
in establishing an efficient recombinant protein expression system (Zhang et al.
2006). Gasser et al. identified new protein chaperone genes, including CUP5,
SSA4, BMH2, KIN2, SSE1, and BFR2 at the transcriptional level. The overexpres-
sion of these genes significantly enhanced the secretion of 2F5Fab antibody in P.
pastoris, with the final titer of 2F5Fab reaching up to 47.27 mg/L (Gasser et al.
2007). Stadlmayr et al. established a cDNA overexpression library in P. pastoris
and identified three new protein chaperones as the secretion-enhancing factors,
which increased the expression level of the model protein by up to 45% (Stadl-
mayr et al. 2010). Huang et al. identified six significantly upregulated genes related
to recombinant protein production using comparative proteomic analysis. In par-
ticular, the co-expression of TPX, FBA, and PGAM increased the expression level
of the reporter gene by 2.46-, 1.58-, and 1.33-fold, respectively (Huangfu et al.
2015). Noteworthy, owing to the different properties of foreign proteins, there is a
dearth of generally applicable engineering approaches. In other words, the optimal
protein chaperone or secretory factors can be different case by case and should be
evaluated individually. For example, overexpression of Pdi in P. pastoris failed to
increase the production of A33scFv antibody protein and overexpression of BiP
even reduced the yield of glucose oxidase by 10-fold (Heide et al. 2002).
3.1.2 Industrial Enzymes

In recent years, industrial enzymes have been increasingly leveraged in the chem-
ical, food and beverage, pharmaceutical, cosmetic, and textile industries. Owing
to the increasing demand for industrial enzymes, the development of production
strategies has accelerated. In this regard, the effectiveness of P. pastoris as a host
for high-level expression of recombinant proteins has attracted increasing atten-
tion, because of the presence of strong methanol-inducible promoters (more than
30% of the total proteins) and HCDF (higher than 200 g/L biomass). In addi-
tion, P. pastoris has a strong ability to secrete target proteins into the fermentation
270 W. Sun et al.
medium, facilitating downstream purification at a much lower cost. Therefore, P.

pastoris has been regarded as a favorable host for large-scale production of indus-
trial enzymes. Representative industrial enzymes produced in P. pastoris using
HCDF are listed in Table 4.
As a type of hydrolase, lipase demonstrates high regioselectivity and stereose-
lectivity and can catalyze ester hydrolysis and transesterification. Therefore, lipases
are widely used in food, cosmetics, and pharmaceuticals. Zheng et al. cloned
Table 4 Representative industrial enzymes expressed in P. pastoris

Product Strain Vector Scale Titer Application References
(L) (g/L)
β-Mannanase GS115 pPIC9K 5 0.17 Production of Li et al.
manno-oligosaccharides (2018)
Invertase KM71 pPIC9 7 2.5 Producing inverted Veana
sugar et al.
(2014)
Hyaluronidase GS115 pPIC9K 3 0.42 Degradation of Jin et al.
hyaluronic acid (2014)
Xylanase X-33 pPICZαA 50 9.88 Bakery, beverages, Li et al.
starch processing (2015b)
Pectinase GS115 pAO815 – 0.35 Brewing, papermaking Peng et al.
(2019)
Glucose GS115 pPIC9 40 0.83 Bakery, eggs, starch Meng
oxidase processing et al.
(2014)
Formate X-33 pPICZαA 3 0.05 Cofactor regeneration, Duman
dehydrogenase CO2 reduction (2020)
Peroxidase DSMZ pUC57 14 0.94 Diagnostics, Majeke
70,382 bioremediation, lignin (2020)
degradation
L-Glutamate GS115 pHBM905A 20 11 Transformation YaPing
oxidase of L-glutamic acid, et al.
industrial fermentation, (2017)
biosensors
Laccase X-33 pGAPZA 5 0.495 Dye decolorization, Kittl et al.
beverages (2012)
Hydrolases X-33 pPICZαA 5 0.61 Triacylglycerol lipase Zheng
et al.
(2019)
Lipase B SMD1168 – 10 2 Resolution of chiral Jahic et al.
compounds (2003)
Tannin acyl KM71 pPIC9K 7 0.074 Detannification of food Zhong
hydrolase et al.
(2004)
Phytase GS115 pPICZαA 10 9.58 Animal feeding Li et al.
(2015c)
the Aspergillus oryzae lipase gene (AOL) to yield the plasmid pPICZαA-AOL,
which was subsequently integrated into the genome of P. pastoris X-33. Using the
methanol feeding strategy, AOL with a specific activity of 432 U/mg was obtained
in a 5 L bioreactor (Zheng et al. 2019). To increase the yield of lipase, Zhang et al.
employed a fusion expression strategy by fusing small ubiquitin modifying pro-
tein (SUMO) with Aspergillus niger lipase (ANL) to obtain SANL. The resultant
chimeric gene was cloned into pPIC9K for heterologous expression in P. pastoris
GS115. The highest activity of SANL was ~960 U/mL in a 3 L fermenter, which
was 1.85-fold higher than that of its parent ANL (Zhang et al. 2019a).
Although the expression of recombinant proteins is mainly induced by
methanol, a co-substrate culture strategy has been found to increase the yield of
industrial enzymes. Berrios et al. cloned the Rhizopus oryzae lipase gene (ROL)
and constructed the plasmid pPICZαA-ROL for heterologous expression in P. pas-
toris X-33. The engineered strain was continuously cultured with methanol and
glycerol as co-substrates in a 1.5 L BioStatAplus bioreactor. The results showed
that using glycerol as a co-substrate at 22 and 30 °C could increase the volumet-
ric productivity of recombinant lipase and reduce the consumption of methanol
(Berrios et al. 2017). In addition, the co-substrate culture could also be applied
for the industrial production of phytase. As an animal feed additive, phytase can
decompose phytic acid and greatly reduce the input of animal feed. Li et al. engi-
neered phytase production in P. pastoris by modifying PAOX1 and the α factor
signal peptide and increasing gene copy numbers. Phytase activity as high as
2,119 U/mL with a corresponding titer of 0.75 g/L was achieved, which was 4.12-
fold higher than that of the parent strain. In a 10 L fermenter, using glycerol and
methanol as co-substrates for fed-batch fermentation, the titer and enzyme activity
of phytase could be further improved to as high as 9.58 g/L and 35,032 U/mL,
respectively (Li et al. 2015c).
Besides the traditional strategies in engineering secretion signals and modify-
ing PAOX1 promoter, genome-scale metabolic models can be employed to regulate
the metabolic fluxes from a systems perspective, to improve the expression level
of recombinant proteins. Saitua et al. employed the dynamic flux balance analy-
sis (dFBA) framework to establish a dynamic genome-scale metabolic model, to
simulate recombinant protein expression process in P. pastoris (Saitua et al. 2017).
Starting with seven state variables including glucose, biomass, and fermentation
quantity, they analyzed the kinetics of substrate assumption and distribution of
metabolic flux. On this basis, Nocon et al. optimized the dFBA algorithm and pre-
dicted gene targets (including both gain- and loss-of-function targets), to enhance
the production of recombinant proteins. Overexpression targets were identified to
reside in the pentose phosphate pathway and the TCA cycle, whereas knockout tar-
gets were found to belong to several branch points of glycolysis (Fig. 2). Five out
of the nine predicted targets were found to increase the expression level of cytoso-
lic human superoxide dismutase (hSOD). More importantly, most of the same
genetic modifications led to enhanced expression of bacterial β-glucuronidase,
indicating the general applicability of the identified metabolic engineering targets
(Nocon et al. 2014).
272 W. Sun et al.
Fig. 2 Implementation of a genome-scale metabolic model to predict gene overexpression and

knockout targets of the central metabolism for increased production of recombinant proteins in
P. pastoris (Nocon et al. 2014). Overexpressed genes are shown in green and deleted genes are
shown in red. ZWF1: glucose-6-phosphate dehydrogenase; SOL3: 6-phosphogluconolactonase;
GND2: phosphogluconate dehydrogenase; MDH1: malate dehydrogenase; TPI1: triose-phosphate
isomerase; ADH2: alcohol dehydrogenase; ALD4: aldehyde dehydrogenase; PDA1: pyruvate dehy-
drogenase; PDC1: pyruvate decarboxylase; RPE1: ribulose 5-phosphate 3-epimerase; GPD1:
glycerol-3-phosphate dehydrogenase; GUT2: glycerol-3-phosphate dehydrogenase; GDH3: gluta-
mate dehydrogenase
3.2 Bulk Chemicals
With the rise of synthetic biology, yeast has become an important cell factory to
produce fine chemicals. Currently, S. cerevisiae is the preferred host to produce
a wide range of chemicals, including, but not limited to: glycerol, L-propanediol,
lactic acid, succinic acid, and isoprene. Comparative metabolomics indicated that
the intermediate metabolites in P. pastoris could cover more than 90% of those
in S. cerevisiae, indicating great potential for chemical production in P. pastoris
(Carnicer et al. 2012). Currently, the production of S-adenosyl-L-methionine (Chu
et al. 2013), xylitol (Louie et al. 2021), hyaluronic acid (Oliveira et al. 2016),
gluconic acid (Liu et al. 2016), and lactic acid (Lima et al. 2016) has been
reported, confirming the possibility of producing simple and complex chemicals

in P. pastoris.
To achieve efficient and cost-effective production of chemicals, a combination
of metabolic engineering modification modifications and bioprocess optimization
is generally employed. Cheng et al. constructed the glucose-D-arabitol-D-xylulose-
xylitol pathway to produce xylitol from glucose for the first time. The D-arabitol
dehydrogenase gene from Klebsiella pneumoniae and the xylitol dehydrogenase
gene from Gluconobacter oxydans were cloned into pPIC9K for subsequent inte-
gration into the genome of the GS225 strain, a derivative strain of P. pastoris
GS115 strain after adaptive evolution. The yield of xylitol was 0.078 g/g glucose
when it was fermented in a 3 L fermenter (Cheng et al. 2014).
Mixed carbon source fermentation may be more beneficial for promoting prod-
uct production, and this strategy has been used in the production of a variety of
chemicals, such as glucaric acid. As an organic acid, glucaric acid is considered to
be one of “the most valuable chemicals from biomass” and plays an important role
in the synthesis of many biodegradable substances. Much attention has been paid
to the production of glucaric acid using a microbial cell factory. Liu et al. over-
expressed the inositol oxygenase gene (MIOX) and urinate dehydrogenase gene
(UDH) from Pseudomonas aeruginosa KT2440 for glucaric acid production in
P. pastoris. As MIOX was determined to be rate-limiting, fusion expression of
MIOX with UDH with a flexible linker was employed to improve the conversion
efficiency. With glucose and myo-inositol as the co-substrates in fed-batch fermen-
tation, the engineered P. pastoris strain was able to produce glucaric acid at a titer
as high as 6.61 ± 0.30 g/L (Liu et al. 2016).
2-Phenylethanol (2-PE) is widely used in cosmetics and high-end perfumes
because of its rose flavor. In a recent study, Kong et al. overexpressed the 2-
ketoacid decarboxylase gene (ARO10), aldehyde reductase gene (ADH6), and
aromatic aminotransferase gene (ARO8) from S. cerevisiae, together with feedback
inhibition-insensitive mutant genes, 3-deoxy-D-arabino-heptulosonate-7-phosphate
synthase (aroGfbr ) and chorismate mutase/prephenate dehydratase (pheAfbr ) from
E. coli, under the control of the strong constitutive promoter, PGAP , and achieved
de novo biosynthesis of 2-PE from glucose for the first time. Using shake flask
fermentation for 36 h, 1,169 mg/L of 2-PE was found to accumulate in the engi-
neered P. pastoris strain (Kong 2020). Notably, the titer of 2-PE synthesized by P.
pastoris was higher than those achieved in E. coli and S. cerevisiae, indicating that
P. pastoris has excellent potential as a host strain to produce chemicals.
As mentioned earlier, although P. pastoris has been used to synthesize various
compounds, most of the engineered strains still use glucose and other fermentable
sugars as substrates (Pena et al. 2018). While it is often added as an inducer,
methanol has rarely been used as a substrate. Owing to the increasing concerns
about sustainability and the abundant availability of methanol, the use of methanol
as a substrate to produce chemicals has become a research hotspot. Cai et al.
used methanol as the sole carbon and energy source to produce Monachine J and
lovastatin in metabolically engineered P. pastoris, with titers reaching 60.0 mg/L
274 W. Sun et al.
and 14.4 mg/L, respectively. After bioprocess optimization, including the employ-
ment of a co-culture strategy and fed-batch fermentation with glycerol as the
co-substrate, the yield of Monachine J and lovastatin reached 594 mg/L and
251 mg/L, respectively. In terms of methanol conversion, Yamada et al. integrated
D-LDH into the rDNA loci of P. pastoris. Multicopy integration of the D-LDH
expression cassette was achieved following post-transformational gene amplifica-
tion and selection on gradually increasing zeocine concentrations. The optimally
engineered P. pastoris strain produced D-lactic acid with a titer of 3.48 g/L by
means of test-tube fermentation for 96 h, with methanol as the sole carbon source.
This is the first report on the establishment of P. pastoris as a microbial cell factory
for the conversion of methanol to lactic acid (Yamada et al. 2019). This study pro-
vides a basis for the application of gene integration strategy at the rDNA loci in P.
pastoris for the construction of microbial cell factories for the production of value-
added chemicals. Although research on the synthesis of chemicals from methanol
is still in its infancy and the titer is still not high enough for industrial produc-
tion, the challenges in methanol conversion should be able to be addressed using
metabolic engineering and synthetic biology approaches in the future. P. pastoris is
believed to play an increasingly important role in biorefinery and biomanufacturing
in the near future.
3.3 Natural Products
Key enzymes of secondary metabolite (natural product) biosynthetic pathways

are often found to have low expression levels and/or limited enzymatic activities,
which becomes the bottleneck for efficient biosynthesis of high-value natural prod-
ucts. Considering the advantages of high-level expression and post-translational
modifications of complex eukaryotic proteins (i.e., cytochrome P450s, CYPs), P.
pastoris is a promising microbial cell factory for the synthesis of complex bio-
logically active molecules. Currently, natural products synthesized in P. pastoris
mainly include terpenoids, polyketides, and flavonoids (Table 5).
Terpenoids are hydrocarbons that are widely found in plants and microor-
ganisms. Many terpenoids have important physiological activities and therefore
are important research targets for the development of new drugs. The biosyn-
thetic pathways of a series of terpenoids, such as lycopene, carotene, astaxanthin,
(+)-nootkatone, and dammarenediol-II, have been successfully constructed in P.
pastoris. (+)-Nootkatone is a sesquiterpene compound of great commercial value,
with a grapefruit aroma and various biological activities. Although the existing
chemical synthesis technology of (+)-nootkatone can meet industrial and commer-
cial needs, heavy metals and flammable compounds are involved in the synthesis
process. Therefore, the synthesis of (+)-nootkatone using biological methods is
a new trend in the near future (Fig. 3). Through co-expression of the prem-
naspirodiene oxygenase (HPO) from Hyoscyamus muticus and the cytochrome
P450 reductase from A. thaliana, trans-nootkatol was produced by hydroxylation
of (+)-valencene, and trans-nootkatol was further oxidized to (+)-nootkatone by
Table 5 Production of natural products in P. pastoris cell factories
Nature product Host Genes Promoter Yield References
Lycopene X-33 crtE, crtB, crtI PGAP 1.141 μg/g DCW Araya-Garay et al. (2012a)
β-Carotene X-33 crtE, crtB, crtI, crtL PGAP 339 μg/g DCW Araya-Garay et al. (2012a)
Astaxanthin X-33 crtE, crtB, crtI, crtL, crtW, crtZ PGAP 3.7 μg/g DCW Araya-Garay et al. (2012b)
Nootkatone CBS7435 his4 ku70 CPR, HPO, ADH, truncated HMG1 PAOX1 17 mg/L Wriessnegger et al. (2014)
208 mg/L
(fed-batch)
Dammarenediol-II GS115 ERG1, ERG7 PAOX1 1.073 mg/L DCW Liu et al. (2015)
Repression PTHL11
PgDDS PAOX1
Menaquinone-4 GS115 SaGGPPS PGAP 0.24 mg/g DCW Sun et al. (2019)
HsUBIAD1 PAOX1
IDI PGAP
Recent Advances in Synthetic Biology Applications …
6-MSA GS115 NpgA, ATX PAOX1 2.2 g/L (fed-batch) Gao et al. (2013)
Citrinin GS115 pksCT, MPL1, MPL2 PAOX1 0.6 ± 0.1 mg/L Xue et al. (2017)
MPL4, MPL6
MPR7
Lovastatin GS115 LovA, LovB PAOX1 24.6 mg/L Liu et al. (2018)
LovC, LovD 250.8 mg/L (fed-batch)
LovF, LovG
(continued)
275
276
Table 5 (continued)
Nature product Host Genes Promoter Yield References
NpgA
17-Hydroxyprogesterone GS115 Human CYP17 cDNA PAOX1 Not reported Kolar et al. (2007)
Note CrtE: geranylgeranyl diphosphate synthase; CrtB: phytoene synthase; CrtI: phytoene desaturase; CrtL: lycopene β-cyclase; CrtW: xanthophylls; CrtZ:
β-carotene hydroxylase; CPR: A. thaliana cytochrome P450 reductase; HPO: premnaspirodiene oxygenase of H. muticus; ADH: alcohol dehydrogenase; trun-
cated HMG1: truncated hydroxy-methylglutaryl-CoA reductase; ERG1: 2,3-oxidosqualene synthase; ERG7: responsible for conversion of 2,3-oxidosqualene to
lanosterol; PgDDS: dammarenediol-II synthase from Panax ginseng; SaGGPPS: geranylgeranyl diphosphate synthase from the archaebacterium Sulfolobus aci-
docaldarius; HsUBIAD1: a human homologue of E. coli prenyltransferase menA and mammalian mitochondrial prenyltransferase COQ2 ; IDI: IPP isomerase;
NpgA: A. nidulans phosphopantetheinyl transferase; ATX: Aspergillus terrus 6-methylsalicylic acid synthase; pksCT: M. purpureus citrinin polyketide synthase;
MPL1: serine hydrolase; MPL2: oxygenase. MPL4: dehydrogenase. MPL6: short-chain dehydrogenase. MPL7: oxidoreductase. MPR1: a transporter. LovA: a
cytochrome P450 monooxygenase; LovB: lovastatin nonaketide synthase; LovC: enoyl-reductase; LovD: acyl-transferase; LovF: lovastatin diketide synthase;
LovG: thioesterase; Human CYP17 cDNA: catalyzing the conversion of progesterone to 17-hydroxyprogesterone as well as 16-hydroxyprogesterone
W. Sun et al.
Fig. 3 Synthetic pathway of (+)-nootkatone in P. pastoris. (+)-Valencene is synthesized from far-

nesyl pyrophosphate by valencene synthase (ValS). HPO/CPR converts (+)-valencene into trans-
nootkatol, which is further converted by the endogenous alcohol dehydrogenase (ADH) to form
(+)-nootkatone. Enzymes that can convert trans-nootkatol to (+)-nootkatone naturally exist in P.
pastoris. Overexpression of the endogenous ADH and the truncated hydroxymethylglutaryl coen-
zyme A reductase (tHMG1) from S. cerevisiae, as well as the endogenous Rad52 significantly
increased the production of (+)-nootkatone. Exogenous genes are shown in red and bold arrows
represent overexpressed endogenous genes. HPO: premnaspirodiene oxygenase from H. muticus;
CPR: cytochrome P450 reductase from A. thaliana
the endogenous dehydrogenase of P. pastoris. Further introduction of valencene

synthase (ValS) and the truncated S. cerevisiae hydroxy-methylglutaryl CoA reduc-
tase resulted in the construction of a strain capable of de novo production of
(+)-nootkatone from glucose. The production of (+)-nootkatone reached a titer
of 17 mg/L in shake flasks and 208 mg/L in the bioreactors (Wriessnegger et al.
2014). Notably, overexpression of RAD52 and the optimization of the medium
have increased the production of trans-nootkatone by 5-fold (Wriessnegger, et al.
2016).
Polyketides are a class of secondary metabolites with various structures and
biological activities. With 6-methylsalicylic acid (6-MSA) being the first polyke-
tide produced in P. pastoris, the biosynthetic pathways of citrinin, monacolin, and
other polyketides have also been successfully reconstituted in this host. Synthe-
sized by a relatively small gene cluster (Fig. 4), citrinin serves as a representative
for polyketide biosynthesis in P. pastoris (Shimizu et al. 2007; Sakai et al. 2008).
A total of seven foreign genes was heterologously expressed to construct the cit-
rinin biosynthetic pathway, including the citrinin polyketide synthase gene PksCT
(CitS) from Monascus purpureus, the phosphoubiquitin transferase gene NpgA
from Aspergillus nidulans, as well as the cluster genes, including MPL1 (CitA),
MPL2 (CitB), MPL4 (CitD), MPL6 (CitE), and MPL7 (CitC) from M. purpureus.
278 W. Sun et al.
Fig. 4 Reconstitution of the citrinin biosynthetic pathway in P. pastoris. CitS (pksCT ): polyketide
synthase; CitA (MPL1): serine hydrolase; CitB (MPL2): iron II oxidase; CitD (MPL4): aldehyde
dehydrogenase; CitE (MPR1): short-chain dehydrogenase
After 24 h of cultivation with methanol as the sole carbon source, citrinin was
produced up to a concentration of 0.6 mg/L (Xue et al. 2017).
4 Recent Advances in Engineering P. kudriavzeii
P. kudriavzeii is a non-conventional yeast that can be found in various fermented

foods (Choi et al. 2017; Vuyst et al. 2016; Qin et al. 2016), cocoa beans (Delgado-
Ospina, et al. 2020), fruits (Park, et al. 2018), wastewater (Pajot et al. 2011), etc.
Other names include Issatchenkia orientalis, Candida glycerinogenes, and Candida
krusei (Douglass 2018). It is a multistress-tolerant yeast that can grow at low pH
(Xiao et al. 2014; Park et al. 2018; Sun et al. 2020; Toivari et al. 2013; Hisamatsu
et al. 2006), high temperature (as high as 50 °C) (Park et al. 2018; Yuangsaard
et al. 2013; Chamnipa et al. 2018), and high concentrations of salt conditions
(Isono et al. 2012); thus, it has been engineered to produce organic acids such
as D-xylonic acid (Toivari et al. 2013), succinic acid (Xiao et al. 2014), D-lactic
acid (Park et al. 2018), and itaconic acid (Sun et al. 2020). For example, Toivari
et al. engineered P. kudriavzevii VTT C-79090T to express a D-xylose dehydro-
genase gene from Caulobacter crescentus at the PDC1 locus, resulting in 146 g/L
D-xylonate production at pH 3.0 (Toivari et al. 2013). Xiao et al. engineered I.
orientalis SD108 by overexpressing three native genes (i.e., encoding pyruvate car-
boxylase, malate dehydrogenase, and fumarase) and fumarate reductase that was
previously codon-optimized for expression in S. cerevisiae via genome integration,
enabling production of succinic acid at a titer of 11.63 g/L in shake flask (Xiao
et al. 2014). In addition, by replacing the gene encoding pyruvate decarboxylase
1 with the gene encoding D-lactate dehydrogenase from Lactobacillus plantarum
followed by adaptive evolution, the engineered P. kudriavzevii NG7 strain was
able to produce D-lactic acid at a titer of 135 g/L (pH 3.6) and 154 g/L (pH 4.7)
(Park et al. 2018). Our group also engineered P. kudriavzevii YB4010 to produce
1.23 g/L itaconic acid at pH 3.9 in fed batch fermentation by overexpressing a
cis-aconitic acid decarboxylase gene from Aspergillus terreus and a native mito-
chondrial tricarboxylate transporter in the strain with the isocitrate dehydrogenase
gene deleted (Sun et al. 2020). P. kudriavzevii can also produce ethanol at a high
salt concentration (50 g/L Na2 SO4 ) at pH 2.0 or a temperature as high as 43 °C
(Isono et al. 2012). Other applications have been demonstrated in wine fermenta-
tion (Mónaco et al. 2014, 2016), production of potential probiotics (Greppi et al.
2017), biological control (Bajaj et al. 2013), and bioremediation for heavy metal
removal (Li et al. 2016b; Zhang et al. 2019b). To date, the registered P. kudriavzeii
strains are mainly diploid (Xiao et al. 2014; Xi et al. 2021), although triploid and
aneuploid strains have also been reported (Douglass 2018).
Prior to the creation of episomal plasmids, engineering works in P. kudriavzeii
were usually performed by directly transforming a linear fragment carrying the
target gene(s) and a selection marker flanked by homologous arms of the target
integration site (Park et al. 2018). URA3 is often used as a selection marker because
of the relatively easy protocol for marker recycling. Considering that the strain is
a diploid, a single-round integration will likely create a heterozygous strain, and
a second-round integration to the wild-type allele is recommended to improve
genetic stability. Recently, Tran and Cao et al. created an episomal plasmid con-
sisting of an ARS and LEU2 originating from S. cerevisiae, P. kudriavzeii URA3,
and a GFP reporter gene. The percentage of GFP+ cells in the culture grown from
a single colony was approximately 60% after 24 h (Cao et al. 2020). They iso-
lated a centromere-like (CEN-L) sequence from the P. kudriavzeii genome with the
assistance of an in silico GC3 analysis to identify the “GC3 valley” on each chro-
mosome, followed by sequence alignment to identify the conserved regions (Cao
et al. 2020). As a CEN is responsible for faithful chromosome segregation and
plays a critical role in stabilizing plasmids, the newly constructed plasmid includ-
ing CEN-L led to an increased percentage of GFP+ cells, to 81% after cultivation
for 24 h and to 67% after cultivation for 120 h.
RNA sequencing is usually implemented to identify strong constitutive promot-
ers and terminators. Cao et al. grew P. kudriavzeii under four growth conditions
(YNB medium with or without lignocellulosic biomass inhibitors under aerobic
or anaerobic conditions) and analyzed the transcriptome. Thirty-five promoters
of the most highly expressed genes identified based on RNA-sequencing data
were selected and cloned with the GFP reporter gene and TEF1 terminator on
an episomal plasmid containing ARS. Strong, medium, and weak constitutive pro-
moters were categorized based on flow cytometry. For terminator identification,
14 terminators of the strong promoters identified above were selected for further
comparison. Double reporter genes (i.e., GFP and mCherry) were placed between
the TDH3 promoter and the PGK1 terminator. Each of the candidate terminator
sequences was cloned between the GFP and the mCherry open reading frames
(ORFs) with a random sequence or no sequence inserted between the two ORFs
as controls. Quantitative PCR was used to calculate the transcriptional ratios of
280 W. Sun et al.
mCherry and GFP. Thirteen of the 14 candidate terminators had ratios below 0.03
and were categorized as strong terminators. Moreover, similar to S. cerevisiae, P.
kudriavzeii has a relatively high HR efficiency. An HR-mediated DNA assembly
method was developed to facilitate rapid plasmid construction in a single step. Co-
transforming five linear DNA fragments, with 70–80 bp overlaps designed between
the adjacent fragments, directly into I. orientalis SD108 led to the successful con-
struction of a 14.5 kb plasmid containing the xylose utilization pathway with an
assembly efficiency of 100% (Cao et al. 2020).
Genome editing tools have been developed for P. kudriavzeii. The promoter
used to transcribe the gene encoding sgRNA needs to be an RNA polymerase
(RNAP) III promoter because a typical RNAP II promoter used to transcribe
proteins will make the gene undergo post-transcriptional modifications such as
5 -end capping and 3 -end polyadenylation, which may inactivate the Cas9/gRNA
complex (Gao and Zhao 2014). Tran and Cao et al. chose a series of RNAP III
promoters including tRNALeu , tRNASer , 5S rRNA, RPR1 (the RNA component of
RNase P, the 250 bp upstream sequence of RPR1), and fusions of 5S rRNA or
RPR1’ (the 250 bp upstream sequence of RPR1 and the first 120 bp of RPR1)
with tRNALeu . An iCas9 (containing D147Y and P411T) that possesses a higher
activity than Cas9 from Streptococcus pyogenes was tagged with a nuclear local-
ization sequence and expressed by an episomal plasmid, together with each of
the sgRNA cassettes. Targeting ADE2, LEU2, HIS3, and TRP1 in I. orientalis
SD108 showed that RPR1’-tRNALeu led to the highest single-gene disruption effi-
ciency of 97–100% and was therefore used to create double and triple knockouts.
Double-gene knockouts of ADE2/TRP1 and ADE2/HIS3 were attained with effi-
ciencies of 72.8% and 89.9%, respectively, whereas triple-gene knockout efficiency
for ADE2/HIS3/SDH2 was approximately 47% (Tran et al. 2019). In parallel, our
group also carried out a similar study by evaluating ADE2 disruption efficiencies
with five versions of Cas9 (including S. pyogenes Cas9, iCas9, a codon-optimized
Cas9 version for Homo sapiens, Candida albicans, and Scheffersomyces stipitis)
and RPR1 (the 311 bp upstream sequence of RPR1) as the sgRNA promoter in
P. kudriavzevii YB4010. The highest efficiency (42%) was achieved using iCas9
(Sun et al. 2020).
Another interesting area for exploring P. kudriavzevii as a production host for
organic acids is the identification of their transporters. Previously, our group iden-
tified a mitochondrial tricarboxylate transporter, Pk_MttA, which can potentially
transport citrate and cis-aconitate from the mitochondria to the cytosol, thereby
increasing itaconic acid production in P. kudriavzevii YB4010 (Sun et al. 2020).
A recent genome sequencing and transcriptome analysis of P. kudriavzevii CY902
led to the identification of two JEN family carboxylate transporters (PkJEN2-1
and PkJEN2-2), which can import succinate into cell (Xi et al. 2021). Substrate
specificity analysis showed that both PkJEN2-1 and PkJEN2-2 are dicarboxylate
importers for succinate, L-malate, and fumarate. In addition, PkJEN2-1 can import
α-ketoglutarate, whereas PKJEN2-2 can also uptake citrate but not α-ketoglutarate.
The structural basis of PkJEN2-2 specificity toward tricarboxylate substrates was
studied using model-based structure analysis and rational design. By inactivating
both transporters, enhanced extracellular succinate accumulation can be achieved

in the late stages of fermentation. This study highlights an important direction for
future studies to improve organic acid production in P. kudriavzevii.
5 Conclusions and Perspectives
A panel of synthetic biology tools has been developed to establish P. pastoris

as a microbial cell factory for the efficient production of recombinant proteins,
chemicals, and natural products. Although P. pastoris has been widely employed
for high-level expression of heterologous proteins, its application in the biosyn-
thesis of chemicals and natural products is still limited to a few examples. On
the other hand, although P. pastoris can utilize methanol as the sole carbon and
energy source, the methanol-to-chemical conversion efficiency is still rather low,
as most of the methanol is converted to CO2 via the dissimilatory pathway for
energy generation. Thus, the redirection of methanol flux toward the assimilatory
pathway rather than the dissimilatory pathway is a prerequisite for establishing P.
pastoris as a cell factory for chemical production from methanol. In other words,
the methanol conversion pathway for the assimilation of methanol and biosynthe-
sis of the desired product should be carefully engineered. Particularly, Lu et al.
constructed a three-step synthetic acetyl-CoA (SACA) pathway for the synthesis
of acetyl-CoA from formaldehyde by combining an engineered glycolaldehyde
synthetase variant (GALS), acetyl phosphate synthetase (ACPS), and phosphate
acetyltransferase (PTA). SACA represents the shortest pathway for acetyl-CoA
biosynthesis ever reported and is promising for the efficient production of acetyl-
CoA-derived compounds from C1 carbon sources (Lu et al. 2019). Considering
the capability of formaldehyde formation and the role of acetyl-CoA as an impor-
tant biosynthesis precursor, the SACA pathway is expected to be employed in P.
pastoris to produce value-added compounds from methanol in the near future.
The complexity and our insufficient understanding of the cellular metabolic
and regulatory networks have largely limited our capability of designing effi-
cient Pichia cell factories. For example, the expression of recombinant proteins
is affected by central metabolisms in P. pastoris (Nocon et al. 2014). One strat-
egy is to establish genome-scale metabolic models, such as PpaMBEL1254 (Sohn
et al. 2010), iPP668 (Chung et al. 2010), and iLC915 (Caspeta et al. 2012),
to describe the cellular metabolic network from a systems biology perspective.
Genome-scale metabolic models have been employed to guide the design of P.
pastoris cell factories with increased expression of hSOD, human lysozyme, and
antibody fragment Fab-3H6 (Cankorur-Cetinkaya et al. 2018). Unfortunately, the
complexity of cellular metabolic and regulatory networks is still beyond the reach
of the genome-scale metabolic models. For example, the overexpression of a non-
intuitive gene, RAD52, encoding a protein responsible for DNA recombination was
found to be beneficial for the expression of CYPs and correspondingly the produc-
tion of trans-nootkatone (Wriessnegger, et al. 2016). Similar to that of P. pastoris,
282 W. Sun et al.
a genome-scale metabolic model, iIsor850, has been developed for P. kudriavze-

vii (i.e., I. orientalis SD108). This model contains 850 genes, 1826 reactions, and
1702 metabolites (Suthers 2020). Biomass composition data and the estimated ATP
maintenance requirements were collected by cultivating I. orientalis SD108 in a
chemostat under carbon limitation, to improve the predictive power of the model.
The consistency of the model predictions was validated using assessment of sub-
strate utilization and gene knockouts. This model was used to propose engineering
strategies for enhanced succinic acid production using the OptKnock framework.
This model will be beneficial for the metabolic engineering of P. kudriavzevii for
other organic acid production.
However, so far, only a small portion of genes is included in the genome-scale
metabolic models, and the predictions are not sufficiently accurate and far from
perfection. Therefore, an alternative strategy is to employ synthetic biology-based
genome-scale engineering, which can perturb many genes in a combinatorial man-
ner, extending our limited knowledge on cellular metabolism and regulation. Such
a creation-driven-understanding technology aims to identify non-intuitive engi-
neering targets, to increase the expression of recombinant proteins, as well as the
production of desirable compounds. Currently, CRISPR-based genome-scale engi-
neering tools have been developed for E. coli (Garst et al. 2017), S. cerevisiae (Lian
et al. 2017, 2019), and mammalian cells (Gilbert et al. 2014), and are expected to
be established in P. pastoris and P. kudriavzevii in the near future, which can
be employed for the construction of efficient cell factories in a high-throughput
manner.
Acknowledgements YZ, JG, and JL were supported by the National Key Research and Develop-
ment Program of China (2018YFA0901800). WS and ZS were supported by the U.S. Department
of Energy, Office of Science, Biological and Environmental Research through Ames Laboratory.
Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under
Contract No. DE-AC02-07CH11358.
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47
Kluyveromyces marxianus
as a Platform in Synthetic Biology
for the Production of Useful
Materials
Noppon Lertwattanasakul, Mochamad Nurcholis,

Nadchanok Rodrussamee, Tomoyuki Kosaka, Masayuki Murata,
and Mamoru Yamada
Abstract
In Kluyveromyces marxianus, a thermotolerant yeast that has already been uti-
lized for producing various useful materials, recent advances including complete
genome sequencing and transcriptome analysis have provided valuable infor-
mation on its physiological and metabolic characteristics including its capacity
for assimilation of various sugars and tolerance to high temperatures, which
are distinct from Saccharomyces cerevisiae. These prominent properties enable
the development of high-temperature fermentation (HTF) technology for indus-
trial applications. In addition, the yeast is able to integrate DNA fragments
into its genome, which makes it easy to introduce foreign genes or gene sets
N. Lertwattanasakul
Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
M. Nurcholis
Department of Food Science and Technology, Faculty of Agricultural Technology, Brawijaya
University, Malang 65145, Indonesia
N. Rodrussamee
Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand
T. Kosaka · M. Yamada (B)
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University,
Yamaguchi 753-8515, Japan
T. Kosaka · M. Murata · M. Yamada
Graduate School of Science and Technology for Innovation, Yamaguchi University,
T. Kosaka · M. Yamada
Research Center for Thermotolerant Microbial Resources, Yamaguchi University,
https://doi.org/10.1007/978-3-030-89680-5_11
294 N. Lertwattanasakul et al.
into the genome. Moreover, DNA editing technology and simulation models
are in place. Therefore, K. marxianus is a new platform in synthetic biology for
producing useful materials.
1 Introduction
Microbial production of fuels and chemicals from biomass and other renewable
carbon sources is an attractive alternative to petroleum-derived production. Saccha-
romyces cerevisiae is the organism of choice because of its high rate of production
and tolerance to ethanol titers upwards of 120 g L−1 (Qiu and Jiang 2017; Nielsen
et al. 2013). These outstanding phenotypes, among others, may have led to the
widespread study of S. cerevisiae and its development as a model eukaryotic host
for chemical biosynthesis.
Kluyveromyces marxianus is a Crabtree-negative yeast and favors respiration
over fermentation. This yeast is also industrially relevant because of its wide
substrate spectrum including pentose sugars, fast-growth characteristics, and ther-
motolerance to ~50 °C (Varela et al. 2017; Löbs et al. 2016), making it a promising
host for industrial biotechnology to produce renewable chemicals from agricultural
biomass feedstocks. Native strains of K. marxianus are also known to synthesize
ethyl acetate at rates above 2 g L−1 h−1 in aerated bioreactors (Löser et al. 2013,
2015). In recent years, increasing interest has been shown in several new applica-
tions including production of biomolecules (Hughes et al. 2017; Lin et al. 2017),
biocatalysts (Wang et al. 2017; Simoness et al. 2017), and heterologous protein
expression (Lee et al. 2017; Gombert et al. 2016). However, major genetic engi-
neering limitations have kept this yeast from replacing the commonly used yeast
S. cerevisiae in industrial applications.
2 Assimilation Capacity of Various Substrates
One of the features of K. marxianus that are suitable for industrial applications
is its ability to utilize a broad range of substrates. The major common feature of
sugar utilization by K. marxianus is the ability to assimilate lactose and inulin
as a carbon source, a feature that is absent in S. cerevisiae (Lane and Morrissey
2010). The ability has evolved by acquiring LAC genes, LAC12 and LAC4, that
are accountable for the uptake of lactose and subsequent cleavage into galactose
and glucose, respectively, and the inulinase gene INU1, which liberates fructose
molecules from oligo- or poly-sugars with β-(2,1)-linked fructose units at the
terminal (Rouwenhorst et al. 1988).
Consistent with the capacity of this yeast to utilize a wide variety of substrates,
there are a number of sugar transporters, including 27 putative sugar transporters,
encoded by the genome of K. marxianus DMKU 3-1042 (Lertwattanasakul et al.
2015). The ability for utilization of a wide variety of carbon sources by various
strains of K. marxianus compared to S. cerevisiae has been shown (Nonklang et al.
Kluyveromyces marxianus as a Platform in Synthetic Biology … 295
2008) (Fig. 1). These carbon sources include monosaccharides of six-carbon sugars
(galactose, fructose, and mannose), monosaccharides of five-carbon sugars (xylose,
xylitol, and arabinose), disaccharides (sucrose, lactose, and cellobiose), trisaccha-
ride (raffinose), polysaccharide (inulin), and a non-fermentable carbon source of
glycerol. Besides, K. marxianus DMB1 can utilize sorbitol, which is not generally
utilized by K. marxianus strains, as a non-fermentable carbon source (Goshima
et al. 2013b). K. marxianus can utilize these carbon sources, while S. cerevisiae
cannot utilize cellobiose, xylose, xylitol, arabinose, glycerol, sorbitol, and lactose.
Ethanol is one of the classic bulk chemical products of yeasts. Much interest has
been shown in K. marxianus for the possibility of it being a platform for ethanol
production because of not only its capability of utilizing a broad range of substrates
but also its fermentation ability at high temperatures. These properties lead us to
develop a stable HTF technology with several benefits as described below and
to utilize not only first-generation substrates but also second- or third-generation
substrates.
Fig. 1 Metabolic pathways for synthesis of high-value chemicals in K. marxianus. Abbreviated

versions of how these processes that lead to the formation of classes of metabolite are shown,
along with examples cited in the main text. Reactions are depicted as unidirectional, although many
are reversible. Important enzymatic steps are indicated by the key, as follows: 1. pyruvate dehy-
drogenase; 2. pyruvate decarboxylase; 3. alcohol dehydrogenase; 4. alcohol acyl transferase; 5.
xylose reductase; 6. xylitol dehydrogenase; 7. xylulokinase; 8. inulinase; 9. β-galactosidase; 10.
endopolygalacturonase
Related to conversion from lactose to ethanol, which has been received much
attention, expression of genes for β-galactosidases in K. marxianus is induced
by its natural inducers, galactose, and lactose. However, the production of β-
galactosidases seems to be dependent on the substrate concentration. In K.
marxianus CBS6556, the maximum activity of β-galactosidase is obtained at low
concentrations of the inducer carbohydrates, in the range between 0.5 and 15 mM.
When shifted to a high concentration of D-galactose or lactose, a repressive mech-
anism is superimposed to the inducing effect of the substrate (Martins et al. 2002).
K. marxianus exhibits glucose repression in galactose utilization, and galactose is
utilized after diminishing glucose in a mixed-sugar medium (Rodrussamee et al.
2011). Moreover, mutation of MIG1, which plays key roles as a regulator complex
in glucose repression, increases the activity of lactose hydrolysis (Zoppellari and
Bardi 2013).
In dairy industries, dairy effluents as high strength wastewater contains a
high concentration of lactose, which is responsible for the high chemical oxy-
gen demand (COD), causing an environmental problem (Karim et al. 2020). Whey
and permeate are effluents from cheese processing, while scotta is effluent from
ricotta processing. Whey is a waste produced by dairy industries in large amounts
(approximately, 10 L per kg of cheese), and it contains 4–6% lactose in addi-
tion to proteins and other nutrients (Guimarães et al. 2010). Many studies on the
conversion of whey to ethanol have been performed because whey is a low-cost
and abundant material with high carbohydrate concentrations (Zafar and Owais
2006; Ozmihci and Kargi 2007; Guimarães et al. 2010; Zoppellari and Bardi 2013;
Roohina et al. 2016). It has been reported that the yield of ethanol from cheese
whey powder produced by K. marxianus DSMZ-7239 is equal to the theoretical
yield of 0.54 g EtOH per g lactose (Ozmihci and Kargi 2007). Moreover, whey
permeate and scotta have the potential to become key inexpensive substrates for
bioethanol production by K. marxianus (Jedrzejewska and Kozak 2011; Sansonetti
et al. 2009; Zoppellari and Bardi 2013). Besides dairy byproducts, third-generation
substrates such as marine biomass have been used for producing ethanol. For
example, the red seaweed Gracilaria verrucosa, which contains glucose and galac-
tose, has been used for producing ethanol, with S. cerevisiae, Candida lusitaniae,
and K. marxianus adapted to high concentrations of galactose (Park et al. 2020).
Among them, K. marxianus has shown the highest ethanol yield coefficient of
0.47 g g−1 .
Conversion of inulin as a D-fructose polymer to ethanol is a notable conversion
process that maximally utilizes the characteristics of K. marxianus, and extensive
studies on inulin have been performed using Jerusalem artichoke (JA), a crop that
contains nearly 20% of carbohydrates, 70–90% of which is inulin (Hu et al. 2012;
Yuan et al. 2012; Kim et al. 2013; Kim and Kim 2014; Charoensopharat et al.
2015). Inulinase encoded by INU1 is very effective in hydrolyzing inulin to D-
fructose monomers, which are then imported into cells by the fructose transporter
Frt1p and catabolized through the glycolytic pathway. Regulation of the expression
of INU1 in K. marxianus has been studied using various strains, and it has been
shown that the production of inulinase depends on the carbon source (Sokolenko
and Karpechenko 2015; Lertwattanasakul et al. 2011; Hoshida et al. 2018). In

K. marxianus NCIM 3231, sucrose and fructose acted as inducers for inulinase
production, though their actions were weaker than that of inulin, whereas in K.
marxianus CDBBL278, no such inulinase induction was found (Cruz-Guerrero
et al. 1995). The expression of INU1 also seems to be under the control of glu-
cose repression (Gupta et al. 1994). K. marxianus is one of the non-conventional
yeast candidates as ideal consolidated bioprocessing (CBP). Ethanol production
from inulin by CBP in K. marxianus Y179 was found to be dependent on inulin
concentrations and aeration levels, with the yeast producing ethanol up to 98 g
L−1 from inulin with an ethanol yield of 0.43 g g−1 at 30 °C (Gao et al. 2015).
Direct ethanol fermentation from fresh JA tubers without inulin hydrolysis by CBP
using K. marxianus DBKKUY-102 achieved a maximum ethanol concentration of
97.46 g L−1 at 40 °C (Charoensopharat et al. 2015).
Lignocellulosic biomass is an attractive source, which does not compete
with land for food production, because it is the most abundant renewable and
inexpensive biomass (Hahn-Hägerdal et al. 2006). The primary components in
lignocellulosic biomass are cellulose (35–50 wt.%, dry basis), hemicellulose (15–
30%), pectin (2–5%), and lignin (12–35%). Cellulose and hemicellulose, which
account for more than 50% of the total mass, can be converted to sugars for their
conversion to ethanol (Kumar et al. 2016). In contrast to cellulose and starch, lig-
nocellulose is composed of a mixture of hexose (glucose, mannose, and galactose)
and pentose (xylose and arabinose) sugars, which can be released by pretreat-
ment and enzymatic hydrolysis steps (Margeot et al. 2009). Cost-competitive
ethanol yields from lignocellulose, require fermentation of both hexose and pen-
tose constituents (Galbe and Zacchi 2007). In order to realize economical ethanol
production from lignocellulose, one of the prerequisites for making lignocellulosic
ethanol processes economically competitive is a robust-fermenting microorganism
that can effectively convert all sugars released from lignocellulose to ethanol with
a high yield and high productivity. Unlike S. cerevisiae, which is unable to uti-
lize pentose sugars as a sole carbon source for growth and fermentation (Kuhn
et al. 1995), K. marxianus can effectively uptake and catabolize the pentoses,
xylose, and arabinose, via the aldose reductase pathway and produce ethanol from
all sugar components in lignocelluloses except for arabinose (Rodrussamee et al.
2011). However, the yeast inefficiently produces ethanol from xylose compared to
hexose and co-produces xylitol and/or acetic acid as byproducts, and the consump-
tion of xylose is exposed to glucose repression (Harner et al. 2015; Nitiyon et al.
2016; Rodrussamee et al. 2011). Xylose fermentation of this strain also requires
microaerophilic conditions for relieving the cofactor imbalance due to different
preferred coenzymes of xylose reductase (XR) and xylitol dehydrogenase (XDH),
having a higher affinity for NADPH and NAD+ , respectively (Harner et al. 2015).
Some strains are capable of fermenting xylose, though inefficiently, even at temper-
atures of more than 40 °C (Suryawati et al. 2008; Kumar et al. 2009; Rodrussamee
et al. 2011). Metabolic engineering strategies may thus be required to improve the
capability for fermentation of xylose, like those adopted for S. cerevisiae (Mat-
sushika et al. 2009). Many attempts have been made to improve the coenzyme
specificities by replacing KmXR and/or KmXDH with the corresponding genes

from other microorganisms with or without modification by site-directed mutage-
nesis (Table 1) such as by using XR of Schefferomyces stipitis (SsXR) (Zhang et al.
2013), XR and XDH of S. stipitis (SsXR and SsXDH) together with xylulokinase
(XK) of S. cerevisiae (ScXK) (Goshima et al. 2013a), and XR of Neurospora crassa
(NcXR) and SsXDH together with overexpression of several downstream K. marx-
ianus genes (Zhang et al. 2015a, b). Recently, Suzuki et al. (2019) developed a
recombinant K. marxianus DMB13 strain by multiple site-directed mutagenesis to
overexpress an NADP+ -dependent KmXDH mutant gene and by overexpression of
the wild-type KmXR and KmXK genes. The recombinant strain rapidly converted
xylose to ethanol after depletion of glucose and achieved the maximum ethanol
yield of 0.402 g g−1 in xylose/glucose co-fermentation at 40 °C. An alternative
approach of expressing a xylose isomerase (XI), which directly converts xylose
to xylulose, has been performed in K. marxianus. Wang et al. (2013) transformed
the xylose isomerase gene XYLA from the fungus Orpinomyces under control of
the GAPDH promoter in K. marxianus YHJ010 in which KmXR and KmXDH
had been disrupted and adapted the transformant to a xylose medium by repet-
itive cultivation. The resultant strain achieved ethanol yields of 0.38 g g−1 and
0.31 g g−1 at 42 °C and 45 °C, respectively, and produced 8.25 g L−1 ethanol
from the corn cob hydrolysate containing 20.04 g L−1 xylose. Similar evolution-
ary adaptation to xylose at 45 °C improved ethanol and xylitol yields in the xylose
medium (Sharma et al. 2016, 2017). A trial of an evolutionary adaptation approach
together with random mutagenesis using ethyl methanesulfonate also improved the
ethanol yield from xylose, being 2.31-fold higher than that of the parental strain
(Kwon et al. 2019). On the other hand, a random kanMX4-insertion mutagene-
sis revealed essential factors for pentose metabolism in K. marxianus, including a
cytochrome oxidase assembly factor (singleton) (KmCOX15), a transcription fac-
tor required for assembly of the Atp9p subunit of mitochondrial ATP synthase
(KmATP25) and cytochrome c heme lyase (KmCYC3), suggest that respiratory
activity is essential for utilization of pentose in (Lertwattanasakul et al. 2013).
Consolidated bioprocessing (CBP) is an alternative and interesting technology
for conversion of lignocellulose into desired products in one step without exoge-
nous enzymes because of its simplicity and potentially low cost (Olson et al. 2012).
Breakdown of lignocellulose into fermentable sugars requires three major types
of enzymes, endo-1,4-β-glucanase (EC 3.2.1.4), cellobiohydrolase (EC 3.2.1.91),
and β-glucosidase (EC 3.2.1.21), and several hemicellulases such as endo-1,4-
β-xylanase (EC 3.2.1.8), endo-1,4-β-mannanase (EC 3.2.1.78), β-xylosidase (EC
3.2.1.27), and hemicellulolytic esterases (Shallom and Shoham 2003; Li et al.
2017). However, no natural microorganisms with both efficient enzyme produc-
tion capability for lignocellulose saccharification and ethanol production ability
are currently available (Hasunuma and Kondo 2012). Since, fortunately, K. marxi-
anus has some capability to utilize cellobiose and xylose, heterologous expression
of other cellulolytic and/or xylanolytic enzymes may expand its capability for the
conversion of lignocellulose to biorefinery products including ethanol.
Table 1 Examples of native and non-native chemicals produced by K. marxianus
Aimed product Modification Source Parental strain Cultivation condition Titer References
L-Lactic acid ↑PfLDH Plasmodium YZB058 Glucose/xylose, 42 °C 103 g/L Kong et al. (2019)
falciparum
↑BmLDH Bacillus
megaterium
↑ScJEN1 Saccharomyces
cerevisiae
↑KmPFK ΔKmDLD1 Kluyveromyces
marxianus
↑Bmldh B. megaterium KM1 – 24 g/L Pecota et al. (2007)
Xylitol ↑ScGAL2 (N376F) S. cerevisiae KCTC 17,555 Glucose/xylose, 30 °C 95.58 g/L Kwon et al. (2020)
↑NcXYL1 Neurospora crassa NBRC1777 Xylose, 45 °C 60.03 g/L Zhang et al. (2014)
2-Phenylethanol ↑KmARO4 (K221L) ↑KmPHA2 K. marxianus CBS 6556 Glucose, 30 °C 1943 ± 63 mg/L Li et al. (2021)
↑KmARO7 (G141S) ↑KmARO10
KmEAT1
↑KmTKL1 ↑KmTAL1 K. marxianus NBRC1777 Glucose, 30 °C 850 mg/L Rajkumar and
↑KmARO3 (feedback resistant) Morrissey (2020)
KmARO8
KmTYR1prΔ::REV1pr
Kluyveromyces marxianus as a Platform in Synthetic Biology …
↑Bbxfpk Bifidobacterium
breve
↑Septa Salmonella
enterica
(continued)
299
300
Table 1 (continued)
↑EcppsA Escherichia coli
↑ScARO10 ↑ScADH2 S. cerevisiae DMKU 3-1042 Glucose, 30 °C 1.0 g/L Kim et al. (2014)
Hexanoic acid ↑atoB E. coli ATCC17555 Galactose, 37 °C 154 mg/L Cheon et al. (2014)
↑bktB Ralstonia eutropha
↑crt ↑hbd Clostridium
acetobutylicum
↑ter Treponema
denticola
↑MCT1 S. cerevisiae
Pyruvate KmPDC1 KmGPD1 K. marxianus YZJ051 Glucose/xylose, 42 °C 29.21 g/L Zhang et al. (2017a,
↑KmMTH1-ΔT b)
↑SsXYL2 Scheffersomyces
stipitis
↑ScGAL2 (N376F) S. cerevisaie
β-Carotene ↑HpCHYb Haematococcus KY3 Galactose, 30 °C 224.4 ± 9.9 mg/g DCW Chang et al. (2015)
pluvialis
Triacetic acid lactone ↑G2PS1 Gerbera hybrida CBS 6556 Xylose, 37 °C 1.24 g/L McTaggart et al.
(2019)
D-Allulose ↑dpe Agrobacterium CICC 1911 Fructose, 55 °C 190 g/L Yang et al. (2018)
tumefaciens
Fatty acid ethyl esters ↑KmATF1 K. marxianus UMPe-1 Glucose, 30 °C 1.53% Campos-García
et al. (2018)
Cellulases ↑EGI ↑EGIA Aspergillus niger KY3 Glucose – Chang et al. (2017)
(continued)
N. Lertwattanasakul et al.
Table 1 (continued)
↑EGIII ↑CBHI Trichoderma
reesei
↑CBHII Chemically
synthesized and
optimized for K.
marxianus
↑NpaBGS Neocallimastix
patriciarum
Tannase – – NRRL Y-8281 Olive pomace, 45 °C 1026.12 U/mg Mahmoud et al.
(2018)
α-Galactosidase – Cyamopsis CBS 6556 Starch 153 mg/L Bergkamp et al.
tetragonoloba (1993)
α-Amylase ↑TAA A. oryzae DMKU 3-1042 – – Hoshida et al.
(2014)
β-Galactosidase ↑KlLAC4 Kluyveromyces NBRC1777 Glucose, 45 °C 20 U/mg Yang et al. (2015)
lactis
β-Glucuronidase ↑gusA E. coli NBRC1777 Glucose, 45 °C 0.8 U/mg Yang et al. (2015)
Superoxide dismutase ↑KlSOD1 K. lactis L3 Glucose, 30 °C 24 kU/L Raimondi et al.
(2010)
β-Glucuronidase ↑gusA E. coli KM1 Glucose, 37 °C 50 nmole 4-MU/min Pecota and Da silva
/mL (2005)
Glucose oxidase ↑GOX A. niger CBS 6556 Glucose, 30 °C 1722 U/g DCW Rocha et al. (2010)
(continued)
301
302
Table 1 (continued)
Esterase ↑est Thermus CBS 6556 Sucrose 56.9 U/g DCW Rocha et al. (2011)
thermophilus
Glucoamylase ↑GAA Arxula L3 Glucose, 40 °C 80 U/mL Raimondi et al.
adeninivorans (2013)
Dengue virus type I – Dengue virus UFV-3 Galactose, 37 °C 1.2 mg/mL Bragança et al.
non-structural protein 1 (2015)
Cytochrome P450 ↑CYP505A1 Fusarium UOFS Y1185 Glucose, 28 °C – Theron et al. (2014)
monooxygenase oxysporum
↑CYP102A1 B. megaterium
Xylose isomerase ↑XYLA Orpinomyces NBRC1777 Xylose – Wang et al. (2013)
Porcine circovirus type 2 – Porcine circovirus FIM-1 Glucose, 30 °C 1.91 g/L Duan et al. (2019)
virus-like particles type 2
Porcine parvovirus – Parvovirinae virus FIM-1 Glucose, 30 °C 2.5 g/L Yang et al. (2021)
virus-like particles
Single-chain antibody ↑scFv HyHEL-10 scFv NBRC1777 Xylose, 30 °C – Nambu-Nishida
et al. (2018)
Ethanol ↑KmXYL2 (NADP+ -dependent) K. marxianus DMB13 Xylose/glucose, 40 °C – Suzuki et al. (2019)
↑PsXYL1 (N272D) Pichia stipitis NBRC1777 Xylose, 42 °C 3.55 g/L Zhang et al. (2013)
↑NcXYL1 N. crassa NBRC1777 Xylose/glucose, 42 °C 6.22 g/L Zhang et al. (2015a,
b)
(continued)
Table 1 (continued)
↑PsXYL2 P. stipitis
↑PsXYL1 ↑PsXYL2 P. stipitis DMB1 Xylose, 45 °C 3.3 g/L Goshima et al.
↑ScXYL3 S. cerevisiae (2013a)
↑AMY A. oryzae NBRC1777 Starch, 48 °C 36.88 g/L Wang et al. (2014)

↑AMY ↑GAM1 Debaryomyces
occidentalis
↑AMY A. oryzae DMKU 3-1042 – – Nonklang et al.
(2008)
↑EGIII ↑CBHI T. reesei KY3 Cellulose, 30 °C 0.9 g/L Chang et al. (2012)
↑EG ↑CBH T. reesei KY3 Cellulose, 40 °C 0.6 g/L Chang et al. (2013)
↑EG ↑CBH A. niger
↑BGL N. patriciarum
↑CDT N. crassa
↑EG A. niger NBRC1777 Cellobiose, 45 °C 43.4 g/L Hong et al. (2007)
↑CBH ↑BGL T. reesei
↑EG T. reesei NBRC1777 Cellulosic β-glucan, 48 °C 4.24 g/L Yanase et al. (2010)
↑BGL A. aculeatus
↑FLO1 ↑FLO5 ↑FLO9 or S. cerevisiae DMKU 3-1042 Glucose, 40 °C 48 g/L Nonklang et al.
↑FLO10 (2009)
(continued)
303
304
Table 1 (continued)
↑BGL Thermoascus NBRC1777 Cellobiose, 45 °C 29.5 g/L Matsuzaki et al.
aurantiacus (2012)
↑ScGAL2 ↑ScGAL2 (N376F) S. cerevisiae KCTC 17555 Glucose/galactose, 30 °C 36.14 g/L Kwon et al. (2020)
↑olpB-ScGPI ↑cipA C. thermocellum 4G5 Avicel, 30 °C 3.09 g/L Anandharaj et al.
S. cerevisiae Phosphoric acid-swollen 8.61 g/L (2020)
cellulose, 30 °C
LDH lactate dehydrogenase, JEN1 proton-coupled monocarboxylate transporter, PFK 6-phosphofructokinase, dld1 putative d-lactate dehydrogenase, GAL2 galactose permease, XYL1 xylose
reductase, XYL2 xylitol dehydrogenase, XYL3 xylulokinase, XYLA xylose isomerase, ARO3/ARO4 DAHP synthase, ARO7 chorismate mutase, ARO8 aromatic aminotransferase, ARO10
phenylpyruvate decarboxylase, PHA2 prephenate dehydratase, eat1 ethanol acetyltransferase, TKL1 transketolase, TAL1 transaldolase, xfpk phosphoketolase, pta phosphotransacetylase,
ppsA phosphoenolpyruvate synthase, ADH2 alcohol dehydrogenase 2, atoB acetyl-CoA acetyltransferase, bktB β-ketothiolase, crt crotonase, hbd 3-hydroxybutyryl-CoA dehydrogenase, ter
trans-enoyl-CoA reductase, MCT1 malonyl CoA-acyl carrier protein transacylase, PDC1 pyruvate decarboxylase, GPD1 glycerol-3-phosphate dehydrogenase, MTH1 negative regulator of the
glucose-sensing signal transduction pathway, CHYb β-carotene hydroxylase, G2PS1 2-pyrone synthase, dpe D-psicose-3-epimerase, ATF1 alcohol acetyl-transferase, EG endoglucanase,
CBH cellobiohydrolase BGL β-glucosidase, LAC4 β-galactosidase, gusA β-glucuronidase, SOD1 superoxide dismutase, GOX glucose oxidase, est esterase, GAA glucoamylase, CYP
cytochrome P450 monooxygenase, AMY amylase, GAM1 glucoamylase, CDT cellodextrin transporter, FLO flocculating, GPI glycosylphosphatidylinositol, cipA scaffoldin, olpB anchoring
protein
A recombinant of K. marxianus NBRC1777 expressing endoglucanase from

Trichoderma reesei and β-glucosidase from Aspergillus aculeatus on its cell sur-
face produced 43.4 g L−1 ethanol, corresponding to 90% of the theoretical yield
from 10% cellobiose (Yanase et al. 2010). Chang et al. (2012) developed a tech-
nique named Promoter-based Gene Assembly and Simultaneous Overexpression
(PGASO), which uses overlapping oligonucleotides for recombinatorial assembly
of gene cassettes with individual promoters to engineer K. marxianus KY3 for
co-expressing three types of cellulase genes for exoglucanase and endoglucanase
from Trichoderma reesei and β-glucosidase from a cow rumen fungus. The engi-
neered strain was found to utilize β-glycan, cellobiose, or carboxymethyl cellulose
as the sole carbon source for growth and directly convert cellobiose and β-glycan
to ethanol. In addition, the co-expression of five cellulase genes for two cellobio-
hydrolases, two endo-β-1,4-glucanases from Trichoderma reesei, and β-glucosidase
from the cow rumen fungus Neocallimastix patriciarum, and a gene for the cel-
lodextrin transporter from N. crassa resulted in direct conversion of cellulose to
ethanol, producing 0.6 g L−1 ethanol from 10% avicel (Chang et al. 2013). Zhou
et al. (2018) attempted heterologous expression and secretion of lignocellulolytic
enzymes, endo-1,4-β-mannanase, endo-1,4-β-endoxylanase, endo-1,4-β-glucanase,
and feruloyl esterase in K. marxianus using the INU1 promoter and the signal
sequence of inulinase.
3 Thermotolerance and High-Temperature Fermentation

Ability
K. marxianus has the interesting feature of producing several useful materials such
as ethanol as its main product (Table 2). The application of HTF at a temperature
of about 40 °C or higher is expected to provide many advantages over general
fermentation at a temperature of about 30 °C or less including reducing operating
costs of cooling systems, minimizing risk of contamination, reducing viscosity of
the fermentation broth, efficiently achieving simultaneous saccharification and fer-
mentation, robustness process against accidental temperature rise even in tropical
countries, operating continuous ethanol removal, and increasing enzyme activity
for biomass hydrolysis (Banat et al. 1998; Fonseca et al. 2008; Hoshida and Akada
2017; Kosaka et al. 2018). Fermenting microbes for HTF must be thermotolerant,
that is, they must grow sufficiently and ferment efficiently at high temperatures.
Yeast strains that have often been isolated as ethanol-producing thermotoler-
ant yeasts are strains of K. marxianus and S. cerevisiae because they are highly
thermotolerant yeasts and efficient ethanol-producing yeasts, respectively (Hoshida
and Akada 2017). K. marxianus DMKU 3-1042 efficiently produced ethanol from
sugar cane juice at a temperature of 40 °C, and the maximal ethanol concentration
was 67.8 g L−1 and the yield was 60.4% of the theoretical yield (Limtong et al.
2007). Five K. marxianus strains isolated in Laos exhibited strong fermentation
abilities in a 16% sugars-containing medium of glucose, sucrose, sugarcane, or
molasses at 40 °C (Keo-oudone et al. 2016). The evolutionary adapted KM-100d
Table.2 Comparison of ethanol production levels and yields among various strains of K. marxi-
anus
Strains Temp Carbon sources Time Ethanol Ethanol References
(°C) (h) production yield (g
(g L−1 ) g−1 )
K. marxianus 30 Xylose 48 1.71 ± 0.45 0.09 ± 0.03 Nitiyon et al.
DMKU 3-1042 (2016)
30 Xylose 72 ~2.60 0.13 Rodrussamee
et al. (2011)
37 Xylose 36 1.29 ± 0.23 0.07 ± 0.01 Nitiyon et al.
(2016)
et al. (2011)
et al. (2011)
40 Sugar cane 60 67.8 0.6 Limtong et al.
juice (2007)
K. marxianus 30 Xylose 48 2.91 ± 0.40 0.15 ± 0.02 Nitiyon et al.
BUNL-21 (2016)
37 Xylose 36 2.58 ± 0.05 0.14 ± 0.00 Nitiyon et al.
(2016)
40 Glucose 24 68.7 0.43 Keo-oudone
et al. (2016)
K. marxianus IMB3 45 Xylose 0.8–1.2 0.08–0.12 Banat et al.
(1998)
K. marxianus FIM1 30 Glucose 48 110 0.55 Mo et al. (2019)
45 Glucose 48 50 0.25 Mo et al. (2019)
K. marxianus 40 Mannose 24 8.52 0.426 Rouhollah et al.
(2007)
K. marxianus SBK1 40 Glucose and 72 23.82 0.35 Kim et al. (2019)
xylose
K. marxianus DMB1 42 Lignocellulosic 25.98 Goshima et al.
hydrolysates (2013b)
K. marxianus CICC 40 Lignocellulosic 42.6 Du et al. (2019)
1727–5 hydrolysates
K. marxianus 37 Inulin 72 ~104.83 0.47 Charoensopharat
DBKKU Y-102 jerusalem et al. (2015)
artichoke
40 Inulin 72 ~97.46 0.45 Charoensopharat
jerusalem et al. (2015)
artichoke
K. marxianus Y179 30 Inulin 36 98 0.43 Gao et al. (2015)
mutant improved ethanol productivity to produce about 50 g L−1 in a glucose-

containing medium at 45 °C (Mo et al. 2019). Rouhollah et al. (2007) co-cultured
P. stipitis and K. marxianus to convert mixed sugars of glucose and xylose to
ethanol and achieved a high ethanol yield (0.42 g g−1 ) and high maximum ethanol
(31.87 g L−1 ) at 40 °C. Goshima et al. (2013b) reported fermentation of ethanol
from lignocellulosic hydrolysates of Japanese cedar and eucalyptus with ethanol
concentrations of 25.98 g L−1 and 13.4 g L−1 at 42 and 45 °C, respectively.
Other yeast strains could also produce ethanol at high temperatures including
Saccharomyces uvarum (Hacking et al. 1984), Saccharomyces carlsbergensis (Szc-
zodrak and Targoński 1988), Ogataea polymorpha (Ryabova et al. 2003), Candida
pseudotropicalis (Hacking et al. 1984), Candida acidothermophilum (Kadam and
Schmidt 1997), and Pichia kudriavzevii (Chamnipa et al. 2018; Koutinas et al.
2016; Oberoi et al. 2012; Pongcharoen et al. 2018; Yuangsaard et al. 2013). Among
these yeasts, some K. marxianus strains are able to produce ethanol at tempera-
tures above 40 °C and to have maximum growth temperatures of 47 °C (Anderson
et al. 1986), 49 °C (Hughes et al. 1984; Nonklang et al. 2008), and even 52 °C
(Banat et al. 1992; Lane and Morrissey 2010). These abilities of K. marxianus are
superior to those of other species that have an optimum fermentation temperature
of about 40 °C, suggesting that K. marxianus is a better species for HTF (Hoshida
and Akada 2017) and for simultaneous saccharification and fermentation (SSF)
(Choudhary et al. 2016).
There have been many studies on SSF using K. marxianus as shown in Table 2.
However, the major problem with ethanol production using K. marxianus strains
is the low production levels of lignocellulolytic enzymes. Heterologous expression
of lignocellulolytic enzymes needs to be improved by optimizing the inulinase
promoter and signal sequence of K. marxianus through mutagenesis. A mutation
improved the secretory expression of lignocellulolytic enzymes, including endo-
1,4-β-glucanase, endo-1,4-β-endoxylanase, and endo-1,4-β-mannanase by up to
threefold (Zhou et al. 2018). On the other hand, a thermotolerant mutant derived
from K. marxianus SBK1 that is capable of simultaneous co-fermentation of glu-
cose and xylose produced 23.82 g L−1 ethanol at 40 °C. A mutant strain that
alleviated catabolite repression fermented mixed sugars simultaneously (Kim et al.
2019). Co-culture of K. marxianus CICC-1727–5 with Spathaspora passalidarum
ATCC MYA-4345 was performed to convert lignocellulosic biomass to ethanol
(Du et al. 2019).
4 Findings from Complete Genome Sequencing

and Transcriptome Analysis
Genomic and transcriptomic studies have shed light on K. marxianus, and an

increasing number of genome sequences of K. marxianus strains are becoming
available. Genomic studies have been performed on KCTC 17555 (Jeong et al.
2012), DMB1 (Suzuki et al. 2014), CCT 7735 (Silveira et al. 2014), NBRC1777
(Inokuma et al. 2015), DMKU 3-1042 (Lertwattanasakul et al. 2015), B0399
308
Table.3 Using K. marxianus for ethanol production in SSF at high temperatures

Strains of K. Biomass Temp. (°C) Pretreatment Enzymes used in EtOH production Yield (YE/S ) References
marxianus saccharification (g L−1 )
L. G 10% Solka-floc 42 – 15 FPU/g 37.6 0.50 g g−1 Ballesteros et al.
(microcrystalline (cellulase) (1991)
cellulose)
Y01070 5% Spruce chip 42 Pretreatment by 37 FPU/g 7.5 31.5 g/100 g Bollók et al.
impregnation with (cellulase) and (2000)
2.5% SO2 and then 38 IU/g
steam at 215 °C for (β-glucosidase)
5 min
NCIM 3358 10% Solka-floc 43 – 40 FPU/g 35.0 2.5–3.5% w/v Krishna et al.
(microcrystalline (cellulase) and (2001)
cellulose) 50 U/g
(β-glucosidase)
Y01070 6% Solka-floc 40 – 15 FPU/g 17.8 0.34 Kádár et al.
6% OCC (cellulase) and 14.1 0.31 (2004)
6% paper sludge 15 IU 8.8 0.33
(β-glucosidase)
IMB4 Switchgrass (4.1% 45 Pretreatment by 15 FPU/g 16.6 NR Suryawati et al.
glucan) hydrothermolysis at (cellulase) (78% theoretical (2008)
200 °C for 10 min yield)a
IMB3 8% Switchgrass 45 Pretreatment by 82.2 FPU/mL 22.5 NR Pessani et al.
hydrothermolysis at (cellulase) (86% theoretical (2011)
200 °C for 10 min yield)a
(continued)
Table.3 (continued)
Strains of K. Biomass Temp. (°C) Pretreatment Enzymes used in EtOH production Yield (YE/S ) References
marxianus saccharification (g L−1 )
ATCC 36907 8% Sunflower 38 Pretreatment by 6% 20 FPU/g 27.88 0.47 g g−1 Camargo et al.
meal H2 SO4 (w/v), at (cellulase) and (2014)
121 °C, for 20 min 13.3 CBU/g
and then 1% NaOH (β-glucosidase)
NRRLY-6860 8% Rice straw 45 Pretreatment by 25 FPU/g 11.5 0.24 g g−1 Castro and
cellulignin dilute acid (100 mg (cellulase) and Roberto (2014)
H2 SO4 ) at 120 °C 25 UI/g
for 30 min (β-glucosidase)
K21 8.05% Taro waste 40 – α-amylase 48.98 NR Wu et al. (2016)
(=40 g L −1 (500–1500 (94.2% theoretical
glucose) units/mg) yield)a
MTCC 1389 5% woody stem of 41 Pretreatment by 3% 12 FPU/g 21.45 0.67 Sivarathnakumar
Prosopis juliflora (v/v) nitric acid, (cellulase) et al. (2019)
and sonication
(40 kHz)
NR not reported; a reported value; OCC old corrugated cardboard
309
(Quarella et al. 2016), UFS-Y2791 (Schabort et al. 2016), and other nine strains:
L01, L02, L03, L04, L05, CBS397, NBRC0272, NBRC0288, and NBRC0617
(Ortiz-Merino et al. 2018). However, complete genome sequences of only two
strains, DMKU 3-1042 and NBRC1777, are currently available. The former
genome of 11.0 Mb is composed of 8 chromosomes in total including mitochon-
drial DNA. Annotation of the genome revealed a total of 4952 genes. A total of
202 tRNAs and 8 rDNAs were identified.
A major potential future application of K. marxianus may be ethanol production
from lignocellulosic biomass, which is an anaerobic or oxygen-limited process
in which both glucose and xylose are present. Detailed transcription start site
sequencing (TSS Seq) to explore the response of K. marxianus DMKU 3-1042
was thus performed under four different conditions: shaking condition in a rich
medium at 30 °C (30D) or 45 °C (45D), a static condition in a rich medium at
30 °C (30DS), and shaking condition in a xylose-containing rich medium at 30 °C
(30X) (Lertwattanasakul et al. 2015).
Under the 30DS condition, K. marxianus may increase the turnover of RNAs
and proteins in addition to suppression of transporters that depend on the mito-
chondrial respiratory activity. Most of the genes for several oxygen-dependent
biosynthetic pathways, such as those for heme, sterols, unsaturated fatty acids,
pyrimidine, and deoxyribonucleotides (Ishtar Snoek and Yde Steensma 2006), are
crucial for cellular metabolism under a static condition. Under the 45D condition,
K. marxianus seems to drastically change metabolic pathways, that is, enhance-
ment of the pentose phosphate pathway (PPP) and attenuation of the TCA cycle
after the fumarate-producing step. Several genes involved in both the DNA repair
pathways of homologous recombination (HR) and non-homologous end-joining
(NHEJ) are upregulated. Heat shock proteins and chaperones, such as Hsp26,
Hsp60, Hsp78, Hsp82, Ssa3, and Cpr6, are crucial for survival at high temper-
atures. The thermotolerance of K. marxianus is thus likely to be achieved by
systematic mechanisms consisting of various strategies. The yeast prevents the
generation of reactive oxygen species (ROS) by minimizing mitochondrial activ-
ity and mainly acquires ATP from glycolysis rather than from the TCA cycle at
high temperatures. Fu et al. (2019) also reported that excess ROS generated dur-
ing a high temperature fermentation condition could be neutralized by NADPH
in K. marxianus. The degree of fatty acid unsaturation may be reduced to adapt
to high temperatures. Genes associated with DNA repair or lipid composition of
the plasma membrane are upregulated. The yeast also produces more ergosterol
to deal with ethanol stress. Under the 30X condition, degradation of lipids in the
peroxisome seems to be stimulated and amino acid synthesis is kept at a low level,
indicating the possibility that fatty acids could be a subsidiary intracellular carbon
source in the xylose medium. Consistently, Schabort et al. (2016) also reported
that peroxisomal fatty acid catabolism is dramatically upregulated in a defined
xylose mineral medium without fatty acids. They also described mechanisms by
which fatty acids are activated and products of β-oxidation are transferred to the
mitochondria.
Notably, oxidative stress-response genes were highly induced under the three
conditions tested, indicating that ROS accumulated in the cytoplasm, mitochondria,
and peroxisome under the 30DS and 30X conditions and in the cytoplasm and
mitochondria under the 45D condition. In conclusion, K. marxianus likely adapts
to the three different growth conditions by distinctive metabolic pathways from
the control condition. Interestingly, the yeast appears to overcome the problem of
ROS, which tend to accumulate under all three conditions. Nicotinamide adenine
dinucleotide phosphate (NADPH) synthesis from several reactions is the key for
cells to cope with ROS.
Marcišauskas et al. (2019) reported the first genome-scale metabolic model of
K. marxianus, iSM996, using TSS data reported by Lertwattanasakul et al. (2015).
The model includes 1913 reactions associated with 996 genes and 1531 metabo-
lites. The iSM996 was used to construct three condition-specific models in YPD
medium while considering the low oxygen and high temperature conditions. The
results suggest that at a high temperature, the cell turns off more genes, thereby
introducing new auxotrophies and utilizing as many resources as possible from the
medium. These findings may be used in the design of growth media at low levels
of oxygen and/or high temperatures.
The global transcriptional response of K. marxianus to multiple inhibitors
including acetic acid, phenols, furfural, and HMF at 42 °C was also studied via
RNA-seq technology (Wang et al. 2018). Genes involved in the glycolysis pathway,
fatty acid metabolism, ergosterol metabolism, and vitamin B6 and B1 metabolic
process were enriched in the downregulated gene set, while genes involved in the
TCA cycle, respiratory chain and detoxification of ROS and transporter coding
genes were enriched in the upregulated gene set in response to the stress with
multiple inhibitors. Redox balance and NAD(P)+ /NAD(P)H homeostasis play an
important role in tolerance to lignocellulose-derived inhibitors.
5 Possible Mechanism of Thermotolerance
K. marxianus can grow well at temperatures over 45 °C, unlike K. lactis, which
belongs to the same genus, or S. cerevisiae, which is a closely related yeast in
hemiascomycetous yeasts. K. marxianus may thus have an intrinsic mechanism to
survive at high temperatures. K. marxianus strains are relatively resistant against
hydrogen peroxide, furfural, and hydroxymethyl furfural (Nitiyon et al. 2016).
Thermotolerance of the yeast may thus overlap with other stress tolerances, assum-
ing a common mechanism of robustness against stressors. K. marxianus, which is
one of the mesophilic yeasts, may have evolved into yeast with thermal resis-
tance due to natural selection pressure in a high-temperature environment, and the
acquisition of thermotolerance might have allowed it to withstand other stresses.
A clue for the mechanism of thermotolerance might be provided by tran-
scriptome analysis, flux analysis, or comparison with a non-thermotolerant yeast.
Transcriptome analysis (Lertwattanasakul et al. 2015) revealed that there are a
tremendous number of significantly upregulated and downregulated genes in K.
marxianus at high temperatures, suggesting a drastic change of metabolism from

that at low temperatures. The metabolic flow inside cells at high temperatures
seems to be different from that at low temperatures. Compared to low temper-
atures, repression of glycolysis and enhancement of PPP activity are thought to
occur at high temperatures and attenuation of the TCA cycle after the fumarate-
producing step is also thought to occur at high temperatures. These changes and
the fact that the intracellular level of ROS increases at high temperatures (Zhang
et al. 2015a, b) lead to the speculation that the former provides NADPH for scav-
enging ROS and that the latter deals with H2 O2 via electron transfer from succinate
dehydrogenase to cytochrome c peroxidase. Consistent with these conjectures, a
higher temperature generates more ROS, which causes DNA damage (Hori et al.
2009). In fact, a change in temperature from low to high temperatures causes a
drastic increase in the level of ROS and similar transcriptional change, suggest-
ing a shift of the metabolic flow from glycolysis to the PPP (unpublished data).
Notably, genes for DNA double-strand break repair and removal of uracil in DNA
molecules are upregulated, suggesting enhancement of double-strand breaks or
deamination of cytosines in DNA at high temperatures. Results of transcriptome
analysis have suggested additional strategies for survival at high temperatures:
alteration of ribosome biogenesis including pre-rRNA processing presumably for
stable and efficient protein synthesis, reduction of mitochondrial ribosome biogen-
esis probably for saving energy, minimization of electron leakage in the respiratory
chain by reduction of its components, and enhanced expression of heat shock
proteins and chaperones.
Lehnen et al. (2019) reported distinct metabolic responses of thermotolerant
Ogataea and Kluyveromyces strains to high temperatures, which were investigated
by 13 C-metabolic flux and physiology analyses, suggesting that there are no highly
conserved metabolic traits among thermotolerant yeasts. However, compared to S.
cerevisiae, both thermotolerant species exhibited high PPP and TCA cycle activ-
ities under all temperature conditions. While the maximum growth temperatures
are similar for the thermotolerant strains, the metabolic network response to high
temperatures is not conserved among the different species. Metabolic flux distribu-
tions in O. polymorpha are irresponsive to high temperatures, while K. marxianus
strains exhibit flux rerouting at elevated temperatures. Mejía-Barajas et al. (2017)
compared two K. marxianus strains and one S. cerevisiae strain isolated from
hot environments with a laboratory yeast strain. One of the K. marxianus strains
exhibited strong thermotolerant traits with a high specific growth rate and biomass
productivity together with shorter duplication time, lower ROS production level,
and lower lipid peroxidation level and also increase in the activity of catalase and
amount of saturated fatty acids in membranes after elevation of temperature.
Although there is still insufficient evidence for concluding the mechanisms of
thermotolerance in yeasts, thermotolerant yeasts may share strategies to maintain
a low level of ROS, which generally increase at high temperatures (Zhang et al.
2015a, b) and are mainly generated by leakage of electrons from the respiratory
chain in mitochondria (Pan 2011), which is enhanced at high temperatures due
to structural instability of the mitochondrial membrane (Tarrío et al. 2008) or by
an increase in respiration rate (Abbott et al. 2009). Low levels of ROS are scav-
enged by non-enzymatic and enzymatic anti-oxidizing agents such as glutathione
(GSH), thioredoxin (TRX), superoxide dismutase, catalase, and peroxidases, but
high levels of ROS cause oxidation of intracellular components, such as DNA,
protein, and lipid and induce apoptosis (Madeo et al. 1999; Scherz-Shouval and
Elazar 2007). Increased activity of the PPP supports the high demand of NADPH
at high temperatures for glutathione reductase-mediated protection against oxida-
tive stress (Konings 1988; Grant 2001; Sugiyama et al. 2000). However, it is not
likely that a higher level of NADPH production is sufficient for thermotolerance
because increased PPP activity (Celton et al. 2012; Frick and Wittmann 2005) in S.
cerevisiae is not capable of supporting growth at elevated temperatures, and ther-
mosensitive K. lactis (Tarrío et al. 2006) and Pichia pastoris (Jorda et al. 2014)
have relatively high PPP activity. Notably, genome-wide analysis of thermotoler-
ant genes supporting cell survival at a critical high temperature (CHT), an upper
limit of temperature, in three bacteria, Escherichia coli (Murata et al. 2011, 2018),
thermotolerant Acetobacter tropicalis (Soemphol et al. 2011), and thermotolerant
Zymomonas mobilis (Charoensuk et al. 2017), revealed that they share thermo-
tolerant genes for membrane stabilization, protection against oxidative stress, and
repair of damage of DNA or proteins, which may contribute to minimization of
ROS generation, avoidance of ROS damage, and recovery from ROS damage,
respectively.
As for thermotolerance, microbes seem to have repair mechanisms for damage
of proteins, lipid, or DNA caused by ROS (Piper 1993) and structural specialties
of the membranes and enzymes, which may be crucial for metabolic activity at
high temperatures (Mejía-Barajas et al. 2018; Fields 2001). Heat shock proteins
including proteinases participate in the renaturation or degradation of unfolded or
damaged proteins. Enhanced expression of genes for heat shock proteins as well
as genes for ROS-scavenging enzymes improves thermotolerance in Z. mobilis
(Anggarini et al. 2016). The susceptibility of lipids to oxidation depends on the
lipid composition and degree of unsaturation (Catalá 2012). The extent of cellular
damage under heat shock conditions is correlated positively with increasing unsat-
uration of fatty acids (Suryawati et al. 2008). Consistently, Steels et al. (1994)
found that the most stress-resistant yeast membranes were enriched in saturated
fatty acids. Membrane fluidity is related to the ratio of saturated to unsaturated
fatty acids (Los and Murata 2004).
To further understand the mechanism of thermotolerance in yeasts, thermal
adaptation followed by detailed analysis of causative mutations or enhanced ther-
motolerance of a thermosensitive yeast by heterologous expression of genes from a
thermotolerant yeast may be useful. Heterologous expression of heat shock genes
from thermophiles has been shown to improve thermotolerance of S. cerevisiae
(Liu et al. 2014). The transcription factors KmHsf1 and KmMsn2 of K. marxianus
can promote both cell growth and ethanol fermentation of S. cerevisiae at high tem-
peratures (Li et al. 2017). These two transcription factors might increase ethanol
production by different mechanisms. In addition, KmMsn2 might also help to cope
with a high temperature by regulating genes associated with lipid metabolism to

change the membrane fluidity.
The thermal adaptation may lead to a decrease in the generation of ROS in
cells that produce higher levels of ROS at higher temperatures, suggesting that
the thermally adapted cells could become robust and resistant to many stressors
(Matsushita et al. 2016). Kosaka et al. (2019) performed a thermal adaptation
of two species and three strains of mesophilic microbes for the improvement of
their CHTs. The results of experiments including analysis of the characteristics of
mutants suggested that these microbes have a genomic potential to endure a 2–3 °C
rise in temperature but possess a limited variety of strategies for thermal adapta-
tion. The thermoadapted mutants bear 2–15 mutations in protein-coding regions
of genes, which are categorized into 6 groups. Most of them are overlapping with
the common classification for thermotolerant genes of E. coli, A. tropicalis, and
Z. mobilis (see above), suggesting that mutations generated by thermal adaptation
enhance physiological functions of the products of thermotolerant genes or their
closely related genes (Kosaka et al. 2019).
In conclusion, the common and basic mechanisms of thermotolerance of
mesophilic microbes including yeasts that have been tested at least are avoid-
ance of ROS generation and damage by ROS and repair systems of ROS-directed
cellular damage. To maintain ROS at a low level, they may have developed com-
mon or unique strategies. The thermotolerance trait may thus be achieved by an
integrated mechanism consisting of various strategies. The thermotolerance of K.
marxianus may also have been achieved by a systematic mechanism including
enhanced production of NADPH by the PPP. Especially, the yeast would mainly
acquire ATP from glycolysis rather than the TCA cycle at high temperatures, which
could prevent the generation of ROS by minimization of mitochondrial activity.
The mechanisms against ROS under a high-temperature condition may allow cells
to endure other stresses because most of the stresses seem to generate ROS. Tran-
scriptome analysis suggests that ROS accumulate at low temperatures under a
static condition compared to a shaking condition or in xylose medium compared
to glucose medium (Lertwattanasakul et al. 2015).
6 High Protein Production Ability
Escherichia coli has so far been primarily used as a host for the production of use-
ful proteins by genetic engineering, probably due to the accumulation of advanced
methodologies including the development of various vectors that are superior to
other microbes (Rosano and Ceccarelli 2014). Although various kinds of cytokines
or growth factors are produced with E. coli and are already commercially available,
E. coli has some drawbacks (Nausch et al. 2013; Feng et al. 2015). For example,
lipopolysaccharide as a constituent of the membrane in E. coli is harmful to the
human body, and great care must therefore be taken, especially in the purifica-
tion of pharmaceutical products (Storeng et al. 1987). The surface antigen protein
of hepatitis B virus (HBsAg) or tissue plasminogen activators (TPA) cannot be
produced as active proteins, and small substances such as peptide hormones are
decomposed in E. coli cells (Pumpen et al. 1984; Elghanam et al. 2012; Xu et al.
2017). Therefore, attempts have been made to use other hosts to compensate for the
disadvantage of E. coli, and yeast is drawing attention as one of the candidates. The
utilization of S. cerevisiae, which is generally used as a host for DNA recombina-
tion research (Mattanovich et al. 2012; Porro et al. 2005), has several advantages:
1. a wealth of genetic knowledge has accumulated, 2. the fundamental mecha-
nisms of replication, transcription and translation have been elucidated, making it
a suitable model of analysis of biological phenomena in higher organisms, 3. the
yeast has a long history of being used industrially as a useful microorganism for
food, feed, and pharmaceutical raw materials, and 4. there is abundant information
on fermentation engineering and culture engineering. However, S. cerevisiae also
has drawbacks for protein production. S. cerevisiae as a Crabtree-positive yeast
consumes oxygen in a limited range in the presence of glucose above a certain
density, despite high levels of dissolved O2 (van Urk et al. 1990). As a result,
S. cerevisiae specializes in ethanol production, reducing the yield of the target
product per sugar. Therefore, the Crabtree effect may be one of the causes of the
small applicable range of material production of S. cerevisiae. In addition, since the
yeast growth temperature is around 30 °C, temperature control during fermentation
is indispensable and costly.
Thermotolerant K. marxianus, which is generally accepted as safe (GRAS), is
a Crabtree-negative yeast that acts on the metabolism from a TCA cycle with a
priority independent of glucose concentration and performs aerobic alcohol fer-
mentation (Blank et al. 2005; Vandijken et al. 1993). Additionally, K. marxianus
is the fastest-growing yeast known so far (Groeneveld et al. 2009), reported growth
rates of K. marxianus, K. lactis, S. cerevisiae and P. pastoris being 0.80 h−1 ,
0.50 h−1 , 0.37 h−1 , and 0.18 h−1 , respectively (Lane and Morrissey 2010; Gao
et al. 2012), and has prominent features including efficient fermentation at high
temperatures, assimilation of various sugars, and advanced genetic tools as men-
tioned above. Therefore, K. marxianus is one of the alternative yeasts that have
great potential in technology to produce various useful proteins under aerobic
conditions.
K. marxianus has promising properties for producing various enzymes such
as β-glucosidase, β-galactosidase, inulinase, endopolygalacturonase, and xylosi-
dase (Fonseca et al. 2008; Lertwattanasakul et al. 2015) (Fig. 1). β-glucosidase
of Kluyveromyces fragilis ATCC 12424 has been cloned and expressed in S. cere-
visiae, and it has been applied for hydrolysis of cellulosic materials (Raynal et al.
1987). The ability to hydrolyze cellulose is useful for further application in ethanol
production from cellulosic biomass. β-Glucosidase is one of three major cellulase
enzymes that are responsible for the regulation of the whole cellulolytic process
to produce fermentable sugars (Zhou et al. 2018). β-Galactosidase can be pro-
duced from a MIG1 mutant of K. marxianus KM-15 for hydrolysis of lactose in
foods to become glucose and galactose (Zhou et al. 2013). This property is very
attractive because it produces lactose-free or low-lactose foods and reduces health
issues of people who suffered from lactose intolerance (Wolf et al. 2018). Inuli-
nase can be produced from the disrupted MIG1 gene and the overexpressed INU1
gene (Zhou et al. 2014). The enzyme is used to hydrolyze inulin by degrading the
β-2,1-fructosyl bond to become fructose (Hoshida et al. 2018). The enzyme can
be applied in food, fermentation, pharmaceutical, and chemical industries (Chi
et al. 2011). Endopolygalacturonase produced by K. marxianus BKM Y-719 is
used for the reduction of viscosity in fruit processing products (Šiekštele et al.
1999). Xylosidase is related to catalysis of the reducing site of xylooligosaccha-
rides and liberates xylose. Efficient liberation of xylose is related to the ability
of a non-conventional yeast to convert xylose to ethanol. Xylosidase activity of
thermotolerant yeast K. marxianus strains NIRE-K1 and NIRE-K3 was found to
be higher than that of Candida tropicalis. An increased amount of xylosidase
was found in adapted K. marxianus cells (Behera et al. 2016). Other possible
enzymes related to biomass utilization have been predicted from the whole genome
sequence (Lertwattanasakul et al. 2015), and they include β-glucosidase (LAC4),
endo-1,3(4)-β-glucanase 1 (DSE4), and endo-1,3(4)-β-glucanase 2 (ACF2) in addi-
tion to three lactose permeases (3 copies of LAC12). Phosphatase, aminopeptidase,
and carboxypeptidase have also been reported as possible enzymes related to
biomass utilization (Nurcholis et al. 2020).
Production of proteins with K. marxianus that have been investigated so far
include enzymes (β-galactosidase, β-glucosidase, inulinase, polygalacturonase, and
others) (Topete et al. 1997; Su et al. 2021; Yarimizu et al. 2015; Hoshida et al.
2018), single-cell proteins (Rajkumar and Morrissey 2020), and antibodies (Duan
et al. 2019; Yang et al. 2021). However, there is a problem in producing a protein
using K. marxianus. K. marxianus cells have a strong cell wall, and it takes a lot
of labor to purify the target protein produced inside cells, and thus the production
amount is limited. Therefore, if the yeast can secrete the protein outside the cell
body, application of highly efficient production and continuous in vitro cultivation
as well as simplification of the industrial process by the ease of purification would
be possible. In order to release the produced protein extracellularly, the N-terminal
signal sequence of a known secretory protein must be connected to the protein of
interest. Zhou et al. (2018) reported that a P10L substitution in the signal sequence
of the INU1 gene increased the secretory expression of lignocellulolytic enzymes
in K. marxianus. The P10L substitution extended the hydrophobic core of the sig-
nal sequence and promoted secretion of mature proteins. Raimondi et al. (2010)
succeeded in extracellular secretion of superoxide dismutase (SOD) by fusion with
an appropriate signal sequence with SOD. In addition to enzymes, the production
of a single-chain Fv antibody (Nambu-Nishida et al. 2018) and virus-like particles
of porcine parvovirus (Yang et al. 2021) and porcine circovirus (Duan et al. 2019)
has been reported.
7 High Ability of Non-homologous End-Joining

for Genetic Engineering
There are two major mechanisms by which cells can repair DNA double-strand
breaks (DSBs), which are intimately related to the classes of genetic recombina-
tion, homologous and non-homologous recombinations (Daley et al. 2005). The
core components of NHEJ required for rejoining of any DSB can be experimen-
tally defined as the proteins needed for simple religation. In the NHEJ pathway,
binding of the Ku heterodimer (Ku70/Ku80) to both ends of the DNA DSB with
the participation of additional proteins, such as Lig4, Nej1, and Lif1, promotes
repair of the DSB (Kooistra et al. 2004; Palmbos et al. 2005; Kegel et al. 2006;
Maassen et al. 2008). Ku, which is conserved from bacteria to humans, is an
indispensable protein for NHEJ (Doherty et al. 2001), and disruption of KU70 or
KU80, results in efficient gene targeting in K. lactis (Kooistra et al. 2004), P. stipitis
(Maassen et al. 2008), and K. marxianus (Abdel-Banat et al. 2010). In yeast lacking
KU70, a high frequency of non-homologous gene integration was abolished and
KmKU70 mutants showed 82–95% homologous gene targeting efficiency using
homologous sequences of 40–1000 bp, indicating that the highly efficient NHEJ
pathway can be used in random gene disruption techniques such as transposon
mutagenesis and plasmid-free gene manipulations in K. marxianus (Abdel-Banat
et al. 2010). In addition, the linear DNA integrative technique can eliminate the
burden of plasmid construction for targeted gene manipulation in K. marxianus.
8 Genome Editing Tools
Synthetic biology leverages the metabolic capacity of microorganisms for the

biosynthesis of simple and complex compounds that are sourced unsustainably
from fossil fuels or that are too expensive to use chemical synthesis on an industrial
scale (Cernak et al. 2018). The yeast S. cerevisiae functions as a major eukary-
ote for synthetic biology but lacks the metabolic potential available in many of
the more than one thousand yeast species identified so far. However, these yeasts
remain difficult to use due to the lack of synthetic biology tools to access the
underlying metabolic networks and physiology (Löbs et al. 2017).
The ability of K. marxianus to grow at high temperatures and utilize a wide
variety of industry-related substrates reflects its potential for biotechnological
applications (Foseca et al. 2008, Lane and Morrissey 2010). However, metabolic
engineering of K. marxianus is limited by the lack of sophisticated genome edit-
ing tools and an incomplete understanding of its genetics, metabolism, and cellular
physiology. A basic requirement of metabolic engineering is the ability to express
native or heterologous genes from expression cassettes. In K. marxianus, stable
plasmid options are limited to the centromeric region and autonomous replicat-
ing sequence of the host genome and a selectable auxotrophic or drug resistance
marker (Hoshida et al. 2014). Transformation with a linear DNA fragment contain-
ing an expression cassette and a selectable marker result in genomic integration
in one of two ways: heterologous DNA is either incorporated into the genome
at a random locus (Kegel et al. 2006) by NHEJ, or the cassette is targeted to
a specific site on the genome by homologous recombination (HR) to the site of
interest (Lieber 2010; Löbs et al. 2017). In S. cerevisiae, HR is the primary DNA
repair pathway, and its high capabilities have made genomic engineering relatively
efficient and have facilitated the development of a wide range of in vivo DNA
assembly tools (Shao et al. 2009; Horwitz et al. 2015). However, in most other
yeasts, NHEJ is the preferred DNA repair pathway and genome engineering with
HR is inefficient. The high functionality of K. marxianus NHEJ can limit genome
editing in many applications. However, NHEJ-mediated functional marker selec-
tion as a novel DNA cloning method has been developed in the yeast (Hoshida
et al. 2014). Some researchers have taken advantage of this ability for multiplexed
gene integration (Cheon et al. 2014), but successful transformants differ signifi-
cantly in hexanoic acid-producing ability, probably due to gene insertion at crucial
genomic loci. Based on available genomic and transcriptomic data, Rajkumar et al.
(2019) characterized a set of native sequences in K. marxianus, including consti-
tutive and inducible promoters and terminators for expression of multiple genes,
for metabolic engineering and synthetic biology. Several known centromeres and
autonomous replication sequences (ARS) are also included in their collection.
These tools will serve as the basis for efficiently building next-generation cell
factories from this alternative yeast. A widely used strategy for enhancing HR in
non-conventional yeasts is the disruption of genes essential for the NHEJ path-
way such as KU70 or KU80. In K. marxianus, the disruption of KU70 and KU80
increased HR rates to 95% and 70%, respectively (Abdel-Banat et al. 2010; Choo
et al. 2014).
An alternative strategy for achieving efficient HR is the introduction of a
genomic DSB using a programmable endonuclease in the presence of a homol-
ogous repair template (Liu et al. 2017). Several programmable tools exist for a
targeted DSB, including dimeric meganucleases, zinc finger nucleases (ZFNs),
transcription activator-like effector nucleases (TALENs), and clustered regu-
larly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9
(CRISPR-Cas9) (Liu et al. 2017). The CRISPR-Cas9 gene-editing system has
been widely used in many yeasts including S. cerevisiae, Schizosaccharomyces
pombe, Yarrowia lipolytica, and Kluyveromyces lactis and recently in K. marxi-
anus (Nambu-Nishida et al. 2017; Löbs et al. 2017; Lee et al. 2018; Juergens
et al. 2018). The CRISPR-Cas9 system has been developed for use with K. marx-
ianus, enabling both NHEJ-based and HR-based genome editing (Cernak et al.
2018; Rajkumar et al. 2019). The genetic loci, ALPHA3 and KAT1, responsible for
mating-type switching in K. marxianus were identified and heterothallic haploid
strains were constructed using the CRISPR-Cas9 system. This is indispensable
for genetic manipulation of the most desired traits, which are likely to depend on
multiple unlinked genetic loci, and remain difficult to identify without the ability
to carry out genetic crosses. Three complex traits, the ability to take up exoge-
nous DNA, thermotolerance, and high lipid production, have been successfully
combined into a single K. marxianus isolate (Cernak et al. 2018).
9 Examples of Production of Other Useful Materials by

K. marxianus
9.1 Flavor Metabolites
A non-conventional yeast can increase the variety and complexity of aroma pro-
files of bakery products such as nuts and fruity aroma (Aslankoohi et al. 2016)
and alcoholic beverages such as wine and beer (Gamero et al. 2020). In addition
to producing ethanol, K. marxianus is capable of producing a variety of volatile
molecules or aromatic esters used as fragrances or flavors (Table 1). The ability
of the yeast to produce acetate esters such as 2-phenyl ethyl acetate (2-PEA) and
isoamyl acetate from 2-phenyl ethanol (2-PE) and isoamyl alcohol is due to the
presence of alcohol acetyltransferase or AATase (Gethins et al. 2015). The ability
to produce 2-phenylethanol from glucose without an additional L-phenylalanine
supplement can be achieved in K. marxianus by genetic engineering via over-
expression of ARO10 for phenylpyruvate decarboxylase and ADH2 for alcohol
dehydrogenase II from S. cerevisiae (Kim et al. 2014). Genes involved in ethyl
acetate biosynthesis in K. marxianus were identified (Löbs et al. 2017). KmAdh2
was found to be critical for aerobic and anaerobic ethanol production. Aerobically
produced ethanol is supplied for the biosynthesis of ethyl acetate catalyzed by
KmAtf. KmAdh7 was found to exhibit activity toward the oxidation of hemiacetal,
a possible alternative route for the synthesis of ethyl acetate.
9.2 Fructose
Fructose is a saccharide used for sweeteners, seasonings, humectant, color and

flavor development, freezing-point depression, and osmotic stability in foods and
beverages such as confectionary, baby food, and high-fructose syrup (Hanover
and White 1993). A high concentration of glucose-free fructose can be produced
by overexpressing GLK1 in an HXK1-knockout mutant or by a RAG5-knockout
mutant of K. marxianus (Zhang et al. 2017a, b; Nurcholis et al. 2019). These
findings suggest that disruption of RAG5 results in reduced kinase activity for
glucose and fructose, reduced glucose uptake due to prevention of the expression
of RAG1 for a glucose transporter, and increased inulinase activity, which in turn
lead to decreased glucose utilization and fructose accumulation (Nurcholis et al.
2019).
9.3 Xylitol Formation
Xylitol has gained increasing attention in recent years due to its use in several
industries such as food, dental products, and pharmaceuticals (Ravella et al. 2012).
The ability of K. marxianus to ferment xylose under oxygen-limited conditions
is weak due to its redox imbalance. Xylose consumption and fermentation can
be enhanced by genetically engineered strains of K. marxianus, in which xylose

reductase (XR) is one of the targeted genes. Replacing the native XR of K. marx-
ianus with the XR from Pichia stipitis significantly improved xylose assimilation
to ethanol and xylitol at high temperatures (Zhang et al. 2013). There are several
reports of xylitol production (Table 1). Hua et al. (2019) obtained a high con-
centration of xylitol, 82.85 g L−1 , from detoxified corncob hydrolysate at 42 °C,
and Du et al. (2019) reported xylitol production from a xylose medium with a
yield of 0.58 g g−1 at 40 °C. Notably, the MIG1-disrupted mutant of K. marxinaus
exhibited increased accumulation of xylitol in a xylose medium (Nurcholis et al.
2019). It is assumed that Mig1 represses xylose utilization but that dysfunction of
Mig1 causes increased xylose utilization, leading to a limitation of NAD+ required
for conversion of xylitol to xylulose. Most xylose-fermenting yeasts, including
K. marxianus, have a xylose metabolic pathway, in which XR converts xylose to
xylitol and further to xylulose under cofactors-balanced conditions, but xylitol oxi-
dation is prevented if NAD+ is insufficient. The XR of K. marxianus NBRC1777
has sole coenzyme specificity, which is activated only by NADPH (Zhang et al.
2011), while the xylitol dehydrogenase (XDH) of K. marxianus strains prefers to
use NAD+ (Lulu et al. 2013).
10 Conclusions
To be a platform of synthetic biology, there are at least three necessary conditions:

excellent potential, sufficient available information, and genetic engineering tools.
In this chapter, with a focus on those three points for K. marxianus, the prominent
properties of K. marxianus including thermotolerance, broad substrate specificity
and protein productivity were introduced in Sects. 2–3, 5–6, and 9, genome and
transcription information was presented in Sect. 4, and genetic engineering tools
including modeling were introduced in Sects. 7 and 8. It was shown that this
yeast has the necessary conditions for use as a platform of synthetic biology, and
the development of synthetic biology that utilizes K. marxianus for enterprises is
expected. Furthermore, in order to accelerate synthetic biology, it is desirable to
enhance metabolic modeling in addition to these three points, but, as with other
organisms, comprehensive metabolic modeling has not yet been established.
The degree of interest of K. marxianus is reflected in the difference in the num-
ber of publications for K. marxianus compared to that for K. lactis (Fig. 2). The
number of publications sharply increased from 2005 to 2014 and the number of
publications has been maintained each year since 2014. On the other hand, there
were many publications for K. lactis from around 1992, but the number has grad-
ually decreased since 2011. The trends in publications for the two species may
imply a shift of trends towards thermotolerance, which is beneficial for indus-
trial applications. On the other hand, although basic research on K. marxianus is
far behind that on S. cerevisiae, several milestones have been achieved so far for
supporting further basic research on K. marxianus (Nurcholis et al. 2020), includ-
ing complete genome sequencing of two strains (Lertwattanasakul et al. 2015;
80
70
60
50
40
30
20
10
0
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
2014
2015
2016
2017
2018
2019
2020
Fig. 2 Changes in the number of publications related to K. marxianus (straight lines) and the
number of publications related to K. lactis (broken lines). Analysis was performed using PubMed
Inokuma et al. 2015), transcriptome analysis under various conditions (Lertwat-

tanasakul et al. 2015; Gao et al. 2015; Schabort et al. 2016; Mo et al. 2019; Fu
et al. 2019; Rollero et al. 2019), transformation with a linear DNA fragment (Non-
klang et al. 2008), genome editing (Nambu-Nishida et al. 2017; Rajkumar et al.
2019), and modeling (Pentjuss et al. 2017; Marcišauskas et al. 2019).
S. cerevisiae has been utilized in food production in human society for a long
time and has recently become a major industrial and model microorganism due
to the many genetic and genomic tools that have become available to under-
stand its biology. However, it has been difficult to expand the capabilities of S.
cerevisiae for the use of various carbon sources, production of various products,
and tolerance to stresses for industrial applications. Other non-conventional yeasts
need to be developed to solve many of these problems. The beneficial characteris-
tics and potentials of K. marxianus were summarized in this review. Due to such
intrinsically outstanding features in addition to the development of biotechnologi-
cal tools, K. marxianus has become one of the most interesting non-conventional
yeasts, comparable to S. cerevisiae, at least for industrial applications. The recent
advances as mentioned above may enable the integration of comprehensive data
and biotechnological tools to promote industrial applications as well as further
basic research.
Acknowledgements This study was supported by the Advanced Low Carbon Technology Research
and Development Program, which was granted by the Japan Science and Technology Agency
(JPMJAL1106) (MM, TK, and MY) and e-ASIA Joint Research Program, which was granted by
Japan Science (JPMJSC16E5) (MM, TK, and MY) and Technology Agency, Ministry of Research,
Technology and Higher Education of the Republic of Indonesia, Agricultural Research Development
Agency of Thailand and Ministry of Science and Technology of Laos, and partially supported by the
Core to Core Program A. Advanced Research Networks, which was granted by the Japan Society
for the Promotion of Science, the National Research Council of Thailand, Ministry of Science and
Technology in Vietnam, National Univ. of Laos, Univ. of Brawijaya and Beuth Univ. of Applied
Science Berlin (NL, MM, TK, and MY), and the Japan Society for the Promotion of Science,
MEXT/JSPS Kakenhi (25250028 and 16H02485 to MY).
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Synthetic Biology in the Candida
(CTG) Clade
Dalal Kasir, Sébastien Besseau, Marc Clastre, Audrey Oudin,

Monzer Hamze, Vincent Courdavault, Marwan Osman,
and Nicolas Papon
Abstract
The continuous bio-race to find an ideal microbial chassis opens the path
toward the Candida CTG clade to enter the competition. Unexpectedly, this
unique CUG-serine coding clade successfully mastered the production of var-
ious nutraceutical, commercial, and pharmaceutical valuable compounds. The
following chapter aims to snapshot the Candida CTG clade’s bioengineering
properties and their biotechnological applications in synthetic biology.
1 Introduction
The ever-increasing progress in technologies and genetic engineering has enabled

this discipline to book a place in the list of revolutionizing scientific fields. Gen-
erally, the application of synthetic biology on “microbial chassis” aims to draw a
detailed roadmap to successfully predict, guide, and control the functionality of
cellular behavior (Leonard et al. 2008). Briefly, the manipulation of the microbial
genomic repertoire by knockin/knockout of certain DNA sequences with known
D. Kasir · M. Hamze · M. Osman

Laboratoire Microbiologie Santé et Environnement (LMSE), Doctoral School of Sciences and
Technology, Faculty of Public Health, Lebanese University, Tripoli, Lebanon
S. Besseau · M. Clastre · A. Oudin · V. Courdavault (B)
Université de Tours, BBV EA 2106, 31 avenue Monge, 37200 Tours, France
M. Osman
Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine,
Cornell University, Ithaca, NY 14850, USA
N. Papon (B)
Université d’Angers, GEIHP, E3142, SFR 4208 ICAT, Angers, France
https://doi.org/10.1007/978-3-030-89680-5_12
338 D. Kasir et al.
behaviors can generate synthetic bio-factories with new desirable properties (Lam
et al. 2010). The decrease in DNA synthesis and sequencing cost, the improvement
of the gene-to-protein relationship decoding, the standardization of DNA assem-
bly modalities, and the implementation of detector–reporter systems are considered
key players in this modern sector (Jensen and Keasling 2015). Various microbial
factories are considered promising candidates in several biotechnological appli-
cations. Among these, the mycotic CTG clade corresponds to a subdivision of
restricted ascomycetous yeast group that displays a distinctive genetic code (Papon
et al. 2014). In these species, a mistranslation reassigned the universal canon-
ical leucine CUG codon to serine predominantly (Papon et al. 2014). Despite
the presence of numerous Candida species that pose a significant health chal-
lenge (due to their ability to develop muco-cutaneous and/or systemic infections),
the CTG clade encompasses several species with strong biotechnological applica-
tion prepotency (Defosse et al. 2018a; Turner and Butler 2014). Currently, these
mycotic microorganisms are considered as suitable bio-producer candidates for
industrial enzymes, single-cell protein, bioethanol, vitamins, sweeteners, lipids,
and metabolites with pharmaceutical and nutritional values (Johnson 2013). Dis-
playing extraordinary potentials is one of the reasons that bring these yeasts into
the discipline of synthetic biology, namely tolerate niche osmolarity changes, grow
on a variety of carbon sources (including pentoses, n-alkanes, fatty acids, and
phenols), metabolize inexpensive substrate, and stand for extreme environmental
stresses (Papon et al. 2014; Johnson 2013). The genetic toolbox of CTG clade
yeasts is progressively prepared to generate a stand-alone bio-factory via recipient
strain construction, selectable marker adaptation, control (inducible or repress-
ible) gene expression system formation, and transformation protocol optimization
(Papon et al. 2012). Such efforts with the recent advancements in omics studies and
metabolic modeling improve the understanding of CTG clade metabolic behavior,
bringing it to the list of microbial competitors in the field of synthetic biology
(Papon et al. 2014). The following chapter will cover the taxonomy, the main
applied classical genetic tools, omics resources, and the major biotechnological
potentials in Candida CTG clade.
2 Taxonomy
Similar to all microbial taxonomic relationships, the phylogeny of Candida

species was reformulated after the introduction of advanced molecular tech-
niques. Recently, whole genome analysis-based studies steered the subdivision
of Saccharomycotina into 8 clades including in particular: (i) the Saccharomyc-
etaceae clade including Candida glabrata and yeasts of Saccharomyces genus
and (ii) CTG clade comprising Candida albicans, Candida dubliniensis, Can-
dida tropicalis, Candida parapsilosis, Candida maltosa, Candida famata, Candida
lusitaniae, Candida oleophila, Candida auris, Diutina rugosa, Lodderomyces
elongisporus, Metschnikowia fructicola, Meyerozyma guilliermondii, Millerozyma
farinosa, Spathaspora passalidarum, Yamadazyma tenuis, and Scheffersomyces
Synthetic Biology in the Candida (CTG) Clade 339
Fig. 1 Phylogeny of Saccharomycotina and CTG clade yeasts. The topology represents the cur-
rent vision of the relations between clades and other species [according to (Dujon and Louis 2017;
Shen et al. 2016; Chen et al. 2000; Tsui et al. 2008)]. The human opportunistic species are shown
in red. The phylogeny of Saccharomycotina presented here in 8 clades is not definitive nor used
by all authors (Dujon and Louis 2017). Only a few species of the CTG clade are indicated in the
phylogenetic tree on the right. Adapted from Defosse et al. (2018a)
stipitis (Fig. 1) (Fitzpatrick et al. 2006; Butler et al. 2009; Nosek et al. 2009;
Defosse et al. 2018b). The unique genetic decoding ability of CTG clade was
characterized for the first time by Kawaguchi et al. (1989) in D. rugosa. Gener-
ally, the deviation of this clade’s coding system greatly supports the “ambiguous
intermediate theory,” which hypothesized that codons are reassigned through enig-
matic decoding in which a mutation in tRNA can expand its decoding array
capacity (Santos et al. 2004). Mühlhausen et al. (2014) described the cause for
the translation codon shift in CTG clade by demonstrating the polyphyletic rela-
tionship among the Candida genus using phylogenomic analyses of 26 motor
and cytoskeletal proteins. The evolutionary driving force mainly led to the emer-
gence of two groups with codon bias; one used the standard CUG translation,
while the other is an alternative yeast codon usage (AYCU) (Mühlhausen and
Kollmar 2014). Comparative and molecular phylogenomics studies showed that
tRNACAG Ser emerged (272 ± 25 million years ago) prior to the separation between
the Saccharomyces and Candida genera (170 ± 27 million years ago); such appear-
ance drives the common ancestor of CTG clade to lose the cognate tRNACAG leu
to the newly mutant tRNACAG Ser (Defosse et al. 2018b; Santos et al. 2011).
Comparative genomics analyses of CTG-Ser clade species revealed that CTG
codons are most frequently aligned with Ser rather than Leu amino acid (Riley
et al. 2016). Furthermore, their predicted tRNACAG Ser mainly has the three sery-
lation features: (i) a guanine at position 33 (G33) in the anticodon loop (may
lessen the rates of leucylation), (ii) Ser identity element (positioned in the vari-
able loop), and (iii) G discriminator base at position 73 (Riley et al. 2016). The
genomic comparison of ascomycete yeasts gives a snapshot of their biotechnolog-
ical exploitations, which reveals a heterogeneous distribution of metabolic traits,
with restriction to a single clade (such as methylotrophy), and patches distribu-
tion for others (such as D-xylose utilization) (Riley et al. 2016). Functionally
speaking, the incorporation of serine in polypeptides originating from CUG-loaded
340 D. Kasir et al.
mRNA drives the diversification in the primary structure of translated proteins that
possibly promotes the CTG clade yeasts to adapt to challenging environmental
conditions (Defosse et al. 2018b; Santos et al. 2011).
3 Classical Genetic Tools
The ability to generate mutants is considered a cornerstone in synthetic biology

not only to decipher the proteomic function of certain genes but also to create
sophisticated bio-systems with the potency to produce beneficial compounds. Early
researchers introduced mutations into the genomes of tested strains by exposing
them to either UV or chemical mutagens, causing inaccurate and undesirable muta-
tions. The innovation of genome modification techniques over the past decade
revolutionized the propensity to accurately regulate a vast array of genes at the
genome level (Ren et al. 2020). Genome-scale engineering represents the keystone
methodology to precisely manipulate the microbiological factories’ operation, gen-
erating a reproducible bio-system (Ren et al. 2020). The following section will
cover the main standard and advanced genetic tools applied in Candida CTG clade
yeasts.
3.1 Selection Markers
Selection markers are considered an integral part of the construction of transfor-

mation vectors since they allow the screening for successful transformants. The
choice of selection markers is dependent on the genomic repertoire of the trans-
formant host strains (Hinchliffe et al. 1993). Löbs et al. (2017) recently described
the procedure of generation and utilization of auxotrophic markers for engineered
yeast. Typically, random mutagenesis or homologous recombination facilitates the
generation of stable auxotrophic strains, which allows further advanced genome
editing (random/targeted integration, Cre-lox, HisG/lacZ, and CRISPR–Cas9) to
be applied in the targeted yeast species (Löbs et al. 2017). Generally, in Can-
dida, the standard dominant selection genes are divided into two main groups:
metabolic (nutritional) markers and drug-resistant cassettes (Papon et al. 2012). In
the metabolic-marker-mediated yeast transformation, the formation of selection
markers undergoes three main steps: (i) generation and isolation of an aux-
otrophic recipient strain, (ii) identification of the interrupted metabolic pathway
by “medium complementation” with the appropriate amino acid or base, and (iii)
restoration of the metabolically recessive mutation by using the gene encoding the
functional “repairing” enzyme (Papon et al. 2012). Currently, numerous metabolic
genes are used as selectable markers in Candida CTG clade, such as HIS1, ARG4,
URA3, ADE2, MET2, LYS4, and ARO4 (Samaranayake and Hanes 2011; Vyas et al.
2015; Kosa et al. 2007; Dmytruk et al. 2011).
However, the extensive investigation and optimization of drug-resistant cas-
settes facilitate the functionality of such markers in the CTG clade (Papon
et al. 2012). Four main codon-optimized genes constitute the drug-resistant

markers in Candida spp.: (i) hygromycin phosphotransferase (HPH; confers
resistance to hygromycin B), (ii) streptothricin acetyltransferase (SAT-1; con-
fers resistance to nourseothricin), (iii) glyoxalase (ble; confers resistance to
bleomycin/phleomycin/zeocin), and iv) inosine 5`-monophosphate dehydrogenase
(IMH; confers resistance to mycophenolic acid) (Papon et al. 2012). Furthermore,
new dominant selectable markers for C. famata were recently developed by Brati-
ichuk et al. (2020) termed BSD (Aspergillus terreus origin) encoding for blasticidin
S deaminase, thus conferring resistance against blasticidin (Table 1).
3.2 Reporter Genes
Reporter genes are considered a robust tool in genetic analysis, reporting success-
ful transformation and monitoring protein interaction and localization (Papon et al.
2012; Expression et al. 2002; Jones and Thomas 2003). Furthermore, apart from
reporting successful recipients of an episomal system, various reporter genes facil-
itate the dual-labeling approach in different Candida CTG clade yeasts which aids
in studying protein co-expression and co-localization (Courdavault et al. 2011; Rei-
jnst et al. 2011). Among the reporter genes used in Candida genetic manipulation
are:
3.2.1 Fluorescent Reporters

Generally, they are implicated in tracking protein localization/interaction and
monitoring promoter activity in a broad range of fungal species (Papon et al.
2012). Numerous reengineered fluorescent proteins have been developed, aim-
ing for codon optimization and brightness improvement (Cormack et al. 1996,
1997). Initially, the green fluorescent protein (GFP; Aequorea victoria origin)
was successfully integrated into C. albicans-manipulating cassettes after integra-
tion of chromophore mutations and exhibited strong fluorescence emission in
comparison with the wild-type strains (Morschhäuser et al. 1998). Later, cer-
tain genetic manipulations (site-directed mutagenesis) of the yeast-enhanced GFP
synthetic gene yEGFP3 facilitated the production of two variants, yellow fluores-
cent protein (YFP) and cyan fluorescent protein (CFP) (Papon et al. 2012). Both
GFP variants were reengineered to enhance brightness, provide further emission
wavelengths, and promote multicolor imaging of differential gene expression and
protein localization in C. albicans (Gerami-Nejad et al. 2001).
3.2.2 Enzymatic Reporters

In Candida CTG clade, two enzymatic systems are mainly applied, Luciferase
and β-Galactosidase: Luciferase, encoded from certain bioluminescent genes to
produce different varieties of luciferase enzyme (Roda et al. 2004). For a light
emission, a substrate called luciferin is added to the manipulation platform (Roda
et al. 2004). The luciferase system was notably recognized in Candida genetic
342
Table 1 Molecular toolbox for Candida CTG clade engineering

Candida CTG Selectable Reporter Genetic editing tool References
clade spp. marker genes Plasmid Recombinase Tet-system CRISPR-Cas
C. albicans HIS1 yEGFP3 pAYCU267 caFLP tTA CaCas9 Defosse et al. (2018a), Samaranayake and
ARG4 GFP pAYCU268 ecaFLP cartTA CRIME Hanes (2011), Reijnst et al. (2011), Cormack
URA3 CaGFPc pAYCU228 cre et al. (1997), Morschhäuser et al. (1998),
ADE2 YFP Gerami-Nejad et al. (2001, 2009), Enjalbert
CaHygB CFP et al. (2009), Uhl and Johnson (2001), Staib
caSAT-1 Venus et al. (1999, 2000a), Morschhäuser et al.
caSAT1 YFP (1999), Reuss et al. (2004), Park and
CaNAT1 yEmRFP Morschhäuser (2005), Dennison et al. (2005),
IMH3 DsRFP Nakayama et al. (2000), Roemer et al. (2003),
RLUC Vyas et al. (2015), Huang and Mitchell (2017),
FLUC Shen et al. (2005), Basso et al. (2010), Zhang
gLUC59 and Konopka (2010), Leuker et al. (1992),
LAC4 Köhler et al. (1997), Srikantha et al. (1996),
lacZ Doyle et al. (2006a, b), Keppler-Ross et al.
(2008)
C. dubliniensis HIS1 GFP pAYCU267 FLP – – Defosse et al. (2018a), Staib et al. (2000b),
ARG4 pAYCU268 Mancera et al. (2019), Wirsching et al. (2001))
LEU2 pAYCU228
MPAR
C. tropicalis URA3 LAC4 pAYCU267 FLP – CRISPR/Cas9 Defosse et al. (2018a) Lombardi et al. (2019),
HYG# lacZ pAYCU268 Mancera et al. (2019), Hara et al. (2001),
IMH3r pAYCU228 Xiang et al. (2014), Beckerman et al. (2001)
(continued)
D. Kasir et al.
Table 1 (continued)
C. parapsilosis URA3 LAC4 pAYCU257 ecaFLP – CRISPR/Cas9 Defosse et al. (2018a), Kosa et al. (2007),
MET2 pAYCU281 CpFLP Lombardi et al. (2019), Ding and Butler
LYS4 pAYCU230 (2007), Lombardi et al. (2017) Shen et al.
IMH3r pAYCU210 (2005), Gácser et al. (2007), Nguyen et al.
CpSAT1 pAYCU212 (2009)
CaNAT1
C. maltosa URA3 LAC4 pAYCU267 – – – Defosse et al. (2018a), Papon et al. (2012),
pAYCU268 Masuda et al. (1994)
pAYCU228
C. famata BSD LAC4 pAYCU272 – – CRISPR/Cas9 Defosse et al. (2018a), Dmytruk et al. (2011),
Synthetic Biology in the Candida (CTG) Clade
Dh IMH3 pAYCU273 Bratiichuk et al. (2020), Spasskaya et al.

Sa ble pAYCU244 (2021), Dmytruk et al. (2014), Ishchuk et al.
ARO4 (2008)
C. lusitaniae URA3 GFP pAYCU257 – – CRISPR/Cas9 (RNPs) Defosse et al. (2018a), Grahl et al. (2017),
NAT YFP pAYCU211 Norton et al. (2017), Lin et al. (2011), Gabriel
IMH3.2 pAYCU256 et al. (2014), Defosse et al. (2018b)
pAYCU281
pAYCU230
pAYCU210
pAYCU212
C. oleophila HYG# lacZ – – – – Segal et al. (2002), Yehuda et al. (2002)
C. auris NAT1 mCh pAYCU257 – – CRISPR/Cas9 (RNPs) Defosse et al. (2018a), Grahl et al. (2017)
D. rugosa zeo-n ND pAYCU267 – – – Defosse et al. (2018a), Tang et al. (2003)
pAYCU268
pAYCU228
343
(continued)
344
Table 1 (continued)
L. elongisporus IMH3.2 YFP pAYCU254 – – – Defosse et al. (2018a, b)
M. guilliermondii MET2 yEGFP3 pAYCU211 – – – Defosse et al. (2018a), Courdavault et al.
HYG# YFP pAYCU256 (2011), Millerioux et al. (2011), Obando
SAT1 CFP pAYCU281 Montoya et al. (2014), Foureau et al. (2013)
URA5 yEmRFP pAYCU230
pAYCU210
pAYCU212
M. farinosa ble ND pAYCU230 – – – Defosse et al. (2018a), Wang et al. (2006)
pAYCU210
pAYCU212
M. fructicola ND CFP pAYCU211 – – – Defosse et al. (2018a)
pAYCU256
pAYCU230
pAYCU210
pAYCU212
S. stipitis ble yEGFP3 pAYCU257 Cre – CRISPR/Cas9 Defosse et al. (2018a, b), Laplaza et al. (2006),
IMH3.2 pAYCU211 Cao et al. (2017b), Passoth et al. (2003)
pAYCU256
pAYCU230
pAYCU210
pAYCU212
S. passalidarum IMH3.2 GFP pAYCU270 – – – Defosse et al. (2018a, b)
pAYCU271
pAYCU229
Y. tenuis ND ND pCtAKR – – – Wohlbach et al. (2011)
D. Kasir et al.
engineering especially after developing a codon-optimized luciferase gene (Gaus-

sia princeps origin) fused to the gene encoding glycosylphosphatidylinositol-linked
cell wall protein (Pga59p) (Moreno-Ruiz et al. 2009; Enjalbert et al. 2009). This
construction overcomes the low cell permeability to luciferin by expressing Pga59p
product to the cell surface of the engineered strain (Papon et al. 2012).
The enzyme β-Galactosidase encoded by lacZ gene is used to generate high
versatile reporting approach in several bioengineered models (Papon et al. 2012;
Smale 2010). Two main detector substrates are used in β-Galactosidase system, o-
nitrophenyl galactoside (ONPG) and 5-bromo-4-chloro-3-indolyl β-D-galactoside
(X-gal) triggering the appearance of yellow (soluble) and blue (insoluble) prod-
ucts, respectively (Möckli and Auerbach 2004; Juers et al. 2012; Clark et al. 2019).
Notably, in contrast to the previous optimization process applied in Candida tool-
box, the codon-optimized version lacZ gene (Streptococcus thermophiles origin)
developed by Uhl et al. (2001) showed an equivalent activity to the wild-type
sequence, which justifies the preferable application of S. thermophiles lacZ gene
in numerous studies targeting C. albicans.
3.3 Auto-replicating Sequence
The discovery of the microbial system’s transformation modality (firstly reported

by Frederick Griffith in 1928) revolutionized the vision, the orientation, and the
plasticity of scientific research (Griffith 1928; Schindler 2020). The transformation
phenomenon enables yeast to acquire new genetic traits by either the integration
of non-replicative plasmids or the acquisition of an independent replicative epi-
some (Orr-Weaver et al. 1981). Moreover, the random gene integration modality
demonstrated its efficacy in generating CUG-Ser yeast successful transformants,
mediated by electroporation and lithium acetate approaches (Gordon et al. 2019).
In addition to plasmid’s capacity to transform traditional genes (selectable markers,
reporter genes, and gene of interest), they are also capable of transforming a full
gene manipulating system into various recipient strains (Lombardi et al. 2019). In
the CTG clade, the episomal transformation was prone to certain difficulties due
to their alternative translation behavior. Optimization of selectable markers and
reporter genes represents the key players in building a successful transformation
construct. The recent identification of centromere sequences in non-conventional
yeasts allowed the creation of a building stable episome construct for heterologous
expression in S. stipitis (Cao et al. 2017a). Additionally, Defosse et al. (2018a)
developed an episomal module to genetically manipulate CTG yeast clade with
DNA-interchangeable capacity (customization property; as an action of commonly
used and rare-cutter restriction enzymes) called pAYCU plasmids (Fig. 2), con-
sisting of two main gene cassettes: 1) drug-resistant cassette and 2) reporter gene
expression cassette. The main advantage of this flexible construct is the numerous
possibilities offered for both, the selection marker and the reporter gene cassettes
(Defosse et al. 2018a). SAT1, HPH # , and MgIMH3.2 are examples of ectopically
expressed selectable markers in the designed episomal construct (Defosse et al.
346 D. Kasir et al.
Fig. 2 Schematic illustration of the standard pAYCU plasmid constructs (Defosse et al. 2018a)
2018a). Regarding reporter genes, the synthetic construct offers a broad spectrum
of ORF-adapted fluorescent protein alternatives including yeast-enhanced green
(yeGFP), yellow (yeYFP), cyan (yeCFP), monomeric cherry (yemCherry), the
surface-exposed Gaussia princeps luciferase (gLUC59), and lacZ gene (StlacZ)
(Courdavault et al. 2011; Enjalbert et al. 2009; Uhl and Johnson 2001) (Fig. 2).
Various combinations of pAYCU episome construct are offered to adapt the genetic
transformation and manipulation in Candida spp. (Defosse et al. 2018a) (Table
1). Nowadays, the plasmid transformation is no longer restricted to the conven-
tional ectopic gene manipulation but rather is extended to markerless gene editing
modalities (Lombardi et al. 2019; Wang et al. 2018). Wang et al. (2018) recently
reported the construction of a “suicide plasmid” named pPICPJ-mazF, facilitating
scarless metabolic manipulation in C. tropicalis to increase the conversion rate of
oils into long-chain dicarboxylic acids (DCAs). The future perspective for Candida
gene manipulation will be mainly shifting from standardized conventional plasmid
transformation to genetic circuit design with directed functional behavior (Papon
et al. 2014; Røkke et al. 2014).
3.4 Recombinases
The emergence of the site-specific recombinases upgraded genetic engineering to

the next level in numerous prokaryotic and eukaryotic genomes (Papon et al.
2012). Their ability to promote safe targeted genetic modifications (without the
addition of toxic compounds such as 5-fluoroorotate applied in URA3 blaster sys-
tems) grants it access to the list of adopted powerful molecular tools (Papon et al.
2012). The intensive efforts over the last two decades facilitated the application of
two main recombinase systems in the CTG clade, the flippase (FLP) recognition
target (FRT) and the Cre recombinase (Cre) locus of crossing over (x), P1 (loxP)
site-specific recombinases (Papon et al. 2012; Nunes-Düby et al. 1998; Austin
et al. 1981; Sternberg and Hamilton 1981).
3.4.1 FLP-FRT System

Generally, the system is composed of two functional elements, FLP and FRT
sites (Holkers et al. 2006). Its mode of action is primed by FLP that catalyzes
high-fidelity recombination between two FRT sites (Holkers et al. 2006). More-
over, FLP can mediate DNA-FRT-flanked segment excision and inversion in direct
and inverted repeat configurations, respectively (Holkers et al. 2006). Since the
FLP-FRT modality is considered as one of the first engineered recombinase strate-
gies in Candida, several codon optimization processes and transcription regulation
exchanges were carried out to optimize its expression in Candida spp. (Papon
et al. 2012). For instance, Staib et al. (1999) and Morschhäuser et al. (1999) suc-
cessfully developed caFLP (Saccharomyces cerevisiae origin) under the control
of SAP2 promoter (inducible, expressing genes according to the stage of mycotic
infection) to guide the excision of FRT-flanked IMH3, CDR4, and MDR1 (the last
two genes were sequentially disrupted) genes in C. albicans strains. Notably, an
improved enzymatic activity was recognized in C. albicans for the new codon-
optimized FLP recombinase, called ecaFLP, which was expressed under SAP2
and MAL2 promoters, and in a tetracycline-inducible system (Staib et al. 2000a;
Michel et al. 2002; Sánchez-Martínez and Pérez-Martín 2002; Reuss et al. 2004;
Park and Morschhäuser 2005). A more interesting capability was spotted on FRT-
flanked-MAL2 promoter-driven ecaFLP coupled with the caSAT1 marker cassette,
facilitating the selection of nourseothricin-resistant strains. The FLP-mediated
manipulation modality could offer an excision property for the ectopic cassette
driven by culturing the transformants in a maltose-enriched medium (selection
marker recycling) (Reuss et al. 2004). Worth mentioning, the MAL2 promoter-
driven FLP manipulation strategy was successfully integrated into C. parapsilosis
upon replacing the promoter with a species-specific MAL2 transcription regulatory
region (Ding and Butler 2007).
3.4.2 Cre-LoxP System

Typically, this system (originating from P1 bacteriophage) is composed of two
main elements, causing recombination (Cre) and loxP flanked (floxed) DNA seg-
ment (McLellan et al. 2017). It was recently engineered to direct an efficient
genetic manipulation in CTG clade (Papon et al. 2012). Dennison et al. (2005)
reported successful sequential disruption of the ADE2 and MET15 genes in C.
albicans mediated by the codon-optimized version of cre gene (driven by MET3
promoter). Furthermore, Laplaza et al. (2006) reported the construction of another
effective version of mutagenized cre gene that allows functional genomics and
metabolic engineering in Candida CTG clade. More recently, Shahana et al. (2014)
constructed a new Clox system with an efficacy-improved cre gene (containing
348 D. Kasir et al.
synthetic intron). Interestingly, this system facilitates multi-marker recycling, with

open choices for selection markers (NAT1, URA3, HIS1, ARG4) (Shahana et al.
2014).
3.5 Tetracycline-Regulatable Dual System
The myriad potency of certain bacteria to resist tetracycline antibiotics (as an

action of their special molecular toolbox) opens the path toward developing a
powerful system, allowing the control of gene expression in prokaryotes and
eukaryotes (Papon et al. 2012; Berens and Hillen 2004). The two key players in
this system are the TetR transactivator and the tetO operator (from the promoter of
tetracycline-resistant genes) (Papon et al. 2012; Berens and Hillen 2004). Remark-
ably, this system can control the genetic switches via two modalities, Tet-off and
Tet-on (Sprengel and Hasan 2007). In CTG clade, the Tet-off system consists of
the tTA, hybrid codon-modified TetR (E. coli origin) fused to ScHPA4-TA (S. cere-
visiae origin; encoding Hap4 activation domain), and the TR promoter (Papon
et al. 2012; Nakayama et al. 2000). The tetracycline-deficient medium enables
the binding of tTA transactivator on the TR promoter, allowing an active expres-
sion of the gene of interest (Papon et al. 2012), whereas tetracycline-enriched
medium acts as a negative regulator for gene expression (Papon et al. 2012). On
the contrary, the Tet-on system behaves oppositely, where the addition of tetra-
cycline triggers the expression of the gene of interest (Park and Morschhäuser
2005). To perform such modality in CTG clade yeast, a reverse tetracycline-
controlled transactivator (cartTA) and the Ptet promoter are needed (Papon et al.
2012; Park and Morschhäuser 2005). Typically, the cartTA is the result of fus-
ing carTetR nucleotide sequence to the codon-optimized sequence, ScGAL4-TA (S.
cerevisiae origin, encoding Gal4 activation domain) (Papon et al. 2012; Park and
Morschhäuser 2005). Notably, the modification performed in the carTetR sequence
reversed the effect of tetracycline on the expressed transactivator cartTA (Papon
et al. 2012) (Fig. 3). In fact, the emergence of this regulatable system facilitates
the development of gene replacement and conditional expression (GRACE) strat-
egy in C. albicans (Roemer et al. 2003). Additionally, it demonstrated its efficacy
in regulating the gene expression of C. tropicalis toolbox (Bijlani et al. 2018).
3.6 CRISPR–Cas9 System
The recent breakthrough discovery of clustered regularly interspaced short palin-

dromic repeats (CRISPR)/CRISPR-associated system (Cas) greatly facilitated the
birth of a new genome-editing era (Jinek et al. 2012). The natural implication of
CRISPR–Cas9 in bacterial adaptive immunity against invading bacteriophages sup-
ports its theoretical potency to target any “suspected DNA” (DiCarlo et al. 2013).
The recent advancement in bioengineering succeeded in translating the extraordi-
nary potentials (accuracy, precision, and flexibility) of CRISPR–Cas9 in the area of
Fig. 3 Tet-off-/Tet-on-regulatable dual system in CTG clade (Nakayama et al. 2000)
genome editing (Heidari et al. 2017; Stoneman et al. 2020). Typically, the selec-
tion of a single-guide RNA (sgRNA) target site is considered as the prime step
in designing each CRISPR–Cas9 experiment (Stoneman et al. 2020). Upon the
assembly of the sgRNA and Cas9 (endonuclease) enzyme complex, the guide RNA
directs Cas9 for manipulating its complementary region in the genome (Stoneman
et al. 2020).
Experimentally speaking, this novel tool demonstrates its ability to meticulously
reduce the transformation steps and minimize the off-target effects and the overall
number of “unwanted” genomic changes, especially hyperploidy events (Marton
et al. 2020; Chandrasegaran and Carroll 2016). Cao et al. (2017b) demonstrated
the potency of using CRISPR technology in non-conventional yeasts, namely
S. stipitis to overcome the genome editing’s limited efficacy, where the rate of
ade2-knockout and trp1-knockout was boosted up from <1% to 80%. In addi-
tion, both centromere (CEN) and autonomously replicating sequence (ARS) of the
350 D. Kasir et al.
CRISPR–Cas9 episome system were able to trick the yeast cell and solve both,
segregation bias and directing plasmid replication (Cao et al. 2017b). The overall
titer of the target component was improved 3-folds through using the above-
mentioned stable minichromosome-like expression construct (Cao et al. 2017b).
Recently, a new species, “the Cinderella of the non-conventional yeast,” namely
C. famata, joined the list of CRISPR–Cas9-manipulated microorganisms (Prista
et al. 2016; Spasskaya et al. 2021). Spasskaya et al. (2021) demonstrated the
integral role of 26S proteasome in the extremophilic nature of this interesting
species (halo- and osmotolerant capacity) by developing scarless highly efficient
single (pDhCRISPR-1)- and dual-guide (pDhCRISPR-2) CRISPR systems. The
targeted mutagenesis of DhRpn4-binding sites (Rpn4: induced transcription factor,
regulates proteasomal gene expression under stress conditions) leads to C. famata
proteasomal deregulation, which generated strains sensitive to various environ-
mental stresses (such as gene-proteotoxic, oxidative stress, and conditions of high
salinity and osmolarity) (Spasskaya et al. 2021). Moreover, this cutting-edge tool
allowed mastering the generation of diploid mutant strains such as C. albicans,
rather than only disrupting a single allele as a result of conventional mutagenesis
strategies that limit the efficacy of constructing null mutations (both alleles are
modified at once) (Vyas et al. 2015). However, the application of CRISPR/Cas9-
based gene editing in CTG clade is somehow cumbersome because of the
prerequisite of Cas9 gene recoding to overcome the “leucine/serine translation
exchange” issue; thus, codon optimization is crucially needed in CRISPR-CTG
clade applications (Vyas et al. 2015). Different studies have focused on the Can-
dida codon optimization; for instance, Vyas et al. (2015) constructed two systems:
solo and duet, expressing Candida/Saccharomyces codon-optimized version of
Cas9 (CaCas9) that evades the usage of CUG codon with compatibility to all
CTG species. Ironically, Grahl et al. (Grahl et al. 2017) described an alternative
approach to circumvent the need for species-specific manipulation constructs by
applying expression-free CRISPR genome editing in C. lusitaniae, C. glabrata, and
C. auris. The alternative construct is composed of purified Cas9 protein and gene-
specific and scaffold RNAs referred as RNA–protein complexes (RNPs) (Grahl
et al. 2017).
The transient system is typically used as a safeguard to avoid the nega-
tive feedback (cell toxicity and strain fitness drop) of CRISPR/Cas9 constitutive
expression (Min et al. 2016). In 2017, Norton et al. (2017) developed the first
transient CRISPR–Cas9 expression with gene deletion efficiencies up to 81%
in Ku70 and LIG4 (non-homologous end joining (NHEJ) factors) in C. lusita-
niae-deficient strains. Huang and Mitchell (2017) generated a marker recycling
model called CRISPR–Cas9-induced marker excision (CRIME) in C. albicans
to avoid the paucity of drug-resistant makers. Methodologically speaking, the
selection marker that is used to delete the target gene is flanked by directly
repeated sequences. The CRISPR–Cas9 system is used dually to delete the gene
of interest subsequently to the deletion of the marker itself by double-strand break
(DSB) induction, leading to marker excision by recombination between the direct
repeats (Huang and Mitchell 2017). Furthermore, Nguyen et al. (2017) devel-
oped two recyclable CRISPR-mediated approaches, namely the LEUpOUT system
(applicable to LEU2/leu2 parental strain) and the HIS–FLP system (applicable
theoretically to any nourseothricin-sensitive C. albicans strain), to allow scarless
genome manipulation of C. albicans without the integration of permanent markers
or the application of a cloning step. Furthermore, Lombardi et al. (2017) applied
an episome-based CRISPR–Cas9 system in C. parapsilosis; the adopted plasmid
system called pRIBO theoretically offered the ability to manipulate any genetic
background with the possibility of sequential genome editing in the same back-
ground since the plasmid is escaped easily in the absence of selection. Afterward,
the former system was further improved into markerless-gene manipulation sys-
tem adapting various Candida spp., namely pCP-tRNA and pCT-tRNA applied
on C. parapsilosis (and their sister species, Candida orthopsilosis and Candida
metapsilosis) and C. tropicalis, respectively (Lombardi et al. 2019).
Interestingly, since bioinformatics is the keystone in scientific speaking lan-
guages, a breakthrough in CRISPR–Cas9 application area was made by Stoneman
et al. (2020) who developed recently CRISpy-pop, a flexible Python-based Web
application with a user-friendly graphical interface. The substantial aim of this
application is to theoretically design CRISPR/Cas9-driven genetic manipulations
on individual strain or population of strains to predict the most efficient combina-
tion (sgRNA and strain) (Stoneman et al. 2020). Currently, this powerful tool can
cover both bacteria (Zymomonas mobilis) and yeasts (S. cerevisiae), with a future
perspective to integrate new biological models (Stoneman et al. 2020).
4 Omics Resources
The complexity of the living cell hindered the scientists’ ability to see the
whole picture behind any given phenotype for a long period of time, since they
mainly adopted the “one–one analysis” (one gene/protein at a time) in different
biomedical researches, neglecting the myriad interactions between genes, pro-
teins, carbohydrates, lipids and metabolites that are eventually translated into a
certain phenotype (Stagljar 2016). The innovations of technological techniques
have significantly facilitated the conjugation of the “omics” suffix to both, the
genomic and post-genomic levels (transcriptomics, proteomics, and metabolomics)
that decode holistically the physiological and biochemical framework of numer-
ous organisms (Hasin et al. 2017; Babar et al. 2018; Narad et al. 2018; Patra
et al. 2021). Since omics experiments (high-dimensional biology) are hypothesis-
generating rather than hypothesis-driven studies (traditional experiment approach),
it could aid in opening the path toward building a nonbiased picture of the biolog-
ical system (Horgan and Kenny 2011). Generally, systemic biology represents an
interdisciplinary platform for mathematical analysis and computational modeling
approaches that translate the complexity of a defined biological system via either
top-down or bottom-up modalities (Patra et al. 2021).
352 D. Kasir et al.
The application of the state-of-the-art multi-omics approaches is a cornerstone

in synthetic biology, not only for reducing the time needed for bio-product candi-
dates to be commercially available but also by allowing the utilization of numerous
strategies for process optimization via metabolic engineering modality (Roldão
et al. 2012). The significance of the implantation of omics system in microbial cell
factory optimization relies basically on the ability of such multi-directory approach
to decode the underlined intra- and inter-cellular network communication, aiding
in a greater understanding of principal cellular elements’ properties and functions
and thus retrofitting and controlling the optimum niche for high quality and yield
production (Roldão et al. 2012). Generally speaking, the adopted omics approaches
differ according to the application field. In industrial biotechnology and bioprocess
engineering, five main omics modalities are applied, including the following.
4.1 Genomics
It represents a snapshot of the production repertoire of a defined microbe. In gen-

eral, genomics approach offers the potency to analyze and study comprehensively
microorganisms’ genomes to understand the function/interaction of genes within
the entire genome network (Roldão et al. 2012). The whole-genome sequencing is
undoubtedly crucial in screening and identifying the gene pool that leads to spe-
cific phenotype expression. Additionally, such a platform can allow predicting the
ideal growth conditions for targeted bio-factories (Herrgård and Panagiotou 2012).
4.2 Transcriptomics
An integrative approach provides a precise view of all the microorganism’s pos-

sible expression profiles under specific environmental conditions. Additionally,
it directs the spotlight on important gene interactome, possible gene targets for
metabolic engineering, and functional diversity among yeast strains (Roldão et al.
2012). The continuous development in this field facilitated the accurate prediction
of microbe’s expression profile under heterogeneous environmental conditions.
The recent construction of Reaction Inclusion by Parsimony and Transcript Distri-
bution (RIPTiDe) platform aids greatly in implementing the transcriptome analysis
in identifying the ideal metabolic pathways that lead to producing a desirable
compound regardless of the external niche (Jenior et al. 2020).
4.3 Proteomics
This modality represents the third stage in the holistic understanding of a cell’s
biological processes. It offers in-depth characterization of microorganisms’ pro-
tein profiles (including their location, structure, and function) since the transcript
and protein levels are weakly related due to the specific regulatory mechanisms
associated with the translational control (posttranslational modifications and trans-

lation efficiency) (Maier et al. 2009; Olivares-Hernández et al. 2011, 2010). The
variable nature of proteomics in response to environmental conditions imposes the
need for a constant tracking system for such a dynamic platform (Raghavachari
2011).
4.4 Metabolomics
These approaches constitute the fourth stage of the “biological puzzle-solving” that
provide a snapshot of metabolite concentrations (Pickford et al. 2019). Generally,
the metabolomics approach aims to study quantitatively the microorganism’s cel-
lular metabolite profile under specific culture conditions (Roldão et al. 2012). The
construction of mathematical models is considered one of the efficient tools in plot-
ting and understanding the metabolic profile in a defined organism by integrating
data science platforms such KBase, which involves semi-automated reconstruction
approaches (such as RAVEN, Merlin, and ModelSEED) (Arkin et al. 2018). It
displays a detailed map for the underlying molecular interaction that can evalu-
ate the efficacy of future network response under specific genetic/environmental
interventions (Roldão et al. 2012). A corrected meticulously comprehensive view
of microbe’s metabolic trait could be achieved by adding species-specific software
databases such as BRENDA, KEGG, and MetaCyc (Patra et al. 2021).
4.5 Fluxomics
Since metabolomics neglects the control and the functional regulation of metabolic
networks, the fluxomics platform acts as a complementary approach to intensively
understand the microorganism’s phenotype by determining the rates of metabolic
reactions (Pickford et al. 2019; Ratcliffe and Shachar-Hill 2005). Thereby improv-
ing our ability to predict the effect of genetic manipulation on the entity of the
microorganism’s physiological behavior, which leads to the formation of micro-
bial systems with improved metabolic capacity (Yu et al. 2012). The offered flux
balance analysis (FBA) in the fluxomics discipline greatly facilitates the reading
of metabolite flow through a defined metabolic model (Orth et al. 2010; Price
et al. 2004). The FBA analysis involves several advantages, such as the analysis
of phenotypic behaviors and flux coupling, the evolution of metabolic status with
environmental and genetic changes, and the prediction of metabolic engineering
foci (Patra et al. 2021).
The high-throughput potency of omics to comprehensively study the microbial
system is directly dependent on the available pool of technologies to scientifically
decode such huge data. The genomics platform constituted various technologies to
systematically analyze the Candida genome such as Candida Gene Order Browser
(CGOB), multiplex PCR, whole-genome sequencing, flow cytometry, slot blot
hybridization, microarray, Avadis, FuncExpression, High-Throughput GoMiner,
and GREAT (Roldão et al. 2012; Sampaio et al. 2005; Asadzadeh et al. 2020;
354 D. Kasir et al.
Maldonado et al. 2018; Chang et al. 2015; Loeffler et al. 2000; Campa et al. 2008;
Maguire et al. 2013).
Regarding transcriptomics, numerous techniques and sites are established to
facilitate integrative analysis of Candida transcripts, e.g., dual RNA sequenc-
ing, CLASSIFY, FunCluster, GARBAN, GOdist, dChip, ErmineJ, GoMiner,
Matrix2png, TM4, MAGIC, GeneSpring, Array-Pro Analyzer, and ArrayStar
(Roldão et al. 2012; Schulze et al. 2016).
For the Candida proteomics and metabolomics profiling, different method-
ological approaches are developed to perform a holistic analysis of yeast
proteomic and metabolomic repertoire, e.g., sodium dodecyl sulfate polyacry-
lamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis
(2DE), differential in-gel electrophoresis (DIGE), electrospray ionization-mass
spectrometry (ESI–MS), matrix-assisted laser desorption/ionization-MS (MALDI-
MS), MALDI-time-of-flight (MALDI-TOF), liquid chromatography-MS (LC–
MS), high-performance liquid chromatography (HPLC), gas chromatography-MS
(GC–MS), ultra-performance liquid chromatography-MS (UPLC-MS), AMDIS
software, Pathway Activity Profiling (PAPi) algorithm, UPLC-QTOF-MS Perseus
software, SIMCA, and FiatFlux (Wang et al. 2009, 2020; Larbi and Jefferies 2009;
Gräslund et al. 2008; Kim et al. 2005; Awad et al. 2018; Karkowska-Kuleta et al.
2020; Cabrera et al. 2010; Christen and Sauer 2011; Han et al. 2012).
The fluxomics yeast profiling is still restricted due to the disciplines nascent
state, leaving behind some constructed tools to examine the microbial pheno-
typic expression such as 13 C metabolic flux analysis (13 C MFA), OptKnock,
OptReg, OptStrain, optimal metabolic network identification (OMNI), OptGene,
thermodynamics-based MFA, COBRA toolbox, and OpenFLUX (Pharkya and
Maranas 2006; Henry et al. 2007; Feng et al. 2010; Pharkya et al. 2004; Her-
rgård et al. 2006; Blazeck and Alper 2010; Schellenberger et al. 2011; Veras et al.
2019).
5 Candida Metabolic Engineering
From adapting modern gene-editing tools to the implication in different industrial

and medicinal approaches, Candida CTG clade yeasts continuously demonstrate
their potency to update and track the latest scientific trends. The recent intensive
efforts in optimizing/advancing the CTG clade molecular toolbox and provid-
ing a series of synthetic constructs facilitate the rewiring of various Candida
spp. in several biotechnological applications such as bioremediation, biocontrol,
biofuel, vitamins, and industrial enzyme production (Defosse et al. 2018a, b;
Papon et al. 2012). The current deviation of bioengineering orientation toward
the notion of microbial consortia (rather than sufficing only with the monoculture
application) modernizes the vision of culturing several microbial factories into
bio-consortia (offers an ideal niche for efficient production), the Candida CTG
clade makes no exception for this modern application (McCarty and Ledesma-
Amaro 2019; Kouzuma et al. 2015; Brenner et al. 2008; Bhatia et al. 2018).
The microbial community takes advantage of “the power of the group” which
offers multiple boosters for a higher production rate such as having a robust bio-
community, tolerating various environmental challenges, reducing the “metabolic
load,” allowing the exchange of resources, activating bio-communication (between
species), and expanding the metabolic fitness (Stenuit and Agathos 2015; Tsoi
et al. 2018; Bassler and Losick 2006). Interestingly, C. famata was recently
reported as a potential candidate to enter the microbial consortia construction for
the industrial production of riboflavin (Bhatia et al. 2018). The application of such
bio-consortium is expected to increase in the near future especially with the suc-
cessful integration of CRISPR/Cas9 system in the consortium building process
(Wang et al. 2016a). The following section will cover the current main metabolic
engineering applications and biotechnological potentials of Candida CTG clade
yeasts (Table 2).
5.1 Ethanol Production
The world’s modern and technological lifestyle posed many challenges in sus-
taining energy sources especially with the existence of ever-increasing concerns
about economic and environmental consequences (Pereira et al. 2015). This stim-
ulates collaborative research efforts aiming to produce renewable, eco-friendly
biofuel sources using microbial factories (Du et al. 2019). Lignocellulose’s (con-
sists of cellulose, hemicellulose, and lignin) bioconversion into ethanol is currently
being considered as an alternative source to petroleum-based fuels (Rodrussamee
et al. 2018). The efficient fermentation of lignocellulose sugars represents a lim-
iting factor in the production process, especially xylose sugar (most abundant in
hemicellulose) (Rodrussamee et al. 2018). However, since CTG clade species are
considered as xylose growers (utilized as their sole carbon source) with some hav-
ing the ability to ferment xylose naturally into ethanol, they represent an attractive
superior microbial platform in such an industrial section (Papon et al. 2014). The
comparative genomic studies for xylose fermenters revealed the presence of ampli-
fication in sugar transporters and cell surface proteins, which may explain their
exceptional sugar environment (Wohlbach et al. 2011). Interestingly, Wohlbach
et al. reported an increase in the flux of the xylose assimilation pathway with
the absence of xylitol accumulation in strains engineered with pCtAKR (Candida
tenuis aldo/keto reductase), suggesting their involvement in stimulating NADH
recycling and glycerol production (Wohlbach et al. 2011). The insertion of opti-
mized (to frequently used codons in S. cerevisiae) exo-inulinase gene INU1 from
M. guilliermondii (INU1Y ) in S. cerevisiae resulted in Y13 recombinant strains
with inulinase activity up to 43.84 U/mL and 126.30 mg/mL ethanol production
from 300.0 g/L inulin (Liu et al. 2014). In addition, the fusion of S. stipitis with
S. cerevisiae (the microbe of choice in the ethanol industry) protoplasts gener-
ates a hybrid (unstable) strain with enhanced xylose-based ethanol production in
comparison with S. stipitis wild strain (Ruchala et al. 2020; Yoon et al. 1996). Fur-
thermore, the generation of S. stipitis Δhxk1 and 2-deoxyglucose-resistant mutants
has resulted in the derepression of xylose utilization in both engineered strains
356
Table 2 Current biotechnological potentials of the Candida CTG clade yeasts

Produced compound Candida CTG clade spp. (parental Modification Titer References
strain)
Adipic acid C. tropicalis KCTC 7212 ΔAOX4:AOX5 12.1 g/L Ju et al. (2020)
Citric acid C. oleophila ATCC 20177 Submerged fermentation 20.7 g/L Kim et al. (2015)
C. oleophila ATCC 20373 60.1 g/L
C. tropicalis ATCC 20115 45.0 g/L
C. oleophila ATCC 20177 Nitrogen limitation condition 74.2 g/L Anastassiadis et al. (2005)
Dicarboxylic acids C. tropicalis 1798 ΔCART 32.84 g/L Wang et al. (2018)
M. guilliermondii Δku70 KU141F1 ↑CYP52A12 ND Werner et al. (2017)
Ethanol S. passalidarum ATCC MYA-4345 Fermentation at 30 °C 31.9 g/L Du et al. (2019)
Fumaric acid S. stipitis CBS6054 ΔPsfum1 ΔPsfum2 ↑YMAE1 4.67 g/L Wei et al. (2015)
Pyruvate carboxylase, malate
dehydrogenase, and fumarase codon
optimization
Lipid C. tropicalis SY005 ↑CtRAP1 0.37 g/g Chattopadhyay et al. (2020)
L-lactic acid S. stipitis CBS6054 ↑LDH 58 g/L Ilmén et al. (2007)
2-phenylethanol M. guilliermondii YLG18 Optimization fermentation condition 3.20 g/L Yan et al. (2021)
In situ product recovery (ISPR)
Riboflavin C. famata VKMY-9 ↑SEF1 ↑IMH3 ↑RIB1 ↑RIB7 1.026 g/L Dmytruk et al. (2011)
C. famata AF-4 ↑RIB1 ↑RIB7 16.4 g/L Dmytruk et al. (2014)
C. famata VKMY-9 Sef1 C. famata: P SEF1 C. albicans 0.028–0.040 g/L Andreieva et al. (2020)
/C. tropicalis + C. famata SEF1 ORF
S-adenosyl-l-methionine S. stipitis BS 5776 Δ Erg6p 0.052 g/g Križanović et al. (2015)
(continued)
D. Kasir et al.
Table 2 (continued)
Produced compound Candida CTG clade spp. (parental Modification Titer References
strain)
Sophorolipids C. albicans O-13–1 Semi-continuous fermentation by 477 g/L Zhang et al. (2018)
using a novel bioreactor with
DVDSB
Shikimate S. stipitis FPL-UC7 ↑aro4K220L ↑aro1D900A ↑tkt1 3.11 g/L Gao et al. (2017)
Xylitol C. tropicalis LXU1-NXRG ↑At5g17010 1.14 g/L Jeon et al. (2013)
C. guilliermondii FTI 20,037 (ATCC Permeabilized with Triton X-100 2.65 g/L Cortez et al. (2016)
201,935)
ND not determined
Synthetic Biology in the Candida (CTG) Clade
357
358 D. Kasir et al.
(Sreenath and Jeffries 1999; Dashtban et al. 2015). Remarkably, comparative

metabolite profiling of xylose-fermenting yeasts showed a significant capability
of S. stipitis to produce ethanol more effectively than S. cerevisiae SR8 at xylose
(as the sole carbon source) concentration of 40 g/L with negligible by-product
(xylitol and glycerol) formation (Shin et al. 2019). Genome shuffling in S. stipi-
tis was recognized as a competent approach in xylose-based ethanol production,
which facilitates the design of TJ2-3 strain that produced 21.9 g/L of ethanol (Shi
et al. 2014).
Papon et al. (2014) summarized three modalities to augment the ethanol produc-
tion using engineered CTG clade yeasts: (i) comparative physiological analysis of
xylose growers and xylose fermenters, (ii) omics studies and metabolic modeling,
and (iii) construction of molecular tools. Yeast metabolic engineering in xylose-
based ethanol production is mainly focused on improving sugar uptake and the
initial assimilation steps (Vleet and Jeffries 2009). For instance, the heterologous
expression of xylose transporter gene At5g17010 (Arabidopsis thaliana origin)
in C. tropicalis strain (LXU1-NXRG) blocked the glucose suppression effect on
xylose assimilation, with an increase in the xylose uptake rate by 37–73% (depend-
ing on the glucose/xylose mixture ratio) (Jeon et al. 2013). The potency of C.
tropicalis in producing a high yield of ethanol is also associated with its intrinsic
tolerance toward generated biomass pretreatment inhibitors such as furfural (Wang
et al. 2016b). The underlined furfural detoxification mechanism is correlated to the
regulated expression of alcohol dehydrogenase 1 (ADH1) gene; such capability
was demonstrated after the heterologous expression of ctADH1 in Escherichia coli
BL21, resulting in a furfural degradation rate up to 1.59-fold (Wang et al. 2016b).
Rodrussamee et al. (2018) reported the efficient activity of newly isolated thermo-
tolerant S. passalidarum CMUWF1–2 in xylose–ethanol bioconversion. The strain
reached 0.43 g, 0.40 g, and 0.20 g ethanol/g xylose productivity rate at 30 °C, 37
°C and 40 °C, respectively. Worth mentioning, this strain shows remarkable resis-
tance ability to glucose repression phenomenon (Rodrussamee et al. 2018). Also,
S. passalidarum ATCC MYA-4345 strain succeeded lately in conferment glucose
and xylose with peak ethanol production up to 31.9 g/L (Du et al. 2019).
5.2 Lipid Production
The microbial lipid production has revolutionized both the industrial sector that
relies intensively on non-renewable, environmentally hazardous resources of fos-
sil fuel and the bio-medicinal area that recently integrated the lipo-molecules in
numerous scientific applications (Li et al. 2020; Younes et al. 2020). Naturally
speaking, the lipid accumulation in oleaginous microorganisms can take two path-
ways, the “de novo” (fermentation on hydrophilic substrates such as sugars and
related substrates) and the “ex novo” lipid synthesis (fermentation on hydrophobic
substrates such as oils and alkane; greatly depends on the carbon source) (Yan et al.
2020; Huang et al. 2017). For example, sophorolipids, and low-toxic, biodegrad-
able, and ecofriendly biosurfactants have gained interest in various therapeutic
biological applications since they exhibit antineoplastic, anti-bacterial, and anti-

inflammatory properties (Li et al. 2020). The application spectrum of sophorolipids
was extended to additional fields such as petroleum, soil remediation, food, cos-
metics, detergents, and pharmacy (Li et al. 2020; Wang et al. 2019; Hirata et al.
2009). Interestingly, C. albicans O-13-1 was reported to produce sophorolipids up
to 477 g/L with a productivity rate equal to 1.59 g/L by applying a semi-continuous
fermentation strategy and the usage of a novel bioreactor with dual ventilation
pipes and dual sieve plates (DVDSB) (Zhang et al. 2018). Another example of
Candida CTG clade contribution in lipo-synthesis was recently reported by Chat-
topadhyay et al. (2020) who constructed a novel strategy to boost lipid production
in C. tropicalis SY005. The procedure highlighted the usage of two approaches,
FLP/FRT-based recombination system and pCtINT1 integrative episome that facil-
itate the ectopic expression of CtRAP1 transcription factor (under the control
of GAL1p promoter), which upregulated the expression of FAS1, FAS2, PAH1,
and DGAT (play a role in fatty acid and triacylglycerol biosynthetic pathways)
(Chattopadhyay et al. 2020). The prescribed approach aids in increasing lipid accu-
mulation up to 60% (0.37 g/g dry cell weight) in comparison with the wild-type
strain (Chattopadhyay et al. 2020).
Additionally, the CTG clade yeasts are also presented as competent candidates
in the adipic acid industry, which is mainly involved in manufacturing nylons,
pH adjustment, adhesives, resins, and synthetic lubricants (Polen et al. 2013;
Castellan et al. 1991). Ju et al. (2020) developed metabolically engineered C. tropi-
calis AOX4:AOX5 (encoding acyl-CoA oxidases) mutant strain with adipic acid
maximum production, 12.1 g/L and 0.1 g/L productivity rate. The analysis of a
genome-scale metabolic model of C. tropicalis (iCT646) deciphers the presence of
52 unique reactions (some belong to the lipid metabolic pathways) in this oleagi-
nous yeast relative to the genome-scale models of S. cerevisiae, Yarrowia lipolytica,
and C. glabrata (Mishra et al. 2016). Such profile may explain the capability of
C. tropicalis in catalyzing the oxidation pathway, thus facilitating the synthesis
of DCA (Mishra et al. 2016). Wang et al. (2018) reported the construction of C.
tropicalis ΔCART strain mutant with sophisticated metabolic behavior to effec-
tively produce long-chain DCA (intermediate, involved in the chemical industry).
The application of pPICPJ-mazF-based markerless editing system facilitates the
CART gene (encoding carnitine acetyltransferase) knockout and the replacement
of the NADPH–cytochrome P450 reductase gene promoter with the constitutively
expressed promoter pGAP (Wang et al. 2018). Such manipulation improved the
yield of long-chain DCA up to 32.84 g/L (11.4-fold relative to the parental strain)
(Wang et al. 2018). Werner et al. (Werner et al. 2017) reported M. guilliermondii
Δku70 KU141F1 as a promising biocatalyst in DCA production. The overexpres-
sion of CYP52A12 (encoding cytochrome P450 monooxygenase, catalyzing the
ω-oxidation of fatty acids and alkanes) showed a significant increase in DCA pro-
duction after 72 h (Werner et al. 2017). However, the introduction of Δpox1Δpox2
double mutant (POX1 and POX2 encoding for the main acyl-CoA oxidases,
catalyzing the oleic acid degradation in the β-oxidation pathway) in the strain
360 D. Kasir et al.
KU141F1 showed higher DCA degradation (relative to the parental strain), sug-
gesting the coexistence of non-peroxisomal fatty acid catabolism pathway (Werner
et al. 2017).
5.3 Vitamin Production
The current diet–vitamin insufficiency posed an urgent demand for compensatory

production of these valuable nutraceuticals compounds that shape good health and
mental strength (Revuelta et al. 2016; Ledesma-Amaro et al. 2013). To overcome
this drawback, a determinant orientation toward scaling up vitamin productivity via
adopting specialized microbial factories (especially yeast) dominated in synthetic
biology field recently (Ledesma-Amaro et al. 2013; Varga and Maraz 2002). For
instance, vitamin B2 (riboflavin) is a metabolic precursor of flavin mononucleotide
(FMN) and flavin adenine dinucleotide (FAD) (both act as coenzymes in various
enzymatic redox reactions), firstly discovered in 1879 as a yellow pigment from
milk (named lactoflavin) (Dmytruk et al. 2011; Liu et al. 2020). The production of
this vitamin is stimulated in the bioengineering area mainly due to its nutritional
and pharmaceutical values (Abbas and Sibirny 2011). There is a specialized group
of yeast species with an ability to produce riboflavin, termed “flavogenic yeast”
including, C. famata, C. albicans, M. guilliermondii, Schwanniomyces occidentalis,
and others (Papon et al. 2013). For instance, C. famata which is considered a
potential riboflavin producer demonstrates a noticeable enhancement in riboflavin
synthesis especially with RIB1 + (GTP cyclohydrolase II), RIB7 + (RF synthase)
expression profile (Dmytruk et al. 2014). The two former metabolic enzymes
(under iron starvation) aid in 62-fold productivity enhancement compared to other
tested candidates that give only a 3.1- or 16-fold productivity rate (with differ-
ent expression profiles) (Liu et al. 2020; Dmytruk et al. 2014). Furthermore, the
metabolic manipulation of C. famata dep8-conventionally mutated strain via the
introduction of additional copies of SEF1, IMH3, RIB1, and RIB7 aids in a 4.1-fold
increase in riboflavin production (Dmytruk et al. 2011). Similarly, M. guillier-
mondii which were firstly described by Tanner et al. (1945) in 1945 for their
ability to overproduce vitamin B2 under iron deficiency were found to upregu-
late riboflavin production under iron-enriched medium using rib80, rib81, hit1,
and red1–red6 mutant strains (Papon et al. 2013). Andreieva et al. (2020) recently
generated a species–hybrid vitamin B2 inducing construct that restores riboflavin
overproduce ability in sef1 mutant strain, involving the fusion of C. albicans
(flavinogenic) or C. tropicalis (non-flavinogenic) promoter SEF1 with SEF1 ORF
of C. famata. In addition, they demonstrated the positive feedback of Sfu1 (GATA-
type transcription factor) and/or vacuolar ATPase subunit A (VMA1) repression on
vitamin B2 upregulation (Andreieva et al. 2020).
5.4 Other Valuable Compound Bio-production
The biotechnological potentials of Candida CTG clade are not constrained only to
the former cited applications; rather, numerous species show additional capabilities
in producing valuable bio-molecules such as industrial enzymes and therapeutic
compounds (Papon et al. 2013; Roa Engel et al. 2008; Križanović et al. 2015).
Various Candida CTG clade yeasts demonstrate a remarkable capacity in pro-
ducing citric acid, an organic acid commonly used in food and pharmaceutical
industries (Singh Dhillon et al. 2011). Anastassiadis et al. (2005) reported a suc-
cessful continuous citric acid fermentation by Candida oleophila ATCC 20177
strain under nitrogen limitation condition with synthesis rate up to 74.2 g/L. Fur-
thermore, Kim et al. described considerable citric acid productivity by submerged
fermentation of C. oleophila ATCC 20177, C. tropicalis ATCC 20115, and C.
oleophila ATCC 20373 strains up to 20.7 g/L, 45.0 g/L, and 60.1 g/L, respectively
(Kim et al. 2015).
M. guilliermondii appears to be one of the most efficient candidates in the
xylose-xylitol conversion process with considerable variation in xylitol yield based
on the accredited strain (Papon et al. 2013). The generated xylitol is beneficial in
various nutraceuticals (chewing gum, candies, wafer fillings, and chocolate) and
pharmaceuticals (protein extraction stabilizer and antineoplastic properties) indus-
tries (Papon et al. 2013; Ur-Rehman et al. 2015). Cortez et al. (2016) reported
an effective xylitol production from D-xylose using M. guilliermondii (permeabi-
lized with Triton X-100) with a yield up to 0.80 g/g and 2.65 g/L volumetric
productivity. Furthermore, M. guilliermondii showed high potentials in producing
2-phenylethanol (2-PE), aromatic compound (rose scent) used in cosmetics, per-
fume, and food industries (Yan et al. 2020). Yan et al. (2021) recently reported
newly identified M. guilliermondii strain YLG18 as a promising candidate for 2-
PE high production from L-phe. Upon optimization process, this strain was able
to synthesize 2-PE up to 3.20 g/L (Yan et al. 2021).
S. stipitis shows promising potentials in producing various valuable compounds
among which is fumaric acid, a C4-dicarboxylic acid applied in nutraceutical,
pharmaceutical, and chemical industries (Roa Engel et al. 2008). Wei et al. (2015)
described the development of an optimized strain PSYPMFfS with the following
properties, overexpression of heterologous reductive fumaric acid synthetic path-
way (Rhizopus oryzae FM19), codon modification, blockage of the conversion of
fumaric acid (double-deletion of fumarase genes (Psfum1 and Psfum2), and over-
expression of fumaric acid heterologous transporter (YMAE1). The obtained strain
produced fumaric acid titer up to 4.67 g/L from xylose (Wei et al. 2015). Addi-
tionally, Ilmén et al. (2007) described an efficient production of l-lactic acid by
an engineered S. stipitis strain; the random integration of heterologous LDH gene
enhanced the yield of lactate up to 58 g/L from 100 g/L xylose. The produced lactic
acid can be applied in various industrial applications such as biodegradable plas-
tics and textile fiber manufacturers (Ilmén et al. 2007). The systematic metabolic
engineering driven by flux facilitates the design of S. stipitis-mutant strains with
improved biomass yield up to 44% (in ZWF1 overexpressed mutant) (Unrean et al.
362 D. Kasir et al.
2016). Further application of metabolic evolution results in maximum biomass

of 9.81 g/L (Unrean et al. 2016). Remarkably, S. stipitis was reported to be an
effective producer of S-adenosyl-l-methionine (SAM), a metabolic intermediate in
enzymatic reactions which participates in several biological processes, commonly
applied in medicine as a chemotherapeutic agent in the treatment of numerous
pathologies, including depression, liver disease, Lesch-Nyhan disease, Alzheimer’s
disease, and diarrhea (Križanović et al. 2015; Chen et al. 2016). Križanović et al.
(2015) showed that S. stipitis strain M12 with disrupted Erg6p (responsible for
C-24 methylation in ergosterol biosynthesis) resulted in higher accumulation of
SAM up to 52.48 mg/g CDW. The production potency of S. stipitis is not lim-
ited to the former molecules; rather, it demonstrates a remarkable capability in
producing shikimate, a building block for several industrial, pharmaceutical, and
nutraceutical applications (Gao et al. 2017). Gao et al. (2017) reported the success-
ful generation of a recombinant S. stipitis FPL-UC7 platform, carrying aro4K220L,
aro1D900A, and tkt1 (encoding enzymes involved in the shikimate pathway) and
strong promoters and terminators, synthesizing shikimate up to 3.11 g/L (7-folds
higher relative to S. cerevisiae shikimate yield).
5.5 Bioremediation
Nowadays, environmental pollution is considered one of the most challenging

worldwide health-related problems especially with the excessive application of
pesticides and chemical fertilizers in various agricultural practices (Dangi et al.
2019). The ever-increasing of industrial development is significantly contributing
to the liberation of toxic organic and inorganic compounds and heavy metals that
pose a disastrous effect on the whole ecosystem (Cheng 2016; Larsson 2014).
Bioremediation, a promising newly developed eco-friendly practice, aims to break
the pollution cycle through the usage of “bio-cleaners,” which are certain microbes
(bacteria, fungi, and algae) with specialized ability to remove or neutralize contam-
inants present in the environment (Dangi et al. 2019). Remarkably, the fungi have
many advantages over other adopted microbes in bioremediation process (Defosse
et al. 2018b). Higher resistance toward toxins, natural self-regulation of accumu-
lated pollutants, quick nutrients capturing, and biomass development are among the
advantages offered by the mycoremediation approach (Defosse et al. 2018b; Treu
and Falandysz 2017). The Candida CTG clade is no exception for such poten-
cies (Defosse et al. 2018b). For instance, soil contaminated with chromium (a
toxic metal) was efficiently restored after the implication of C. maltosa that par-
tially reduced the two forms of chromium metal (trivalent Cr(III) and hexavalent
Cr(VI) (Ramírez-Ramírez et al. 2004). Furthermore, the live and dead biomasses
of C. tropicalis were capable to limit the phytoavailability of the mutagenic form
of chromium Cr(VI) (Bahafid et al. 2013). Coimbra et al. (2009) demonstrated
the capability of both M. guilliermondii and C. tropicalis to effectively remove
hydrophobic contaminants (petroleum and motor oil) with the implementation of
the substrates, soybean oil, and soybean oil + glucose. Recently, García-Béjar
et al. (2020) demonstrated that the bioremediatory activity of C. albicans and C.
parapsilosis ranged from 50 to 64% in removing aflatoxin B1, and the combina-
tion of the aforementioned species with C. tropicalis displayed a removal capacity
ranging from 56 to 68% for zinc metal.
5.6 Biocontrol
The spread of phytopathogens and crop pests in agricultural industries poses a

major challenge to food security and the balance of the ecosystem since it repre-
sents one of the main causes of worldwide crop losses (Babbal and Khasa 2017;
Marian and Shimizu 2019). Despite the periodical application of chemical pesti-
cides, the estimated loss of the annual global crop yields is up to 40% (Messing and
Brodeur 2018). Microbial biocontrol, a fast-growing practice, aims to use microbes
(fungi, bacteria, yeast, and viruses) with the ability to control the spreading of
such pests in a safe and eco-friendly manner (Babbal and Khasa 2017). Interest-
ingly, different Candida CTG spp. are being considered as potential candidates
for the application of microbial biocontrol approaches (Defosse et al. 2018b).
Various Candida spp. are granted the access to the list of registered biocontrol
yeasts for their adaptation properties such as secretion of hydrolytic enzymes
(proteases, chitinases, glucanases) and volatile compounds (implicated in anti-
mycotic activity), biofilm formation, high osmotolerance, induction of resistance
in the plant/fruit (to control post-harvest decay), and guide of hyphal parasitism
(Freimoser et al. 2019).
For instance, Guerrero et al. (2014) reported a significant suppression of Peni-
cillium expansum spore germination on the post-harvest apple fruit after the
application of three strains of C. oleophila (L06, L07 smooth, and L07 rough)
with an inhibitory rate up to 97%. The suggested mode of action for the effi-
cient bio-controllability of C. oleophila is mainly associated with the production of
exo-β1,3-glucanase enzyme (Guerrero et al. 2014). Interestingly, numerous antag-
onistic yeast-based commercial products are being developed for the control of
post-harvest pathogens, among which are C. oleophila-based products, namely
Aspire (controls decay on stone fruit, pome, citrus, strawberry) and Nexy (con-
trols decay on pome, banana, citrus) (Zhang et al. 2020). The biocontrol potency
is not exclusive to C. oleophila. Recently, Sun et al. (Sun et al. 2021) described the
synergistic bio-controllability action of M. guilliermondii Y-1 (1 × 108 cells/mL)
and melatonin (100 μmol/L; possibly implicated in improving the strain Y-1 colo-
nization ability) against apple gray mold (caused by Botrytis cinerea). A significant
reduction in disease incidence and lesion diameter is achieved with the aforemen-
tioned combination. Furthermore, the co-culture of M. guilliermondii KL3 and
Aureobasidium pullulans GE17 was able to suppress the spore germination of both,
Penicillium digitatum (green mold) and P. expansum (blue mold) by the efficacy
of 86–95% (Agirman and Erten 2020).
364 D. Kasir et al.
6 Concluding Remarks and Perspectives
The importance of CTG clade yeasts in the field of synthetic biology is not
doubtable anymore; all the identified capabilities in this unique clade are just pre-
senting the tip of submerged potentials. The future vision of Candida metabolic
engineering will mainly deviate to the Candida consortium rather than the sin-
gle strain notion with the application of more species-directed (species–hybrid)
and synthetic standardized gene rewiring modules. The unlimited efforts to stan-
dardize the advanced gene-editing tools are the keystone in deciphering the
competent species. Thus, the continuous optimization and integration of both,
gene-engineering strategies and omics platforms, will facilitate building special-
ized potentially unexplored CTG clade yeast strains with desirable properties in
additional biotechnological fields.
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Synthetic Biology of Yeasts Tools and Applications (2022)

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What is the book about?

What is the book about?

What topics are covered in the first part of the book?

What topics are covered in the first part of the book?

Farshad

ISBN 978-3-030-89679-9 ISBN 978-3-030-89680-5 (eBook)

(CTG) Clade”, covers applications of synthetic biology to produce diverse and

Harzevil, Gilan, Iran Farshad Darvishi Harzevili

Synthetic Biology Tools and Cellular Engineering

Applications of Yeast Synthetic Biology

Application of Yeast Synthetic Biology for the Production of Citrus

Pathway design • Pathway analysis • Metabolic engineering • Yeast

Advancements in metabolic engineering and synthetic biology have enabled accel-

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 3

promise of a more sustainable (bio)economy (Voigt 2020). To keep pace with

2 In Silico Pathway Prediction and Design

Retro-biosynthesis and stoichiometry-based optimization methods have been

2.1 Data- and Knowledge-Bases for Metabolic Reaction

2.2 Pathway Prediction Using Retro-Biosynthesis Tools

Firstly, a distinction is made between retro-biosynthesis and classical retro-

various criteria based on structural (chemical) similarity, reaction promiscuity, and

Table 1 Retro-biosynthesis tools for metabolic pathway prediction

2.3 Stoichiometry-Based Optimization Methods for Pathway

Given a universal metabolic reaction network, the ‘pathway design’ problem

2.3.1 Pathways with Desired Stoichiometric Properties

Table 2 Optimization-based methods for metabolic pathway design

2011). In practice, these pathway enumeration methods rely on the solution of

2.3.2 Pathways with Maximum Thermodynamic Favorability

2.3.3 Pathways with Minimum Enzymatic Cost

3 Case Studies of Metabolic Pathway Prediction

In this section, selected case studies illustrate different pathway engineering

impact of the latter applications is especially highlighted in the context of harness-

3.1 Balancing Redox Cofactor Supply for Improving Substrate

farnesene. Implementation of other computationally predicted major metabolic

3.2 Increasing Cytosolic Acetyl-CoA Availability for Metabolic

Cytosolic acetyl-CoA is a key metabolite for the production of a range of valuable

3.3 Engineering a Heterologous CBB Cycle for CO2 Fixation

In an early effort from Guadalupe-Medina et al. (2013), a heterologous Calvin–

4 Challenges and Outlook

Paulina Korpys-Woźniak, Monika Kubiak, Monika Borkowska,

P. Korpys-Woźniak · M. Kubiak · M. Borkowska · E. Celińska (B)

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 27

1 General Concept of Modularity

Fig. 1 General outline of bioparts assembly and combinatorial cloning

G2 can be changed, but the linking/docking sites remain unchanged—this

By definition, entering a modular cloning approach is a long-term strategy of

In contrast, an assembly technique developed only 2 years later, USER®

several advancing modifications improving flexibility of the method, (ii) S. cere-

destination vectors. In 2017, customized collections of standardized bioparts for

3 Restriction Digestion/Recombination-Based Assembly

Restriction digestion/recombination-assisted bioparts assembly relies on flanking

BioBrick assembly (BB) (Knight 2003) of standardized bioparts, otherwise known

There is no specific scaffold of linking/docking in BB technique. While it saves

of the Registry of Standard Biological Parts (http://partsregistry.org/ Main_Page),

Introduction of GoldenGate (GG) cloning strategy to molecular biology portfolio

Likewise, the GG standard was modified to generate a cloning system GoldenBraid

in a complete construction of ~13 kbp by the native recombination machinery of

Recently, (Liu et al. 2019) exploited the GG assembly method to construct

Gateway™ cloning technology (GW) relies on a system of short att sequences

In its basic format, GW is used for single-fragment subcloning, but can be