Tir 

et al. Microbial Cell Factories (2022) 21:157

https://doi.org/10.1186/s12934-022-01882-6 Microbial Cell Factories

RESEARCH Open Access

From strain engineering to process

development: monoclonal antibody production
with an unnatural amino acid in Pichia pastoris
Nora Tir1,2, Lina Heistinger1,2,6, Clemens Grünwald‑Gruber3, Leo A. Jakob4, Stephan Dickgiesser5,
Nicolas Rasche5 and Diethard Mattanovich2* 

Abstract 
Background:  Expansion of the genetic code is a frequently employed approach for the modification of recombinant
protein properties. It involves reassignment of a codon to another, e.g., unnatural, amino acid and requires the action
of a pair of orthogonal tRNA and aminoacyl tRNA synthetase modified to recognize only the desired amino acid. This
approach was applied for the production of trastuzumab IgG carrying p-azido-l-phenylalanine (pAzF) in the industrial
yeast Pichia pastoris. Combining the knowledge of protein folding and secretion with bioreactor cultivations, the aim
of the work was to make the production of monoclonal antibodies with an expanded genetic code cost-effective on
a laboratory scale.
Results:  Co-translational transport of proteins into the endoplasmic reticulum through secretion signal prepeptide
change and overexpression of lumenal chaperones Kar2p and Lhs1p improved the production of trastuzumab IgG
and its Fab fragment with incorporated pAzF. In the case of Fab, a knockout of vacuolar targeting for protein degrada‑
tion further increased protein yield. Fed-batch bioreactor cultivations of engineered P. pastoris strains increased IgG
and ­IgGpAzF productivity by around 50- and 20-fold compared to screenings, yielding up to 238 mg ­L−1 and 15 mg ­L−1
of fully assembled tetrameric protein, respectively. Successful site-specific incorporation of pAzF was confirmed by
mass spectrometry.
Conclusions:  Pichia pastoris was successfully employed for cost-effective laboratory-scale production of a monoclo‑
nal antibody with an unnatural amino acid. Applying the results of this work in glycoengineered strains, and taking
further steps in process development opens great possibilities for utilizing P. pastoris in the development of antibodies
for subsequent conjugations with, e.g., bioactive payloads.
Keywords:  Pichia pastoris, Monoclonal antibody, Unnatural amino acid, Strain engineering, Bioreactor cultivation

Background Particularly after the formulation of the official defini-

In the past two decades, the demand for site-specific tion of “click” chemistry [1], research has focused on the
incorporation of reactive chemical groups in a wide range incorporation of unnatural amino acids (uAAs) into pro-
of proteins has been growing to gain novel properties. teins with reactive groups that cannot be found in nature
and react with biological systems. Such reactions are
bioorthogonal and, frequently, they proceed in one step,
*Correspondence: [email protected]
2
without the formation of side products. These desirable
Department of Biotechnology, Institute of Microbiology and Microbial
Biotechnology, University of Natural Resources and Life Sciences, Muthgasse
features have opened doors to various applications and
18, 1190 Vienna, Austria manipulations of endogenous and heterologous proteins
Full list of author information is available at the end of the article [2, 3].

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Tir et al. Microbial Cell Factories (2022) 21:157 Page 2 of 16

Several conditions need to be fulfilled to successfully of this yeast. Protein folding and secretion pathways in
incorporate an uAA at the desired position in a target P. pastoris were discussed in detail by Damasceno et  al.
protein. The uAA to be site-specifically incorporated [16] and Delic et al. [17]. Furthermore, successful incor-
must not be recognized by endogenous tRNAs and tRNA porations of uAAs into recombinant proteins produced
synthetases (RSs). In other words, the technology used by P. pastoris were previously reported [18, 19]. In the
and the uAA must be orthogonal in the host cell. The present study, we combined our knowledge of P. pastoris
most common approach to achieving orthogonality is with the ongoing requirements for improving monoclo-
using a tRNA and its cognate RS from an organism phylo- nal antibody production with an expanded genetic code
genetically distant from the expression host. Altogether, to relieve some of the major bottlenecks in the secretion
the codon reassignment by which an uAA is site-specif- of trastuzumab antigen-binding fragment (Fab) and full-
ically incorporated instead of a canonical amino acid is length IgG with site-specifically incorporated p-l-azido-
called the expansion of the genetic code. It is frequently phenylalanine (pAzF).
employed for the incorporation of uAAs into monoclo-
nal antibodies and antibody formats, predominantly of Results
the IgG1 subclass, to enable site-specific click reactions Influence of ­tRNACUA​/RS expression on pAzF incorporation
with small molecule payloads. The resulting products are yield
known as antibody-drug conjugates (ADCs) and extend Genes for the orthogonal pair of amber suppressor tRNA
the usage of immunotherapeutics. An overview of Food ­(tRNACUA​) and pAzF-specific RS ­(RSpAzF) were derived
and Drug Administration-approved ADCs currently on from E. coli tyrosyl-tRNACUA​ and tyrosyl-RS, as reported
the market is given by Tong et al. [4]. previously [18, 20, 21]. Methanol-inducible promoters of
The growing need for fast and cost-effective antibody varying strength were chosen for each gene based on the
development and production requires finding suitable knowledge of the methanol utilization pathway in P. pas-
expression hosts. Unlike the production of antibody frag- toris [15, 22]. Enhanced green fluorescent protein (eGFP)
ments, for which bacterial systems can be used, tetra- was used as a reporter protein for pAzF incorporation in
meric IgGs with suitable glycosylation patterns require small-scale screenings and detection was done by flow
eukaryotic hosts. The simplest and inexpensive eukary- cytometry. The codon for tyrosine-40 in the sequence
otic hosts are yeasts that have already shown the poten- was replaced with the amber stop codon (­eGFPY40X). For
tial for full-length IgG production [5, 6]. By combining eGFP expression, the strong methanol-inducible pro-
glycoengineering [7], optimization of protein folding and moter for alcohol oxidase 1 was used. All cloning was
secretion [8, 9], and process development [10, 11], yeast performed using GoldenPiCS [23] and the corresponding
research is gaining more attention for the fast and cheap schemes are shown in Additional file 1: Fig. S1.
production of recombinant therapeutics. The industrial Promoter strengths in the presence of methanol were
yeast Pichia pastoris (syn. Komagataella spp.) is one of previously determined by microarray analysis and
the well-established microbial hosts for recombinant decreases in the following order [22]: dihydroxyacetone
protein production. Like other yeast species, it requires synthase 2 ­ (PDAS2) 
> alcohol oxidase 1 ­ (PAOX1) > dihy-
inexpensive media for growth, can produce proteins with droxyacetone synthase 1 ­ (PDAS1) > peroxisomal mem-
post-translational modifications including human-type brane-associated protein 20 (­PPMP20), where the relative
glycosylation [12], and has a higher secretion capacity ­PPMP20 strength has not been published yet. Although
than prokaryotes. Moreover, P. pastoris, unlike Saccha- less than 5% of the overall difference in stop codon sup-
romyces cerevisiae, has a Crabtree-negative phenotype pression was observed between the cultures expressing
[13, 14], facilitating its cultivation at high cell densities to both ­tRNACUA​ and ­RSpAzF under the strongest methanol-
obtain higher product titers. Due to its methylotrophic inducible promoters in P. pastoris, it was evident that a
metabolism and genes tightly regulated by methanol, strong expression was required for ­tRNACUA​ (Fig.  1). It
it is commonly used for the inducible production of has already been established that increasing the t­ RNACUA​
large amounts of heterologous proteins [15]. In addi- gene copy number above 3 does not improve the stop
tion, P. pastoris secretes very few endogenous proteins codon suppression and that the addition of a  polymer-
in the supernatant, which reduces the effort to purify the ase II promoter in front of the polymerase III promoter
secreted product. further increases expression levels [21]. In this work, the
Research on (heterologous) protein folding and secre- construct with 3 tandem copies of ­tRNACUA​, each lack-
tion in P. pastoris has advanced significantly in the last ing the CCA trinucleotide at its 3′ terminus, flanked by
decade. A growing need for cost-effective production the SUP4 region from S. cerevisiae was used. In addition,
of recombinant therapeutics and industrial enzymes the choice of different methanol-inducible promoters for
pushes the need for strain and metabolic engineering each gene placed in the same vector further increases
 Tir et al. Microbial Cell Factories (2022) 21:157 Page 3 of 16

Fig. 1  Amber suppression in ­eGFPY40pAzF depends on the promoter combination in the ­tRNACUA​/RSpAzF pair. Mean values above the boxes are
percentages calculated from the relative amounts of ­eGFPY40pAzF to eGFP produced per cell from three to eight biological replicates and are shown
as horizontal lines in the boxes. Whiskers represent minimal and maximal values. The table below the graph shows the identity of the promoter
combinations

genome stability when prolonged cultivation times are and yield, supporting the previous observation by Gas-
needed. Therefore, the combination of the ­ PDAS2 for ser et al. [8]. However, overall strong expression of both
­tRNACUA​ ­(PDAS2_3xSUP4-tRNACUA​) and ­ PAOX1 for RS chains was required for the best product titer and yield.
­(PAOX1_RSpAzF) was used in all other experiments. The Therefore, the combination used in all further experi-
optimum pAzF concentration in the culture medium was ments with Fab and IgG was ­PAOX1 for the light chain
determined as 2 mM, leading to the maximum suppres- ­(PAOX1_LC) and P ­ DAS2 for the heavy chain ­(PDAS2_HC).
sion with the least possible impact on biomass growth
(Additional file 2: Table S1). Changing the secretion signal prepeptide to improve
protein secretion
Tuning light and heavy chain expression Aside from promoter selection, the effects of the pre-
Monoclonal antibody production in microbial hosts is Ost1-pro-MFα secretion signal and the standard
challenging and product-dependent. In shake-flask culti- pre-pro-secretion signal for the mating factor α (pre-pro-
vations, most of the secreted product is reduced antibody MFα) on the secretion of both trastuzumab Fab and IgG
formats rather than fully-assembled tetrameric antibod- were compared. The mean titer and yield of Fab fused to
ies [6]. In order to monitor product quality and quantity, the pre-pro-MFα were almost twofold higher than that
P. pastoris secreting trastuzumab Fab fragment fused to of Fab fused to the pre-Ost1-pro-MFα secretion signal
the prepeptide for oligosaccharide transferase 1 and the (Fig.  3). In the case of IgG, titer and yield of pre-Ost1-
propeptide for mating factor alpha of S. cerevisiae (pre- pro-MFα fused protein were about 1.3-fold and 1.1-fold
Ost1-pro-MFα) was used [24, 25]. In the screenings for higher than those of pre-pro-MFα fused one, respec-
Fab expression, different methanol-inducible promot- tively. However, when examining supernatants from the
ers for each chain were tested (Fig.  2). When the same screenings by SDS-PAGE and western blot (Additional
choice of promoters was used in the opposite order, the file  1: Fig. S2), significantly more endogenous proteins
combination with a stronger promoter for the light chain were observed in the cultures producing Fab and IgG
resulted in more than a twofold higher product titer fused to the pre-pro-MFα secretion signal. Since protein
Tir et al. Microbial Cell Factories (2022) 21:157 Page 4 of 16

Fig. 2  Titers and yields of the wild-type trastuzumab Fab depend on the promoter combination. Mean values above the boxes were calculated
from seven to ten biological replicates and are shown as horizontal lines. Dots and triangles show each data point and whiskers show minimal and
maximal values. The yields were calculated as the amount of protein per wet cell weight (WCW). The table below the graph shows the identity of
the promoter combinations

Fig. 3  Influence of secretion signals on Fab and IgG titers (left) and yields (right). Mean values above the boxes were calculated from seven to nine
biological replicates and are shown as horizontal lines. Dots and triangles show each data point and whiskers show minimal and maximal values.
The yields were calculated as the amount of protein per wet cell weight (WCW)
 Tir et al. Microbial Cell Factories (2022) 21:157 Page 5 of 16

purification from bioreactor cultivations was the final on IgG secretion. However, no change in IgG titer and
goal, the pre-Ost1-pro-MFα secretion signal was chosen yield was observed in this work.
for all other experiments. A positive impact of overexpression of the peptidyl-
Additionally, no band corresponding to the tetrameric prolyl isomerase CPR5 in S. cerevisiae producing a
protein ­(HC2-LC2) was observed in both cases. On the full-length human IgG was reported [9]. Therefore,
screening scale, this is not unusual and depends on the it appeared as a promising target for improving IgG
exact identity of IgG. Nevertheless, it was still possible to production in P. pastoris. Nevertheless, a reduction
detect protein by ELISA because the antibody combina- of 30% in both IgG titer and yield was observed upon
tion used binds the heavy chain and detects any species CPR5 overexpression. As with PDI1 overexpression, the
with assembled Fab domain, namely HC-LC, ­HC2-LC, observed effect could be a consequence of the protein
and, if present, H
­ C2-LC2. burden in the ER or protein misfolding due to the con-
stitutive CPR5 expression.
Strain improvement by helper factor overexpression Kar2p is the ER chaperone of the Hsp70 family
The choice of targets for overexpression and knockout whose overexpression generally increases recombi-
in this work was based on the previous experience with nant protein secretion. Similar to the observation in
Fab and, to a certain extent, IgG production in yeast. The S. cerevisiae [9], KAR2 overexpression under the glyc-
list with the target description and references is given in eraldehyde-3-phosphatase promoter ­ (PGAP) did not
Table 1. improve IgG titer and yield. Lhs1p functions as a nucle-
Overexpression of the transcription factors HAC1i otide exchange factor (NEF) that interacts with Kar2p
(intronless HAC1) and YAP1 did not improve IgG titer [30]. Reduced IgG titer and yield by 60% were observed
and yield (Fig.  4). Overexpression of PDI1 reduced IgG upon LHS1 overexpression under the porine 1 pro-
titer and yield by 30%, in contrast to some of the reports moter ­(PPOR1), which has 50% strength compared to
for recombinant protein production in P. pastoris [8, 27]. ­PGAP [23]. This is likely due to the chaperone disbalance
Together with the lack of improvement of IgG secretion in the ER, although positive effects were observed upon
upon HAC1i and YAP1 overexpression, this observation LHS1 overexpression in S. cerevisiae producing recom-
points towards other bottlenecks of P. pastoris as a mon- binant human albumin [29]. In this work, the balance
oclonal antibody production host. between Kar2p and Lhs1p activity was crucial for the
Sbh1p is a subunit of the Sec61p ER translocation com- production of both Fab (not shown) and IgG. Co-over-
plex (in addition to Sec62p, Sec63p, Sec71p, and Sec72p) expression of the two ER chaperones increased IgG
that interacts with the exocyst and the ribosome dur- titer and yield around 1.6-fold, confirming the previous
ing co-translational translocation into the ER. It was observation that the bottleneck in P. pastoris producing
expected that SBH1 overexpression has a positive effect Fab is the ER import [31]. The finding was then applied
to the strains producing the IgG variant carrying pAzF

Table 1  Overview of overexpression and knockout targets tested in this work for IgG producing P. pastoris 
Gene Name description Product description Cellular localization References

YAP1 Yeast activator protein 1 Transcription factor involved in the regulation of tran‑ Nucleus and cytoplasm [26]
scription in response to oxidative stress
HAC1 Homologous to Atf/Creb1 Transcription factor involved in the regulation of the Nucleus [27]
unfolded protein response
PDI1 Protein disulfide isomerase 1 Multifunctional protein disulfide isomerase and protein ER lumen [8]
disulfide oxidoreductase
SBH1 Sec61 beta homolog 1 Beta subunit of Sec61 ER translocation complex ER membrane [28]
CPR5 Cyclosporin-sensitive proline rotamase 5 Rearranges peptidyl-prolyl bonds; transcriptionally ER lumen [9]
induced in response to the UPR
KAR2 Karyogamy gene 2 ATPase involved in protein import into the ER, mediates ER lumen [6]
protein folding, and regulates UPR
LHS1 Luminal Hsp70 protein 1 UPR regulated chaperone of the ER lumen involved in ER lumen [29]
polypeptide translocation and folding
YPT7a Yeast protein transport 7 Rab family GTPase involved in membrane trafficking Vacuolar, endosomal, Golgi [28]
and mitochondrial mem‑
brane
a
Knockout target
Tir et al. Microbial Cell Factories (2022) 21:157 Page 6 of 16

Fig. 4  Influence of helper factor overexpression on IgG production. Titers are shown in light grey boxes and yields in dark grey boxes. Mean values
above the boxes were calculated from seven to ten biological replicates (dots and triangles) relative to the parent strain without overexpression and
are shown as the horizontal line in each box. Whiskers show minimal and maximal values

in the light chain at position Y173 and compared with ­IgGpAzF production in bioreactors. In all cases, improv-
Fab-producing analogs. ing co-translational translocation into the ER made
major improvements in product secretion.
Disruption of vacuolar targeting further increased
recombinant protein secretion Importance of process control for IgG production in P.
An additional bottleneck in IgG secretion was tackled pastoris
by deleting the gene for Ypt7p, a Rab-family GTPase The strains co-overexpressing KAR2 and LHS1 were used
involved in membrane trafficking and mediates endo- for fed-batch bioreactor cultivations to produce full-
some-to-vacuole transport for protein degradation length trastuzumab IgG and I­ gGpAzF. Due to the low pro-
[32]. YPT7 deletion strains producing Fab and IgG ductivity, three bioreactors were employed for ­IgGpAzF

DASGIP® system. The cultivation process was adapted

were generated using the CRISPR/Cas9 approach with production and one for IgG production in the parallel
a template DNA containing YPT7 flanking regions. Fab
titer was increased by around twofold, and IgG titer by from the one previously published by Zavec et  al. [11]
around 1.8-fold upon YPT7 deletion (Fig. 5). The addi- and the feeding scheme is shown in Additional file  1:
tive effect was observed when YPT7 knockout was Fig. S3. Briefly, selected strains were grown in BSMG
combined with KAR2 and LHS1 co-overexpression. Fab until total glycerol consumption (around 22 h) to gener-
titer and yield were increased by up to fourfold, while ate biomass. The feeding strategy involved an 8-h linear
for IgG, there was no additional improvement. addition of 50% glucose, followed by an 18-h linear addi-
YPT7 deletion and co-overexpression of KAR2 and tion of methanol with a linear decrease in glucose feed
LHS1 had an additive effect on F ­abpAzF production, rate to adapt the cells to methanol, and continued with
resulting in a 2.3-fold and a 2.7-fold increase in titer a 72-h methanol-only phase for protein production at
and yield, respectively (Fig. 6a). In the case of I­ gGpAzF, low growth conditions. Supernatants from four sampling
KAR2 and LHS1 co-overexpression alone increased points were analyzed for IgG content and productivity
protein titer up to threefold and yield up to fourfold (Additional file 1: Figs. S4 and S5). At the end of the cul-
(Fig.  6b). In the Δypt7 strain, the co-overexpression tivations, supernatants were harvested and used for pro-
increased protein titer up to twofold and yield up to tein purifications.
2.5-fold. For this reason, wild-type strains co-over- Protein A affinity chromatography was used for the
expressing KAR2 and LHS1 were used for IgG and first purification step to separate IgG fragments from
 Tir et al. Microbial Cell Factories (2022) 21:157 Page 7 of 16

Fig. 5  Influence of YPT7 disruption and KAR2 and LHS1 co-overexpression on Fab and IgG production. Titer fold changes are shown in light grey
boxes and yield fold changes in dark grey boxes. Mean values above the boxes were calculated from three to ten biological replicates (dots and
triangles) relative to the wild-type strain expressing either Fab or IgG and are shown as the horizontal line in each box. Whiskers show minimal and
maximal values

yeast proteins. Fully assembled IgGs were isolated by development of the yeast as a cost-effective production
size-exclusion chromatography in the next step (Fig.  7). platform of monoclonal antibodies with an expanded
From the SEC profiles, it was possible to recalculate genetic code for the subsequent generation of antibody-
actual tetrameric protein amounts, resulting in around drug conjugates.
15 mg ­L−1 for ­IgGpAzF and 238 mg ­L−1 for IgG. Collected Since P. pastoris has genes for methanol utilization
fractions were further analyzed by SDS-PAGE with sil- expressed under tightly controllable methanol-inducible
ver staining and western blotting (Additional file  1: Fig. promoters, the choice of promoters for expression of all
S6) and those containing pure tetrameric IgGs were used heterologous genes in this work was based on methanol
for mass spectrometry analysis to confirm site-specific metabolism [15]. The gene for the t­ RNACUA​ was derived
pAzF incorporation (Fig.  8). However, from intact mass from E. coli tyrosyl-tRNA, as described previously [21].
analysis of reduced samples (Additional file 1: Fig. S7), an It was also established that adding an RNA polymerase II
additional signal for the light chain insensitive to PNGase (Pol II) promoter in front of the ­tRNACUA​ gene inserted
treatment was observed with an extra 1027  Da corre- within the yeast suppressor tRNA (SUP4) flanking region
sponding to a propeptide fragment (EEGVSLEKR). with a binding site for RNA polymerase III increased
its expression levels. Furthermore, triple tandem copies
of the flanked ­tRNACUA​ yielded the highest amber sup-
Discussion pression [21]. Therefore, Pol II promoters were tested
Monoclonal antibody production in microbial hosts is for the ­tRNACUA​ construct in this work. The modified
rising in popularity with a growing demand for affordable ­RSpAzF was derived from E. coli tyrosyl-RS [33]. Metha-
therapeutics, particularly for treatments of various can- nol-inducible promoters for each gene of the orthogonal
cers. P. pastoris has already proven to be a promising host ­tRNACUA​/RSpAzF pair were used in different combina-
cell for recombinant protein production due to its high tions to screen for the highest pAzF incorporation into
secretion capacity with low amounts of secreted endoge- ­eGFPY40X as a reporter protein. It was observed that a
nous proteins, strong inducible promoters, and inexpen- stronger promoter is needed for t­RNACUA​ expression
sive cultivation media. In this work, a P. pastoris strain to improve pAzF incorporation yield when reverse pro-
was engineered to improve the production of trastu- moter combinations were compared. Further improve-
zumab IgG carrying site-specifically incorporated pAzF ments may be achieved by optimizing the ­ tRNACUA​
as a model uAA. The aim was to further contribute to the
Tir et al. Microbial Cell Factories (2022) 21:157 Page 8 of 16

Fig. 6  FabpAzF and ­IgGpAzF productivity in strains co-overexpressing KAR2 and LHS1 with YPT7 disruption. Titers (left) and yields (right) of a ­FabpAzF
and b ­IgGpAzF. Mean values above the boxes were calculated from three to ten biological replicates and are shown as horizontal lines. Dots and
triangles show each data point and whiskers show minimal and maximal values. The yields were calculated as the amount of protein per wet cell
weight (WCW)

flanking region and using synthetic promoters for both combinations with strong promoters for both chains
genes. resulted in the highest Fab expression. The expression
Expression levels of both chains constituting tetrameric could be further optimized by varying light-to-heavy
IgG were tuned using its Fab fragment, which expres- chain gene copies or screening for synthetic promot-
sion and detection are well established in P. pastoris. A ers. However, it should be taken into consideration that
stronger methanol-inducible promoter for the light chain gene loss can occur due to homologous recombination
was more favorable for Fab expression when reverse pro- during long cultivation times if the gene copy number is
moter combinations for each chain were compared. The increased. For this reason, two different promoters and
 Tir et al. Microbial Cell Factories (2022) 21:157 Page 9 of 16

a
200
1.62

150
1.00
1.00
UV280 [mAU]

100 0.77 0.62

0.62
0.77

50
0.24
0.24 0.24
0.24

0
0 20 40 60 80 100 120

Elution volume [mL]

-50

b
900
1.00
1.00
800

700

600
UV280 [mAU]

500

400

300
0.15
0.15
200

100 0.02 0.10

0.02 0.10 0.04
0.04 0.02
0.02

0
0 20 40 60 80 100 120
-100 Elution volume [mL]
Fig. 7  Size-exclusion chromatography elution profiles after protein A affinity purification. a ­IgGpAzF and b IgG sample. Integrated peaks were
analyzed by SDS-PAGE with silver staining and western blot and pooled. Numbers above the peaks are areas below the curve relative to the area of
the peak corresponding to full-length IgG

terminators were chosen for the expression of Fab and pastoris, it seemed promising to use a different secre-
IgG chains. tion signal for Fab and IgG. Interestingly, the effect of
The long practice of using the S. cerevisiae MFα secre- the pre-Ost1-pro-MFα compared to the pre-pro-MFα
tion signal for recombinant protein secretion in P. pas- secretion signal on Fab and IgG secretion was signifi-
toris was challenged. From work by Barrero et  al. [24], cantly different. The hybrid signal reduced Fab secretion
where a hybrid secretion signal of the oligosaccharide almost twofold and increased IgG secretion 1.3-fold. In
transferase 1 prepeptide and the MFα propeptide was order to conclude if this is the general case, more IgGs
used to improve recombinant protein secretion in P. and their respective Fab fragments should be compared.
Tir et al. Microbial Cell Factories (2022) 21:157 Page 10 of 16

Fig. 8  Tandem mass spectrum of the light chain tryptic peptide carrying the site-specific mutation. a IgG light chain tryptic peptide with tyrosine
at position 173 and b ­IgGpAzF light chain tryptic peptide with pAzF at position 173 (mass increment of 25 Da). All +1y and +1b ions are listed above
the corresponding sequence in the upper right corner

Nevertheless, in both cases, a significant amount of formation. Surprisingly, overexpression of foldase Cpr5p,
endogenous proteins was found in culture supernatants which showed an improvement of IgG production in S.
when proteins were fused to the pre-pro-MFα secretion cerevisiae [9], and protein disulfide isomerase Pdi1p had
signal compared to the pre-Ost1-pro-MFα. Additionally, adverse effects on IgG secretion, reducing protein titer
it would be of great interest to see if the pre-Ost1-pro- and yield by 40%. Pdi1p is one of the most abundant pro-
MFα can improve the secretion of other glycoproteins teins in the ER that catalyzes disulfide bond formation
compared to the pre-pro-MFα and if it can serve as a and isomerization of incorrectly formed disulfide bonds
starting point for secretion signal sequence engineering. [34, 35]. A similar effect was observed for IgG produc-
As a next step in improving recombinant protein tion in S. cerevisiae [9] and scFv production in P. pastoris,
secretion, strain engineering was performed by over- where an increase of Kar2p levels upon Pdi1p overpro-
expressing genes involved in protein folding and oxida- duction was reported [36]. Moreover, it was shown that
tive stress regulation that have shown positive effects most unsecreted Fab was degraded in the cytosol before
in S. cerevisiae and P. pastoris. Since the effect of over- reaching the ER, making ER-associated degradation
expression of these factors on Fab production has been (ERAD) an explanation of the reduced IgG titers in PDI1
studied previously and tetrameric IgG cannot be eas- overexpressing strain less likely [31]. However, no pro-
ily assembled on the screening scale, IgG was used as teasome activity was assayed in this work to test if the
a model protein. In addition to improving the titer and reduced titers were due to ERAD.
yield of any IgG format detectable by ELISA, the aim of Overexpression of Hac1p and Yap1p did not improve
strain engineering was to improve tetrameric IgG assem- IgG secretion. Hac1p is a transcription factor regu-
bly. As only reduced IgG fragments were observed in lating the UPR genes and its overexpression showed
supernatants from the screenings, the first bottleneck to improvements in the production of secreted recombi-
tackle was the folding of each chain and disulfide bridge nant proteins [8, 37, 38]. YAP1 is another transcription
 Tir et al. Microbial Cell Factories (2022) 21:157 Page 11 of 16

factor involved in oxidative stress relief [39] for which an opal and ochre stop codons did not improve pAzF incor-
improved recombinant protein production by P. pastoris poration (not shown).
was reported [26]. However, these effects significantly Upon mass spectrometry analysis of purified IgG and
vary between target products. Furthermore, overexpres- ­IgGpAzF fractions, intact mass measurements showed an
sion of a component of the ER translocon Sbh1p and the additional light chain signal with an extra 1027 Da. Since
ER chaperone Kar2p had no effect on IgG secretion. Posi- the signal was present in samples treated and untreated
tive effects of Sbh1 overexpression on Fab production by with PNGase, we concluded that it belonged to the pro-
P. pastoris [28] and α-amylase by S. cerevisiae [40] were peptide fragment (EEGVSLEKR), which was cleaved off
reported. Since co-overexpression of all translocon subu- upon tryptic digest and was not detected by peptide mass
nits on full-length IgG production has not been investi- fingerprinting. This leaves some room for further inves-
gated yet, more detailed experiments are needed to get tigation if the observed leftover is due to cell lysis, caus-
a clear picture of IgG import into the ER and the role of ing some IgG to have the partially processed light chain
the translocon components [41]. While overexpression in the supernatant, or a mixture of IgG with fully and
of Lhs1p, a nucleotide exchange factor of Kar2p, which partially processed light chain being secreted through-
enables ADP to ATP exchange and binding of Kar2p to out the cultivation. Additionally, process optimization
the next substrate, reduced IgG production by around by employing methanol feed dependent on the levels of
60%, co-overexpression of Kar2p and Lhs1p improved dissolved oxygen [43] could further reduce cellular stress
IgG production by around 60%. The observed effect is in which frequently causes incomplete secretion signal
agreement with previous findings that a major bottleneck processing.
in Fab, and therefore, IgG, secretion is the ER import
[31]. Conclusions
In addition to overexpression of the common helper In this work, a new contribution to strain engineering
factors, strains with disrupted vacuole sorting were of P. pastoris for the production of monoclonal antibod-
prepared by knocking out YPT7 [28]. In these strains, ies with the expanded genetic code was made. By using
Fab and IgG production was increased by around two- a promising fusion secretion signal, pre-Ost1-pro-MFα
fold. Combining the knockout and co-overexpression [24, 25], co-overexpressing KAR2 and LHS1, and employ-
of KAR2 and LHS1 further increased Fab production by ing controlled cultivation conditions, full-length trastu-
around 3.7-fold, while it resulted in only an additional zumab IgG with pAzF in the light chain was produced
10% increase in IgG production compared to the knock- in titers up to 15  mg  ­L−1. Production of trastuzumab
out alone. In the case of pAzF-carrying variants, ­FabpAzF Fab with pAzF in the light chain was further improved
production showed the same trend as Fab. On the other by deleting YPT7, causing the disruption of vacuolar
hand, the best I­gGpAzF production was achieved in the targeting and increasing product titer and yield almost
strain co-overexpression KAR2 and LHS1, with up to fourfold. Additional improvements could potentially be
threefold higher productivity than in the parent strain. achieved by disrupting nonsense-mediated mRNA decay
Productivity of IgG and ­IgGpAzF formats detectable by and, in the case of IgG, glycoengineering of P. pastoris to
ELISA in bioreactor cultivations was around 50- and obtain a more human-like glycosylation pattern. Further-
20-fold higher than in shake-flask cultivations, respec- more, to reduce the observed propeptide leftover in part
tively. Using a KAR2 and LHS1 co-overexpressing strain of the secreted product, an alternative methanol-feed
for IgG and ­IgGpAzF production in fed-batch bioreactor strategy should be employed, such as pulse-feed con-
cultivations yielded significant amounts of tetrameric trolled by the levels of dissolved oxygen. Lastly, in this
proteins, up to 238 mg ­L−1 and 15 mg ­L−1, respectively, work, the major bottleneck of IgG production was found
as calculated from the SEC profiles in the second puri- to be the ER import, which is in agreement with previous
fication step. Unlike in the screenings, the most abun- research on Fab production in P. pastoris [31].
dant species secreted by the strain producing IgG was
the tetrameric protein. The strain producing I­gGpAzF Materials and methods
secreted predominantly free heavy chain since the light Preparation of P. pastoris strains
chain translation was reduced due to the internal amber All plasmids were prepared in a series of GoldenGate
codon. The ratio of produced tetrameric I­gGpAzF to the assemblies as described previously [23] and cloning of
free heavy chain could be increased by downregulation ­tRNACUA​/RSpAzF and Fab/IgG expression plasmids is
or disruption of nonsense-mediated mRNA decay [42]. shown in Additional file 1: Fig. S1. Primers used for clon-
Trials to downregulate or replace the eukaryotic peptide ing heterologous genes, amplification of overexpression
release factor 1 with variants that preferentially recognize targets and gene deletions are listed in Additional file 2:
Table S2. All the P. pastoris strains used in this study were
Tir et al. Microbial Cell Factories (2022) 21:157 Page 12 of 16

derived from the wild-type CBS2612 strain [44] and are 28 h, the precultures were washed and 2 mL of the main
listed with their respective antibiotic resistances in Addi- cultures were inoculated to a starting ­OD600 of 8. All the
tional file 2: Table S3. The strain used as a positive con- cultures expressing heterologous proteins with pAzF
trol in eGFP screenings expressed the protein under the were supplemented with 2 mM pAzF dissolved in 2 mM
­PAOX1 ­(PAOX1_eGFP), the same as the strain expressing NaOH and the negative controls with 2  mM NaOH.
eGFP with the internal amber stop codon at the position Methanol shots were given to the cells for induction:
Y40 ­(PAOX1_eGFPY40X). The P­ AOX1_eGFPY40X strain also 0.5% was given 3  h post-inoculation and three shots of
expressed the orthogonal pair of the suppressor ­tRNACUA​ 1% 19, 27, and 43  h post-inoculation. The cultures were
and engineered ­RSpAzF [3], for which combinations of harvested 48 h post-inoculation by taking 1 mL cultures
methanol-inducible promoters were tested in this work and centrifuging them at 13 000 rpm for 5 min. Wet cell
­(PX_RSpAzF_PY_3xSUP4-tRNACUA​). The ­tRNACUA​ expres- weight was measured from cell pellets and the superna-
sion cassette contained three tandem copies of the tRNA tant was analyzed by ELISA (Fab and IgG cultures). For
lacking the 3′-CCA terminus and each copy was flanked eGFP expression measurements, cultures from the end of
by the SUP4 region from S. cerevisiae [21]. The strain the screenings were diluted in pre-cooled PBS (0.24 g ­L−1
used for Fab screenings expressed both chains under ­KH2PO4, 1.8 g ­L−1 ­Na2HPO4∙2H2O, 0.2 g ­L−1 KCl, 8 g ­L−1
different methanol-inducible promoters ­(PX′_LC_PY′_HC- NaCl, pH 7.4) to an O­ D600 of 0.4 in 96 microtiter plates.
Fab). Each chain was fused to the pre-Ost1-pro-MFα
secretion signal [24, 25] lacking the C-terminal EAEA Flow cytometry analysis
sequence. The final producer strain expressed IgG Flow cytometry analysis was performed in a CytoFLEX S
fused to the same secretion signal. The light chain was benchtop flow cytometer (Beckman Coulter). The excita-
expressed from the ­PAOX1 and the heavy chain from the tion wavelength for eGFP was 488 nm and for detection,
­PDAS2 ­(PAOX1_LC_PDAS2_HC-IgG). The strain expressing a 525/40  nm bandpass filter was used. The sample flow
Fab and IgG variant with the amber codon in the light rate was set to 30 µL ­min−1, and the number of recorded
chain at position Y173 ­ (PAOX1_LCY173X_PDAS2_HC-Fab; events was 10,000. For statistical analysis, geometric
­PAOX1_LCY173X_PDAS2_HC-IgG) also expressed ­tRNACUA​ means of the fluorescence and the forward scatter signal
under the ­ PDAS2 and the synthetase under the ­ PAOX1 heights were taken from which a mean relative cell-asso-
­(PAOX1_RSpAzF_PDAS2_3xSUP4-tRNACUA​). Helper factors ciated product (PRel) was calculated as reported previ-
PDI1 (PP7435_Chr4-0107), HAC1i (PP7435_Chr1-0700), ously [45, 46]. The percentage of stop codon suppression
YAP1 (PP7435_Chr4-0370), and KAR2 (PP7435_Chr2- in ­eGFPY40X was calculated from the ratio of the PRel of
1167) were constitutively overexpressed under the glyc- each clone and PRel of two to three biological replicates of
eraldehyde-3-phosphate dehydrogenase promoter ­(PGAP). the positive control.
LHS1 (PP7435_Chr1-0059) was overexpressed under the
promoter for mitochondrial porin 1 ­(PPOR1_LHS1) and Enzyme‑linked immunosorbent assay
CPR5 (PP7435_Chr1-0581) and SBH1 (PP7435_Chr2- ELISA was performed as described previously [31],
0220) under the promoter for the translational elonga- except for the coating antibodies used. For Fab samples,
tion factor EF-1α ­(PTEF_CPR5; ­PTEF_SBH1). The YPT7 mouse anti-human IgG Fab (Invitrogen) was used and for
(PP7435_Chr4-0052) deletion strains were prepared IgG samples, anti-human IgG (γ-chain specific, Sigma)

the standard for IgG was the commercial Herceptin™

from parents producing both variants of Fab and IgG and was used. The standard for Fab was as described, while
a positive clone from each was selected for KAR2 and
LHS1 co-overexpression. (Roche). Stop codon suppression yields were calculated
from the product yields with pAzF for each clone rela-
Screenings in 24 deep‑well plates tive to two to three biological replicates expressing corre-
All the screenings were performed in 24 deep-well sponding wild-type proteins. Titer and yield fold changes
plates with shaking at 280  rpm at 25  °C. The minimal in overexpression screenings were calculated from the
medium used for main cultures was ASMv6, as previ- ratios of titers and yields for each clone relative to the
ously described [11] and controlled glucose release was average of titers and yields of two to three biological rep-
achieved using the EnPump200 kit (EnPresso) to the licates of the wild-type strains.
final polysaccharide concentration of 25  g  ­L−1 and 0.3%
of the corresponding enzyme. For the precultures, 2 mL Silver staining and western blot

samples) or NuPAGE® Novex 4-12% Bis-Tris (for IgG

of selective YPD media (10 g ­L−1 yeast extract, 20 g ­L−1 For SDS-PAGE, either NuPAGE 12% Bis-Tris (for Fab
soy peptone, 2% v/v glucose) were inoculated with single
colonies from the selective YPD agar (YPD medium with samples) gels were used. Samples were prepared by boil-
2% agar) plates after incubation for 2 days at 30 °C. After ing 13 or 15  µL supernatants with 5  µL loading buffer
 Tir et al. Microbial Cell Factories (2022) 21:157 Page 13 of 16

(4×, NuPAGE) with or without 2  µL reducing agent medium. The batch volume of 300 mL was inoculated to
(10×, NuPAGE) or 5 mM N-ethylmaleimide (NEM, Ther- an ­OD600 of 1 and 2  mM pAzF were added. The batch
mofisher), respectively, at 100 °C for 1 min. Samples were phase of the bioreactor cultivations took approximately
run in 1×MOPS (NuPAGE) at 180 V for 1 h. After SDS- 24  h. The second phase of linear feed with 50% glucose
PAGE run, gels were fixed in a fixing solution (10%  v/v (rate of feed calculated as y = 0.225x + 1.95, x: time [h])
acetic acid, 50%  v/v ethanol) with gentle agitation over- was initiated when the total glycerol was consumed. After
night at 4  °C. The next day, the fixing solution was dis- 8  h, the third phase was initiated by the linear increase
carded, and gels were incubated in an incubation solution in 100% methanol (y = 0.028x + 0.6) and the simultane-
(68.2  g  ­L−1 sodium acetate, 3.2  g  ­L−1 ­Na2S2O3·5H2O, ous linear decrease in 50% glucose (y = 3.75 − 0.111x).
30%  v/v ethanol, 0.25%  v/v glutaraldehyde) with gentle After 18  h of the co-feed phase, the fourth phase was
agitation for 30 min at room temperature. Gels were then initiated by the linear feed with 100% methanol alone
washed three times with distilled water for 10 min. Silver (y = 0.028x + 1.1) [11]. During the bioreactor cultivations,
nitrate solution (0.1%  w/v A­ gNO3, 0.02%  v/v formalde- samples were taken at six time points. Two milliliters
hyde) was added and gels were incubated for 20  min at were taken in three technical replicates for each bioreac-
room temperature. After the binding, gels were briefly tor and centrifuged at 4  °C with a speed of 13,000  rpm
washed with distilled water and a developing solution for 5 min. Pellets were washed with 1 mL 0.1 M HCl and
(5.0 g ­L−1 ­Na2CO3, 0.01% v/v formaldehyde) was added. dried in preweighted tubes at 105  °C for determination
Gels were incubated at room temperature until protein of yeast dry mass (YDM). Supernatants were used for
bands were of desired intensity. Further development the determination of protein concentration. The cultiva-
was quenched with 50 mM EDTA and gels were scanned. tions were terminated 90 h post-induction by harvesting
Gels that were not used for silver staining were blotted on supernatants of each bioreactor at 4  °C with a speed of
Trans-Blot turbo mini 0.2 µm nitrocellulose membranes 10000×g for 30  min. The harvested supernatants were
(Bio-Rad) in the Trans-Blot Turbo transfer system (Bio- used for protein purification.
Rad). Membranes were immediately incubated in block-
ing buffer (PBS pH 7.4, 0.05% Tween, 2% BSA) at 4  °C
Protein purification
with gentle agitation overnight. The next day, membranes
Bioreactor supernatants were concentrated by tangen-
were washed three times for 5  min with washing buffer
tial flow filtration and the buffer was changed to PBS (pH
(PBS pH 7.4, 0.05% Tween). Primary antibodies diluted in
7.4). Affinity purification was performed using the HiTrap
the blocking buffer were incubated for 1 h at room tem-
Protein A HP column. The column was equilibrated with
perature with a washing step in between. Secondary fluo-
12 column volumes (CVs) binding buffer (PBS, pH 7.4) at
rescent antibodies were incubated in the same way, and
the rate of 1 mL ­min−1. The sample was loaded at the rate
after the final washing step, fluorescence was detected by
of 0.5  mL  ­min−1 and washed with the binding buffer at
the Chemidoc MP imaging system (Bio-Rad).
1  mL  ­min−1 for 15 CVs. Elution steps were done at the
rate of 1  mL  ­min−1 using 0.1  M glycin-HCl (pH 3.5) as

The bioreactor cultivations were done in a DASGIP® Par-

Fed‑batch bioreactor cultivation the elution buffer, starting with a 10 CVs linear gradient
from 0 to 50% elution buffer, followed by 5 CVs with 50%
allel Bioreactor System (Eppendorf AG) in 1 L bioreactor elution buffer. A linear gradient from 50 to 100% elution
vessels. Process parameters were controlled remotely by buffer was applied for 10 CVs or until the absorbance
the DASGIP control software (Eppendorf AG, Germany). at 280  nm started rising. Elution at 100% elution buffer
During the batch phase, pH was maintained at 5.5, while with pH 2.5 was applied for the last 12 CVs. Each col-
during the feed phases, at 5.0 by 25% ­NH4OH. The tem- lected fraction was neutralized with 100  mM Tris-HCl.
perature was held at 25 °C during the whole process. Dis- Fractions that showed increased absorbance, washed
solved oxygen (DO) was controlled by a DO-cascade, fractions and unbound fractions were analyzed by SDS-
which consisted of a sequential increase of stirring speed PAGE. Those containing full-length IgG were pooled and
from 200 to 1250 rpm, ingas flow rate from 6 to 50 sL ­h−1 buffer was exchanged to PBS (pH 7.4) using Amicon spin
and ingas oxygen concentration between 21 and 100%, filters. The second step of size-exclusion purification was
depending on individual strain demand. Precultures were performed using HiLoad Superdex 16/600 column. Equi-
inoculated from 1 mL working cell banks in 50 mL selec- libration and washing buffer was PBS (pH 7.4). The flow
tive YPG in 300 mL shake flasks and cultivated at 25 °C rate applied was 0.5 mL ­min−1. Fractions belonging to the
overnight with mixing at 180  rpm. Before the bioreac- same peak were pooled and analyzed by SDS-PAGE and
tor inoculation, ­OD600 was measured and the appropri- western blot.
ate volume of the precultures was washed with the batch
Tir et al. Microbial Cell Factories (2022) 21:157 Page 14 of 16

Mass spectrometry analysis

T4 mediated restriction/ligation as shown above BB1_3x SUP4-tRNACUA​
Fractions containing pure full-length IgGs were analyzed . In the case of Fab/IgG with the amber stop codon at the position Y173
by mass spectrometry. The proteins were reduced with in the light chain, a custom fusion site (black and grey boxes) was created
to replace 5′ TAC of Y173 to 5′ TAG as shown in the insert above BB1_LC.
2.5  mM DTT and incubated at 37  °C for 30  min. Addi- Overhangs of the PCR product carried recognition sites for BsaI (white
tionally, an aliquot of each sample was de-N-glycosylated boxes) for BB1 assembly. BB1s were assembled into BB2s containing fusion
by the addition of 2.5  mU PNGase F and incubated at sites 1 and 4 flanked by the fusion sites A and B or B and C in BbsI/T4
mediated restriction/ligation. BB2s carried single expression cassettes and
40 °C for 2 h. were assembled into BB3s with fusion sites A and C to accommodate two
Two µg of the protein was directly injected into an LC- expression cassettes. Details about the fusion site choice were elaborated
ESI-MS system (LC: Agilent 1290 Infinity II UPLC). A by Kolb et al. [1]. The figure was generated on Biorender.com. Figure S2.
SDS-PAGE with silver staining (left) and western blot (right) analysis of the
gradient from 15 to 80% acetonitrile in 0.1% formic acid supernatants from the screenings: a Fab and b IgG producing strains. Two
[using a Waters BioResolve column (2.1 × 5  mm)] at a biological replicates of each strain were used for the analyses and one of
flow rate of 400 µL ­min−1 was applied (15-min gradient each was used for the reduced protein analysis (DTT “+” wells). The protein
ladder was loaded in the first well, and the corresponding protein stand‑
time). Detection was performed with a Q-TOF instru- ards were loaded in the second well. The pre-pro-MFα secretion signal
ment (Agilent Series 6560 LC-IMS-QTOFMS) equipped is abbreviated as “ppM” and the pre-Ost1-pro-MFα as “pOpM”. The green
with the Jetstream ESI source in positive ion, MS mode bands show the heavy chain and the red ones the light chain. Due to the
high standard concentration, in-gel fragmentation is observed. Figure S3.
(range: 100–3200  Da). Instrument calibration was per- Feeding strategy for methanol-inducible fed-batch bioreactor cultiva‑
formed using an ESI calibration mixture (Agilent). Data tions. A defined minimal medium containing 2% glycerol is used in the
were processed using MassHunter BioConfirm B.08.00 batch phase, where cells generate biomass before methanol induction.
Upon total glycerol consumption, cells are slowly fed with 50% glucose
(Agilent) and the spectrum was deconvoluted by MaxEnt. (blue line) for eight hours, after which the glucose feed slows down and
Alternatively, the samples were digested with trypsin 100% methanol (red line) is slowly added to pre-condition the cells. This
(Promega, Trypsin Gold) after S-alkylation with co-feed phase takes 18 h. The last cultivation phase takes 3 days and only
100% methanol is slowly fed to the cells. Figure S4. IgG and ­IgGpAzF titers
iodoacetamide. The digested samples were loaded on (bars) and yields (lines) produced by the KAR2 and LHS1 co-overexpressing
a nanoEase C18 column (nanoEase M/Z HSS T3 Col- strains during the methanol induction phase of fed-batch bioreactor
umn, 100  Å, 1.8  µm, 300  µm × 150  mm, Waters) using cultivations. The yields were calculated as the amount of protein per yeast
dry mass (YDM). Mean values above the bars were calculated from three
0.1% formic acid as the aqueous solvent. A gradient technical replicates for IgG samples and one technical replicate from
from 3.5% B (B: 80% ACN, 20% A) to 40% B in 30  min three bioreactors for the I­gGpAzF samples. Error bars represent standard
was applied, followed by a 5-min gradient from 40% B deviations and are not shown for the yields to avoid graph crowdedness.
Figure S5. a SDS-PAGE with silver staining and b western blot of super‑
to 95% B that facilitated the elution of large peptides at natants from the fed-batch bioreactor cultivations of the IgG and ­IgGpAzF
a flow rate of 6 µL ­min−1. Detection was performed with producing strains. The cultivation medium for IgG production did not
an ion-trap MS (amazon speed ETD, Bruker) equipped contain pAzF (marked with minuses), while the media for ­IgGpAzF produc‑
tion contained 1 mM pAzF (marked with pluses). The first and the last lane
with the standard ESI source in positive ion, DDA mode contain the protein ladder. The lane with the commercial trastuzumab
(= switching to MSMS mode for eluting peaks). MS- standard is labeled with “St” and the numbers are the sample numbers.
scans were recorded (range: 150–2200  Da) and the 8 The red bands show the light chain (LC), while the green bands show the
heavy chain (HC). The main fragmentation products are assigned on the
highest peaks were selected for fragmentation. Instru- right. Figure S6. Analysis of selected fractions after ­IgGpAzF and IgG purifi‑
ment calibration was performed using an ESI calibration cation by size-exclusion chromatography. a SDS-PAGE with silver staining
mixture (Agilent). and b western blot. One microgram of total protein was loaded in each
well so the band intensity does not reflect the relative protein amounts in
The analysis files were converted (using Data Analy- the samples. The first and the middle lane contain the protein ladder. The
sis, Bruker) to mgf files, which are suitable for perform- lane with a commercial trastuzumab standard is labeled with “St”. The red
ing an MS/MS ion search with X!-Tandem. The files were bands show the light chain, while the green bands show the heavy chain.
Figure S7. Deconvoluted MS spectra of the reduced ­IgGpAzF a, b and IgG
searched against a P. pastoris database including the fol- c, d and treated with PNGase b, d. The molecular weight of the light chain
lowing sequences, adding 25.00648  Da at Y as a poten- without pAzF is 23443.1 Da and 23468.1 Da of the pAzF-carrying variant
tial modification to allow the identification of the pAzF (in the gray box). The molecular weight of the unglycosylated heavy chain
is 49284.65 Da (in the golden box).
incorporation.
Additional file 2: Supplementary tables. Table S1. Concentration test
for optimal amber suppression in ­eGFPY40X by pAzF. Wet cell weight was
Supplementary Information determined at the end of 24 deep-well plate screenings by weighing the
The online version contains supplementary material available at https://​doi.​ pellets from 1 mL culture aliquots. Negative control cultures contained
org/​10.​1186/​s12934-​022-​01882-6. varying amounts of NaOH while the suppression cultures contained vary‑
ing amounts of pAzF, prepared as a stock solution of 100 mM in 100 mM
Additional file 1: Supplementary figures. Figure S1. Cloning scheme of NaOH. The mean relative cell-associated product was calculated from the
plasmids containing a ­tRNACUA​/RSpAzF pair and b Fab/IgG heavy and light ratio of the geometric mean of fluorescence intensity and forward scatter
chains using GoldenPiCS. Backbone 1 (BB1) carried either promoter (fusion as described by Kolb et al. [1] and Dumas et al. [2]. Average values and the
sites 1 and 2; green arrows), recombinant gene (fusion sites 2 and 3; blue corresponding standard deviations were calculated from three biological
and yellow arrows), or terminator (fusion sites 3 and 4; gray arrows). Three replicates. Table S2. List of primers prepared for this study. Table S3. List
tandem copies of SUP4-tRNACUA​ were generated in a single BB1 assembly of strains prepared in this study.
from PCR products carrying custom fusion sites (FS) in overhangs in BsaI/
 Tir et al. Microbial Cell Factories (2022) 21:157 Page 15 of 16

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ral Resources and Life Sciences, Vienna Core Facility Mass Spectrometry, 21. Chen S, Schultz PG, Brock A. An improved system for the generation and
Muthgasse 18, 1190 Vienna, Austria. 4 Department of Biotechnology, Institute analysis of mutant proteins containing unnatural amino acids in Saccha-
of Bioprocess Science and Engineering, University of Natural Resources romyces cerevisiae. J Mol Biol. 2007;371:112–22.
and Life Sciences, Muthgasse 18, 1190 Vienna, Austria. 5 ADCs & Targeted NBE 22. Prielhofer R, Cartwright SP, Graf AB, Valli M, Bill RM, Mattanovich D, Gasser
Therapeutics, Merck Healthcare KGaA, Frankfurter Str. 250, 64293 Darmstadt, B. Pichia pastoris regulates its gene-specific response to different carbon
Germany. 6 Present Address: Department of Biology, Institute of Biochemistry, sources at the transcriptional, rather than the translational, level. BMC
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23. Prielhofer R, Barrero JJ, Steuer S, Gassler T, Zahrl R, Baumann K, Sauer M,
Received: 18 May 2022 Accepted: 26 July 2022 Mattanovich D, Gasser B, Marx H. GoldenPiCS: A Golden Gate-derived
modular cloning system for applied synthetic biology in the yeast Pichia
pastoris. BMC Syst Biol. 2017;11:1–14.
24. Barrero JJ, Casler JC, Valero F, Ferrer P, Glick BS. An improved secretion sig‑
nal enhances the secretion of model proteins from Pichia pastoris. Microb
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