PERSPECTIVE

published: 11 June 2019

doi: 10.3389/fbioe.2019.00139

Factors Influencing Recombinant

Protein Secretion Efficiency in
Gram-Positive Bacteria: Signal
Peptide and Beyond
Chong Peng 1,2,3,4 , Chaoshuo Shi 4 , Xue Cao 4 , Yu Li 1,2,3,4 , Fufeng Liu 1,2,3,4 and Fuping Lu 1,2,3,4*
1
Key Laboratory of Industrial Fermentation Microbiology, Education Ministry of China, Tianjin, China, 2 National Engineering
Laboratory for Industrial Enzymes, Tianjin, China, 3 Tianjin Engineering Research Center of Microbial Metabolism and
Fermentation Process Control, Tianjin, China, 4 College of Biotechnology, Tianjin University of Science and Technology,
Tianjin, China

Signal peptides are short peptides directing newly synthesized proteins toward the
secretory pathway. These N-terminal signal sequences are ubiquitous to all prokaryotes
Edited by: and eukaryotes. Signal peptides play a significant role in recombinant protein production.
Thomas Bartholomäus Brück, Previous studies have demonstrated that the secretion amount of a given target protein
Technical University of
Munich, Germany
varies significantly depending on the signal peptide that is fused to the protein. Signal
Reviewed by:
peptide selection and signal peptide modification are the two main methods for the
Amir Feizi, optimization of a recombinant protein secretion. However, the highly efficient signal
Chalmers University of peptide for a target protein with a specific bacterial expression host is not predictable
Technology, Sweden
Daniela Monti, so far. In this article, we collect several signal peptides that have previously performed
Italian National Research Council well for recombinant protein secretion in gram-positive bacteria. We also discuss several
(CNR), Italy
factors influencing recombinant protein secretion efficiency in gram-positive bacteria.
*Correspondence:
Fuping Lu
Signal peptides with a higher charge/length ratio in n-region, more consensus residues at
[email protected] the−3 and−1positions in c-region and a much higher proportion of coils are more likely to
perform well in the secretion of recombinant proteins. These summaries can be utilized to
Specialty section:
the selection and directed modification of signal peptides for a given recombinant protein.
This article was submitted to
Bioprocess Engineering, Keywords: signal peptide, recombinant protein, secretory pathway, gram-positive bacteria, secretion efficiency
a section of the journal
Frontiers in Bioengineering and
Biotechnology
INTRODUCTION
Received: 21 February 2019
Accepted: 23 May 2019 In both eukaryotic and prokaryotic cells, all proteins are synthesized in cytoplasm. Proteins that
Published: 11 June 2019
are destined to enter into the secretory pathway are usually endowed with an N-terminal signal
Citation: sequence: the signal peptide (SP). SPs are short peptides and usually have a length of 16–30 amino
Peng C, Shi C, Cao X, Li Y, Liu F and acids. After directing proteins to their specific locations, SPs are removed by signal peptidases
Lu F (2019) Factors Influencing
(Blobel and Dobberstein, 1975; von Heijne, 1990, 1998; Molhoj and Dal Degan, 2004). Research
Recombinant Protein Secretion
Efficiency in Gram-Positive Bacteria:
on SPs is quite appealing in the field of protein secretion mechanism. Additionally, research about
Signal Peptide and Beyond. SPs is valuable in medical research such as disease diagnosis and treatment. For example, mutation
Front. Bioeng. Biotechnol. 7:139. in the preproinsulin signal peptide is associated with the onset of diabetes (Bonfanti et al., 2009). A
doi: 10.3389/fbioe.2019.00139 new identified variant in SP of the human luteinizing hormone receptor (LHCGR) affects receptor

Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 1 June 2019 | Volume 7 | Article 139
Peng et al. Factors Influencing Protein Secretion Efficiency

biogenesis and would cause Leydig cell hypoplasia (Vezzoli et al., 2011). Tat-dependent proteins are transported across
et al., 2015). Jarjanazi et al. (2008) carried out a comprehensive lipid bilayers in a folded state (Figure 1B). The energy for
literature survey and retrieved 26 disease associated mutations translocation comes from the proton motive force (PMF). In
in the signal peptide domains of 21 human proteins gram-positive bacteria with high GC-content genomes, the Tat
(Jarjanazi et al., 2008). translocase consists of TatA, TatB, and TatC. In low-GC gram-
Signal peptides also play a decisive role in the industrial positive bacteria, the Tat system is composed of TatC and a
production of recombinant proteins. There is a tremendously bifunctional TatA protein (Goosens et al., 2014). These two and
strong market demand for recombinant proteins such as other different types of secretion machinery have been well-
industrial enzymes and biopharmaceutical proteins (Walsh, reviewed in several excellent articles (Palmer and Berks, 2012;
2018). Different prokaryotic and eukaryotic expression systems Freudl, 2013; Goosens et al., 2014; Ates et al., 2016; Green and
have been developed to produce recombinant proteins. Among Mecsas, 2016; Tsirigotaki et al., 2017; Owji et al., 2018). Readers
them, bacterial systems are most attractive because they are can refer to these reviews for a better understanding of the
simple to manipulate and cost-effective (Terpe, 2006). However, protein secretory mechanisms in gram-positive bacteria.
the accumulation of recombinant proteins in the cytoplasm Based on the export pathways of the preproteins and the
will lead to the formation of inclusion bodies or protein signal peptidase cleavage sites, signal peptides can be classified
degradation via proteases (Mergulhao et al., 2005; Anne et al., into several categories, among which Sec-type signal peptides
2016). The recombinant protein folding may also be disturbed and twin-arginine signal peptides are more abundant and well-
by endogenous proteins. If the recombinant protein is secreted studied (Tjalsma et al., 2000, 2004). Signal peptides from different
out of the cell, the above bottlenecks in the mass production proteins show a common structure. Generally, a signal peptide
of recombinant proteins can be avoided, and the downstream is composed of three distinct domains: a positively charged n-
recovery process of protein production will also be considerably region (1–5 residues long), a central, hydrophobic h-region (7–15
simplified. Thus, developing an efficient secretion system will residues long), and a c-region (3–7 residues) with the cleavage
contribute a lot in the high yield of recombinant proteins (Quax, site of signal peptidase (von Heijne, 1985, 1990). The general
1997). It has been shown that using different homologous or structure of signal peptides is shown in Figure 1C. A highly
heterologous signal peptides can affect the yields of recombinant conserved twin-arginine motif (SRRXFLK, where X is often, but
proteins (Degering et al., 2010; Low et al., 2013; Hemmerich not always, a polar amino acid residue) is located at the n/h-
et al., 2016; Kleiner-Grote et al., 2018). Selecting a proper signal region boundary of Tat-specific signal peptides (Berks, 1996;
peptide to increase the secretion efficiency becomes a common Berks et al., 2000). Several bioinformatic tools have been built
methodology to optimize the production of recombinant protein. and maintained by different research groups to predict signal
Gram-positive bacteria usually consist of only one cell peptides, such as SignalP (Petersen et al., 2011), Phobius (Kaell
membrane. The secretion of a target protein in gram-positive et al., 2007), PrediSi (Hiller et al., 2004) for Sec-type signal
bacteria is thought to be more efficient (Freudl, 2013; Anne peptides and TatP (Bendtsen et al., 2005), Tatfind Server (Rose
et al., 2016). Various gram-positive bacteria, especially the et al., 2002), PRED-TAT (Bagos et al., 2010) for twin-arginine
generally recognized as safe (GRAS) gram-positive model signal peptides (Caccia et al., 2013).
bacterium Bacillus subtilis (Sewalt et al., 2016), are widely This article is a brief review of factors that influence signal
utilized for expression of recombinant proteins in biotechnology peptide secretion efficiency for recombinant protein in gram-
(Sone et al., 2015; Anne et al., 2016; Freudl, 2018). Several positive bacteria, especially in B. subtilis. We summarize several
different protein export systems have been identified in gram- experimental achievements in the screening of a proper signal
positive bacteria to date, including the general secretion (Sec) peptide for a given protein. We also discuss the differences
pathway, the twin-arginine translocation (Tat) pathway and between good-performing and bad-performing signal peptides
type VII/WXG100 secretion systems. Figures 1A,B are the for different recombinant proteins in B. subtilis. Additionally,
schematic figures of Sec and Tat export pathways in gram- other factors including the pro-region of recombinant
positive bacteria. Sec-dependent proteins are translocated to the protein and the expression host are also summarized in the
plasma membrane either co- or post-translationally (Figure 1A). last part.
In the co-translational export mode, precursor proteins are
recognized at the ribosome by the signal recognition particle
(SRP) and then targeted to the transmembrane SecYEG channel OPTIMIZATION OF RECOMBINATION
by SRP and FtsY, the SRP membrane receptor (Elvekrog and PROTEIN SECRETION BY SIGNAL
Walter, 2015). In the post-translational export mode, the post- PEPTIDE SCREENING
translationally interacting proteins (PIP’s), such as the general
chaperones GroELS, DnaK-DnaJ-GrpE, trigger factor, the CsaA Generating a signal peptide library has proven to be a
protein and the soluble form of SecA, keep the fully synthesized practicable approach for the optimal secretion of recombinant
precursor proteins in an unfolded secretion-competent state proteins in Gram-positive expression hosts. The first effort
(Wu et al., 1998; Herbort et al., 1999). Then the motor to systematically search the best-performing signal peptide
protein SecA translocates the preproteins through SecYEG using for heterologous protein secretion was performed a decade
metabolic energy from ATP hydrolysis (Schiebel et al., 1991). In ago. In this study, a signal peptide library consisting of
addition, SecDF enhances the release of preproteins (Tsukazaki 173 predicted Sec-type SPs from B. subtilis strain 168 was

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Peng et al. Factors Influencing Protein Secretion Efficiency

FIGURE 1 | Two major gram-positive bacterial export pathways and signal peptides with different secretion efficiencies. (A) The general secretion (Sec) protein export
pathway in gram-positive bacteria. (1). In the co-translational export mode, preproteins are recognized at the ribosome by the signal recognition particle (SRP). Then
the SRP membrane receptor FtsY binds to the ribosome-nascent chain (RNC)-SRP complex. SRP and FtsY target the preproteins to the transmembrane
(Continued)

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Peng et al. Factors Influencing Protein Secretion Efficiency

FIGURE 1 | SecYEG channel. (2). In the post-translational export mode, precursor proteins are fully synthesized and are kept in an unfolded secretion-competent
state by the post-translationally interacting proteins (PIP’s), such as the general chaperones GroELS/DnaK-DnaJ-GrpE/trigger factor, the CsaA protein and the soluble
form of SecA. Then the motor protein SecA translocates the preproteins through SecYEG using metabolic energy from ATP hydrolysis. SecDF enhances the release
of preproteins. (B) The twin-arginine translocation (Tat) export pathway in Gram-positive bacteria. After being synthesized, the Tat-dependent pre-protein folds rapidly
into its native conformation, sometimes with the help of cofactors. The energy for translocation comes from the proton motive force (PMF). In gram-positive bacteria
with high GC-content genomes, the Tat translocase consists of TatA, TatB, and TatC. In low-GC gram-positive bacteria, the Tat system is composed of TatC and a
bifunctional TatA protein. (C) The general structure of signal peptides. Adapted by permission from Springer Nature Customer Service Center GmbH: Springer Nature,
Nature Biotechnology (Molhoj and Dal Degan, 2004), copyright 2004. (D) Cumulative distributions of the charge/length ratio of n-region in good-performing and
bad-performing signal peptides. (E) Boxplots of the total hydrophobic values of signal peptides and the hydrophobic values in h-regions. (F) Sequence logos of
c-region aligned by their cleavage sites in good-performing and bad-performing signal peptides. (G) Boxplots of the proportions of helices, strands, and coils in
good-performing and bad-performing signal peptides. (H) ROC curves of models trained with 1 parameter (dark red), 26 parameters (dark green), and 29
parameters (purple). The data used in the upper half of (D–H) are from Brockmeier et al.’s study (2006). Good-performing SPs are the top 36 SPs showing high
cutinase activity (top 25% of all SPs). Bad-performing SPs are the 39 SPs showing no cutinase activity (the lower 27% of all SPs). The data used in the bottom half of
(D–H) are from Zhang et al.’s study (2016). For the 114 Sec-type signal peptides with promoter P43, the top 20 SPs with Xylanase activity > 100 units/ml are selected
as good-performing signal peptides. The last 18 SPs with Xylanase activity < 1 units/ml are selected as bad-performing signal peptides.

constructed (Brockmeier et al., 2006). The functionality of versa. This work indicates that promoters do not affect the
each SP was studied using cutinase from Fusarium solani secretion performance of signal peptides. If a signal peptide
pisi as the reporter protein. B. subtilis TEB1030 was used to performs well when using promoter A in the expression of the
express the SP-cutinase fusions. This study reveals that the target protein, it will also perform well when promoter B is
enzymatic activities of cutinase vary significantly when different used (Zhang et al., 2016).
SPs were fused to the protein. A similar conclusion was Signal peptide library construction followed by high-through
also obtained in the comprehensive analysis of signal peptide screening has also been reported in the secretion of several
functionality from Lactobacillus plantarum. Mathiesen et al. recombinant proteins (Degering et al., 2010; Tsuji et al., 2015; Cai
(2009) constructed a library of 76 Sec-type signal peptides et al., 2016; Hemmerich et al., 2016). Featured with high efficiency
from L. plantarum WCFS1. Staphylococcal nuclease (NucA) and high coverage of SPs, this method has screened many good-
was used as the reporter protein. This screening showed performing signal peptides for different recombinant proteins.
considerable variation in the levels of secreted NucA (Mathiesen Table 1 shows the signal peptides that have previously performed
et al., 2009). In another experiment, 405 candidate signal well in gram-positive bacteria. Apart from the signal peptide
peptides were predicted in the completely sequenced genome library-based method, there are also plenty of researches, too
of Corynebacterium glutamicum R. Then each of the SPs was numerous to be entirely listed, in which a few signal peptides are
fused to a heterologous α-amylase (AmyE) from Geobacillus involved (Freudl, 2018; Kalbarczyk et al., 2018; Owji et al., 2018).
stearothermophilus. A total of 108 SPs were shown to mediate If the secretion efficiencies of these SP-protein combinations are
detectable secretion of AmyE from the expression host C. gathered up in specific database, they will be of great value for
glutamicum R. Eleven of these samples exhibited 50- to 150- signal peptide selection and further data analysis.
fold higher secretion level than that of the signal peptide
derived from the well-known corynebacterial secretory protein
PS2 (Watanabe et al., 2009). PRIMARY AND SECONDARY STRUCTURE
A promoter is defined as the region of DNA sequence that OF SIGNAL PEPTIDES WITH DIFFERENT
initiates the gene transcription (Wrighton, 2018). Promoters SECRETION EFFICIENCIES
are often used together with signal peptides as regulatory
elements for the expression and production of recombinant All researches mentioned in the previous section come to the
proteins (Guan et al., 2016; Gu et al., 2017; Maffei et al., unanimous conclusion that the secretion levels of recombinant
2017; Cui et al., 2018). Zhang et al. (2016) performed an protein differ significantly when different SPs are fused to the
experimental screen of 138 signal peptides from B. subtilis for protein. In other words, the physicochemical properties of SPs
the production of an alkali-tolerant xylanase (XynBYG) from may affect the secretion levels of recombinant proteins. To
Bacillus pumilus BYG. They used B. subtilis WB700 as the further explore the factors that determine the secretion efficiency
expression host. Two promoters (Pglvm and the constitutive of SPs, biologists would also perform some statistical analysis
promoter P43) were separately used in the expression of the between the yields of target proteins and signal peptide characters
protein. The yields of XynBYG using Pglvm promoter were such as lengths, charges, pI values, D-scores from SignalP and so
higher than using the P43 promoter, which indicated that on (Zhang et al., 2016; Fu et al., 2018). In this section, we will
Pglvm promoter is more efficient than the P43 promoter for try to investigate the differences between good-performing and
XynBYG expression. In further analysis, an obvious correlation bad-performing signal peptides by in silico analysis of SPs.
with a Pearson correlation coefficient of 0.97 was observed The in silico analysis are performed with 143 Sec-type signal
between the yields of XynBYG driven by the two promoters. peptides in Brockmeier et al.’s (2006) study and 114 Sec-type
In other words, good-performing SPs would have higher signal peptides in Zhang et al.’s (2016) study. For Brockmeier
secretion efficiency than bad-performing SPs no matter which et al.’s data, the top 36 SPs (25% of all SPs) showing high cutinase
promoter is used in the expression of the protein, and vice activity are selected as good-performing signal peptides. The 39

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Peng et al. Factors Influencing Protein Secretion Efficiency

TABLE 1 | Examples of several signal peptides that perform well in gram-positive bacteria.

Signal peptide Recombinant protein Host Yield Ranking Reference

Signal sequence Origin Protein Origin

MKNMSCKLVVSVT B. subtilis, Cutinase F. solani pisi B. subtilis 4.67 [U/mL] 1/173 Brockmeier
LFFSFLTIGPLAHA Epr TEB1030 et al., 2006
MAKPLSKGGILVKKVLIAGA B. subtilis, Aminopeptidase B. subtilis B. subtilis WB600 88.59 1/20 Guan et al.,
VGTAVLFGTLSSGIPGLPAADAQVAKA YncM Zj016 [U/mL] 2016
MKKFNFKTMLLLVLASCVFGVV L. NucA S. aureus L. plantarum 35.84 1/78 Mathiesen
VNVTTSLGPQTAITAQA plantarumWCFS1 (nuclease) WCFS1 [U/mL] et al., 2009
MKEVRFWGLLLGL L. AmyA L. L. plantarum 3.4 1/18 Mathiesen
FVCLGAVIPLVSKA plantarumWCFS1 (amylase) amylovorus WCFS1 [102 mU/mL] et al., 2009
NRRL
B-4549
MQINRRGFLKA C. AmyE G. C. glutamicum 288.3 1/31 Watanabe
TAGLATIGAASMFMPKANA glutamicum R (α-amylase) stearothermophilus [U/mL] et al., 2009
MRSKKLWISLLF B. Nattokinase B. subtilis B. licheniformis 31.99 1/81 Cai et al.,
ALTLIFTMAFSNMSA licheniformis natto 10F-3 [FU/mL] 2016
WX-02, AprE
MKNMSCKLVVSVTL B. subtilis, Cutinase F. solani pisi C. glutamicum 13.1 [U/mL] 1/64 Hemmerich
FFSFLTIGPLAHA Epr et al., 2016
MKKFPKKLLPIAVL B. subtilis, XynBYG B. pumilus B. subtilis WB700 327.2 1/138 Zhang et al.,
SSIAFSSLASGSVPEASA PhoB (alkaline BYG [U/mL] 2016
active
xylanase)
MRSKKLWISLLFAL B. subtilis168, Alkaline B. B. subtilis WB600 7574.08 1/35 Our lab
TLIFTMAFSNMSVQA AprE protease alcalophilus [U/mL]
TCCC11004
MRIFKKAVFVIMI B. subtilis Alkaline B. B. 19835.7 1/86 Our lab
SFLIATVNVNTAHA 168, DacB protease alcalophilus amyloliquefaciens [U/mL]
TCCC11004 111018

SPs (27% of all SPs) showing no cutinase activity are selected shown that increasing the positive charge in n-region reduced
as bad-performing signal peptides. For the 114 Sec-type signal the protein secretion (Ravn et al., 2003; Jonet et al., 2012; Gao
peptides with promoter P43 reported by Zhang et al., the top et al., 2016). We suspect that positively charged residues in
20 SPs (with Xylanase activity > 100 units/ml) are selected as h-region and c-region of SP and the mature protein may lead to
good-performing signal peptides. The last 18 SPs (with Xylanase the contradictory results.
activity < 1 units/ml) are selected as bad-performing signal Figure 1E shows the boxplots of the total hydrophobic values
peptides. The analysis results are displayed in Figure 1. in signal peptides and the hydrophobic values in h-regions.
Figure 1D shows the cumulative distributions of the The Kyte-Doolittle hydrophobic scale is used in the current
charge/length ratio of n-region in good-performing and bad- study (Kyte and Doolittle, 1982). The Wilcoxon Rank Sum Test
performing signal peptides. The two panels of Figure 1D reveal reveals that hydrophobic values show no statistically significant
that the charge/length ratio of n-region in good-performing differences between good-performing and bad-performing signal
SPs is higher than that in bad-performing SPs. Previous studies peptides (P-values > 0.05). Previous studies showed that
also proved the importance of positively charged residues in interfering in the h-region hydrophobicity has various effects
the n-region during the initial step of protein secretion across on protein secretion. For example, reducing the hydrophobicity
the membrane. Substitution of positively charged residues with of Staphylococcus aureus SP completely abolished the secretion
uncharged or negatively charged residues would reduce the of mature protein (Mordkovich et al., 2015). Increasing the h-
protein synthesis rate and transport rate (Inouye et al., 1982; region hydrophobicity promoted the secretion of the heavy chain
Nesmeyanova et al., 1997). Increasing the positive charge of of monoclonal antibody in Escherichia coli (Zhou et al., 2016).
n-region has been demonstrated to improve secretion efficiency Substitution of Gly with Cys and Leu in the PhoE SP shifted
in both gram-positive (Takimura et al., 1997; Ng and Sarkar, protein secretion from SecB to SRP-dependent pathway (Adams
2013) and gram-negative bacteria (Ismail et al., 2011). However, et al., 2002). It is more likely that the order of residues and the
it is notable that the increase in the positive charge is not always secondary structure they formed in h-region regulate the protein
favorable. The plots in Figure 1D show the prediction power of secretion efficiency (Zhang et al., 2016; Han et al., 2017).
the charge/length ratio of n-region can be up to 1, and it might We also generate the sequence logos of c-region in good-
be not helpful when the value is above 1. Other studies have performing and bad-performing signal peptides with the

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Peng et al. Factors Influencing Protein Secretion Efficiency

WebLogo service (Crooks et al., 2004) (Figure 1F). The sequence OTHER FACTORS INFLUENCING PROTEIN
logos are aligned by their cleavage sites. Data from both SECRETION EFFICIENCY IN ACTION
Brockmeier et al.’s and Zhang et al.’ study show that residues
at the−3 and−1positions relative to the signal peptidase The experimental researches of signal peptide screening also
cleavage site are more consensus in good-performing SPs show that the secretion efficiency is at least in part dependent on
than in bad-performing SPs. Alanine residues are more likely the protein that is secreted. In Brockmeier et al.’s study, a subset
to appear at positions−3 and−1 in good-performing signal of signal peptides in the SPs library was fused to a cytoplasmatic
peptides. Early studies have also shown that the presence of esterase of metagenomic origin. Surprisingly, the best signal
Ala residues at positions−3 and−1 resulted in a considerable peptide for cutinase secretion was inefficient for esterase and
improvement in recombinant protein secretion (Ravn et al., 2003; vice versa (Brockmeier et al., 2006). Similarly, in Mathiesen
Guan et al., 2015). et al.’s study, lactobacillal amylase (AmyA) was also used as the
Figure 1G shows the boxplots of the proportions of helices, reporter protein with a selected set of SPs. No correlation was
strands and coils in good-performing and bad-performing SPs. observed between the signal peptide performance with NucA and
The secondary structure of signal peptides are predicted by with AmyA. The secretion efficiency of a given signal peptide is
PSIPRED (Buchan et al., 2013). For Brockmeier et al.’s data changeable when it is fused to different proteins (Mathiesen et al.,
(2006), the Wilcoxon Rank Sum Test suggests that good- 2009). The ∼30 residues downstream of the signal sequence,
performing signal peptides have a much higher proportion of termed the “pro-region,” has also been shown to be critical for
coils (the upper half of Figure 1G). However, the P- values protein secretion (Andersson and von Heijne, 1991; Low et al.,
in Zhang et al.’s data (2016) are not significant enough (the 2013; Musik et al., 2019). Our suspicion is that the pro-region
bottom half of Figure 1G). In a recent study, a native Sec-type influences protein secretion efficiency through its intervention to
signal peptide and its modified counterpart were used to secrete the interaction between the signal peptide and signal peptidase.
Candida antarctica Lipase B (CALB) in E. coli. The molecular Degering et al. (2010) constructed a signal peptide library
dynamic simulation shows that the native signal peptide contains consisting of 173 signal peptides from B. subtilis and 220 signal
an alpha-helix structure, whereas the designed one consists peptides from Bacillus licheniformis to improve the production of
only coils and turns. The secondary structure of designed subtilisin protease BPN’ from Bacillus amyloliquefaciens ATCC
signal peptide creates a more stable interaction with the signal 23844. Three different Bacillus expression strains (B. subtilis
peptidase. Their results showed that the designed signal peptide TEB1030, B. licheniformis DSM13/MW3, and B. licheniformis
increased the secretion of CALB (Ghahremanifard et al., 2018). strain H402) were used as expression hosts. Both homologous
According to the above analysis, we suspect that the and heterologous signal peptides fused to the target protein
secondary structure is critical to the secretion efficiency can direct protease secretion. Strikingly, the majority of SP-
of a signal peptide. Coils help to enhance the interaction BPN’ fusions showed similar relative levels of protease secretion
between signal peptides and signal peptidases. The positive in all three Bacillus expression strains (Degering et al., 2010).
charge of n-region, the hydrophobicity of h-region and the However, in another study, distantly related organisms are used
Ala residues at the−3 and−1positions in c-region may exert as expression hosts (Hemmerich et al., 2016). In this research, a
indirect effects on the secretion efficiency of the signal signal peptide library consisting of about 150 SPs from low-GC
peptide through their effects on the secondary structure of the firmicutes B. subtilis was constructed. Cutinase from F. solani pisi
signal peptide. used by Brockmeier et al. (2006) was also selected as the model
To test if it is possible to predict SPs performance based on enzyme. The SP-cutinase fusions were successfully transferred
the above sequence and structure features, we developed three to high-GC actinobacterium C. glutamicum ATCC13032 as
support vector machine (SVM)-based models for each of the alternative secretion host. The protein secretion levels with the
two data sets. The models were implemented with the software same SP in Brockmeier et al.’s (2006) study (B. subtilis as secretion
toolbox LIBSVM 3.23 (Chang and Lin, 2011). In model 1, only host) and in this study (C. glutamicum as secretion host) were
1 parameter, the charge/length ratio of n-region, was used. In compared. Interestingly, no correlation was observed between
model 2, a total of 26 parameters including the charge/length the two sets of data. Videlicet, the cutinase secretion levels
ratio of n-region, the hydrophobic values in h-region, the length directed by the same signal peptide differ dramatically with B.
of SP, the length of N/H/C region and the frequencies of 20 subtilis and C. glutamicum as secretion hosts. The results of the
amino acids in each SP (20 features) were used. In model 3, two studies show that the phylogenetic distance of expression
the proportions of helices, strands and coils in SP (3 features) hosts may affect the secretion performance of specific SP-protein
together with the 26 feathers in model 2 were used. The ROC combinations (Hemmerich et al., 2016).
curve in 10-fold cross-validation tests for each model is presented
in Figure 1H. The AUC scores of the three models are between
0.53 and 0.61 for Brockmeier et al.’s data (2006). For Zhang CONCLUSION AND PERSPECTIVES
et al.’s data (2016), the AUC scores are between 0.71 and 0.74.
Given the immaturity of these models, it would deserve a try Secreting recombinant protein out of the cell can improve the
to predict SP performance with machine learning methods if yield and simplify the purification process. A highly efficient
more features and more accurate algorithms are added to the signal peptide is of great value in the construction of secretory
prediction models. expression system. Signal peptide library construction followed

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Peng et al. Factors Influencing Protein Secretion Efficiency

by high-through screening has been successfully applied in the DATA AVAILABILITY

selecting of appropriate signal peptides for a target protein. This
technology and other genetic engineering tools such as CRISPER Publicly available datasets were analyzed in this study. This
can be further implemented on bacterial systems for the good- data can be found here: https://www.sciencedirect.com/science/
performing SPs selection and recombinant proteins production. article/pii/S0022283606009272 and https://link.springer.com/
In silico analysis of good-performing and bad-performing article/10.1007%2Fs00253-016-7615-4.
signal peptides reveals that good-performing signal peptides
have a higher charge/length ratio in n-region and more AUTHOR CONTRIBUTIONS
consensus residues (alanine amino acids are preferred)
at the−3 and−1positions in c-region. Moreover, good- FpL conceived and designed the study. CP performed the study
performing signal peptides have a much higher proportion and drafted the manuscript. CS, XC, and YL took part in the
of coils. Except for the signal peptide properties itself, the data collection. FfL took part in the data analysis. All the authors
pro-region of the target protein and the expression host edited the manuscript and approved the final manuscript.
may also influence the secretion efficiency. We speculate
that the interaction between the signal peptide and signal FUNDING
peptidase is critical to the recombinant protein secretion
efficiency. The primary and secondary structure, as mentioned The present work was supported by The National Key
above, would most likely influence the secretion efficiency of Research and Development Program of China (Grant No
the signal peptide through their effects on the interaction 2017YFB0308401) and the public service platform project of
between the signal peptide and signal peptidase. We fine strain selection and fermentation technology for industrial
hope more experimental data can be generated and more microbe (Grant No. 17PTGCCX00190).
regularities about secretion efficiencies can be summed up
by bioinformatic approaches. The bioinformatic databases ACKNOWLEDGMENTS
and concluded laws will become great contributors to the
selection and directed modification of signal peptides for a given The authors would like to thank Prof. Feng Gao in Tianjin
recombinant protein. University for the invaluable assistance and inspiring discussions.

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