1. Introduction
Bacillus subtilis has many attributes such as food security, easy genetic manipulation, and efficient protein secretion ability, which make it a popular host for recombinant protein production [
1,
2,
3]. It also has long been used as a model organism for molecular research, as well as an industrial workhorse for the production of valuable enzymes [
4]. Many strategies including host strain modification, promoter and signal peptide optimization, and fermentation optimization have been used in the construction of cell factory of
B. subtilis to improve the production level of individual proteins [
1,
5,
6,
7,
8]. Even so, as the expression of heterologous protein is a complex process in which cells have their own unknown regulation law in every step of protein synthesis, most of the heterologous proteins are produced at very low levels or even inexpression in the recombinant
B. subtilis strains [
1,
9,
10,
11].
Generally, except codon optimization, the initial steps to enhance the yield of enzymes are to optimize molecular tools of the expression system, such as promoters and signal peptides (SPs). However, the optimal expression molecular tools for any given secretory protein cannot currently be predicted through experience or in silico so far [
1]. N-terminal signal peptides (SPs) are responsible for guiding the target proteins into the typical secretion pathways (Sec and TAT) in
B. subtilis [
12,
13,
14]. Many studies have been performed to improve the extracellular expression of the specific protein by screening an efficient signal peptide [
15,
16,
17,
18,
19]. However, no research had pay attention to another property of signal peptide is that the N-terminal SP sequence is also the initiation of transcription and translation of the target genes so far. While the transcription and translation initiation are the major rate-limiting steps for protein synthesis [
20]. Verma et al. had report that the N-terminal amino acids encoded by codons 3 to 5 impact protein yield [
20]. Besides, the effect is independent of tRNA abundance, translation initiation efficiency, or overall mRNA structure [
20]. According to this theory, we could bold assume that some low efficient secretion SPs for one target protein identified through detection of extracellular protein content may not about its specific secretory structure but limited by its transcription and translation initiation of the N-terminal sequences. Likewise, the optimum SP for a target protein determined by detecting extracellular protein content may concurrently has suitable N-terminal sequences with more efficient transcription and translation initiation.
Based on the hypothesis, after identifying the optimum SP (LipA) of five SPs belonging two different secretion pathways (Sec and TAT) for the extracellular expression of an alkaline pectin lyase APL in B. subtilis, the N-terminal first 5 and 7 amino acids sequences were substituted to reveal their effects on the expression and secretion of APL. It is verified by a series of experimental designs that LipA guiding to relatively higher extracellular expression of APL in B. subtilis not only because its secretion ability but also accounting for its persistently high transcriptional level of the N-terminal amino acids. The highest extracellular APL activity produced by the engineered B. subtilis strain was up to 12,295 U/ml whcih is the highest heterologous expression yield of alkaline pectinase in prokaryotic expression system as far as reported before.
4. Discussion
B. subtilis is generally considered as a good prokaryotic expression host for heterologous proteins based on its advances in extracellular proteins secretion. Over the past decades, in order to make the efficient secretory expression of foreign proteins at a large scale in
B. subtilis, more and more signal peptides were explored to improve the extracellular expression of different proteins. In this study, five signal peptides which had been verified to have efficient secretion rate were detected to guide the extracellular expression of APL in
B. subtilis. Among them, the signal peptides YncM and PelB are belonging to the typical Sec pathway, while LipA, WapA, and AmyX are belonging to the TAT pathway with characteristic double arginines at the N-terminal of their sequences (
Table 1). Based on comparing the extracellular APL activities of the recombinant strains connecting different signal peptides at the N-terminal of mature peptide of APL, the TAT-type signal peptides LipA and WapA showed remarkably higher secretory expression than the Sec-type signal peptides YncM and PelB (
Figure 1). While another TAT-type signal peptide AmyX made obviously low expression both in and out cells (
Figure 1). Thus, it could not simply estimate any secretion pathway or signal peptide type is more suitable for the secretory expression of APL in
B. subtilis. Meanwhile, because the signal peptides were on the N-terminal of the target proteins, the beginning of the gene transcript and protein translation of the target proteins would also be related to the sequence properties of different signal peptides. As is well-known that the initiation of transcript and translation is a major rate-limiting step for protein synthesis [
20]. So, the N-terminal signal peptides theoretically may not only determine the secretion rate but also make critical roles in the expression level of the target proteins. However, the effects of the N-terminal signal peptides on the expression level of the heterologous genes have few reported studies so far.
Verma et al. had employed a library of more than 250,000 reporters combined with in vitro and in vivo protein expression assays to assess the influence of early elongation on protein synthesis. They reported that the identity of the amino acids encoded by codons 3 to 5 impact protein yield [
20]. Inspired by that study, we selectively switched the N-terminal first 5 amino acid sequences of different signal peptides to assess their influences both on the expression and secretion of the target protein APL (
Table 1). Besides, in order to also evaluate the effects of typical double arginines on the protein secretion, the N-terminal first 7 amino acid sequences substitutions were designed simultaneously (
Table 1). When the N-terminal 5 and 7 amino acids of LipA were substituted with that of YncM, respectively, the extracellular activities of APL were declined remarkably (
Figure 2A). Besides, the recombinant signal peptide LipA (YncM N7) which broken the typical double-arginine sequence made relatively higher extracellular expression than the recombinant signal peptide LipA (YncM N5) which kept the typical double-arginine sequence of the TAT-type signal peptide (
Figure 2A, B). It indicated that the specific N-terminal amino acids sequence is the critical factor for the extracellular expression of the target protein rather than the secretion type. Meanwhile, the N-terminal 5 and 7 amino acids of YncM and AmyX were substituted by that of LipA led to increasing of the extracellular activities of APL (
Figure 2A). First of all, it could conclude that the N-terminal sequence of LipA has even greater advantages than those of YncM and AmyX in the synthesis of APL in
B. subtilis. Besides, the N-terminal 5 and 7 amino acids sequence was not the only factor affecting the expression of the target proteins. When it combined with the remnant sequences of YncM and AmyX led to significant different effect on the gene expression of APL (
Figure 2). The recombinant signal peptides AmyX (LipA N5) and AmyX (LipA N7) made very high expression levels of APL which even higher than that guided by LipA (
Figure 2). This interesting finding would make a significant strategy for improving different heterologous proteins synthesis in
B. subtilis. However, based on current research results in this study, the N-terminal amino acids sequence of the signal peptides did not make significant effect on the extracellular secretion rate of the target proteins (
Figure 2A).
As shown in
Figure 2A, YncM (LipA N5) and AmyX (LipA N5) made relatively more significant influences in the expression level of APL than YncM (LipA N7) and AmyX (LipA N7), respectively. Thus, the engineered strains containing the recombinant signal peptide YncM (LipA N5) and AmyX (LipA N5) were chose to further evaluate the APL expression difference during the whole fermentation process. As shown in
Figure 3, LipA and YncM (LipA N5) mainly produced extracellular APL activities even at the beginning fermentation phase at 6 h, while AmyX (LipA N5) mainly produced intracellular APL activities during the whole fermentation process and the extracellular activity began increasing after 15 h fermentation. Inferred from this result that LipA N5 just has the effect on improving the gene expression and it may destroy the typic double arginines specific sequence thus led to AmyX (LipA N5) lost secretory function. As for a portion of extracellular APL activity detected, it is likely caused by cell lysis in the late fermentation period. To test this hypothesis, LipA N5 was directly attached to the N-terminal of the mature peptide of APL. As shown in
Figure 4, LipA N5 indeed remarkably enhanced the expression of APL. As the strain containing the mature peptide of APL has been detected extracellular APL activity after 12 h fermentation, thus as the same the extracellular activity of the strain containing APL (LipA N5) is also due to cell lysis after 12 h fermentation (
Figure 4). So, the hypothesis that LipA N5 only having the influence on the expression of target proteins was proved.
To sum up above results, the recombinant signal peptide YncM (LipA N5) made increased expression with no effect on its secretion, while the recombinant signal peptides AmyX (LipA N5) made remarkably increased expression with secretion function lost (
Figure 5A). Through detecting the relative transcriptional level of each engineered strain, the recombinant proteins with LipA N5 sequence at their N-terminal showed relatively higher transcriptional level than their control strains (
Figure 5C). Moreover, the high expression level strains also showed consistently high transcription levels during the fermentation process (
Figure 5C). Thus, the specific N-terminal sequence LipA N5 was identified for the potential of improving the expression of heterologous proteins in
B. subtilis. Meanwhile, it is also suggested that researchers should also consider the effect of N-terminal sequence on expression when selecting appropriate signal peptides in
B. subtilis.
LipA was identified the optimum signal peptide for the heterologous extracellular expression of APL with both high expression level based on its N-terminal specific sequence and efficient secretion rate though TAT pathway in
B. subtilis. Fed-batch culture in a 5-L reactor was performed to test the industrial fermentation capacity of the engineered strain. The highest extracellular APL activity (12,295 U/ml) obtained at 60 h fermentation which further exceeded the total soluble APL activity (10,181 U/mL) expressed in the recombinant
Escherichia coli strain we had structed [
22]. So far, it is the highest heterologous expression yield of alkaline pectinase in prokaryotic expression system as far as reported before [
22].
Author Contributions
Conceptualization, H.Z. and M.Z.; methodology, J.Z.; software, M.Z. and J.T.; validation, X.Z., J.T., and X.F.; formal analysis, J.Z.; investigation, H.Z.; resources, J.Z., and J.X.; data curation, M.Z.; writing—original draft preparation, M.Z.; writing—review and editing, H.Z.; visualization, J.Z.; supervision, H.S., and H.Z.; project administration, H.S.; funding acquisition, H.Z., M.Z., Y.Z., and W.B. All authors have read and agreed to the published version of the manuscript.