Method for improving secretory expression level of expansin in bacillus subtilis
By modifying recombinant strains of Bacillus subtilis, overexpressing the swelling hormone encoding gene and secretion cofactors, and optimizing the secretion pathway, the problems of low and unstable swelling hormone production were solved, achieving efficient and stable swelling hormone secretion and expression, supporting industrial production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- YIXING INST OF FOOD & BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-01-23
- Publication Date
- 2026-05-01
AI Technical Summary
Existing technologies for producing swelling agents from Bacillus subtilis suffer from problems such as low yield, unstable yield, and difficulty in purification, making it difficult to achieve industrial-scale production.
By constructing recombinant Bacillus subtilis, modifying the host bacteria and overexpressing the expansionin encoding gene and the secretion cofactor encoding gene, the expression was regulated by signal peptides such as AspB signal peptide and YwcI signal peptide, and a secretion cofactor expression vector was constructed to optimize the secretion pathway and improve the protein secretion level.
The expression level of swelling hormone was increased to 232 mg/L in shake flasks and to 1.3 g/L in 5 L fermenters, achieving efficient and stable secretion of swelling hormone and laying the foundation for large-scale industrial production.
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Abstract
Description
A method to increase the expression level of swelling hormone secretion in Bacillus subtilis Technical Field
[0001] This invention relates to the field of genetic engineering technology, and in particular to a method for increasing the expression level of expansin secretion in Bacillus subtilis. Background Technology
[0002] Expansins are a family of key non-enzymatic proteins found in plant cell walls. They disrupt hydrogen bonds between cellulose microfibrils and matrix polysaccharides (such as hemicellulose), inducing irreversible physical relaxation of the cell wall without hydrolytic activity, thus regulating plant cell expansion and morphogenesis. In recent years, they have shown broad application prospects in agricultural production and the efficient utilization of biomass resources. In agricultural production, expansins can serve as key functional factors regulating plant growth and development and enhancing environmental adaptability, potentially promoting early crop growth, improving root absorption capacity, and enhancing stress resistance and yield stability, thereby meeting the practical needs of sustainable agriculture. In biomass resource utilization, expansins can significantly improve the accessibility of cellulase to substrates, providing new ideas for the saccharification and conversion of lignocellulose biomass and improving resource utilization. In summary, expansins have become a hot topic in interdisciplinary research in biotechnology and agronomy, and in-depth exploration of them holds broad application prospects.
[0003] Currently, swelling agents are mainly obtained through natural plant extraction, bio-extraction, and microbial production. Traditional plant extraction methods are limited by factors such as long plant growth cycles, complex raw material composition, and extremely low protein content, resulting in cumbersome extraction processes, high costs, and difficulty in achieving industrial-scale production. Bio-extraction methods are also difficult to purify and have low yields, making industrial-scale production difficult. In contrast, microbial fermentation methods offer milder production conditions and higher production efficiency, showing great promise for applications. Current research has used Pichia pastoris as a host for the secretion and expression of swelling agents. Although a certain yield can be achieved, this system uses methanol as a carbon source, and the byproducts generated during metabolism can negatively impact its growth; moreover, methanol is toxic, posing safety hazards in industrial applications. In addition, the long cultivation time and high fermentation costs limit its widespread application.
[0004] Bacillus subtilis possesses excellent characteristics such as a clear genetic background, mature gene manipulation techniques, rapid growth rate, and absence of endotoxins. It also exhibits a very strong protein secretion capacity, making it an ideal strain for strain modification to achieve efficient production of swelling hormones. However, current research on swelling hormone production by Bacillus subtilis is still in its early stages, facing problems such as low yield, unstable yield, and difficulties in purification. Summary of the Invention
[0005] Therefore, the technical problem to be solved by the present invention is to overcome the deficiency of the lack of efficient synthesis of swelling hormones by Bacillus subtilis in the prior art, and to provide a new modification method that can promote the secretion and expression of swelling hormones from microorganisms and plants, thus laying the foundation for the construction of an efficient expression system of swelling hormones in Bacillus subtilis.
[0006] The first objective of this invention is to provide a recombinant Bacillus subtilis strain modified based on a host bacterium, the modification including overexpression of an expansin-encoding gene and a secretion cofactor-encoding gene, wherein the expansin-encoding gene is expressed by the promoter shown in SEQ ID NO.1.
[0007] Furthermore, the expression of the expansionin-encoding gene is regulated by a signal peptide, including but not limited to the AspB signal peptide and the YwcI signal peptide.
[0008] Furthermore, the AspB signal peptide sequence is shown in SEQ ID NO.2.
[0009] Furthermore, the sequence of the YwcI signal peptide is shown in SEQ ID NO.3.
[0010] Furthermore, the amino acid sequences of the expansionin BsEXLX1 and LeEXP2 are shown in SEQ ID NO.4 and SEQ ID NO.5.
[0011] Furthermore, the secretory cofactors include, but are not limited to, GroEL, DnaJ, GrpE, StoA, SipS, SipV, SipT, and SipU.
[0012] Furthermore, the amino acid sequences of GroEL are shown in SEQ ID NO.6, DnaJ in SEQ ID NO.9, GrpE in SEQ ID NO.22, StoA in SEQ ID NO.21, SipS in SEQ ID NO.26, SipV in SEQ ID NO.25, SipT in SEQ ID NO.24, and SipU in SEQ ID NO.27.
[0013] Furthermore, the secretion cofactor is promoted by promoter P veg Start the expression.
[0014] Furthermore, when the amino acid sequence of the swelling factor is as shown in SEQ ID NO.4, the secretion cofactor is GroEL or DnaJ;
[0015] When the amino acid sequence of the swelling hormone is as shown in SEQ ID NO.5, the secretion cofactor is GrpE, StoA, SipS, SipV, SipT, or SipU.
[0016] Furthermore, the host bacteria include, but are not limited to, Bacillus subtilis G800.
[0017] Furthermore, the overexpression is performed in a free manner (plasmid).
[0018] A second objective of this invention is to provide the application of the recombinant Bacillus subtilis in the preparation of swelling agents.
[0019] A third objective of this invention is to provide a method for synthesizing expansion agent, which uses the recombinant Bacillus subtilis for fermentation production.
[0020] A fourth objective of this invention is to provide a method for increasing the secretion of a target product in Bacillus subtilis, comprising the steps of replacing the promoter preceding the target gene (either a genomic replacement or a replacement within the expression frame of the overexpressed target gene) with the promoter shown in SEQ ID NO.1 and overexpressing the secretion cofactor gene.
[0021] Furthermore, the target product includes, but is not limited to, swelling hormone, and correspondingly, the target gene includes, but is not limited to, the swelling hormone encoding gene.
[0022] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:
[0023] This invention first obtains the optimal mutant P by constructing a time-series promoter random mutation library with four different growth stages. SpoVG-TB-1 The application of the superior promoter mutants to the fermentation production of expansin (BsEXLX1 and LeEXP2) improved protein expression levels to a certain extent. Building upon this, the present invention also constructed a cofactor expression vector for the Bacillus subtilis secretion pathway. Overexpression of cofactors in previously successfully secreting expansin in Bacillus subtilis optimized the efficient secretion of expansin. In shake flasks, the secretion level of BsEXLX1 increased from 195 mg / L to 232 mg / L, and the protein secretion of LeEXP2 increased by approximately three times compared to the control. Further scale-up fermentation in a 5 L fermenter validated the results, achieving a further increase in expansin secretion levels, with protein expression reaching 1.3 g / L and LeEXP2 secretion reaching 43.7 mg / L. The expansin protein produced by the recombinant strains constructed in this invention exhibits stable expression and high extracellular secretion levels, laying the foundation for the further large-scale industrial production of expansin. Attached Figure Description
[0024] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.
[0025] Figure 1 is a schematic diagram of the promoter screening principle.
[0026] Figure 2 shows the flow cytometry screening process.
[0027] Figure 3 shows the sieve pattern of the 96-well plate for the starter.
[0028] Figure 4 shows the fluorescence intensity of the optimal promoter.
[0029] Figure 5 shows the yield of each mutant applied to the recombinant expression of BsEXLX1.
[0030] Figure 6 shows the yield of each mutant applied to the recombinant expression of LeEXP2.
[0031] Figure 7 is a schematic diagram of the secretion mechanism of Bacillus subtilis.
[0032] Figure 8 shows the yield of BsEXLX1 secretory cofactor overexpression.
[0033] Figure 9 shows the yield of LeEXP2 secretory cofactor overexpression.
[0034] Figure 10 shows the SDS-PAGE analysis of BsEXLX1 during the fermentation process in a 5 L fermenter.
[0035] Figure 11 shows the fermentation curve of the BsEXLX1 in a 5 L bioreactor.
[0036] Figure 12 shows the Western blotting analysis during the fermentation process in a 5 L fermenter of LeEXP2.
[0037] Figure 13 shows the fermentation curve in the 5 L bioreactor of LeEXP2. Detailed Implementation
[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.
[0039] The solution involved in this invention is as follows:
[0040] This invention first screens out the optimal mutant promoter P. SpoVG-TB-1This invention, when applied to the fermentation production of expansion hormones (BsEXLX1 and LeEXP2), has shown a certain degree of improvement in protein expression levels. Secondly, this invention provides an expression vector for cofactors related to the secretion pathway in Bacillus subtilis, specifically the pcrF20NM2 expression vector, which uses P... veg To drive the expression of cofactor-encoding genes by promoters, the main cofactors related to the secretion process in Bacillus subtilis include molecular chaperones (such as GroES / EL, DnaK / J, PrsA, Tig, etc.), redox cofactors (such as BdbA, BdbB, BdbC, BdbD, CcdA, etc.), signal peptidases (such as sipT, sipV, sipS, etc.), and transport proteins (such as SecA, SecDF, FtsY, etc.). Specifically, this invention constructs cofactor expression vectors in the Bacillus subtilis secretion pathway and achieves overexpression of related cofactors in the secretion pathway based on existing engineered strains, thereby improving the limitations of target proteins in secretion, transport, folding, maturation, and stability, and thus increasing the extracellular secretion level of expansin. In shake flasks, the secretory expression level of BsEXLX1 increased from 195 mg / L to 232 mg / L, and the protein secretion of LeEXP2 increased by about 3 times compared to the control. Building upon this foundation, further scale-up validation was achieved using a 5L fermenter, resulting in a further increase in the expansin secretion level, with protein expression reaching 1.3 g / L and LeEXP2 secretion reaching 43.7 mg / L. This provides a technological basis and industrialization potential for subsequent large-scale fermentation production and application development. The expansin produced by the recombinant strain constructed in this invention exhibits stable and high expression levels, demonstrating broad application prospects.
[0041] The strains involved in this invention are as follows:
[0042] Bacillus subtilis G800 is described in the literature “A genetic toolkit for efficient production of secretory protein in Bacillus subtilis”.
[0043] The sequence involved in this invention is shown below:
[0044] P SpoVG-TB-1 The nucleotide sequence is shown in SEQ ID NO.1.
[0045] SP AspB The nucleotide sequence is shown in SEQ ID NO.2.
[0046] SP YwcI The nucleotide sequence is shown in SEQ ID.3.
[0047] The amino acid sequence of BsEXLX1 is shown in SEQ ID NO.4.
[0048] The amino acid sequence of LeEXP2 is shown in SEQ ID NO.5.
[0049] The amino acid sequence of GroEL is shown in SEQ ID NO.6.
[0050] The amino acid sequence of GroES is shown in SEQ ID NO.7.
[0051] The amino acid sequence of the DnaK is shown in SEQ ID NO.8.
[0052] The amino acid sequence of DnaJ is shown in SEQ ID NO.9.
[0053] The amino acid sequence of PrsA is shown in SEQ ID NO.10.
[0054] The amino acid sequence of CsaA is shown in SEQ ID NO.11.
[0055] The amino acid sequence of the DbsA is shown in SEQ ID NO.12.
[0056] The amino acid sequence of FtsY is shown in SEQ ID NO.13.
[0057] The amino acid sequence of BdbA is shown in SEQ ID NO.14.
[0058] The amino acid sequence of BdbB is shown in SEQ ID NO.15.
[0059] The amino acid sequence of BdbC is shown in SEQ ID NO.16.
[0060] The amino acid sequence of the BdbD is shown in SEQ ID NO.17.
[0061] The amino acid sequence of TrxA is shown in SEQ ID NO.18.
[0062] The amino acid sequence of YneN is shown in SEQ ID NO.19.
[0063] The amino acid sequence of ResA is shown in SEQ ID NO.20.
[0064] The amino acid sequence of StoA is shown in SEQ ID NO.21.
[0065] The amino acid sequence of GrpE is shown in SEQ ID NO.22.
[0066] The amino acid sequence of the Tig is shown in SEQ ID NO.23.
[0067] The amino acid sequence of the SipT is shown in SEQ ID NO.24.
[0068] The amino acid sequence of the SipV is shown in SEQ ID NO.25.
[0069] The amino acid sequence of the Sips is shown in SEQ ID NO.26.
[0070] The amino acid sequence of the SipU is shown in SEQ ID NO.27.
[0071] The amino acid sequence of SecA is shown in SEQ ID NO.28.
[0072] The amino acid sequence of the SecDF is shown in SEQ ID NO.29.
[0073] Example 1: Plasmid pHT-P hag , P LytR , P spoVG , P mmgA Construction of -sfGFP
[0074] The plasmids were constructed in E. coli DH5α, and the four promoter-starting plasmids were constructed from promoter P. hag ,P LytR , P spoVG , P mmgA It consists of sfGFP and uses the commercially available pHT plasmid as the expression vector. The plasmid construction diagram is shown in Figure 1, and the information related to the time-series promoter is shown in Table 1.
[0075] Table 1. Information related to sequential promoters
[0076]
[0077] Table 2 Primer sequence listing
[0078]
[0079] Example 2: Construction of a random mutant library
[0080] Using the plasmid obtained in Example 1 as a PCR template, random saturation mutations were performed on the conserved regions of each promoter using degenerate primers. The resulting plasmids were then transformed into *E. coli* for cloning to obtain mixed plasmids, which were used to construct a random mutant library. The mutant library was then transformed into *Bacillus subtilis* for further screening. The primer list is shown in Table 3.
[0081] Table 3 Random Mutant Primer Sequence List
[0082]
[0083]
[0084] Example 3: High-throughput screening of promoter random mutation libraries
[0085] The bacterial colonies obtained in Example 2 were scraped and cultured at 220 rpm and 37°C for 6 h. After culture, the bacterial cells were centrifuged at 4000 rpm and 4°C for 5 minutes, the supernatant was discarded, and the bacterial pellet was washed three times with PBS. The bacterial suspension was diluted to an OD600 of approximately 0.3. Sorting was performed using a flow cytometer (BD FACSAria III) with the following parameters: 488 nm excitation laser and 530 ± 30 nm emission light, 70 μm nozzle, sheath pressure of 70 psi, and a data acquisition rate of 3000 events / s. Data were analyzed using BD FACSDiva software (v8.0). The sorted cells were collected in LB medium, activated at 220 rpm and 37°C for 1 hour, and then plated onto LB agar plates. The fluorescence intensity during initial screening using flow cytometry is shown in Figure 2. The obtained plate strains were inoculated into 96-well plates with 200 μL of seed culture medium (containing 5 mg / L chloramphenicol) per well, and cultured at 37°C with shaking at 700 rpm for 48 h. The bacterial concentration and total fluorescence intensity were measured every 12 h. The relative fluorescence intensity of sfGFP was measured using a Cytation 3 microplate reader (Berten Instruments, Inc., USA) according to the method described in Yang S, Liu Q, Zhang Y, et al. Construction and characterization of broad-spectrum promoters for synthetic biology [J]. ACS Synthetic Biology, 2018, 7(1): 287–291. Among approximately 630 hag promoter mutants, four strains exhibited significantly enhanced fluorescence intensity, with the highest fluorescence level being approximately four times that of the wild type. Among approximately 3000 SpoVG promoter mutants, about 200 showed enhanced fluorescence, with the optimal mutant reaching approximately twice the fluorescence intensity of the wild type. Among approximately 400 LytR promoter mutants, four strains showed significantly enhanced fluorescence expression, with the strongest mutant increasing its fluorescence intensity to approximately five times that of the wild type. Furthermore, among approximately 900 mmgA promoter mutants, about 70 strains showed upregulated fluorescence expression, with the highest fluorescence intensity of the mutant approximately four times that of the wild type. The dot plot of the time-series promoter screening process is shown in Figure 3, and the fluorescence intensity of the optimal time-series promoter mutants is shown in Figure 4.
[0086] Example 4: Applying promoter mutants to recombinant expression of swelling hormone
[0087] The superior promoter mutants obtained in Example 3 were applied for validation, using intumin (BsEXLX1, Genbank accession number: CAB13755.1 and LeEXP2, Genbank accession number: AAC64201.1) as reporter proteins. Each promoter mutant was applied to the recombinant expression of BsEXLX1. All selected promoter mutants increased the secretory expression level of BsEXLX1 to varying degrees. Among them, P... SpoVG-TB-1 The mutant exhibited the highest recombinant expression level, reaching a protein concentration of approximately 145 mg / L. Applying the optimal mutant to recombinant expression of LeEXP2, Western blotting results showed P... SpoVG-TB-1 The mutant showed the highest level of recombinant expression. Figure 5 shows the yield of recombinant expression of BsEXLX1 when each mutant was applied, and Figure 6 shows the protein expression of LeEXP2 when the optimal mutant was applied.
[0088] in:
[0089] The recombinant expansin BsEXLX1 was constructed from Bacillus subtilis by replacing the promoter preceding the BsEXLX1 coding sequence with various mutant promoters, ligating it to the pHT plasmid, and introducing it into Bacillus subtilis G800. The recombinant expansin LeEXP2 was constructed from Bacillus subtilis by replacing the promoter preceding the LeEXP2 coding sequence with various optimal mutant promoters (hag-TB-1, LytR-TB-1, mmgA-TB-1, SpovG-TB-1), ligating it to the pHT plasmid, and introducing it into Bacillus subtilis G800.
[0090] The fermentation conditions were as follows: Recombinant Bacillus subtilis seed culture was inoculated into the fermentation medium at a volume percentage of 5%, and cultured at 37℃ and 220 rpm for 36 h. The fermentation medium consisted of: 12 g / L peptone, 24 g / L yeast extract, 4 mL / L glycerol, 2.2 g / L KH₂PO₄, and 12.3 g / L K₂HPO₄.
[0091] Example 5: Construction of a vector for expressing Bacillus subtilis secretory accessory proteins
[0092] Figure 6 shows the relevant schematic diagram of the secretory protein process in Bacillus subtilis, illustrating the complete secretory expression pathway of BsEXLX1 in recombinant Bacillus subtilis. Molecular chaperones (such as GroES / EL, DnaK / J, PrsA, etc.) synchronously assist the correct folding of nascent polypeptide chains intracellularly, effectively avoiding the risk of protein degradation by intracellular enzymes. Transmembrane transport-related proteins (such as SecA, SecDF, etc.) and signal peptidases (such as SipsS, SipsT, SipsU, etc.) can significantly improve the rate of precursor protein penetration through the cell membrane and the cleavage release efficiency of the signal peptide. In addition, redox accessory proteins (such as BdbA / B / C / D, CcdA, TrxA, etc.) ensure the formation of active disulfide bonds after secretion.
[0093] Constructing cofactor expression plasmids: The vector uses pcrF20NM2 as the backbone (refer to Liu Y, Liu L, Li J, Du G, Chen J. Synthetic Biology Toolbox and Chassis Development in Bacillus subtilis. Trends Biotechnol. 2019 May;37(5):548-562. doi: 10.1016 / j.tibtech.2018.10.005.), with P veg The expression of genes encoding secretion cofactors driven by promoters is used to increase the extracellular secretion level of target proteins (such as swellin).
[0094] Example 6: Construction of strains overexpressing secretory cofactors
[0095] Construction of plasmid pHT-P spoVG-TB1 -SP AspB -BsEXLX1 was introduced into Bacillus subtilis G800 to obtain B. subtilis G800S1AE strain.
[0096] Construction of plasmid pHT-P spoVG-TB1 -SP YwcI -LeEXP2 was introduced into Bacillus subtilis G800 to obtain B. subtilis G800S1YL.
[0097] The helper factor expression plasmids obtained in Example 5 were transformed into *B. subtilis* G800S1AE and *B. subtilis* G800S1YL strains, respectively, and then inoculated into seed medium containing chloramphenicol (5 mg / L) and cultured overnight at 37°C with shaking at 220 rpm. Then, at a 5% inoculum (v / v) transfer to fermentation medium containing chloramphenicol (5 mg / L) and cultured at 37°C with shaking at 220 rpm for 36 h, resulting in engineered strains capable of further enhancing the expression level of expansin. Figure 8 shows the yield of BsEXLX1 secretion expression, where control represents the yield of *B. subtilis* G800S1AE strain; Figure 9 shows the yield of LeEXP2 secretion expression, where control represents the yield of *B. subtilis* G800S1YL strain.
[0098] Example 7: Scale-up culture of expansion agent in a 5 L fermenter
[0099] The engineered strain with the optimal expression level of expansin obtained in Example 6 was scaled up in a 5 L fermenter and inoculated into seed medium containing chloramphenicol (5 mg / L). The culture was incubated overnight at 37°C with shaking at 220 rpm to obtain the primary seed culture. Then, a 5% (v / v) inoculum was transferred to 100 mL of fermentation medium containing chloramphenicol (5 mg / L) and incubated at 37°C with shaking at 220 rpm for 12 h to obtain the secondary seed culture. This seed culture was then inoculated into a 5 L fermenter containing 1.5 L of fermentation medium at a 5% (v / v) inoculum. Throughout the fermentation process, the culture temperature was controlled at 37°C, pH 7.0, and dissolved oxygen (DO) level maintained at approximately 30%. The glucose concentration in the fermentation broth was controlled by adjusting the flow rate of the feed pump.
[0100] Figure 10 shows the SDS-PAGE analysis of BsEXLX1 during fermentation in a 5 L fermenter, and Figure 11 shows the relevant curves during the fermentation process. Figure 12 shows the Western blotting analysis of LeEXP2 during fermentation in a 5 L fermenter, and Figure 13 shows the relevant curves during the fermentation process.
[0101] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.
Claims
1. A recombinant Bacillus subtilis, characterized in that, The modification is based on a host bacterium and includes overexpression of a gene encoding an expansin and a gene encoding a secretory cofactor, wherein the expansin-encoding gene is expressed by the promoter shown in SEQ ID NO.
1.
2. The recombinant Bacillus subtilis according to claim 1, characterized in that, The gene encoding the expansionin is expressed under the regulation of a signal peptide.
3. The recombinant Bacillus subtilis according to claim 2, characterized in that, The sequence of the signal peptide includes the sequence shown in SEQ ID NO.2 or SEQ ID NO.
3.
4. The recombinant Bacillus subtilis according to claim 1, characterized in that, The amino acid sequence of the swelling agent is shown in SEQ ID NO.4 or SEQ ID NO.5; and / or, the secretion cofactor is at least one of GroEL, DnaJ, GrpE, StoA, SipS, SipV, SipT, and SipU.
5. The recombinant Bacillus subtilis according to claim 4, characterized in that, When the amino acid sequence of the swelling hormone is as shown in SEQ ID NO.4, the secretion cofactor is GroEL or DnaJ; and / or, when the amino acid sequence of the swelling hormone is as shown in SEQ ID NO.5, the secretion cofactor is GrpE, StoA, SipS, SipV, SipT, or SipU.
6. The recombinant Bacillus subtilis according to claim 4 or 5, characterized in that, The amino acid sequences of GroEL are shown in SEQ ID NO.6, DnaJ in SEQ ID NO.9, GrpE in SEQ ID NO.22, StoA in SEQ ID NO.21, SipS in SEQ ID NO.26, SipV in SEQ ID NO.25, SipT in SEQ ID NO.24, and SipU in SEQ ID NO.
27.
7. The recombinant Bacillus subtilis according to claim 1, characterized in that, The overexpression mode is free expression.
8. The use of the recombinant Bacillus subtilis according to any one of claims 1-7 in the preparation of swelling agent.
9. A method for synthesizing an expansion agent, characterized in that, The recombinant Bacillus subtilis as described in any one of claims 1-7 is used for fermentation production.
10. A method for increasing the secretion of a target product in Bacillus subtilis, characterized in that, The steps include replacing the promoter before the target gene with the promoter shown in SEQ ID NO.1 and overexpressing the secretory cofactor gene.