A self-inducible gene expression system in Bacillus subtilis based on a two-component quorum sensing element

Abstract

The present invention discloses a Bacillus subtilis autoinducing gene expression system based on a two-component quorum sensing element, belonging to the field of genetic engineering technology. A long-lasting and high-expression autoinducing gene expression system is developed. The present invention provides an autoinducing gene expression system, wherein the expression element comprises a constitutive promoter and a target promoter, the RBS sequence of the agrA gene and / or the RBS sequence of the agrD gene, the gene agrA encoding a response regulator protein, the gene agrD encoding the precursor of the signal molecule AIP, the gene agrB encoding a signal processing protein, and the gene agrC encoding a sensor protein that binds to the signal molecule and initiates signal transduction. The present invention provides a new approach and method for designing a recombinant protein expression system based on Bacillus subtilis.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and mainly relates to a Bacillus subtilis self-inducing gene expression system based on a two-component quorum sensing element. Background Art

[0002] With the advancement of genetic engineering and synthetic biology, genetic manipulation of cells has become increasingly important through the construction of gene circuits within them. This has played a significant role in synthetic biology research. Traditional expression control systems can achieve regulation at the transcriptional level, but require exogenous addition of inducers. Common inducers include lactose, isopropylthiogalactoside (IPTG), and xylose. However, the variety of inducible expression systems remains limited, and their performance still falls short of the requirements for large-scale industrial production. Furthermore, the addition of exogenous inducers significantly increases production costs. Furthermore, all metabolic activities within cells are dynamic and complex, and these regulatory strategies are coupled to the growth of the underlying cells. Therefore, constructing artificial static regulatory systems within cells often interferes with the underlying cells. For example, overexpression of exogenous genes can disrupt normal host growth, and the accumulation of toxic intermediates can be toxic to cells, even leading to cell death. Sometimes, the production of specialized compounds is required. For example, when synthesizing cytotoxic products, it is often necessary to grow the host to a high cell density before switching from growth to production mode to minimize the toxic effects of the product on cells. Therefore, the development of self-inducible gene expression regulatory circuits remains very important for metabolic engineering and protein engineering. Summary of the Invention

[0003] The purpose of the present invention is to develop a long-lasting and high-expression autoinducible gene expression system.

[0004] The present invention provides an expression element of an autoinducing gene, which is composed of a constitutive promoter and a target promoter, the RBS sequence of the agrA gene and / or the RBS sequence of the agrD gene, the gene agrA encoding a response regulatory protein, the gene agrD encoding a signal molecule AIP precursor, the gene agrB encoding a signal processing protein, and the gene agrC encoding a sensor protein that binds to the signal molecule and initiates signal transduction.

[0005] It is further defined that the RBS sequence of the agrD gene is shown as SEQ ID NO.16 or SEQ ID NO.17; the RBS sequence of the agrA gene is shown as SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20 or SEQ ID NO.21.

[0006] It is further defined that the constitutive promoters are constitutive promoter Pveg and constitutive promoter P43, the target promoter is P3 promoter, and the vector is pHT01.

[0007] It is further defined that the connection order is: the constitutive promoter P43 is connected to the agrA gene RBS and the agrA gene in sequence, the P3 promoter is connected to the multiple cloning site, the constitutive promoter Pveg is connected to the agrD gene RBS and the agrD gene in sequence, the constitutive promoter Pveg is placed upstream of the P43 promoter, and the agrB gene and the agrC gene are connected to the multiple cloning site.

[0008] The present invention provides a recombinant vector containing the expression element of the above-mentioned autoinducing gene.

[0009] The present invention provides a method for preparing the above-mentioned expression element, characterized in that the specific steps of the method are as follows: a vector containing a constitutive promoter, the RBS sequence of the agrA gene and / or the RBS sequence of the agrD gene, the gene encoding the response regulatory protein agrA, and the gene encoding the signal molecule AIP precursor agrD is transformed into a microbial cell containing the agrB gene and the agrC gene.

[0010] The present invention provides an expression system of an auto-inducing gene and a microbial cell containing the expression element of the auto-inducing gene.

[0011] It is further defined that the microbial cells are Gram-positive bacterial cells.

[0012] It is further defined that the microbial cell is Bacillus subtilis.

[0013] The present invention provides applications of the above-mentioned expression element or expression system in the fields of food, chemical industry and pharmaceuticals.

[0014] Beneficial Effects: The Staphylococcus aureus Agr system is a two-component system that mediates quorum sensing. It uses the receptor histidine kinase to sense the cyclic peptide signaling molecule AIP, which activates the target promoter through a phosphorylation cascade, thereby regulating the expression of the target gene. Based on the research on the Agr two-component signaling system, Agr can be reconstructed in Bacillus subtilis and designed as an autoinducible gene expression system.

[0015] Bacillus subtilis is an important platform for efficient industrial enzyme expression and a key metabolic engineering platform. Constructing a dynamic gene expression system coupled to growth in B. subtilis is crucial for its application in metabolic engineering. Regulating gene expression using endogenous quorum sensing systems can severely impact cell growth and other important physiological processes. Therefore, heterologous reconstructing an artificial quorum sensing system with good orthogonality is of great significance.

[0016] The present invention first reconstructs the Staphylococcus aureus Agr two-component system in Bacillus subtilis, constructing the autoinducing gene expression system AgrQS, driven by an autoinducing peptide (AIP). Using RBS engineering, the expression levels of the key protein component, AgrA, and the signaling molecule, AgrD, are regulated individually and in combination, enabling the modulation of the activation timing, temporal dynamics, and expression output levels of the autoinducing system. This invention provides a new approach and method for designing recombinant protein expression systems based on Bacillus subtilis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 :Schematic diagram of the construction of the Bacillus subtilis self-inducing AgrQS expression system based on the agr quorum sensing element.

[0018] Figure 2 :Functional verification of AgrQS; a is the fluorescence intensity detection of the heterologous constructed AgrQS system; b is the SDS-PAGE protein expression detection;

[0019] Figure 3 : Through RBS engineering, the expression level of agrA was regulated and the effect of changes in agrA expression level on the temporal dynamics of AgrQS system activation was confirmed;

[0020] Figure 4 : The first screening of transformants of the co-regulation system of agrD and agrA expression levels;

[0021] Figure 5 : Second screening of transformants of the co-regulation system of agrD and agrA expression levels;

[0022] Figure 6 : Verify the role of co-regulation of agrD and agrA expression levels in the temporal dynamics of the AgrQS system. DETAILED DESCRIPTION

[0023] The culture medium involved in the following examples is as follows:

[0024] 1. Culture medium:

[0025] LB solid medium: Add 1.5-2% agar powder to LB liquid medium for bacterial screening, activation and plate culture, etc.

[0026] SP-Ⅰ culture medium (10 mL): 4.9 mL SPA solution, 4.9 mL SPB solution, 100 μL 50% glucose solution, and 100 μL 100× CAYE solution, mix well and prepare before use.

[0027] SP-Ⅱ medium (4 mL): 4.92 mL SP-Ⅰ medium, 40 μL 50 mmol·L-1 CaCl2 solution, and 40 μL 250 mmol·L-1 MgCl2 solution were mixed and prepared before use.

[0028] The final concentrations of the antibiotics involved in the present invention are: ampicillin (Amp) 100 μg·mL-1 chloramphenicol (Chl) 10 μg·mL-1

[0029] 2. SDS-PAGE protein detection:

[0030] Remove the glycerol stock, streak onto LB agar plates, and incubate overnight at 37°C in a 37°C incubator. Pick a single colony from the plate and inoculate it into a small test tube containing 3 mL of LB liquid medium. Incubate overnight at 37°C, 200 rpm. Transfer the inoculum to a large test tube containing LB at a 1% inoculum concentration and incubate at 37°C, 200 rpm, for 12 hours. Pipette 200 μL of the culture and centrifuge at 12,000 rpm for 2 minutes. Discard the supernatant and thoroughly resuspend the cells in 200 μL of lysozyme solution. Incubate in a 37°C metal bath for 40-60 minutes until the solution is clear. Then, add 50 μL of 5× loading buffer and boil in a boiling water bath for 10 minutes. Load the sample for analysis.

[0031] 3. Detection of expression of reporter gene sfGFP

[0032] sfGFP expression level: Single colonies were picked from the plate and transferred to a small tube containing 3 mL of LB medium. The culture was incubated overnight at 37°C at 200 rpm. A 1% inoculum of the inoculum was transferred to a larger tube containing LB medium and incubated at 37°C at 200 rpm. Samples were collected at various times to measure OD600 and sfGFP fluorescence intensity. At specific times, appropriate amounts of the fermentation broth were centrifuged at 13,000 rpm for 1 minute to retain the pellet. The precipitate was washed two to three times with 1× PBS and resuspended in an equal volume. 200 μL of the culture was transferred to a 96-well translucent microplate and fluorescence intensity was measured using a microplate reader. The excitation and emission wavelengths were set to 495 nm and 525 nm, respectively.

[0033] 4. Preparation of JM109 Competent Cells and Chemical Transformation

[0034] Take a tube of 60-90 μL of E. coli competent cells, add about 2-5 μL of recombinant plasmid, mix gently, and place on ice for 25-30 minutes; heat shock at 42°C for 90 seconds, and immediately place on ice for 2-3 minutes; add an appropriate amount of recovery medium, incubate at 37°C, 200 rpm for 45-60 minutes; after centrifugation, aspirate an appropriate amount of bacterial liquid and apply it to a screening plate containing appropriate resistance.

[0035] 5. Preparation of B. subtilis 168 Competent Cells and Chemical Transformation

[0036] Transfer the stored glycerol stock or a single colony from a plate to a tube containing 2 mL of SP-I and incubate overnight at 37°C, 200 rpm. Transfer 100 μL of the seed solution to a tube containing 5 mL of SP-I and incubate at 37°C, 200 rpm for 4-6 hours. Then transfer 0.2 mL of the culture to a tube containing 2 mL of SP-II and continue incubating at 37°C, 200 rpm for another 90 minutes. Add 10 μL / mL of 10 mmol / L EGTA and incubate on a shaker for 10 minutes. Aliquot 500 μL of the culture into sterile centrifuge tubes, add an appropriate amount of DNA fragment, and incubate at 37°C, 200 rpm for 90-120 minutes. After centrifugation, spread the culture onto a plate containing the resistant strain.

[0037] 6. Plasmid Extraction

[0038] Specific operation process reference Instructions for the plasmid extraction kit.

[0039] Example 1: Construction and verification of the AgrQS autoinducible gene circuit in Bacillus subtilis

[0040] 1. Using a plasmid containing the agr quorum sensing system (agrQS) as a template, the agrB and agrC elements were amplified and cloned into the pAX01 vector. The agrB and agrC elements were then transformed into B. subtilis 168 and integrated into the lacA site of the Bacillus subtilis genome to obtain recombinant Bacillus subtilis BsBC with agrB and agrC integrated at the lacA site.

[0041] 2. In the Agr system, the gene encoding the precursor of the signal molecule AIP, agrD, was placed downstream of the constitutive promoter Pveg, and agrA was placed downstream of the constitutive promoter P43. The P3 target promoter regulated the expression of the downstream reporter gene sfGFP (linked to the pHT01 vector), constructing the recombinant plasmid pHT-max-DA-P3-sfGFP. The recombinant plasmid was transformed into recombinant Bacillus subtilis BsBC harboring agrB and agrC. The plates were plated with ampicillin, and positive transformants were screened by colony PCR. (Sequences of P3, P43, Pveg, agrA, agrB, agrC, agrD, and sfGFP are shown in Table 1.) The original RBS sequence contained in the plasmid is (5'-ACACGCACCTCGAACGCCACGCAATATATACGAAGGAGGTCCGAC-3', SEQ ID NO. 21).

[0042] Pick up the constructed transformant and the original strain B. subtilis 168 into a small test tube with 3 ml LB liquid medium, 37 ° C, 200 rpm -1 After overnight culture, transfer the inoculum to a large test tube containing LB at a 1% inoculum volume and culture at 37°C, 200 r / min-1. Take samples at different times to measure OD600 and sfGFP fluorescence intensity. (Specific steps: Take an appropriate amount of fermentation liquid at a specific time, 13,000 r / min-1 -1 Centrifuge for 1 minute to retain the precipitate, wash 2-3 times with 1× PBS solution, and resuspend the cells in an equal volume. Transfer 200 μl of the bacterial solution to a 96-well translucent microplate and measure fluorescence intensity using a microplate reader. (The program settings were: excitation wavelength 495 nm and emission wavelength 525 nm, respectively.) After the culture, the proteins produced by the recombinant Bacillus subtilis were subjected to SDS-PAGE gel electrophoresis. Specifically, the cells were first centrifuged at 37°C, 200 rpm, and 400 μl of the solution was added. -1 1 ml of Bacillus subtilis cells cultured for 24 h were collected by centrifugation; the cells were then treated at 37° C. and 0.05% lysozyme for 30 min to fully break the cell wall of Bacillus subtilis, and the treated sample was boiled and prepared into a protein sample.

[0043] The results of SDS-PAGE electrophoresis were as follows: Figure 1 As shown, the integrated strain containing the recombinant plasmid (pHT-agrD-agrA-P3-sfGFP) activated the expression of sfGFP, while the strain without the integrated gene did not express sfGFP. The fluorescence intensity detection ( Figure 2 -a) and SDS-PAGE( Figure 2 -b), indicating that in Bacillus subtilis, all components of the heterologous AgrQS system can function as expected and have efficient heterologous protein expression levels.

[0044] Table 1 Nucleotide sequence information of P3, P43, Pveg, agrA, agrB, agrC, agrD and sfGFP

[0045]

[0046]

[0047] Example 2: Optimizing the signaling dynamics of AgrQS by regulating the levels of the transcriptional regulator AgrA

[0048] The constructed reporter gene sfGFP containing agrD and agrA and regulated by the P3 promoter was cloned into the pHT01 vector (pHT-max-DA-P3-sfGFP) plasmid as a template. The primers P43-RBSm-AF / R in Table 2 were used for whole-plasmid PCR to construct the RBS library of agrA and the plasmid containing sfCFP was used as the expression system (construction primers are shown in Table 2).

[0049] The constructed recombinant plasmid (using pHT-max-DA-P3-sfGFP as a template and reverse amplification with the primers listed in Table 2 to obtain an amplification product containing a degenerate RBS sequence, which was then digested with DpnI to remove the template, resulting in the recombinant plasmid) was transformed into Escherichia coli for amplification. After amplification, the plasmid was extracted and transformed into the recombinant B. subtilis 168 strain BsBC, which incorporates agrB and agrC. The plasmid library was then plated onto LB solid medium to generate a library of RBS mutants expressing AgrA. Single colonies exhibiting green fluorescence were selected from the library and cultured overnight in small test tubes for fluorescence intensity measurement. Ten transformants with high expression levels were selected and transferred to larger test tubes for 12 hours. The relative fluorescence intensity was measured at 4, 6, 8, 10, and 12 hours.

[0050] The results are as follows Figure 2 As shown in Table 3, the temporal dynamics of the AgrQS system regulated by RA2 and RA7 for AgrA were broadened, and the activation time point was delayed from 4 h in the original system to 6-8 h, enabling self-induced gene expression under high bacterial density.

[0051] The RBS sequence of RA2 is CTATGTGAGT (SEQ ID NO. 20); the RBS sequence of RA7 is GTTTGTCAGT (SEQ ID NO. 21).

[0052] Table 2 Primers for RBS mutation of agrA

[0053]

[0054] Note: B in the table indicates a degenerate sequence containing C, G, and T; D indicates a degenerate sequence containing A, G, and T; V indicates a degenerate sequence containing A, C, and G; H indicates a degenerate sequence containing A, C, and T; S indicates a degenerate sequence containing C and G; R indicates a degenerate sequence containing A and G; Y indicates a degenerate sequence containing C and T; and M indicates a degenerate sequence containing A and C.

[0055] Table 3 Total fluorescence intensity of 10 transformants and WT after culture in large test tubes

[0056]

[0057] Note: The original RBS (WT) refers to the transformant obtained by transformation with the system constructed in Example 1.

[0058] Example 3: Simultaneous RBS engineering of the signal synthesis gene agrD and the signal sensor gene agrA in the Agr element to optimize the signal output dynamics of AgrQS

[0059] The pHT-max-DA-P3-sfGFP plasmid was used as a template, and the primers vegRBSmut-DA-F / R in Table 4 were used for whole-plasmid PCR to construct the RBS library of agrD, and the plasmid containing sfGFP was used as the expression system (primers for construction are shown in Table 4).

[0060] The constructed recombinant plasmid (using pHT-max-DA-P3-sfGFP as a template, reverse amplification with the vegRBSmut-DA-F / R primers in Table 4, and cloning into the pHT01 vector to obtain a recombinant plasmid containing a degenerate RBS, pHT-veg-RBS-mut-DA) was transformed into Escherichia coli for amplification. After plasmid pHT-veg-RBS-mut-DA (a mixture of various sequences) was extracted and used as a template for whole-plasmid PCR using primers P43-RBS-mut-DA-F / R in Table 4 to construct a library of RBS mutants of agrA and a plasmid expressing sfGFP (using pHT-veg-RBS-mut-DA as a template, amplification with primers P43-RBS-mut-DA-F / R in Table 4, a plasmid library containing RBS mutations, and DpnI digestion to remove the template to obtain recombinant plasmid 1).

[0061] The constructed recombinant plasmid 1 was transformed into the recombinant B. subtilis 168 with integrated agrB and agrC. The obtained recombinant Bacillus subtilis was cultured in LB medium at 37°C. Nearly 200 single colonies showing green fluorescence were picked out and cultured in a 96-well deep-well plate at 37°C and 300 rpm for 24 hours. The relative fluorescence intensity was measured. The results are shown in Figure 2. Figure 3 and as shown in Table 5. 15 transformants with high expression intensity were obtained by preliminary screening, and the transformants were transferred to small test tubes for further culture overnight and the fluorescence intensity was detected again.

[0062] The results are as follows Figure 3 As shown in Table 6, 7 transformants with higher expression were selected from this group and transferred to large test tubes again, cultured for 12 hours, and the relative fluorescence intensity was measured at 4h, 6h, 8h, 10h, and 12h. Figure 3As shown in Table 7, the gene expression intensity mediated by P3 in the 1-B11 and 1-E2 mutants varied significantly, suggesting that signal output intensity was regulated. Furthermore, the temporal dynamics of expression were delayed to varying degrees, altering the expression dynamics of the AgrQS autoinduction system, enabling spontaneous induction under high-density conditions.

[0063] Figure 4 This was the first screening of transformants from the co-regulation system for agrD and agrA expression levels. In Example 3, we investigated the impact of co-regulation of the agrD and agrA dual-gene expression system on system output performance. As an initial screening in deep-well plates, we obtained a series of dual-gene combinations with significantly different activation levels.

[0064] Figure 5 This is the second screening of transformants of the co-regulatory system of agrD and agrA expression levels; the above combinations were screened and 15 combination expressions with potential functions that meet the requirements were obtained, and their levels of action were different.

[0065] Figure 6 The goal was to validate the effect of co-regulation of agrD and agrA expression levels on the temporal dynamics of the AgrQS system. The 15 combinations described above were screened, and six were identified for further scale-up in Erlenmeyer flasks. The temporal dynamics of expression in these combinations were determined. The results showed that co-regulating the expression of both genes could affect the output expression level of the AgrQS system and delay the activation of the system from the original 4 hours after inoculation to 6-8 hours.

[0066] The high expression mutants 1-B11 and 1-E2 obtained by stepwise expansion culture were sequenced and identified to obtain their RBS sequences (Table 8). Table 4 RBS mutation primers for agrA and agrD

[0067]

[0068] Note: B in the table indicates a degenerate sequence containing C, G, and T; D indicates a degenerate sequence containing A, G, and T; V indicates a degenerate sequence containing A, C, and G; H indicates a degenerate sequence containing A, C, and T; S indicates a degenerate sequence containing C and G; R indicates a degenerate sequence containing A and G; Y indicates a degenerate sequence containing C and T; and M indicates a degenerate sequence containing A and C.

[0069] Table 5 Total fluorescence intensity of 15 transformants screened out by 96-well plate culture after overnight culture

[0070] Mutant strain name Relative fluorescence intensity (au / OD600) 1-B11 2118858 1-D8 2045687 1-E2 2010658 1-A6 1506717 1-A5 1502365 1-D12 1033677 1-B12 1030405 2-A4 618066.8 1-F3 464575.6 1-C10 276093.5 2-A1 268596.3 2-A3 148038 1-F10 144219.1 2-A10 95670.05 1-D1 89727.85

[0071] Table 6 Total fluorescence intensity of 15 transformants screened by micro-test tube culture and WT after overnight culture

[0072] Mutant strain name Relative fluorescence intensity (au / OD600) 1-B11 1781932 WT 1731664 1-E2 1664551 1-A5 1663916 1-D8 1301642 2-A4 1295082 1-D12 1285453 1-B12 1213249 1-A6 1095091 1-F3 736618.1 2-A3 666544.1 1-C10 626347.6 2-A1 606305.8 1-D1 303000.8 2-A10 295987.8 1-F10 213779.5

[0073] Table 7 Total fluorescence intensity of 7 transformants screened out by large test tube culture and WT culture

[0074]

[0075] Note: WT refers to the transformant obtained by transformation of the system constructed in Example 1

[0076] Table 8 RBS sequences of high expression mutants

[0077]

[0078]

[0079] Example 4. Bacillus subtilis autoinducible gene expression system with two-component quorum sensing element

[0080] 1. Using a plasmid containing the agr quorum sensing system (agrQS) as a template, the agrB and agrC elements were amplified and cloned into the pAX01 vector. The agrB and agrC elements were then transformed into B. subtilis 168 and integrated into the lacA site of the Bacillus subtilis genome to obtain recombinant Bacillus subtilis BsBC with agrB and agrC integrated at the lacA site.

[0081] 2. In the Agr system, the gene agrD encoding the precursor of the signal molecule AIP is placed downstream of the constitutive promoter Pveg, and agrA is placed downstream of the constitutive promoter P43. The P3 target promoter regulates the downstream gene to be expressed and is cloned into the pHT01 vector to construct the recombinant plasmid pHT-max-DA-P3-sfGFP (the protein to be expressed is replaced with sfGFP to achieve high expression).

[0082] 3.1 Using pHT-max-DA-P3-sfGFP as a template, reverse amplification was performed with primers B11D-F / R to obtain a recombinant plasmid containing the RBS (SEQ ID NO. 16) expressing agrD in the 1-B11 mutant. This was then transformed into Escherichia coli for amplification. After plasmid extraction, primers B11A-F / R were used as a template to obtain a plasmid containing the RBS shown in SEQ ID NO. 18. The recombinant plasmid was obtained after digestion with DpnI to remove the template.

[0083] The recombinant plasmid was transformed into recombinant Bacillus subtilis BsBC harboring agrB and agrC, plated on LB plates containing ampicillin, and positive transformants were screened by colony PCR. (Sequences for P3, P43, Pveg, agrA, agrB, agrC, agrD, and sfGFP are shown in Table 1).

[0084] Table 9 Clone 1-B11 mutant recombinant plasmid cloning primers

[0085]

[0086] Alternatively, in step 3.2, reverse amplification was performed using pHT-max-DA-P3-sfGFP as a template and primers E2D-F / R to obtain a recombinant plasmid containing the RBS (SEQ ID NO. 17) expressing agrD in the 1-E2 mutant. This was then transformed into E. coli for amplification. The plasmid was extracted and used as a template using primers E2A-F / R to obtain a plasmid containing the RBS shown in SEQ ID NO. 19. The recombinant plasmid was obtained after digestion with DpnI to remove the template.

[0087] The recombinant plasmid was transformed into recombinant Bacillus subtilis BsBC harboring agrB and agrC. The plates were then plated with ampicillin and positive transformants were screened by colony PCR. (Sequences for P3, P43, Pveg, agrA, agrB, agrC, agrD, and sfGFP are shown in Table 1.)

[0088] Table 10 Primers for cloning recombinant plasmids of 1-E2 mutant strains

[0089]

Claims

1. An expression system for an autoinducing gene, characterized in that: The invention relates to a microbial cell containing an expression element of an autoinducing gene, wherein the expression element is composed of constitutive promoters Pveg and P43 and target promoter P3, RBS sequences of agrA and agrD genes, a gene encoding a response regulator protein agrA, a gene encoding a signal molecule AIP precursor agrD, a gene encoding agrB, a gene encoding a signal processing protein, and agrC, a gene encoding a sensor protein that binds to a signal molecule and initiates signal transduction; the microbial cell is Bacillus subtilis; On the pHT01 vector, the constitutive promoter P43 is linked to the RBS sequence of the agrA gene and the agrA gene in sequence, the P3 promoter is linked to the multiple cloning site, and the constitutive promoter Pveg is linked to the RBS sequence of the agrD gene and the agrD gene in sequence. The constitutive promoter Pveg is placed upstream of the P43 promoter. The agrB gene and agrC gene were integrated into the lacA locus of Bacillus subtilis; The RBS sequence of the agrD gene is shown as SEQ ID NO.16 or SEQ ID NO.17; the RBS sequence of the agrA gene is shown as SEQ ID NO.18 or SEQ ID NO.

19.

2. Application of the expression system according to claim 1 in the fields of food, chemical industry and pharmaceuticals.

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