Recombinant bacillus subtilis for highly expressing target gene and application of recombinant bacillus subtilis

Abstract

The invention relates to recombinant bacillus subtilis for highly expressing a target gene and application of the recombinant bacillus subtilis. The preparation method comprises the following steps: firstly, integrating T7RNA polymerase from bacteriophage to an amyE site of a bacillus subtilis genome, screening out four integration sites, namely ginU, prtG, glpT and sfrAA with the highest expression activity from 28 sites in different regions of the bacillus subtilis genome, and integrating a target gene expression cassette driven by a PT7 promoter and transcriptional suppressor protein LacI at the sites; secondly, carrying out mutant screening on the LacI to screen out three different mutation sites, and synthesizing a large amount of target proteins under the condition of not needing an inducer. Finally, the method is applied to integrated expression of green fluorescent protein, hyaluronidase and hyaluronic acid genomes, and high fluorescent activity, high hyaluronidase activity and stable production of hyaluronic acid are respectively obtained. In the fermentation expression process, antibiotics and inducers do not need to be added, so that the method becomes a powerful tool for synthetic biology, enzyme engineering and related applications.

Description

Technical Field

[0001] The present invention relates to the field of synthetic biotechnology, in particular to a recombinant Bacillus subtilis for highly expressing a target gene and an application thereof. Background Art

[0002] Due to the urgent need for green manufacturing and biomanufacturing, bioengineering has received more and more attention. Many technologies such as genetic engineering, protein engineering and synthetic biology have flourished and gradually matured. Microorganisms play an important role as hosts for gene expression and regulation. In these fields, due to the convenience of plasmid experimental operation and the advantages of high yield, people usually use plasmids as vectors to express genes or pathways. However, there are two major disadvantages of plasmid expression systems. First, plasmids, as non-genomic DNA, usually show poor genetic stability (plasmid loss), resulting in a decrease in the yield of recombinant bacteria in the later stage of fermentation or difficulty in increasing it. In addition, on an industrial production scale, the large-scale use of antibiotics will undoubtedly increase costs.

[0003] Integrating exogenous genes into the host's chromosome for expression can well avoid several disadvantages of plasmid expression. First, genes inserted into the genome can be expressed continuously and stably without any resistance screening. In addition, after the gene is integrated and expressed in the host, it is not necessary to add maintenance plasmids to replicate in the cell, thus significantly reducing the growth and metabolic pressure of the host cell. Moreover, with the rapid development of gene editing technology, more and more gene editing technologies have been developed, such as CRISPR-Cas9, CRISPR-Cpf1, and Transposon-Associated CRISPR-Cas System. These provide diversified and convenient technical strategies for integrated expression. Compared with plasmid free expression, integrated expression shows many advantages, but there is still an obvious disadvantage of insufficient expression. Many scholars integrate the genome through multiple copies and increase the gene dosage to increase the expression. In addition, studies have shown that the expression of genes in the genome depends on the location of the genes on the chromosomes. The T7 protomer expression system derived from bacteriophage is a widely used method for recombinant protein production. It is favored for its direct gene manipulation, high expression level and precise regulation of target gene expression. Integrating the T7 expression system into Bacillus subtilis has practical value and significance. Summary of the invention

[0004] To solve the above problems, the present invention provides a recombinant Bacillus subtilis that highly expresses a target gene and its application. The present invention provides a Bacillus subtilis system that can achieve efficient integration and expression by integrating T7 RNA polymerase in Bacillus subtilis, screening for integration sites, and mutation modification of the transcription inhibitor protein LacI, and uses target genes encoding phospholipase D and hyaluronidase and hyaluronic acid gene clusters for verification, and determines the feasibility of the system for efficient expression of exogenous genes.

[0005] The present invention is achieved through the following technical solutions:

[0006] The first object of the present invention is to provide a recombinant Bacillus subtilis that highly expresses a target gene, wherein a T7 RNA polymerase encoding gene expression cassette is integrated into the amyE site of the Bacillus subtilis host genome, and a P is integrated into the high integration strength site of the host genome. T7 A promoter-driven target gene expression cassette; the high integration strength site is the zinU site, prtG site, glpT site or sfrAA site of the Bacillus subtilis host genome;

[0007] The target gene expression cassette includes a transcription inhibitor protein LacI mutant; the transcription inhibitor protein LacI mutant takes the transcription inhibitor protein LacI with an amino acid sequence as shown in SEQ ID NO.6 as a parent, and mutates one or more amino acids at positions 270, 284 and 297 of the parent.

[0008] In one embodiment of the present invention, the valine at position 270 is mutated to alanine;

[0009] and / or, the proline at position 284 is mutated to alanine;

[0010] and / or, the proline at position 284 is mutated to valine;

[0011] And / or, the glycine at position 297 is mutated to alanine.

[0012] In one embodiment of the present invention, the nucleotide sequence of the amyE site is shown in SEQ ID NO.1;

[0013] And / or, the nucleotide sequence of the zinU site is as shown in SEQ ID NO.2;

[0014] And / or, the nucleotide sequence of the prtG site is shown in SEQ ID NO.3;

[0015] And / or, the nucleotide sequence of the glpT site is shown in SEQ ID NO.4;

[0016] And / or, the nucleotide sequence of the sfrAA site is as shown in SEQ ID NO.5.

[0017] In one embodiment of the present invention, the P T7 The promoter-driven target gene expression cassette is composed of P T7-lac The gene is composed of a promoter, an RBS sequence, a target gene, a transcription inhibitor protein LacI mutant gene and a T7 terminator.

[0018] In one embodiment of the present invention, the P T7-lac The sequence of the promoter is shown in SEQ ID NO.7;

[0019] And / or, the sequence of the RBS sequence is shown in SEQ ID NO.8;

[0020] And / or, the sequence of the T7 terminator is shown as SEQ ID NO.9.

[0021] In one embodiment of the present invention, the Bacillus subtilis host is Bacillus subtilis 168, Bacillus subtilis WB600 or Bacillus subtilis SCK6.

[0022] The second object of the present invention is to provide the application of the recombinant Bacillus subtilis in biosynthesis.

[0023] In one embodiment of the present invention, the synthesized product includes phospholipase D, hyaluronidase or hyaluronic acid.

[0024] In one embodiment of the present invention, the recombinant Bacillus subtilis containing any one of claims 1-6 is cultured.

[0025] In one embodiment of the present invention, the recombinant Bacillus subtilis is cultured in a seed culture medium to obtain a seed solution, and then the seed solution is inoculated into a fermentation culture medium for fermentation production.

[0026] In one embodiment of the present invention, the Bacillus subtilis T7 integrated expression system is used to achieve the integrated expression of phospholipase D from Streptomyces antibioticus, and the enzyme activities at zinU, prtG, glpT, and sfrAA sites can reach 378.88U / mL, 184.26U / mL, 127.60U / mL, and 94.28U / mL, respectively.

[0027] In one embodiment of the present invention, the Bacillus subtilis T7 integrated expression system is used to achieve integrated expression of hyaluronidase from Citrobacterfreundii Cf1, and the enzyme activities at the zinU, prtG, glpT, and sfrAA sites can reach 2.60×10 4 U / mL, 1.99×10 4 U / mL, 1.55×10 4 U / mL, 1.02×10 4 U / mL.

[0028] In one embodiment of the present invention, the Bacillus subtilis T7 integrated expression system is used to achieve integrated expression of the hyaluronic acid gene cluster (hasA from Streptococcus zooepidemicus, tuaD, gtaB, glmS, glmU from Bacillus subtilis), and the hyaluronic acid production at the zinU, prtG, glpT, and sfrAA sites can reach 4.34 g / mL, 3.30 g / mL, 3.06 g / mL, and 2.92 g / mL, respectively.

[0029] Beneficial effects of the present invention:

[0030] (1) The present invention uses Bacillus subtilis as a model strain, integrates the phage-derived T7 expression system into Bacillus subtilis, optimizes the efficient integrated expression system of single copy number at the genome level by integration site screening, and mutates the transcription inhibitor protein LacI, thereby achieving efficient recombinant expression of genetic stability that is not dependent on antibiotics and inducers, and the reporter gene expression intensity reaches 134.48% of the plasmid-type expression. At the same time, the constructed system is used to integrate and express phospholipase D, hyaluronidase and hyaluronic acid gene clusters, and the enzyme activity and expression amount of the obtained integrated strain can reach a level equivalent to or higher than that of the plasmid.

[0031] (2) The highly efficient integrated expression system constructed in the present invention can achieve efficient protein recombinant expression without the addition of antibiotics or inducers, and has significantly superior genetic stability. While reducing fermentation costs, it fundamentally avoids antibiotic residues, antibiotic abuse and pollution problems. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0033] Figure 1 It is a schematic diagram of the design principle of the T7 integrated expression system in the present invention;

[0034] Figure 2 The effect of different genomic integration sites on the reporter gene green fluorescent protein (GFP) in the present invention;

[0035] Figure 3 The GFP expression intensity of the LacI mutant in the present invention (a); the time trend of the fluorescence intensity of the LacI combination mutant (b);

[0036] Figure 4 The present invention is the enzyme activity of integrating the phospholipase D gene into different sites of the Bacillus subtilis genome (a); the enzyme activity of integrating the hyaluronidase gene into different sites of the Bacillus subtilis genome (b); and the yield of integrating the hyaluronic acid gene cluster into different sites of the Bacillus subtilis genome (c). DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0038] The present invention first provides a recombinant Bacillus subtilis that highly expresses a target gene. The recombinant Bacillus subtilis comprises the following modifications: integrating a gene expression cassette of T7 RNA polymerase at the amyE site (as shown in SEQ ID NO.1) of the Bacillus subtilis host genome, and T7 The promoter-driven target gene expression cassette and the transcription inhibitor LacI mutant are integrated into the genome zinU, prtG, glpT or sfrAA site; the target gene expression cassette contains the target gene and the P promoter that activates the expression of the target gene. T7 The promoter, RBS sequence, transcription inhibitor protein LacI and terminator are composed. The transcription inhibitor protein LacI is mutated at the following sites: G297A, V270A, P284A, P284V.

[0039] Furthermore, the genomic zinU, prtG, glpT, and sfrAA sites are shown in SEQ ID NO.2-NO.5.

[0040] Furthermore, the target gene expression cassette and / or the P T7-lac The promoter also contains an RBS sequence; T7-lac The sequences of the promoter and RBS are shown in SEQ ID NO.7-8.

[0041] Furthermore, the gene expression frame of the T7 RNA polymerase is integrated through CRISPR-Cas9 technology, and the editing elements required for integration into the amyE site include gRNA; the sequence of the gRNA is shown in SEQ ID NO.10.

[0042] Furthermore, the Escherichia coli host includes Bacillus subtilis 168, Bacillus subtilis WB600 or Bacillus subtilis SCK6.

[0043] The present invention also constructs the efficient integration expression system of single copy number of genome level. Due to the shortcomings such as extra growth burden, genetic instability and antibiotic pollution in plasmid-mediated microbial fermentation, it is crucial to enhance the expression of exogenous genes at the genome level, so as to realize the efficient recombinant expression of genetic stability that is not dependent on antibiotics. Preferably, the present invention uses Bacillus subtilis WB600 as a model strain, selects 28 integration sites in different regions of Bacillus subtilis chromosome, and by characterizing the expression intensity of these 28 integration sites, digs into four sites with the highest expression activity, zinU, prtG, glpT, sfrAA. On this basis, the T7 expression system of phage origin is integrated into these sites. In addition, transcription inhibitor LacI is mutated to construct an efficient integration expression system without adding inducer and antibiotic. Finally, the system constructed is also used to integrate and express phospholipase D, hyaluronidase and hyaluronic acid gene clusters, and the obtained integrated strain enzyme activity and expression amount can reach the level comparable to plasmid.

[0044] Furthermore, the target gene is a gene encoding an exogenous protein (enzyme), including but not limited to phospholipase D, hyaluronidase, hyaluronic acid gene cluster, etc.

[0045] Furthermore, the sequence of the phospholipase D is shown in SEQ ID NO.11, the sequence of the hyaluronidase is shown in SEQ ID NO.12, and the sequence of the hyaluronic acid gene cluster is shown in SEQ ID NO.13-17.

[0046] The present invention also provides a method for efficiently integrating and expressing a target gene, comprising the step of using the recombinant Bacillus subtilis for fermentation.

[0047] Specifically, the present invention provides a highly efficient integrated T7 expression system of Bacillus subtilis, which combines T7 promoter, efficient genomic integration sites zinU, prtG, glpT, sfrAA, transcription inhibitor protein mutant LacI G297A 、LacI V270A 、LacI P284A 、LacI P284V As well as high integration expression intensity Bacillus subtilis chassis cells, an integrated expression system is constructed to achieve efficient integration expression of Bacillus subtilis.

[0048] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0049] The method of the present invention is as follows:

[0050] (1) Selection of genomic integration sites

[0051] In the present invention, 28 genome integration sites for integration expression were selected in different regions of the Bacillus subtilis genome. In order not to destroy the original metabolic pathway and gene expression of Escherichia coli, these 28 integration sites were selected between the genes of the genome. The integration sites are named after the nearby genes.

[0052] (2) Bacillus subtilis gene editing

[0053] Bacillus subtilis WB600 was used as the starting host strain. CRISPR / Cas9 technology was used to knock out and integrate genes in chromosomes. The gene editing plasmid pJOE8999 was constructed, and the N20 sequence was designed as the target gene (integration site) using the CHOPCHOP tool (https: / / chopchop.cbu.uib.no / ), which was combined with gRNA for targeted cutting. The expression of Cas9 in P man The 500bp fragments upstream and downstream of the genome were used as homologous repair arms. After the plasmid was successfully constructed, it was transformed into the host, and the genome was extracted for PCR to determine whether the target gene was successfully knocked out or integrated. The obtained transformants were picked into LB plates and cultured at 52°C for 24 hours. pJOE8999 was removed to obtain a recombinant strain without exogenous plasmids.

[0054] (3) Integration and expression of green fluorescent protein, phospholipase D, hyaluronidase and hyaluronic acid gene cluster recombinant plasmid

[0055] In the present invention, the green fluorescent protein gene gfp is used as a reporter gene and codon optimization is performed. In addition, the present invention also performs codon optimization and expression on the phospholipase D from Streptomyces antibioticus and the hyaluronidase gene from Citrobacter freundii Cf1. The hyaluronic acid gene cluster (hasA from Streptococcus zooepidemicus, tuaD, gtaB, glmS, glmU from Bacillus subtilis) is expressed in an integrated manner.

[0056] When the strain is activated, a single colony is picked from the plate and inoculated into LB medium, and cultured at 37°C and 220rpm for 10-12h. After the seed solution is prepared, 4% fresh seed solution is taken in the clean bench and inoculated into the fermentation medium (sucrose 70g / L, yeast powder 5g / L, peptone 15g / L, K 2 HPO 4 1.7g / L, MgSO 4 0.65 g / L. Sterilize at 121℃ for 20 min. ), culture in a shaker set at 37℃ and 220 rpm. If induction is required, the induction temperature is 30℃. Culture for 8 hours to express the recombinant protein. After collecting cells from the fermentation broth, mix the fermentation broth supernatant with SDS loading buffer. Boil the sample for 15 min and then use it for SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis.

[0057] (4) Methods for detecting the activity of phospholipase D and hyaluronidase and the production of hyaluronic acid

[0058] The activity of phospholipase D was determined by enzyme-linked colorimetry. The reaction mixture (100 μL) consisted of 60 μL substrate solution (10 mg / mL phospholipid, 0.1% (v / v) Triton X-100, 15 mM CaCl2, 40 mM Tris-HCl (pH 7.5)) and 40 μL enzyme sample. Shake at 60°C for 20 min. Add 50 mM EDTA solution (50 mL) and boil for 5 min to terminate the reaction. After cooling, add 500 μL colorimetric solution (40 mM Tris-HCl, 0.75 U choline oxidase, 0.5 U peroxidase), heat at 37°C for 30 min, and measure the absorbance at 505 nm.

[0059] The relative hyaluronan lyase activity of hyaluronidase was determined by UV absorption. 10 μL of purified enzyme was added to 990 μL of hyaluronic acid solution dissolved in 50 mM citrate buffer (pH 5.5) and incubated at 37°C for 30 min. After incubation, the reaction mixture was boiled in water for two minutes and the absorbance of the sample at 232 nm was quantitatively measured to determine its relative activity.

[0060] Purification of hyaluronic acid samples: Add SDS with a final concentration of 0.1% (w / v) to the fermentation broth, mix it and let it stand for 20 minutes to release HA in the cell capsule, collect the fermentation supernatant by centrifugation, add four times the volume of ethanol and treat it at 4°C for 2-4 hours, centrifuge at 6000rpm for 10 minutes to obtain the alcohol precipitation product, add an equal volume of deionized water, and repeat the above operation after re-dissolving. The yield and molecular weight of the HA sample finally obtained were determined. The Bitter-Muir method was used to detect HA yield. The corresponding glucuronic acid content was calculated according to the standard curve, and the corresponding HA yield was calculated according to the glucuronic acid content. The HA molecular weight was determined by gel permeation chromatography-liquid chromatography (GPC) with 0.1MNaNO 3 The mobile phase was 0.5 mL / min in flow rate, a differential detector (RI) was used, an Ohpak SB-806HQ2.0300 chromatographic column was used, and the column temperature was 25°C; the injection volume was 200 μL each time.

[0061] Example 1: Construction of Bacillus subtilis T7 integrated expression system

[0062] (1) Using Bacillus subtilis WB600 as a host organism to efficiently express foreign proteins. In order to overcome the problem of low transformation efficiency of Bacillus subtilis, the xylose-inducible promoter P xylA The genetic cassette containing the comK gene is integrated into the genome under the control of the T7 integrative expression system of Bacillus subtilis. The T7 RNAP expression module and the T7 promoter-driven expression module are used to express T7 RNAP. 43 The promoter was integrated into the amyE site of Bacillus subtilis WB600 to construct Bacillus subtilis WBT7.

[0063] (2) 28 integration sites were selected on the genome of Bacillus subtilis WBT7. In addition, most of the selected integration sites were located between genes, aiming to preserve the original gene expression and metabolic pathways of the strain without causing damage. The green fluorescent protein gene (GFP) was used as a reporter gene and expressed in the form of a constitutive T7 expression cassette. The expression cassette was inserted into the selected 28 sites using CRISPR-Cas9 genome editing technology ( Figure 1 ). The T7 RNA polymerase driver module is composed of P 43 The promoter is controlled and integrated into the amyE site; the T7 expression module is controlled by P T7-lacPromoter driven, integrated into 28 different sites on the genome for screening; after constructing 28 strains of Bacillus subtilis integrated with GFP, their growth was tested, and the results showed that the genome insertion of the reporter gene expression cassette had no significant effect on cell growth. Subsequently, these integrated strains were cultured to express GFP, and the expression level of Bacillus subtilis integrated and expressed at different sites was characterized by detecting the fluorescence intensity of GFP. Figure 2 It can be seen that the relative fluorescence intensity of the zinU site is comparable to the plasmid expression level, and the relative fluorescence intensity of the integration sites such as srfAA, glpT, and prtG reaches about 60–70% of the plasmid expression level;

[0064] (3) Mutation of specific amino acids of LacI was aimed at changing its conformational effect. By modifying the flexibility of key secondary structural elements of the core domain, gene expression under LacI control was increased without the need for additional inducers. The GFP reporter gene was constructed into the zinU site by alanine scanning mutagenesis of 57 key amino acids involved in the core domain peptide exchange pattern of IPTG-LacI and DNA-LacI states. Even in the absence of inducer, certain mutants such as Tyr287, Asp292, Val270, Val271, Gly272, Pro284 and Gly297 still produced significant levels of green fluorescence. Among them, the fluorescence intensities of mutants G297A, V270A and P284A were relatively high, reaching 134.48%, 89.99% and 81.20% of the wild type under the condition of adding inducer, respectively. Through saturation mutagenesis, it was found that G297A, P284V and V270A had better expression levels ( Figure 3 a). Fluorescence measurements during the fluorescence generation process show that ( Figure 3 b), P284V peaked at the middle of growth, while G297A and V270A showed a lag in fluorescence. G297A-P284V-V270A The mutant showed strong fluorescence throughout the fermentation process without the need for additional inducers. This mutant may provide a basis for the application of the T7 integrated expression system of Bacillus subtilis in industrial production.

[0065] Example 2: Integrative expression of heterologous proteins using the Bacillus subtilis T7 expression system

[0066] In order to confirm the potential utility of the T7 integrative expression system in secretory expression of heterologous proteins, two enzymes, phospholipase D and hyaluronidase, were selected for expression. The coding sequence of the phospholipase D gene (from Streptomyces) was optimized to adapt to the use of Bacillus subtilis codons. T7-lacUnder the regulation of the promoter, it was integrated into the zinU site, and the recombinant strain WB600 / pMA5-PLD was used as a control. The signal peptide penP was added to the gene sequence in front of it. The secretase activity reached 339.12U / mL, and the enzyme activity reached 89.5% of the expression amount of the recombinant plasmid pMA5-PLD (378.88U / mL). In addition, integration sites such as prtG (184.26U / mL), glpT (127.60U / mL) and srfAA (94.28U / mL) also showed high activity.

[0067] Hyaluronan lyase is a special class of hyaluronidases that promotes the cleavage of hyaluronic acid by β-elimination of β-1,4 bonds, thereby producing compounds containing unsaturated D-glucuronic acid at the non-reducing end. The final product is an unsaturated HA disaccharide (ΔHA2). Hyaluronan lyase has important application value in the field of biotechnology and can be used to prepare low molecular weight HA. Hyaluronan lyase (derived from Citrobacter freundii Cf1) was integrated into the zinU site under the control of the PT7-lac promoter, with the recombinant strain WB600 / pP43NMK-hylC as a control. By adding a signal peptide, hyaluronan lyase was integrated into the zinU site. The secretase activity reached 2.60×10 4 U / mL, which was 10.6% higher than the expression level of the recombinant plasmid pP43NMK-hylC (2.35×104U / mL). 4 U / mL), glpT (1.55×10 4 U / mL) and srfAA (1.02×10 4 U / mL) and other integration sites showed significant enzyme activity.

[0068] The hyaluronic acid gene cluster (hasA from Streptococcus zooepidemicus, tuaD, gtaB, glmS, glmU from Bacillus subtilis) was expressed in an integrated manner to determine its ability to produce long transcripts from the T7 promoter in Bacillus subtilis and its potential for synthetic biology applications. During shake flask fermentation, HA was successfully produced without IPTG induction, and the fermentation broth had a certain viscosity. The HA titer was highest at 40h of fermentation. After the operon was integrated at the zinU site, its yield was 4.34g / L, which was 40.9% higher than the yield of the pP43NMK expression system (3.08g / L). The integration amounts of the operon at the prtG, glpT and srfAA sites were 3.30g / L, 3.06g / L and 2.92g / L, respectively. Its industrial production potential was further explored at a 5L fermentation scale, and the HA titer obtained was 6.86g / L.

[0069] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A recombinant Bacillus subtilis that highly expresses a target gene, characterized in that: The T7 RNA polymerase encoding gene expression cassette was integrated into the amyE locus of the Bacillus subtilis host genome, and the P T7 Promoter-driven target gene expression cassette; The high integration strength site is the zinU site, prtG site, glpT site or sfrAA site of the Bacillus subtilis host genome; The target gene expression cassette includes a transcription inhibitor protein LacI mutant; the transcription inhibitor protein LacI mutant takes the transcription inhibitor protein LacI with an amino acid sequence as shown in SEQ ID NO.6 as a parent, and mutates one or more amino acids at positions 270, 284 and 297 of the parent.

2. The recombinant Bacillus subtilis according to claim 1, characterized in that The valine at position 270 is mutated to alanine; and / or, the proline at position 284 is mutated to alanine; and / or, the proline at position 284 is mutated to valine; And / or, the glycine at position 297 is mutated to alanine.

3. The recombinant Bacillus subtilis according to claim 1, characterized in that The nucleotide sequence of the amyE site is shown in SEQ ID NO.1; And / or, the nucleotide sequence of the zinU site is as shown in SEQ ID NO.2; And / or, the nucleotide sequence of the prtG site is shown in SEQ ID NO.3; And / or, the nucleotide sequence of the glpT site is shown in SEQ ID NO.4; And / or, the nucleotide sequence of the sfrAA site is as shown in SEQ ID NO.

5.

4. The recombinant Bacillus subtilis according to claim 1, characterized in that The P T7 The promoter-driven target gene expression cassette is composed of P T7-lac The gene is composed of a promoter, an RBS sequence, a target gene, a transcription inhibitor protein LacI mutant gene and a T7 terminator.

5. The recombinant Bacillus subtilis according to claim 4, characterized in that The P T7-lac The sequence of the promoter is shown in SEQ ID NO.7; And / or, the sequence of the RBS sequence is shown in SEQ ID NO.8; And / or, the sequence of the T7 terminator is shown as SEQ ID NO.

9.

6. The recombinant Bacillus subtilis according to claim 1, characterized in that The Bacillus subtilis host is Bacillus subtilis 168, Bacillus subtilis WB600 or Bacillus subtilis SCK6.

7. Use of the recombinant Bacillus subtilis according to any one of claims 1 to 6 in biosynthesis.

8. The use according to claim 7, characterized in that: The products of synthesis include phospholipase D, hyaluronidase or hyaluronic acid.

9. The use according to claim 7 or 8, characterized in that: The recombinant Bacillus subtilis according to any one of claims 1 to 6 is cultured.

10. The use according to claim 9, characterized in that: The recombinant Bacillus subtilis according to any one of claims 1 to 6 is cultured in a seed culture medium to obtain a seed liquid, and then the seed liquid is inoculated into a fermentation culture medium for fermentation production.

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