Engineering strain for synthesizing gamma-poly-L-glutamic acid and application thereof

Abstract

The invention discloses a gamma-PGA synthetase gene cluster pgsBCA '. The gamma-PGA synthetase gene cluster pgsBCA' is formed by sequentially connecting genes pgsB, pgsC and pgsA 'in series, the genes pgsB and pgsC are derived from a gamma-PGA synthetase gene cluster BA-pgsBCA of the bacillus altitudinis; the gene pgsA'is derived from a gamma-PGA synthetase gene cluster BM-pgsB 'C' A 'of bacillus megatherium. Bacillus amyloliquefaciens CF of which a genome knockout polyglutamate synthetase gene cluster is used as a host, and a recombinant polyglutamate synthetase gene cluster pgsBCA'is subjected to free expression through plasmids, so that a gene engineering strain capable of synthesizing gamma-L-PGA is obtained. When the strain is used for producing gamma-L-PGA in a 5L fermentation tank through amplification fermentation, the maximum yield can reach 10.5 g / L. Besides, the gamma-poly-L-glutamic acid is free of cytotoxicity and low in immunogenicity, so that the gamma-poly-L-glutamic acid has a wide application prospect in the fields of preparation of wound dressings, in-vivo absorption materials and the like. The invention provides a new material basis and technical support for application and development of polyglutamic acid in the fields of medicine and related biomedicine.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation synthesis of polyglutamic acid technology, specifically relating to an engineered strain for synthesizing γ-poly-L-glutamic acid and its application. Background Technology

[0002] Monomer configuration is a key factor influencing the functional properties and applications of polymers. Different monomer configurations can significantly alter the chemical properties, biological behavior, and application areas of polymers. Therefore, the regulation of monomer configuration is of great significance for the development of diversified polymer products.

[0003] Taking polylactic acid as an example, poly-L-lactic acid (PLLA) has good biocompatibility and biodegradability, and has been commercially produced. It is widely used as a drug carrier and as a synthetic medical material that can be absorbed by the body (such as medical sutures). Due to its mechanical properties, racemic polylactic acid (PDLLA) is mainly used in the field of engineering plastics. Poly-D-lactic acid (PDLA) is mainly used to prepare drug carriers and medical devices.

[0004] For polyglutamic acid biopolymers, different monomer configurations significantly affect their biocompatibility, degradation behavior, and application areas. Among them, γ-poly-L-glutamic acid (γ-L-PGA), due to its degradation product being L-glutamic acid, exhibits good biocompatibility and low immunogenicity, and shows promising application prospects in areas such as drug carriers, bioadhesives, and medical materials.

[0005] Currently, γ-polyglutamic acid (γ-PGA) is mainly prepared through fermentation by Bacillus microorganisms. However, the γ-PGA obtained by existing fermentation processes is usually dominated by D-glutamic acid monomers, with D-glutamic acid content generally accounting for 60% to 90%. This is insufficient to meet the application requirements for high-proportion or high-purity γ-L-PGA, thus limiting the further development of γ-PGA in the field of medical materials to some extent.

[0006] Currently, there are relatively few microbial strains capable of synthesizing γ-L-PGA, and most of them originate from extremely halophilic or alkaliphilic archaea. These microorganisms generally suffer from harsh culture conditions, long growth cycles, and poor fermentation stability, which are unfavorable for industrial production and large-scale application. Therefore, how to obtain a production system that can stably and efficiently synthesize γ-L-PGA and is suitable for conventional culture conditions remains a pressing technical problem to be solved in this field.

[0007] Although patent CN 113234764 A reports the heterologous synthesis of 2% D-glu configuration γ-PGA in Corynebacterium glutamicum, the synthesis of γ-L-PGA has not yet been achieved, and the yield is low, indicating that it is still some distance away from industrial-scale production.

[0008] Therefore, in order to achieve efficient and controllable synthesis of γ-L-PGA, it is necessary to develop an engineered strain capable of synthesizing γ-L-PGA. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a γ-PGA synthase gene cluster. pgsBCA′ .

[0010] Another technical problem to be solved by the present invention is to provide a gene cluster containing the γ-PGA synthase. pgsBCA′ Recombinant expression vectors or expression cassettes.

[0011] A further technical problem to be solved by the present invention is to provide a gene cluster containing the aforementioned γ-PGA synthase. pgsBCA′ Recombinant strains of recombinant expression vectors or expression cassettes.

[0012] The final technical problem to be solved by this invention is to provide the aforementioned gene cluster containing the γ-PGA synthase. pgsBCA′ Application of recombinant strains with recombinant expression vectors or expression cassettes in the production of γ-poly-L-glutamic acid.

[0013] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0014] The first aspect of this invention provides a γ-PGA synthase gene cluster. pgsBCA′ The gene cluster pgsBCA′ From genes pgsB, pgsC and pgsA′ The genes are connected in series; pgsB and genes pgsC γ-PGA synthase gene cluster derived from Bacillus altissima BA-pgsBCA The gene pgsA′ γ-PGA synthase gene cluster derived from Bacillus megaterium BM-pgsB′C′A′ .

[0015] In some embodiments, the Bacillus hygroscopicus is Bacillus altitudinis CT11; the Bacillus megaterium is Bacillus megaterium 2a-20.

[0016] In some embodiments, the gene pgsB The nucleotide sequence of the gene is shown in SEQ ID NO:1; pgsC The nucleotide sequence is shown in SEQ ID NO:2; the gene pgsA′ The nucleotide sequence is shown in SEQ ID NO:3.

[0017] In some embodiments, the γ-PGA synthase gene cluster pgsBCA′The nucleotide sequence is shown in SEQ ID NO:4.

[0018] A second aspect of the present invention provides a gene cluster containing the γ-PGA synthase. pgsBCA′ The expression cassette or recombinant expression vector.

[0019] In some embodiments, the starting vector of the recombinant expression vector is plasmid Phy300PLK.

[0020] In some embodiments, the promoter of the expression cassette is Pphy, whose nucleotide sequence is shown in SEQ ID NO:5; and the terminator of the expression cassette is Tamy, whose nucleotide sequence is shown in SEQ ID NO:6.

[0021] A third aspect of the present invention provides a gene cluster containing the aforementioned γ-PGA synthase. pgsBCA′ Recombinant strains of recombinant expression vectors or expression cassettes.

[0022] In some embodiments, the originating strain of the recombinant strain is Bacillus amyloliquefaciens.

[0023] In some embodiments, the *Bacillus amyloliquefaciens* has the γ-PGA synthase gene cluster knocked out in its genome. PgsBCA Polysaccharide synthesis operon genes epsA-O and the gene encoding fructooligosaccharide synthase sacB It was named Bacillus amyloliquefaciens CF.

[0024] The Bacillus amyloliquefaciens CF was obtained by knocking out the Bacillus amyloliquefaciens NF genome. epsA-O Gene clusters and sacB The gene was constructed, and the specific construction method has been disclosed in patent CN 116004496 A; the Bacillus amyloliquefaciens NF was obtained by knocking out the γ-polyglutamate synthase encoding gene in the Bacillus amyloliquefaciens NX-2S genome. PgsBCA It was constructed. Bacillus amyloliquefaciens NX-2S has been disclosed in patent CN 106047780 A and was deposited at the China Center for Type Culture Collection on June 23, 2016, with accession number CCTCC No: M 2016346.

[0025] The fourth aspect of the present invention provides the use of the recombinant strain in the production of γ-poly-L-glutamic acid.

[0026] The γ-poly-L-glutamic acid (γ-L-PGA) is a polyamino acid polymerized solely from L-glutamic acid monomers via γ-amide bonds.

[0027] In some embodiments, the method for producing the γ-poly-L-glutamic acid using the recombinant strain includes the following steps: inoculating the recombinant strain into a fermentation medium for fermentation, and separating and purifying the fermentation broth to obtain the γ-poly-L-glutamic acid.

[0028] In some embodiments, the recombinant strain is inoculated into the fermentation medium in the form of a seed culture; the inoculation amount of the seed culture is 1 to 8% v / v.

[0029] In some embodiments, the seed culture is prepared by: inoculating the recombinant strain into LB medium and culturing at 30-37°C and 180-220 rpm for 10-14 h to obtain the seed culture; or, inoculating the recombinant strain into LB medium and culturing at 30-37°C and 180-220 rpm for 10-14 h to obtain the primary seed culture, and inoculating the primary seed culture into the secondary seed medium at an inoculation rate of 1-5% v / v and culturing at 30-37°C and 180-220 rpm for 6-8 h to obtain the seed culture.

[0030] In some embodiments, the fermentation medium and the secondary seed medium comprise glucose 20-80 g / L, L-glutamate sodium 10-80 g / L, ammonium sulfate 5-20 g / L, dipotassium hydrogen phosphate 1-20 g / L, potassium dihydrogen phosphate 0.05-2 g / L, calcium chloride 0.05-2 g / L, zinc sulfate 0-2 g / L, citric acid monohydrate 0.5-5 g / L, and tetracycline hydrochloride 10-50 μg / mL.

[0031] In some embodiments, the fermentation is carried out under the following conditions: a temperature of 30 to 37°C and a stirring speed of 180 to 500 rpm for 48 to 72 hours.

[0032] In some embodiments, the fermentation conditions are as follows: the temperature is 37°C, the stirring speed is 200 rpm for the first 6 hours of fermentation, the stirring speed is gradually increased to 500 rpm during the subsequent fermentation process, the dissolved oxygen in the fermentation medium is not less than 20%, and the fermentation time is 72 hours.

[0033] Beneficial effects:

[0034] This invention uses *Bacillus amyloliquefaciens* CF, a strain whose genome has been knocked out of the polyglutamate synthase gene cluster, as a host, and expresses the recombinant polyglutamate synthase gene cluster in free plasmid. pgsBCA′A genetically engineered bacterial strain capable of synthesizing γ-L-PGA was obtained. Using this strain, the fermentation production of γ-L-PGA was scaled up in a 5 L fermenter, achieving a maximum yield of 10.5 g / L. Further cellular immunoassay experiments revealed that γ-L-PGA exhibited high cellular activity and induced a low level of the inflammatory factor TAF-α. This invention demonstrates the successful and efficient synthesis of γ-L-PGA from Bacillus amyloliquefaciens, and studies have shown that it exhibits a low immune response, showing broad application prospects in the preparation of wound dressings and in vivo absorbable materials. This will provide a solid foundation for the future application of γ-L-PGA in the food and medical device fields. Attached Figure Description

[0035] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0036] Figure 1 This is a linear schematic diagram of the recombinant expression vector Phy300-Pphy-pgsBCA′-Tamy in Example 1.

[0037] Figure 2 The results are obtained from the liquid chromatography detection of the configuration of γ-PGA and γ-polyglutamic acid standards synthesized by the recombinant strain CF-Phy300-Pphy-pgsBCA′-Tamy in Example 2.

[0038] Figure 3 The yield and biomass of γ-L-PGA synthesized by the recombinant strain CF-Phy300-Pphy-pgsBCA′-Tamy in Example 3 were measured in a fermenter.

[0039] Figure 4 The results in Example 4 show the cell activity and TNF-α content after administering different configurations of polyglutamic acid or different chiral glutamic acid monomers to RAW246.7 cells. Detailed Implementation

[0040] The present invention will be further described below with reference to the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0041] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials, such as genome extraction, PCR enzymes, plasmid extraction kits, gel extraction kits, restriction endonucleases, one-step cloning ligases, etc., were all purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0042] In the following examples, the plasmid Phy300PLK was purchased from General Biotechnology (Anhui) Co., Ltd.

[0043] Example 1: Construction of an engineered strain for synthesizing γ-poly-L-glutamic acid

[0044] The gene cluster encoding γ-polyglutamate synthase containing nucleotide sequences as shown in SEQ ID NO:7 BA- pgsBCA (Derived from Bacillus alpineus) Bacillus altitudinis Using CT11 plasmid as a template, high-fidelity DNA polymerase and primers BC-Gaodi-F / R were used for amplification. pgsBC The fragments were purified using a column purification kit. The PCR product was then purified using a gene cluster encoding γ-polyglutamate synthase, containing the nucleotide sequence shown in SEQ ID NO:8. BM-pgsB′C′A′ (Derived from Bacillus megaterium) Bacillus megaterium Using plasmid 2a-20 as a template, high-fidelity DNA polymerase and primer A giant-F / R were used for amplification. pgsA′ The fragments were then purified using a column purification kit. Further, the recovered fragments were purified by overlap PCR using primers BC Highland-F / A Giant-R. pgsBC and pgsA′ Ligation was performed to obtain the recombinant polyglutamate synthase gene cluster. pgsBCA′ .

[0045] Using the *Bacillus amyloliquefaciens* genome as a template, the promoter Pphy fragment was obtained by PCR amplification using primers Pphy-F / R, and its nucleotide sequence is shown in SEQ ID NO:5. Using a laboratory-preserved plasmid as a template, the terminator Tamy was obtained by PCR amplification using primers Tamy-F / R, and its nucleotide sequence is shown in SEQ ID NO:6. The promoter Pphy and terminator Tamy were inserted into plasmid Phy300PLK using enzyme digestion and homologous recombination to obtain plasmid Phy300-Pphy-XhoI-NotI-Tamy. The plasmid was further digested with XhoI / NotI enzymes, and the product was purified using a column purification kit to obtain the linearized expression plasmid backbone.

[0046] Using homologous recombinases, the linearized expression plasmid backbone was further integrated with the recombinant polyglutamate synthase gene cluster. pgsBCA′ Perform recombination and connection, then transform into E.coli In DH5α competent cells, the cells were spread onto LB agar plates containing antibiotics. Single colonies were picked and plasmids were extracted. PCR verification was performed, and the samples were then sent to General Biotechnology (Anhui) Co., Ltd. for sequencing verification. Sequencing was successful, yielding the recombinant expression vector Phy300-Pphy-pgsBCA′-Tamy. A linear schematic diagram of the recombinant expression vector is shown below. Figure 1As shown, the engineered strain CF-Phy300-Pphy-pgsBCA′-Tamy was constructed by demethylating and transforming it into competent Bacillus amyloliquefaciens CF. The method for introducing the recombinant expression vector Phy300-Pphy-pgsBCA′-Tamy into Bacillus amyloliquefaciens CF to construct the engineered strain is the same as that in Example 3 of Patent CN 119752752 A.

[0047] The nucleotide sequences of the primers used in this embodiment are shown in Table 1.

[0048] Table 1 Primer Sequences

[0049]

[0050] Example 2: Fermentation experiment of engineered strain CF-Phy300-Pphy-pgsBCA-Tamy

[0051] The engineered strain CF-Phy300-Pphy-pgsBCA′-Tamy obtained in Example 1 was transferred to LB medium containing 10-50 μg / mL tetracycline hydrochloride and cultured overnight at 37°C and 220 rpm for 12 h. Inoculation was then carried out at a rate of 5% v / v into 500 mL Erlenmeyer flasks containing 100 mL of fermentation medium and cultured at 32°C and 200 rpm for 72 h. After fermentation, polyglutamic acid was isolated and purified from the fermentation broth according to the method described in patent CN 119752752 A. The ratio of D / L-glutamic acid monomers in the γ-polyglutamic acid synthesized by the engineered strain CF-Phy300-Pphy-pgsBCA′-Tamy and in the γ-polyglutamic acid standard (Control, where the molar percentages of D-glutamic acid and L-glutamic acid are both 50%) was determined. The liquid chromatography results of the polyglutamic acid configuration are shown below. Figure 2 As shown, the engineered strain CF-Phy300-Pphy-pgsBCA′-Tamy synthesizes γ-PGA with a D-glutamic acid molar ratio of 0%, therefore it synthesizes γ-L-PGA.

[0052] The fermentation medium has the following formula: 50 g / L glucose, 40 g / L sodium glutamate, 5 g / L ammonium sulfate, 5 g / L dipotassium hydrogen phosphate trihydrate, 1 g / L potassium dihydrogen phosphate, 1 g / L calcium chloride, 1.5 g / L zinc sulfate, 1 g / L citric acid monohydrate, 25 μg / mL tetracycline hydrochloride, pH=7.

[0053] Example 3: Scale-up Fermentation Production in Fermentation Tanks

[0054] The engineered strain CF-Phy300-Pphy-pgsBCA′-Tamy was inoculated into primary seed medium and cultured at 37℃ and 200 rpm for 12 h to obtain primary seed culture. The primary seed culture was then inoculated into secondary seed medium at a rate of 5% v / v and cultured at 37℃ and 200 rpm for 7 h to obtain secondary seed culture. The cultured secondary seed culture was then inoculated into a 5 L fermenter containing 3 L of fermentation medium at a rate of 8% v / v. The pH of the fermentation medium was adjusted to 6.4–7.5 with 2 M sodium hydroxide solution. Fermentation conditions were 37℃, aeration rate of 1.5 vvm, initial fermentation time of 0–6 h, agitator speed of 200 rpm gradually increased to 500 rpm, dissolved oxygen content maintained above 20%, and samples taken periodically. After 72 h of fermentation, the fermentation broth was transferred to the fermentation tank. During fermentation, the contents of residual sugar, residual monosodium glutamate, and γ-L-PGA, as well as the biomass OD, were measured every 12 h. 600 .

[0055] Test results as follows Figure 3 As shown, the biomass OD in the fermentation broth 600 Rapid growth was observed within the range of 12 to 48 hours; γ-L-PGA in the fermentation broth maintained an increasing trend within the range of 12 to 60 hours, and the growth slowed down after 60 hours; the yield of γ-L-PGA reached its maximum at 72 hours, at 10.50 g / L.

[0056] The primary seed culture medium is LB medium.

[0057] The secondary seed culture medium has the following formula: glucose 50 g / L, sodium glutamate 20 g / L, ammonium sulfate 5 g / L, dipotassium hydrogen phosphate trihydrate 5 g / L, potassium dihydrogen phosphate 1 g / L, calcium chloride 1 g / L, zinc sulfate 1.5 g / L, citric acid monohydrate 1 g / L, pH=7.

[0058] The fermentation medium was formulated as follows: 50 g / L glucose, 50 g / L sodium L-glutamate, 5 g / L ammonium sulfate, 5 g / L dipotassium hydrogen phosphate trihydrate, 1 g / L potassium dihydrogen phosphate, 1 g / L calcium chloride, 1.5 g / L zinc sulfate, 1 g / L citric acid monohydrate, 25 μg / mL tetracycline hydrochloride, pH=7.

[0059] Example 4: Cellular immunological evaluation of γ-L-PGA

[0060] The complete culture medium used in the following experiments was Dulbecco modified Eagle's medium (DMEM), supplemented with 10% (v / v) fetal bovine serum (FBS), 100 μg / mL penicillin, and 100 μg / mL streptomycin. Raw264.7 cells were cultured at a constant temperature of 37°C and 5% CO2.

[0061] (1) Cell viability detection

[0062] Raw264.7 cells were seeded in 96-well plates at a density of 1.0 × 10⁶ cells / well. 4 Raw264.7 cells were cultured at 100 cells / well for 24 h until cell adhesion was achieved, then the culture medium was replaced with fresh complete medium. The adhered Raw264.7 cells were divided into four groups: γ-L-PGA, γ-PGA, D-glutamate monomer, L-glutamate monomer, blank control, and lipopolysaccharide (LPS) group (positive control). PBS-dissolved γ-L-PGA, 80 mol% D-glutamate γ-PGA (hereinafter referred to as conventional γ-PGA), D-glutamate monomer, and L-glutamate monomer were added to the γ-L-PGA, γ-PGA, D-glutamate monomer, and L-glutamate monomer groups, respectively. The final concentrations of γ-L-PGA and conventional γ-PGA in the complete medium were 25, 50, 100, 250, and 500 μg / mL, respectively, and the final concentrations of D-glutamate monomer and L-glutamate monomer in the complete medium were 25, 50, and 100 μg / mL, respectively. Cells in the blank control group were not treated in any way. Lipopolysaccharide (LPS) was added to Raw264.7 cells in the LPS group at a final concentration of 1 μg / mL.

[0063] After culturing Raw264.7 cells for 24 h, the cells were washed with PBS, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37 °C for 1 h. The absorbance was measured at 450 nm, and the cell viability of each group relative to the blank control group was calculated.

[0064] Experimental results are as follows Figure 4 As shown in a, 4b, and 4c, neither D-glutamate nor L-glutamate monomers exhibited significant cytotoxicity within the tested concentration range. Figure 4 a). Meanwhile, neither γ-L-PGA nor the conventional γ-PGA formulation produced significant cytotoxic effects on Raw264.7 cells overall. Notably, the γ-L-PGA treatment group exhibited the highest cell survival rate at a concentration of 25 μg / mL, reaching 197.46% ( Figure 4 b), significantly higher than the conventional configuration γ-PGA under the same conditions ( Figure 4c) indicates that γ-L-PGA has better biocompatibility with cells and can promote cell proliferation.

[0065] The conventional configuration of γ-PGA mentioned above was purchased from Nanjing Xuankai Biotechnology Co., Ltd., catalog number 25513-46-6.

[0066] (2) Detection of tumor necrosis factor α (TNF-α)

[0067] Raw264.7 cells were seeded in 24-well plates and cultured for 24 h. Then, γ-L-PGA, conventional γ-PGA (80% D-glutamate molar content), and LPS were added for treatment. The final concentrations of γ-L-PGA and conventional γ-PGA were 50 μg / mL, and the final concentration of LPS was 1 μg / mL. After 24 h of treatment, the cell culture supernatant was collected, and the TNF-α detection kit was used for analysis according to the instructions.

[0068] TNF-α detection results are as follows Figure 4 As shown in Figure d, different configurations of γ-PGA can induce a certain degree of inflammatory response in Raw264.7 cells, with a similar trend to that of LPS. However, at the same drug concentration, the TNF-α secretion level induced by γ-L-PGA was significantly lower than that of conventional γ-PGA. These results indicate that γ-L-PGA has lower immunostimulatory activity and demonstrates higher safety and application potential in medical materials and related biomedical applications.

[0069] This invention provides a concept and method for a polyglutamate synthase mutant and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A gene cluster of γ-PGA synthase pgsBCA′ Its characteristics are, The gene cluster pgsBCA′ From genes pgsB, pgsC and pgsA′ The genes are connected in series; pgsB and genes pgsC γ-PGA synthase gene cluster derived from Bacillus altissima BA-pgsBCA The gene pgsA′ γ-PGA synthase gene cluster derived from Bacillus megaterium BM-pgsB′ C′A′ .

2. The γ-PGA synthase gene cluster according to claim 1 pgsBCA′ Its characteristics are, The gene pgsB The nucleotide sequence of the gene is shown in SEQ ID NO:1; pgsC The nucleotide sequence is shown in SEQ ID NO:2; the gene pgsA′ The nucleotide sequence is shown in SEQ ID NO:

3.

3. The γ-PGA synthase gene cluster according to claim 1 pgsBCA′ Its characteristics are, The γ-PGA synthase gene cluster pgsBCA′ The nucleotide sequence is shown in SEQ ID NO:

4.

4. A gene cluster containing the γ-PGA synthase as described in any one of claims 1 to 3. pgsBCA′ Recombinant expression vectors or expression cassettes.

5. The expression cassette or recombinant expression vector according to claim 4, characterized in that, The starting vector for the recombinant expression vector is plasmid Phy300PLK.

6. A recombinant strain containing the expression cassette or recombinant expression vector as described in any one of claims 4 to 5.

7. The recombinant strain according to claim 6, characterized in that, The recombinant strain originated from Bacillus amyloliquefaciens.

8. The recombinant strain according to claim 7, characterized in that, The aforementioned Bacillus amyloliquefaciens has had the γ-PGA synthase gene cluster knocked out in its genome. PgsBCA Polysaccharide synthesis operon genes epsA-O and the gene encoding fructooligosaccharide synthase sacB .

9. The use of the recombinant strain according to claim 6 in the production of γ-poly-L-glutamic acid.

10. The application according to claim 9, characterized in that, The method for producing the γ-poly-L-glutamic acid using the recombinant strain includes the following steps: inoculating the recombinant strain into a fermentation medium for fermentation, and separating and purifying the fermentation broth to obtain the γ-poly-L-glutamic acid.

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