CN113151136A

CN113151136A - Strain for producing gamma-DL-PGA and method for synthesizing gamma-PGA with different D/L monomer ratios by using same

Info

Publication number: CN113151136A
Application number: CN202110546483.5A
Authority: CN
Inventors: 徐国强; 许正宏; 朱亚鑫; 王籍阅; 郑璞; 张晓梅; 史劲松
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-05-19
Filing date: 2021-05-19
Publication date: 2021-07-23

Abstract

The invention discloses a strain for producing gamma-DL-PGA and a method for synthesizing gamma-PGA with different D/L monomer ratios by one-step fermentation from sugar raw materials, which is a production process for synthesizing the gamma-PGA with different D/L monomer ratios by using a one-step method of fermentation from sugar raw materials, simultaneously expressing a gene cluster capBCA of gamma-PGA synthetase from bacillus licheniformis and a glutamate racemase gene racE of the bacillus subtilis by using C.glutamicum F343 for high yield of L-Glu as a chassis, and synthesizing the gamma-PGA with the D-Glu accounting for 30-50% by using a constitutive promoter to regulate the expression level of the racE endogenously, thereby realizing rational and accurate regulation of the D/L monomer ratio of the gamma-PGA, constructing a synthesis way of the gamma-PGA with different D/L monomer ratios, and saving raw materials and process control cost.

Description

Strain for producing gamma-DL-PGA and method for synthesizing gamma-PGA with different D/L monomer ratios by using same

Technical Field

The invention belongs to the field of synthetic biology and fermentation engineering, and relates to an application of synthetic biology technology, in particular to a strain for producing gamma-DL-PGA and a method for synthesizing gamma-PGA with different D/L monomer ratios, and four corynebacterium glutamicum strains for producing gamma-polyglutamic acid with different D/L monomer ratios are constructed.

Background

γ -polyglutamic acid (γ -PGA) is an anionic polymer polymerized from L-glutamic acid or/and D-glutamic acid monomers, and can be classified into γ -L-PGA (polymerized from only L-Glu monomers), γ -D-PGA (polymerized from only D-Glu monomers), and γ -DL-PGA (polymerized from two monomers, L-Glu and D-Glu). The gamma-PGA has various important physiological functions, such as moisture retention, biocompatibility, complete biodegradation and the like, and has very important application prospect in the fields of functional foods, cosmetics, biomedicines, biological materials and the like.

The gamma-PGA with different monomer ratios of D-Glu/L-Glu (hereinafter referred to as monomer ratio of D/L) has potential new application value. The gamma-PGA containing the L-Glu monomer with high ratio has the characteristics of low immunogenicity, better biocompatibility, tissue affinity and the like, so the gamma-PGA can be widely used as a humectant, a dispersant, a drug delivery agent, a medical biological adhesive and the like, and meanwhile, the L-Glu formed by degrading the gamma-PGA can promote cell growth and tissue repair without toxic or side effect. The gamma-PGA containing high D-Glu monomer ratio is not easy to degrade, is more stable, edible and nontoxic, and can be widely used as an antifreezing agent, a coagulant, a thickener, a metal adsorbent, a biodegradable material and the like.

At present, the gamma-PGA produced by utilizing the fermentation of bacillus is the main production process, but the commonly obtained gamma-PGA monomer mainly comprises D-Glu, so the development of the application of the gamma-PGA is limited. The gamma-L-PGA is produced by a small number of strains, generally extremely halophilic and alkaliphilic archaea, but the strains are not easily cultured. At different Mn²⁺The stereochemical composition of gamma-PGA can be changed at a concentration such that the proportion of D-Glu is 22%67% by weight. However, this method realizes the stereochemical composition of γ -PGA by changing the culture conditions, and has a limited control range.

The method for regulating the D/L monomer ratio of the gamma-PGA by simultaneously expressing the gamma-polyglutamic acid synthetase gene capBCA of the bacillus licheniformis and the glutamic acid racemase gene racE of the bacillus licheniformis is not reported at present, so that the scheme provided by the invention has universal significance for the research of regulating the D/L monomer ratio of the gamma-PGA.

Disclosure of Invention

The invention aims to solve the technical problem of producing gamma-polyglutamic acid with different D/L monomer ratios by utilizing a glutamic acid-independent strain.

The first object of the invention is to provide a strain producing gamma-DL-PGA, the method for constructing the strain producing gamma-DL-PGA comprises the following steps: using C.glutamicum F343 as a chassis, firstly expressing a gene cluster capBCA of gamma-PGA synthetase from bacillus licheniformis, then utilizing a constitutive promoter to express a glutamate racemase gene racE from bacillus subtilis, and utilizing the constitutive promoter to endogenously regulate the expression level of the racE to construct the strain for producing gamma-DL-PGA.

Further, the promoter comprises e/11/12/16-racE, and the strain for producing the gamma-DL-PGA is correspondingly constructed to be C.glutamcum F343 pZM1-capBCA-e/16/11/12 racE.

Further, the construction method of the strain producing gamma-DL-PGA, namely the cloning and heterologous expression of the gamma-polyglutamic acid synthetase gene and the glutamic acid racemase gene, comprises the following steps: firstly extracting a bacillus licheniformis genome, and respectively amplifying gamma-polyglutamic acid synthetase genes capB, capC and capA through PCR; and extracting a bacillus subtilis genome, amplifying a glutamic acid racemase gene rac E by PCR, obtaining a recombinant plasmid with a gamma-polyglutamic acid synthetase gene cluster and a glutamic acid racemase gene by a coda-ligase connection technology and a promoter engineering, and transforming the recombinant plasmid into a competent cell C.glutamicum F343 to construct the strain for producing the gamma-DL-PGA.

Further, the gamma-polyglutamate synthase gene, in one embodiment of the present invention, is from Bacillus licheniformis, available from ATCC under strain number ATCC 9945 a.

Further, the glutamate racemase gene, in one embodiment of the present invention, is derived from Bacillus subtilis, purchased from ATCC under strain number ATCC 6051-U.

Further, the isocaudarner ligation technique, in one embodiment of the invention, is a novel modular synthetic biology tool, epathBrick, used directly in the pathway.

Further, the operon engineering, in one embodiment of the present invention, is a method for regulating the expression of a downstream gene by replacing a promoter. A promoter is a DNA sequence recognized, bound and initiated by RNA polymerase and contains conserved sequences required for RNA polymerase specific binding and transcription initiation. Different promoters have different specific binding with RNA polymerase, resulting in different amounts of transcribed mRNA, and ultimately affecting enzyme expression levels.

The second object of the present invention is to provide a recombinant plasmid, pZM1-capBCA-e/16/11/12-racE, formed during the construction of the gamma-DL-PGA-producing strain.

The third object of the present invention is to provide a genetically engineered bacterium expressing the γ -DL-PGA-producing strain described above. Namely corynebacterium glutamicum, and the gamma-polyglutamate synthetase gene and the glutamic acid racemase are heterologously expressed.

The underpan cell used in the present invention, namely C.glutamcum F343, also called Corynebacterium glutamicum F343, is an industrial strain with high L-Glu production in one embodiment of the present invention, referred to as Zheng P., Liu M., Liu X.D.et al.genome Shuffling improvements and glutamic acid reduction of microorganism and Biotechnology [ J ]. World Journal of microorganism & Biotechnology,2012,28(3): p.1035-1043. the method is obtained by genetic recombination and the L-Glu production can reach 50 g/L.

It is a fourth object of the present invention to provide a method for synthesizing γ -PGA having different D/L monomer ratios, comprising the preparation step using the above γ -DL-PGA-producing strain, the γ -PGA including γ -L-PGA and γ -D-PGA.

Further, the content of the γ -D-PGA in the γ -PGA is 30% to 60%.

Further, the D/L monomer ratio of the gamma-D-PGA and the gamma-L-PGA is 0.4-1.5: 1.

Further, the preparation method using the gamma-DL-PGA-producing strain comprises the following steps: inoculating the seed solution of the strain producing the gamma-DL-PGA to a fermentation culture medium, culturing for 1-2 h at 32 ℃, adding IPTG (isopropyl-beta-D-thiogalactoside) for induction for 1-2 h, and finally culturing for 72 +/-5 h at 37 ℃.

Further, the preparation method using the γ -DL-PGA-producing strain described above comprises the steps of: the culture medium C.glutamicum F343-pZM1-capBCA-e/16/11/12-racE was pipetted 2-5. mu.L of the culture medium from the cryopreservation tube, streaked on LB-Glu (containing 25mg/L kanamycin) plates, and cultured for 24 hours (30 ℃). Selecting single bacterial colony to be cultured in a seed culture medium repeatedly at 32 ℃ and 120rpm for 12h to obtain seed liquid of the strain producing the gamma-DL-PGA. Inoculating the strain into a fermentation medium according to the inoculation amount of 5%, culturing at 32 +/-2 ℃ and 120rpm for 1-2 h, adding IPTG (isopropyl-beta-thiogalactoside) for induction for 1-2 h, and then adjusting the temperature to 37 ℃ for culturing for 72 h.

The sixth object of the present invention is to provide γ -PGA obtained by the above method.

The seventh object of the present invention is to provide the γ -PGA, which can be used in the fields of food, cosmetics, biomedicine, environmental protection, etc.

The invention has the beneficial effects that: (1) the constructed strain can successfully synthesize gamma-polyglutamic acid with different D/L monomer ratios in a culture medium of a saccharine raw material without changing culture conditions, so that the raw material cost is saved, and the economic benefit is improved; (2) the constructed strain can efficiently synthesize gamma-PGA at a high temperature of 37 ℃, so that the consumption of condensed water in the fermentation process in summer is saved, and the production cost is reduced; (3) the constructed strain is used, and gamma-PGA with the D-Glu ratio of 30-60% is synthesized by utilizing a strategy of endogenously regulating the RacE expression level by utilizing a regulating element (RBS and a constitutive promoter), so that the rational and accurate regulation of the D/L monomer ratio of the gamma-PGA is realized.

Drawings

FIG. 1: the influence of the constitutive promoter to the regulation of the expression of the glutamate racemase on the fermentation characteristics of the gamma-PGA;

FIG. 2: the constitutive promoter regulates the influence of the expression level of the glutamate racemase on the D/L monomer ratio and the transcription level of the gamma-PGA.

Detailed Description

The present invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions and results thereof described in the examples are illustrative only and should not be taken as limiting the invention as detailed in the claims.

The measurement and analysis methods used in the examples of the present invention are as follows:

the method for measuring the yield of the gamma-polyglutamic acid comprises the following steps:

sample treatment: centrifuging the fermentation liquor at 12000rpm for 15min, taking the supernatant, diluting by proper times, filtering by a 0.45 mu m filter membrane, taking 500 mu L of the supernatant in a 2mL sampling bottle to be tested. Using a gel permeation chromatography column: TSKgel super Aw 4000, TSKgel super Aw 5000. Column temperature: the sample size was 50uL at 40 ℃. The mobile phase is 0.2M Na₂SO₄The pH of the mobile phase was adjusted to about 4.0 with glacial acetic acid. The detector is as follows: waters liquid phase RID differential detector.

The method for measuring the content of residual sugar and L-Glu comprises the following steps:

centrifuging the fermentation broth obtained at different time points at 12000rpm for 15min, taking supernatant, diluting to appropriate multiple to make its glucose and L-glutamic acid content value in the range of 0-1.0g/L, and measuring glucose and L-glutamic acid content with biosensor.

D/L monomer ratio determination of γ -PGA:

hydrolysis of γ -PGA: Gamma-PGA was dissolved with 2M HCl to a final concentration of 15mg/mL (15mg sample +1mL hydrochloric acid), dissolved at 40 ℃ and then sterilized in an autoclave at 121 ℃ for 50 min. Then, 1mL of 2mol/L NaOH was added to neutralize HCl, and the pH was adjusted to about 7.0.

Derivatization of amino acids: 50 μ L of L, D-Glu standard solution and hydrolysate were taken, 100 μ L of FDAA solution and 20 μ L of NaHCO3 solution were added, the samples were mixed and activated at 40 ℃ for 1h, then the samples were cooled at room temperature for 5min, and then diluted hydrochloric acid solution was added and mixed. And (4) performing gradient dilution on the standard solution, making a standard curve according to data obtained after the gradient dilution, and analyzing the sample by HPLC.

HPLC analysis: mobile phase: acetonitrile/ammonium acetate solution (5/95, pH 5.3, 50 mM); column: column C8, Waters, 3.5 μm, 2.1X 50 mm; a detector: a 340nm ultraviolet detector; temperature: 35 ℃ and 0.6 mL/min.

Preparing a crude enzyme solution and measuring the enzyme activity of the glutamic acid racemase:

preparation of crude enzyme solution: the fermentation broth was centrifuged at 12000rpm at 4 ℃ to collect the cells, which were washed twice with 50mL of 0.85% physiological saline. The collected cells were suspended in 5mL of a standard buffer (0.1mol of Tris-HCl, pH 8.0), lysozyme was added to the suspension and reacted for 2 hours on ice, the suspension was sonicated in ice bath for 10 minutes, centrifuged at 12000rpm for 15 minutes, and the supernatant (crude enzyme solution) was taken out to immediately determine the enzyme activity.

Determination of glutamate racemase enzyme activity: mu.L of the crude enzyme solution was reacted with 100. mu.L of a substrate (0.5g/L L-Glu) at 37 ℃ for 30min, and the reaction was terminated in a boiling water bath. And (4) measuring the content of the D-glutamic acid in the reaction liquid by HPLC.

The unit enzyme activity U is defined as the amount of D-Glu catalyzed by the enzyme per minute, and U/mg represents the unit of enzyme activity contained in 1mg of total protein. Total protein concentration was determined by Bradford.

Reverse transcription and real-time quantitative PCR:

RNA extraction: total RNA was extracted using Trizol method, which was referred to the Shanghai Producer RNA extraction kit instructions.

Reverse transcription and RT-PCR: the method refers to Shanghai Czeri qPCR premix (SYBR Green I) kit instruction. The reverse transcription primers are racE-RT-F (SEQ ID NO.16) and racE-RT-R (SEQ ID NO. 17).

Seed medium (g/L): corn steep liquor 35, glucose 25, K₂HPO₄ 1.5，MgSO₄ 0.6，FeSO₄·7H₂O 0.005，MnCl₂·4H₂O0.005, urea 2.5 (sterilizing), pH 6.8-7.0 per 25mL of the solution in a 250mL triangular flask, and sterilizing at 121 ℃ for 20 min.

Fermentation medium (g/L): corn steep liquor 10, glucose 120, K₂HPO₄ 1.0，MgSO₄ 0.6，FeSO₄·7H₂O 0.002，MnCl₂4H2O 0.002.002, urea 7.0 (sterile), pH 6.8-7.0, 50mL per 500mL Erlenmeyer flask, and sterilizing at 121 ℃ for 20 min.

Comparative example: the construction method of the expression vector recombinant plasmid F343-pZM1-capBCA and the engineering strain thereof comprises the following steps:

taking genome of B.licheniformis as a template, and respectively taking capB-NdeI-F (SEQ ID NO.1), capB-BamHI-R (SEQ ID NO.2), capC-NdeI-F (SEQ ID NO.3), capC-BamHI-R (SEQ ID NO.4), capA-NdeI-F (SEQ ID NO.5) and capA-BamHI-R (SEQ ID NO.6) as primers, and carrying out PCR amplification on target genes capB, capA and capC fragments with enzyme cutting sites. The digested capB fragment of the target gene and a double-restriction (NdeI, BamHI) linearized vector pZM1 are connected overnight by T4 ligase, transformed into competent cells of Escherichia coli JM109, screened on a Kanamycin (Kan) resistant LB plate containing 25mg/L, and the obtained transformants are subjected to colony PCR verification and restriction enzyme digestion verification to obtain correct transformants, and then elements Ptac-lacO-RBS-capB-T7, Ptac-lacO-RBS-capC-T7 and Ptac-lacO-RBS-capA-T7 are sequentially connected onto a pZM1 plasmid by a tailing enzyme technology to finally obtain a recombinant plasmid pZM 1-capBCCA.

And finally, transforming the plasmid into C.glutamicum F343 by electric shock, picking out a transformant on a resistant plate, and carrying out colony PCR verification by using a pZM1-R, pZM1-F primer to successfully construct a recombinant strain C.glutamicum F343-pZM 1-capBCA.

Example 1: the expression vector recombinant plasmid F343-pZM1-capBCA-e/16/11/12-racE and the construction method of the engineering strain thereof:

based on the sequence of the constitutive promoter, the upstream and downstream primers dap-e-racE-F (SEQ ID NO.7), dap-A-16-1-racE-F (SEQ ID NO.10), dap-e11-racE-F (SEQ ID NO.8), dap-e12-racE-F (SEQ ID NO.9), BamHI-racE-R (SEQ ID NO.12) with AvrII and BamHI cleavage sites were designed. Primers (see table 1 for details) were used to PCR amplify glutamate racemase genes with different constitutive promoter sequences using the b.subtilis 6051-U genome as template. After the PCR product and the vector pZM1 were digested with AvrII and BamHI, they were recovered, and then ligated with T4 DNA ligase overnight, transformed into E.coli JM109, spread on an LB solid plate with Kan resistance, and cultured overnight at 37 ℃. Transformants were picked for colony PCR and sequencing verification, and if correct, the plasmid pZM1-e/16/11/12-racE was successfully constructed.

Because of the nature of the plasmids used in the present invention, it is not possible to directly ligate the racE directly behind the capBCA, and it is therefore necessary to construct a plasmid containing the racE and then to ligate the entire fragment to pZM 1-capBCA. Among them, pZM1-capBCA was prepared in the same manner as in the construction of the recombinant plasmid in the comparative example.

Upstream and downstream primers AvrII-racE-F (SEQ ID NO.11) and SalI-racE-R (SEQ ID NO.13) carrying AvrII and BamHI cleavage sites were designed based on the sequence of racE, and the target fragment e/11/12/16-racE was obtained by PCR amplification. The PCR product was digested with AvrII and SalI, and the vector pZM1-capBCA was digested with NheI and SalI, respectively, and recovered. Since NheI and AvrII are a pair of isocaudarner enzymes, the target fragment was ligated to the vector with T4 DNA ligase overnight at 16 ℃, transformed into E.coli JM109, spread on an LB solid plate with Kan resistance, and cultured overnight at 37 ℃. The transformant is picked up for colony PCR and sequencing verification, and if the transformant is correct, the plasmid pZM1-capBCA-e/16/11/12-racE is successfully constructed.

The plasmid was finally shock-transformed into c.glutamicum F343, transformants were selected on resistant plates and colony PCR verified with primers pZM1-R (SEQ ID No.15), pZM1-F (SEQ ID No. 14).

TABLE 1 primer List

C, Corynebacterium glutamicum transformation method:

(1) inoculating a single colony into a seed culture medium, and culturing overnight at 32 ℃ and 120 rpm; inoculating an appropriate amount of the seed culture to a competent medium to obtain an initial OD₆₀₀0.3; culturing at 30 ℃ and 120rpm to OD₆₀₀0.7-0.8, about 4 h; placing the culture medium on ice for 10min, and subpackaging the bacterial solutionCentrifuging in a centrifugal tube for 10min at 4000rpm to obtain a strain; washing with 25ml ice bath 10% glycerol for 4 times; suspension in 1.5ml-2ml 10% glycerol and split into 1.5ml Ep tubes after ice-bath to obtain competent cells.

(2) The electric shock cup is placed with the superclean bench in advance for purging, and the refrigerator is placed.

(3) Melting the competent cells on ice, adding 3-5uLDNA, mixing, adding into an electric shock cup, performing electric conversion at 1.8kV and 5mS, and immediately adding 1mL of BHIS into the electrode cup for suspension;

(4) suspending, transferring to a 1.5mL EP tube, and incubating for 6min at 46 ℃;

(5) after incubation, culturing for 2h at 30 ℃ to recover cells and express resistance;

(6) after incubation, the plates were centrifuged at 12000rpm for 1min and plated on LBHIS plates containing 50ug/mL Kan + resistance for 1-2 d.

Analysis of results

(1): effect of constitutive promoter-based expression of glutamate racemase on fermentation characteristics for production of Gamma-PGA

The successfully constructed engineering strain C.glutamicum F343 pZM1-capBCA-e/16/11/12-racE is fermented, and the influence on the production of gamma-PGA after the expression level of the glutamate racemase is regulated and controlled by a promoter is explored. A series of promoters with different strengths are screened in corynebacterium glutamicum in the early stage of the subject group, and a promoter library is constructed.

After expressing glutamate racemase by using 4 promoters (e, 16, 11, 12, with the promoter e as reference and the intensities of 1, 15, 50, 60, respectively, and the four promoters being gradually increased) with different intensities, biomass (FIG. 1A), glucose consumption (FIG. 1B), L-Glu yield (FIG. 1C), and γ -PGA yield (FIG. 1D) during the fermentation of the strain are decreased as shown in FIG. 1. This is probably because expression of glutamate racemase has an inhibitory effect on the growth of the strain and product synthesis.

After expressing glutamate racemase using a constitutive promoter, γ -PGA synthesized by the strain C.glutamicum F343 pZM1-capBCA-e/16/11/12-racE was 3.92, 2.80, 1.13, 0.64g/L, respectively, and was reduced by 26.45%, 47.47%, 78.80%, 87.99%, respectively, as compared with the results of C.glutamicum F343 pZM1-capBCA in the control comparative example, and the yield was gradually reduced with the increase of promoter strength. On one hand, the expression of foreign protein increases the burden of thallus metabolism, on the other hand, the expression of foreign protein may cause the L-Glu to be racemized into D-Glu by glutamate racemase, and the excessive D-Glu in cells has toxic action on thallus.

(2): effect of constitutive promoter-based expression of glutamate racemase on the D/L monomer ratio of γ -PGA

Precise control of the D/L monomer ratio of γ -PGA can be achieved by controlling the expression level of glutamate racemase from the transcriptional level using 4 constitutive promoters of different strengths, the results of which are shown in fig. 2.

When γ -PGA was synthesized using constitutive promoters e, 16, 11 and 12 having lower strengths and different D/L monomer ratios, the ratios of D-Glu in γ -PGA were 33.82%, 35.45%, 46.09% and 52.53%, respectively, as shown in FIG. 2 (A). The proportion of the D-Glu is gradually increased and kept consistent with the strength of the promoter, so that the D/L monomer ratio of the gamma-PGA in the corynebacterium glutamicum is accurately regulated and controlled, and the D/L monomer ratio reaches a controllable interval of 0.4-1.5: 1.

Meanwhile, the difference of the transcription levels of the glutamate racemase under the control of four different constitutive promoters is determined, and the result is shown in FIG. 2(B), and the expression levels of the glutamate racemase genes racE of the strains capBCA-e-racE, capBCA-16-racE and capBCA-11-racE are respectively reduced by 75.39%, 71.59% and 13.20% compared with the strains capBCA-12-racE.

The results show that the expression of the glutamate racemase gene racE is successfully regulated and controlled by promoters with different strengths, and further the synthesis of the gamma-PGA with different D/L monomer ratios is realized.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

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Claims

1. The strain for producing the gamma-DL-PGA is characterized in that C.glutamicum F343 is taken as a chassis, a gene cluster capBCA of the gamma-PGA synthetase from bacillus licheniformis is firstly expressed, then a constitutive promoter is utilized to express a glutamate racemase gene racE from bacillus subtilis, and the expression level of the racE is endogenously regulated to construct the strain for producing the gamma-DL-PGA.

2. The γ -DL-PGA-producing strain according to claim 1, wherein the promoter comprises e/11/12/16-racE, and the γ -DL-PGA-producing strain constructed correspondingly is C.glutamicum F343 pZM1-capBCA-e/16/11/12 racE.

3. The recombinant plasmid formed during the construction of the γ -DL-PGA-producing strain according to claim 1 or 2.

4. A genetically engineered bacterium expressing the γ -DL-PGA-producing strain of claim 1 or 2.

5. A method for preparing γ -PGA with various D/L monomer ratios, which comprises the step of preparing γ -DL-PGA-producing strains according to claim 1 or 2.

6. The method for synthesizing γ -PGA with different D/L monomer ratio according to claim 5, wherein the γ -PGA comprises γ -L-PGA and γ -D-PGA, wherein the content of γ -D-PGA is 30 to 60%.

7. The method for synthesizing γ -PGA with different D/L monomer ratios according to claim 5, wherein the D/L monomer ratio of γ -D-PGA and γ -L-PGA is 0.4 to 1.5: 1.

8. The method for synthesizing γ -PGA with different D/L monomer ratio according to claim 5, wherein the γ -DL-PGA producing strain according to claim 1 or 2 is prepared by the steps of: inoculating the seed solution of the strain producing the gamma-DL-PGA to a fermentation culture medium, culturing for 1-2 h at the temperature of 32 +/-2 ℃, adding IPTG (isopropyl-beta-thiogalactoside) for induction for 1-2 h, and finally culturing for 72 +/-5 h at the temperature of 37 +/-2 ℃.

9.γ -PGA obtained by the method according to any one of claims 5 to 8.

10. The γ -PGA according to claim 9, which is used in the fields of food, cosmetics, biomedicine, and environmental protection.