Method for non-induction biosynthesis of low-molecular-weight gamma-polyglutamic acid

Abstract

The invention discloses a method for non-induction biosynthesis of low molecular weight gamma-polyglutamic acid, which specifically comprises the following steps: cloning a gamma-polyglutamic acid synthetase gene cluster pgsBCA of bacillus licheniformis to corynebacterium glutamicum F343 with high glutamic acid yield for exogenous expression; a gamma-polyglutamic acid degrading enzyme gene pgdS and pgsBCA from Schlaughua Zehnder bacillus are subjected to tandem expression, so that the biosynthesis of the low-molecular-weight gamma-polyglutamic acid (LMW-gamma-PGA) is realized. Furthermore, natural promoters are screened, constitutive promoters with different intensities are introduced, and the optimal promoter Pdep-A16 is used for replacing the original inducible promoter Ptac, so that synthesis of the LMW-gamma-PGA without an inducer is realized. Finally, continuous fed-batch fermentation is carried out in a 15-L fermentation tank, the yield of the LMW-gamma-PGA reaches 37.42 g / L, and the production intensity is 0.78 g / L / h. And the LMW-gamma-PGA production process without adding an inducer is successfully constructed.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for biosynthesizing low-molecular-weight γ-polyglutamate without induction. Specifically, the method is achieved by screening polyglutamate synthases and degrading enzymes from different sources and expressing them in combination using a constitutive promoter, thereby realizing the synthesis of low-molecular-weight γ-polyglutamate without the need for an inducer. Background Art

[0002] γ-Polyglutamic acid (γ-PGA) is a homopolymeric amino acid composed of glutamic acid monomers linked by γ-amide bonds, forming a linear, fibrous backbone. The abundant carboxyl groups and hydrogen bonds in its structure contribute to its excellent water solubility, superior water retention, and high cation affinity. Depending on the degree of polymerization, the molecular weight of γ-PGA typically ranges from 100 kDa to 2000 kDa. The biological functions and practical applications of γ-PGA are strongly dependent on its molecular weight. High-molecular-weight γ-PGA exhibits high viscosity and can be used as a thickener or biomaterial. Low-molecular-weight γ-PGA (LMW-γ-PGA) (Mw ≤ 200 kDa) exhibits solubility and degradation properties due to differences in chain length, leading to its widespread application in cosmetics, pharmaceuticals, environmental protection, food, and other fields.

[0003] Currently, the commercial production of LMW-γ-PGA primarily relies on direct microbial fermentation, which offers advantages such as inexpensive raw materials, minimal environmental pollution, and high purity of the natural product. However, this method requires exogenous addition of glutamate, which is costly and difficult to control molecular weight. Other traditional production methods include thermal and acid-base degradation, but these methods are complex, require the use of organic reagents, and struggle to precisely control molecular cleavage, resulting in uneven molecular weight distribution and limiting large-scale production. In contrast, enzymatic degradation can specifically hydrolyze γ-PGA under mild conditions to produce LMW-γ-PGA of uniform molecular weight, providing a more controllable and targeted decomposition method and gradually becoming a more ideal method for producing LMW-γ-PGA.

[0004] Previously, researchers successfully used Corynebacterium glutamicum as a base strain to heterologously express γ-PGA synthase, achieving γ-PGA synthesis without the need for glutamate addition, significantly reducing production costs. However, due to the high stability of the Corynebacterium glutamicum genome and the lack of corresponding homologous recombination mechanisms, heterologous expression currently relies on plasmid systems using inducible promoters. This system requires exogenous inducers, increasing production complexity and cost. Furthermore, inducible expression systems have limited regulatory precision, and over-induction may lead to cellular stress responses or the accumulation of toxic proteins, thereby inhibiting cell growth and reducing product accumulation.

[0005] Therefore, screening suitable γ-PGA synthase and degradation enzymes for enzymatic expression, and selecting a constitutive promoter of appropriate strength to replace the inducible promoter can achieve stable and controllable gene expression without the need for inducers, thereby reducing the use of exogenous inducers, which can significantly improve the production efficiency of LMW-γ-PGA and has excellent application value. Summary of the Invention

[0006] The overall goal of the present invention is to achieve LMW-γ-PGA production without exogenous glutamate addition by screening the γ-PGA synthase gene pgsBCA and the degradation enzyme gene pgdS from different sources and expressing them in tandem in Corynebacterium glutamicum F343. Furthermore, by addressing the issue of inducer addition through promoter engineering and optimizing the combined regulation of the γ-PGA synthesis and degradation modules, a recombinant strain with high LMW-γ-PGA production without the need for inducers was constructed. This overcomes the prior art issues of inducer dependence and imprecise molecular weight control, providing a research foundation for industrial production.

[0007] The first object of the present invention is to provide a γ-PGA synthase-degradase gene cluster, the nucleotide sequence of which is shown in SEQ ID NO.13.

[0008] The second object of the present invention is to provide a method for preparing a γ-PGA synthase-degradase gene cluster, the method comprising: connecting the γ-PGA synthase gene cluster pgsBCA and the γ-PGA degradase gene pgdS in series to form a γ-PGA synthase-degradase gene cluster, denoted as pgsBCA-pgdS, wherein:

[0009] The nucleotide sequences of pgsB, pgsC and pgsA in the γ-PGA synthase gene cluster pgsBCA are shown in SEQ ID NOs. 7-9, respectively.

[0010] The nucleotide sequence of the γ-PGA degrading enzyme gene pgdS is shown in SEQ ID NO.10.

[0011] The third object of the present invention is to provide a recombinant plasmid, which is the following (i) or (ii):

[0012] (i) comprising a recombinant gene, wherein the recombinant gene is a γ-PGA synthase-degradase gene cluster as described above or prepared by the method described above;

[0013] (ii) contains a recombinant gene and a constitutive promoter P A16 The recombinant gene is the γ-PGA synthase-degradase gene cluster as described above or prepared by the method described above.

[0014] The fourth object of the present invention is to provide a recombinant strain constructed by transformation with the recombinant plasmid as described above.

[0015] Optionally, in one embodiment of the present invention, C. glutamicum F343 is used as the chassis.

[0016] The fifth object of the present invention is to provide a method for synthesizing LWM-γ-polyglutamic acid, using any of the above-mentioned recombinant strains for fermentation culture.

[0017] Optionally, in one embodiment of the present invention, the method for synthesizing LWM-γ-polyglutamic acid is to utilize a recombinant strain for fermentation culture, wherein the recombinant strain is constructed by transformation of the recombinant plasmid as described above.

[0018] Further optionally, in one embodiment of the present invention, the step of fermenting and culturing the recombinant strain includes: inoculating the seed liquid of the recombinant strain into the fermentation medium, first culturing at 32°C for 4 hours, adding IPTG to induce for 1 hour, and finally culturing at 37°C for 48 hours.

[0019] The sixth object of the present invention is to provide a method for preparing LWM-γ-polyglutamic acid without an inducer, using C. glutamicum F343 as a chassis, heterologously expressing a recombinant plasmid and transforming and constructing a recombinant strain, and fermenting and culturing the obtained recombinant strain, wherein the recombinant plasmid contains a recombinant gene and a constitutive promoter P A16 The recombinant gene is the γ-PGA synthase-degradase gene cluster as described above or prepared by the method described above.

[0020] Optionally, in one embodiment of the present invention, the step of fermenting and culturing the recombinant strain includes: inoculating the seed liquid of the recombinant strain into the fermentation medium, and culturing at 37±2°C for 48±5h.

[0021] Optionally, in one embodiment of the present invention, no inducer is added during the fermentation culture process.

[0022] Optionally, in one embodiment of the present invention, the fermentation medium comprises: 8-12 g·L corn steep liquor -1 , glucose 80-120g·L -1 , K2HPO40.8-1.2g·L -1 , MgSO40.4-0.8g·L -1 , FeSO4·7H2O 0.001-0.003g·L -1 , MnCl2·4H2O 0.001-0.003g·L -1, urea 5.0-9.0g·L -1 , pH 6.8-7.2.

[0023] Further optionally, in one embodiment of the present invention, the fermentation medium comprises: corn steep liquor 8-12 g·L -1 , glucose 120g·L -1 ,K2HPO41.0 g·L -1 , MgSO40.6 g·L -1 , FeSO4·7H2O 0.002g·L -1 , MnCl2·4H2O 0.002g·L -1 , urea 7.0g·L -1 , pH 7.0. Advantages and beneficial effects of the present invention:

[0024] The present invention successfully screened the most suitable source of γ-PGA synthase and degradation enzyme, and expressed them in tandem in Corynebacterium glutamicum, constructed the exogenous synthesis and degradation pathway of γ-PGA, and realized the biosynthesis of LWM-γ-PGA.

[0025] The present invention adopts natural promoter screening and promoter engineering strategies to successfully construct a LWM-γ-PGA recombinant strain that does not require the addition of inducers.

[0026] The present invention successfully carried out 48 h of continuous fed-batch fermentation in a 15-L fermentor, ultimately obtaining 37.42 g / L of LWM-γ-PGA (10≤MW≤200 kDa) with a production intensity of 0.78 g / L / h, achieving large-scale LWM-γ-PGA production and providing strong technical support for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : Schematic diagram of the invention principle.

[0028] Figure 2 : Evaluation of Bacillus fermentation performance; (A) Biomass changes; (B) γ-PGA yield changes; (C) Molecular weight distribution; BAM, B. amyloliquefaciens DSM23117; BL, B. licheniformis 9945a; BS, B. subtilis Ia1a.

[0029] Figure 3 : Evaluation of fermentation performance of recombinant strains; (A) biomass changes; (B) γ-PGA production changes; (C) molecular weight distribution.

[0030] Figure 4 :NMR of fermentation products 1 H proton spectrum qualitative analysis

[0031] Figure 5 : Evaluation of fermentation performance of recombinant strain (containing degradation enzyme); (A) biomass change; (B) γ-PGA yield change; (C) molecular weight distribution.

[0032] Figure 6 : Promoter engineering; (A) Screening of natural promoters; (B) Characterization of fluorescence intensity per unit biomass of different promoter strengths.

[0033] Figure 7 : Evaluation of fermentation performance of recombinant strain (promoter engineering); (A) biomass; (B) γ-PGA production; (C) molecular weight distribution of recombinant strain CG11.

[0034] Figure 8 : Fermentation process parameters and performance evaluation of the recombinant strain in a 15L fermenter; (A) Fermentation process control parameters; (B) Fermentation performance evaluation. DETAILED DESCRIPTION

[0035] like Figure 1 As shown, the present invention clones the γ-polyglutamate synthase gene cluster pgsBCA of Bacillus licheniformis into a high-glutamate-producing Corynebacterium glutamicum F343 for exogenous expression. On this basis, the γ-polyglutamate degradation enzyme gene pgdS from Bacillus spizezenii is expressed in tandem with pgsBCA to achieve the biosynthesis of low molecular weight γ-polyglutamate (LMW-γ-PGA). Further, by screening natural promoters and introducing constitutive promoters of different strengths, the optimal promoter P was selected. dep-A16 This replaced the original inducible promoter Ptac, enabling inducer-free synthesis of LMW-γ-PGA. Finally, continuous fed-batch fermentation in a 15-L fermenter achieved a LMW-γ-PGA yield of 37.42 g / L at a production rate of 0.78 g / L / h. This successfully established an inducer-free LMW-γ-PGA production process.

[0036] The raw materials and materials used in the embodiments of the present invention are as follows:

[0037] (1) Gene clusters pgsBCA of γ-PGA synthases from three different sources

[0038] The nucleotide sequence of the B. subtilis Ia1a synthetase gene pgsB is shown in SEQ ID NO.1, the nucleotide sequence of the synthetase gene pgsC is shown in SEQ ID NO.2, and the nucleotide sequence of the synthetase gene pgsA is shown in SEQ ID NO.3.

[0039] The nucleotide sequence of the synthetase gene pgsB of B. amyloliquefaciens DSM23117 is shown in SEQ ID NO.4, the nucleotide sequence of the synthetase gene pgsC is shown in SEQ ID NO.5, and the nucleotide sequence of the synthetase gene pgsA is shown in SEQ ID NO.6.

[0040] The nucleotide sequence of the synthetase gene pgsB of B. licheniformis 9945a is shown in SEQ ID NO.7, the nucleotide sequence of the synthetase gene pgsC is shown in SEQ ID NO.8, and the nucleotide sequence of the synthetase gene pgsA is shown in SEQ ID NO.9.

[0041] The B. licheniformis 9945a was purchased from ATCC, and the strain number was ATCC 9945a.

[0042] The B. amyloliquefaciens DSM23117 was purchased from DSM, and the strain number was DSM 23117.

[0043] Among them, the B. subtilis Ia1a (NCBI: https: / / www.ncbi.nlm.nih.gov / , BioSample: SAMEA4382852; SRA: ERS1294301) was preserved in the laboratory.

[0044] (2) pgdS genes encoding γ-PGA degradation enzymes from three different sources

[0045] The nucleotide sequence of the B. spizizenii TU-B-10 degradation enzyme gene pgdS is shown in SEQ ID NO.10.

[0046] The nucleotide sequence of the B. stercoris degradation enzyme gene pgdS is shown in SEQ ID NO.11.

[0047] The nucleotide sequence of the B. inaquosorum degradation enzyme gene pgdS is shown in SEQ ID NO.12.

[0048] The B. spizizenii TU-B-10 is derived from the NCBI (https: / / www.ncbi.nlm.nih.gov / ) database, gene ID: 11241199.

[0049] The B. stercoris is derived from the NCBI (https: / / www.ncbi.nlm.nih.gov / ) database, gene ID: 86871806.

[0050] The B. inaquosorum is derived from the NCBI (https: / / www.ncbi.nlm.nih.gov / ) database, gene ID: 76980082.

[0051] In an embodiment of the present invention, a method for preparing the γ-PGA synthase gene cluster pgsBCA includes amplifying the γ-PGA synthase gene cluster pgsBCA derived from B. subtilis Ia1a, B. amyloliquefaciens DSM23117, and B. licheniformis 9945a using designed primers to obtain recombinant fragments pgsBCA(BS), pgsBCA(BAM), and pgsBCA(BL).

[0052] In an embodiment of the present invention, the preparation method of tandemly connecting the γ-PGA synthase gene cluster pgsBCA and the γ-PGA degrading enzyme gene pgdS includes amplifying the γ-PGA synthase gene cluster pgsBCA derived from B. licheniformis 9945a by designed primers, and concatenating them with the γ-PGA degrading enzyme pgdS derived from B. spizizeni TU-B-10, B. stercoris, and B. inaquosorum obtained in the same manner to form pgsBCA-pgdS (BST), pgsBCA-pgdS (BSP), and pgsBCA-pgdS (BI) recombinant fragments.

[0053] In the embodiment of the present invention, the tandem expression adopts homologous recombination connection technology.

[0054] In the embodiment of the present invention, the method for determining the fermentation biomass of the recombinant strain is as follows: an appropriate amount of fermentation broth is diluted and then the absorbance at a wavelength of 600 nm is measured using an ultraviolet spectrophotometer.

[0055] In the embodiment of the present invention, the method for detecting the yield of polyglutamic acid fermented by the recombinant strain is as follows: an appropriate amount of fermentation broth is taken, centrifuged at 12000 rpm for 10 minutes, the precipitate is discarded, the supernatant is appropriately diluted, 100 μL is taken, and reacted with an equal volume of CTAB solution at room temperature for 3 minutes, and the absorbance at a wavelength of 430 nm is measured using a microplate reader.

[0056] In the embodiment of the present invention, the molecular weight detection method of polyglutamic acid fermented by the recombinant strain is as follows: the molecular weight of γ-PGA is detected by gel permeation chromatography (GPC). The sample treated with a 0.45 μm filter membrane is detected using an Agilent 1260 Infinity II liquid phase system RID detector, the gel permeation chromatography column is Shodex OHpak SB-806HQ, the mobile phase is 0.1M Na2SO4, the flow rate is 0.5 mL / min, and the detection wavelength is 210 nm. A standard curve is prepared using a dextran molecular weight standard set (Mp 180; Mp 2700; Mp 5250; Mp 9750; Mp 13050; Mp 36800; Mp 64650; Mp 135350; Mp 300600; Mp 2000000) to calculate the molecular weight of γ-PGA.

[0057] In an embodiment of the present invention, the purification method of polyglutamic acid, a fermentation product of the recombinant strain, is as follows: the fermentation broth is centrifuged at 4°C, 8000 rpm for 15 minutes to remove the bacteria, and the supernatant is added with 3 volumes of ethanol at 4°C overnight. The supernatant is removed by centrifugation at 12000 rpm for 10 minutes, and the mixture is dried in a 40°C oven for 24 hours to obtain a dark yellow crude sample. The sample is fully dissolved in an appropriate amount of deionized water, injected into a dialysis bag with a molecular weight cutoff of 3 kDa, and dialyzed for 24 hours. The sample is then removed from the dialysis bag and freeze-dried to obtain a white or light yellow sample.

[0058] In the embodiment of the present invention, the qualitative method of nuclear magnetic resonance 1H spectrum detection of the fermentation product of the recombinant strain is: 1 H-NMR was measured using a Bruker Spectrometer (Avance III 600 MHZ, Bruker, Switzerland). 30 mg of the purified sample was dissolved in 500 μL of D2O and measured at a frequency of 600 MHz using D2O as the solvent.

[0059] In the embodiment of the present invention, the seed culture medium comprises: 30-40 g·L corn steep liquor -1 , glucose 20-30g·L -1 , K2HPO41.5 g·L -1 , MgSO40.6 g·L -1 , FeSO4·7H2O 0.005g·L -1 , MnCl2·4H2O 0.005g·L -1 , urea 2.5g·L -1 , pH 6.8-7.2.

[0060] In the embodiment of the present invention, the fermentation medium comprises: 8-12 g·L corn steep liquor-1 , glucose 80-120g·L -1 , K2HPO40.8-1.2g·L -1 , MgSO40.4-0.8g·L -1 , FeSO4·7H2O 0.001-0.003g·L -1 , MnCl2·4H2O0.001-0.003g·L -1 , urea 5.0-9.0g·L -1 , pH 6.8-7.2.

[0061] The present invention is further described below with reference to specific examples. The strains and plasmids used in the specific examples are shown in Table 1. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0062] Table 1 Strains and plasmids used in specific examples

[0063]

[0064]

[0065] Example 1: Screening and identification of synthetases from different sources

[0066] The present invention selected the current main natural production strains of γ-PGA, B.subtilis Ia1a, B.amyloliquefaciens DSM23117, and B.licheniformis 9945a, for fermentation performance evaluation. Figure 2 shown.

[0067] Figure 2 In A, the strain derived from B. subtilis grew fastest and entered the stable period at 36 h. The strains derived from B. licheniformis and B. amyloliquefaciens grew relatively slowly and entered the stable period at 40 h. The biomass of the strain derived from B. licheniformis was only 4.55 at 40 h.

[0068] Figure 2 In B, the γ-PGA yield of B. amyloliquefaciens was higher than that of the other two sources at 48 h, reaching 15.25 g / L, and the fermentation broth was also viscous.

[0069] Figure 2 In C, the molecular weight distribution of fermentation products from B. licheniformis and B. amyloliquefaciens was more concentrated, with more than 93% of the molecular weight distribution being above 1000 kDa.

[0070] like Figure 2 As shown in Figure A, pgsBCA from B. licheniformis 9945a, B. amyloliquefaciens DSM 23117, and B. subtilis Ia1a was ligated into pZM1 and transformed into C. glutamicum F343, forming the recombinant plasmids pZM1-BCA(BL), pZM1-BCA(BAM), or pZM1-BCA(BS), respectively. Recombinant strains CG01, CG02, and CG03 were then transformed and used for induced fermentation.

[0071] The steps of inducing fermentation culture of the recombinant strain are as follows: take 3-5 μL of bacterial liquid from the glycerol tube and streak it on the LB plate, and culture it at 30°C for 24 hours. Pick a single colony and inoculate it into 10 mL of LB liquid culture medium containing kana (50 μg / L), and culture it at 30°C and 120 rpm for 12 hours. Transfer it to 25 mL of seed culture medium containing kana according to the inoculation amount of 8%, and culture it at 32°C and 120 rpm for 11 hours. Inoculate it into the fermentation medium according to the inoculation amount of 6%, and culture it at 32°C until OD = 12-15. Add IPTG to induce for 1 hour, increase the temperature to 37°C, and continue fermentation for 48 hours.

[0072] The fermentation performance of the recombinant strain was evaluated after 48 h of fermentation. Figure 3 shown.

[0073] Figure 3 The biomass of the recombinant strain in A was significantly increased compared with that of Bacillus, and the lag phase was greatly shortened. All of them entered the stable phase at 20 hours, and had sufficient time to synthesize γ-PGA.

[0074] Figure 3 The maximum yields of CG01 and CG03 in B can reach 4.92 g / L and 3.32 g / L respectively. Corynebacterium glutamicum is an aerobic bacterium that relies heavily on oxygen to support its normal growth and product synthesis during the fermentation process. The production of γ-PGA affects the viscosity of the fermentation liquid and thus affects the dissolved oxygen, which may be the key factor restricting the yield.

[0075] Figure 3 C is the determination of the molecular weight distribution of the fermentation products of the recombinant strain. Similar to the distribution of Bacillus, the Mw of the produced γ-PGA is all high molecular weight, among which 96.73% of the Mw of the γ-PGA produced by CG01 is greater than 1000 kDa, and the molecular weight distribution is similar to that of B. licheniformis 9945a.

[0076] The results showed that heterologous expression of the pgsBCA gene from B. licheniformis 9945a in C. glutamicum F343 was most effective, resulting in the lowest biomass during fermentation, the highest γ-PGA production, and the highest product yield. Furthermore, the MW of the CG01 product was almost entirely distributed in the high molecular weight range, providing a good foundation for the subsequent synthesis of LMW-γ-PGA from high molecular weight. The structure of the substance was subsequently identified using proton nuclear magnetic resonance (NMR) spectroscopy.

[0077] Figure 4 The 1H NMR spectra of a γ-PGA standard and the purified product from CG01 are shown. The spectra show distinct chemical shifts. The chemical shift for D2O is 4.79 ppm. The H peak with a chemical shift of 4.10 ppm is characteristic of the proton at site A. The hydrogen at sites C (D) (β position) couples with the hydrogen on the chiral carbon atom at site A (α position), generating a doublet with shifts of 2.05 and 1.91 ppm. The H peak at 2.34 ppm is characteristic of the proton at site B. The number of hydrogens at site B (γ position) is identical to the number of hydrogens at sites C (D) (β position), resulting in a single peak with an area equal to the sum of the doublet areas at site B. The spectrum shows the 1H NMR spectrum of the fermentation product from the CG01 strain. Comparison shows that the characteristic peaks of the CG01 fermentation product are consistent with those of the γ-PGA standard, confirming that the product is γ-PGA.

[0078] Finally, pgsBCA from B. licheniformis 9945a was selected as the polyglutamate synthetase of the recombinant strain.

[0079] Table 2 Primers used in Example 1

[0080]

[0081] Example 2: Screening and identification of degradative enzymes from different sources and construction of a tandem system

[0082] During γ-PGA fermentation, rational regulation of the degradation enzymes and the establishment of a "formation-degradation" process can achieve substrate economy and efficiently obtain γ-PGA with a desired molecular weight. Therefore, we co-expressed the degradation enzyme with the synthesis enzyme. pgdS from B. spizizenii TU-B-10, B. stercoris, and B. inaquosorum, along with pgsBCA from B. licheniformis 9945a, were tandemly linked to the expression plasmid pZM1. These recombinant plasmids, pZM1-BCA-S (BLBSP), pZM1-BCA-S (BLBST), and pZM1-BCA-S (BLBI), were generated, respectively. Recombinant strains CG07, CG08, and CG09 were then transformed and their fermentation performance evaluated.

[0083] like Figure 5 As shown in Figure 2, after CG01 expressed the degradation enzymes in series, the viscosity of its fermentation broth was greatly reduced. Figure 5 In A, it was also observed that the biomass of the recombinant strain showed different levels of increase.

[0084] The increase in biomass promoted the synthesis of γ-PGA, such as Figure 5 As shown in B, the best performing CG07 could achieve a yield of 14.05 g / L in 48 h, an increase of 185.6% compared with CG01.

[0085] Molecular weight distribution Figure 5 As shown in Figure C, all γ-PGA produced by fermentation of the recombinant strain CG07 containing pgdS from B. spizizenii TU-B-10 was degraded into LMW-γ-PGA less than 200 kDa. CG09 also showed varying degrees of degradation. In contrast, the yield and molecular weight of CG08 were similar to those of the original strain CG01, suggesting that the degradation enzyme from B. stercoris failed to effectively participate in the degradation reaction.

[0086] The results suggest that the degradation enzyme promotes the production of γ-PGA, but that PgdS from different sources also exhibits specificity for degradation. Subsequent research will be conducted using the recombinant strain CG07 containing pgdS from B. spizizenii TU-B-10.

[0087] Table 3 Primers used in Example 2

[0088]

[0089] Example 3: Construction of constitutive promoter library and promoter replacement

[0090] To reduce the dependence of the expression system on inducers and to co-express pgsBCA and pgdS to improve the production efficiency of γ-PGA, we constructed a constitutive promoter library to regulate the expression intensity of pgsBCA and pgdS.

[0091] We selected the constitutive promoters Pdap-e11, Pdap-A16 and PJ1 reported for use in Corynebacterium glutamicum as research objects, replaced the inducible promoter Ptac on the verification plasmid pZM1-eGFP, and formed three constitutive promoter plasmids pZM1-Pe11-eGFP, pZM1-PA16-eGFP and pZM1-PJ1-eGFP, and successfully constructed recombinant strains CG0709, CG07010 and CG07011 in C. glutamicum F343.

[0092] It should be noted that the present invention aims to study the process of regulating the synthesis-degradation module using components after its construction, so the experimental subject is set as a co-expression system. In addition, the original intention of the present invention is to investigate the effect of using degradation enzymes on the production of low molecular weight or specific molecular weight polyglutamic acid.

[0093] Table 4 Primers used in Example 3

[0094]

[0095] Example 4: Construction of natural promoter library and promoter replacement of γ-PGA synthase recombinant strain

[0096] At the same time, the natural promoter of γ-PGA synthase was explored. Compared with artificial promoters, natural promoters have the advantages of precise regulation and no need for inducers. They are compatible with the host system and can automatically regulate gene expression, avoiding the complexity and cost of relying on exogenous inducers.

[0097] Therefore, we mined and predicted the pgsBCA natural promoter in B.licheniformis 9945a through the BDGP website (BDGP: Neural Network Promoter Prediction), and selected the 8 promoter sequences with the highest scores in Table 5, connected them to the verification plasmid pZM1-eGFP and transformed them into E.coli BL21 (DE3) for fluorescence intensity characterization.

[0098] Specifically, such as Figure 6As shown in A, Ppgs-0 is used as a positive control, and Ppgs1 to 8 are the relative fluorescence intensities of 8 groups of natural promoter sequences. Among them, the fluorescence intensities of Ppgs-1 and Ppgs-8 are significantly higher than those of other sequences, indicating that these two sequences are likely to be the natural promoters of pgsBCA. They were replaced with the inducible promoter Ptac on the verification plasmid pZM1-eGFP by primers to obtain recombinant plasmids pZM1-Ppgs1-eGFP and pZM1-Ppgs8-eGFP, and recombinant strains CG0701 and CG07008 were constructed in C. glutamicum F343.

[0099] The five recombinant strains were then characterized by fluorescence intensity in Corynebacterium glutamicum, e.g. Figure 6 As shown in B, the screened natural promoters Ppgs-1 and Ppgs-8 failed to express normally in C. glutamicum F343, which is inferred to be because the host lacks specific response factors and cannot activate the functions of these promoters.

[0100] Table 5: Natural promoter prediction sequence scores in Example 4

[0101]

[0102] Example 5: Construction of a constitutive promoter library and promoter replacement of γ-PGA synthase-degradase recombinant strains

[0103] We replaced the P in the recombinant plasmid pZM1-BCA-S (BLBST) of CG07 with three constitutive promoters of different strengths. tac Promoter, recombinant plasmids pZM1-PA16-PGA, pZM1-Pe11-PGA and pZM1-PJ1-PGA were obtained, and recombinant strains CG10, CG11 and CG12 were constructed and verified by non-induced fermentation.

[0104] In the present embodiment, the steps for the uninduced fermentation culture of the recombinant strains CG10, CG11, and CG12 are as follows: 3-5 μL of bacterial solution from the glycerol tube was streaked onto an LB plate and incubated at 30°C for 24 hours. A single colony was selected and inoculated into 10 mL of LB liquid medium containing kana (50 μg / L) and incubated at 30°C and 120 rpm for 12 hours. An 8% inoculum was transferred to 25 mL of seed medium containing kana and incubated at 32°C and 120 rpm for 11 hours. A 6% inoculum was inoculated into the fermentation medium and incubated at 32°C to an OD of 12-15. The temperature was raised to 37°C and fermentation continued for 48 hours. Fermentation was performed in a 5L horizontal fermenter with a 2L liquid volume. The pH was controlled by 10% phosphoric acid and 60% ammonia water. The ventilation rate was 1.5 vvm, and the rotation speed was linked to the dissolved oxygen to maintain a DO of 25%.

[0105] from Figure 7 As shown in A, the biomass of CG10 was always maintained at a low level during the fermentation process. Considering that the strain contained P J1 Strong promoter, it is inferred that excessive expression intensity leads to cell metabolic imbalance, thereby restricting cell growth. At the same time, no γ-PGA is produced during the fermentation process.

[0106] The growth of CG12 was not restricted, but the yield was only 1.97 g / L.

[0107] The yield of CG11 containing the weak promoter PA16 reached 11.51 g / L in 48 h, which is close to the yield of CG07 induced fermentation culture in Example 2, indicating that there may be a balance between promoter strength and yield. We tested the molecular weight distribution of CG11, such as Figure 7 As shown in Figure C, the Mw of all γ-PGA was less than 200 kDa, and 6.2% was less than 100 kDa. No residual glutamic acid was detected in the fermentation broth of CG11.

[0108] The CG11 strain of the PA16 was screened out, and the nucleotide sequence of the γ-PGA synthase-degradase gene cluster contained in the corresponding recombinant plasmid is shown in SEQ ID NO.13.

[0109] These results demonstrate that constitutive promoters of varying strengths successfully regulate the co-expression of γ-PGA synthase and degradation enzymes, effectively modulating the transcript level of the degradation enzyme within an appropriate intensity range. This regulatory strategy ensures a balance between γ-PGA synthesis and degradation, optimizes metabolic flux, and avoids the adverse effects of overexpression of the degradation enzyme on product synthesis, thereby improving the overall efficiency of the fermentation process.

[0110] Example 6: Performance evaluation of recombinant strains in a 15 L fermenter

[0111] To further investigate the feasibility of industrial production of LWM-γ-PGA by the recombinant strain CG11, we performed continuous fed-batch fermentation in a 15-L fermentor.

[0112] The initial glucose concentration was set at 40 g / L to reduce the inhibition on cell growth and γ-PGA production. Then, glucose was fed at 4 h with a sugar concentration of 400 g / L. The flow rate was 30 mL / h for 24 h to keep the sugar concentration above 5 g / L.

[0113] like Figure 8As shown in Figure B, LWM-γ-PGA concentration continued to increase after 12 hours, reaching a peak of 37.42 g / L at the end of fermentation at 48 hours, with a productivity of 0.78 g / L / h. During fermentation, residual sugars were completely consumed by 36 hours, but dissolved oxygen and biomass remained stable. LWM-γ-PGA production also continued to increase after 48 hours. During the first 36 hours, a small amount of glutamate accumulated in the fermentation broth. This decreased with sugar consumption, suggesting that glutamate serves as both a precursor for γ-PGA polymerization and a nitrogen source to support continued cell growth. The molecular weight of γ-PGA in continuous fed-batch fermentation was consistently less than 200 kDa, with 98% of the γ-PGA concentration being above 185 kDa.

[0114] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A γ-PGA synthase-degradase gene cluster, characterized in that: The nucleotide sequence of the γ-PGA synthase-degradase gene cluster is shown in SEQ ID NO.

13.

2. A method for preparing a γ-PGA synthase-degradase gene cluster, characterized in that: The γ-PGA synthase gene cluster pgsBCA and the γ-PGA degradase gene pgdS are connected in series to form the γ-PGA synthase-degradase gene cluster, denoted as pgsBCA-pgdS, where The nucleotide sequences of pgsB, pgsC and pgsA in the γ-PGA synthase gene cluster pgsBCA are shown in SEQ ID NOs. 7-9, respectively. The nucleotide sequence of the γ-PGA degrading enzyme gene pgdS is shown in SEQ ID NO.

10.

3. A recombinant plasmid, which is (i) or (ii) below: (i) comprising a recombinant gene, wherein the recombinant gene is a γ-PGA synthase-degradase gene cluster as described in claim 1 or prepared by the method as described in claim 2, (ii) contains a recombinant gene and a constitutive promoter P A16 The recombinant gene is the γ-PGA synthase-degradase gene cluster as described in claim 1 or prepared by the method as described in claim 2.

4. A recombinant strain constructed by transformation with the recombinant plasmid according to claim 3.

5. The recombinant strain according to claim 4, characterized in that The base is C. glutamicum F343.

6. A method for synthesizing LWM-γ-polyglutamic acid, characterized in that: The recombinant strain according to claim 4 or 5 is used for fermentation culture.

7. A method for synthesizing LWM-γ-polyglutamic acid without an inducer, characterized in that: Using C. glutamicum F343 as the chassis, the pgsBCApgdS recombinant plasmid was heterologously expressed and transformed into a recombinant strain, and the obtained recombinant strain was fermented and cultured. The recombinant plasmid contained the recombinant gene and the constitutive promoter P A16 The recombinant gene is the γ-PGA synthase-degradase gene cluster as described in claim 1 or prepared by the method as described in claim 2.

8. The method according to claim 7, characterized in that The step of fermenting and culturing the recombinant strain comprises: inoculating the seed liquid of the recombinant strain into a fermentation medium, and culturing at 37±2° C. for 48±5 hours.

9. The method according to claim 8, characterized in that No inducer is added during the fermentation and cultivation process.

10. The method according to claim 9, characterized in that The fermentation medium includes: corn steep liquor 8-12g·L -1 , glucose 80-120g·L -1 , K2HPO40.8-1.2g·L -1 , MgSO40.4-0.8g·L -1 , FeSO4·7H2O 0.001-0.003g·L -1 ,MnCl2·4H2O 0.001-0.003g·L -1 , urea 5.0-9.0g·L -1 , pH 6.8-7.0.

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