An engineered bacillus amyloliquefaciens strain producing ultra-low molecular weight polyglutamic acid and its application

Abstract

The present invention discloses a biosynthetic method of ultra-low molecular weight polyglutamic acid. The endogenous plasmid P2sip is eliminated, and the polyglutamate hydrolase pgdSA is knocked out to obtain Bacillus amyloliquefaciens NS, and the host bacteria NS-LM are obtained by further coupling and co-expressing the glutamate racemase gene yrpC, the γ-PGA polymerase gene pgsA and the γ-PGA degradation enzyme gene pgdS. The substrate configuration analysis of pgdS from Bacillus subtilis NX-2 determined the positive effect of the stereochemical configuration regulation of γ-PGA on the molecular weight control of γ-PGA. Further, by recombinantly expressing yrpC, pgsA and pgdS, and combining and replacing the pgsA gene from different bacterial sources, the limitation of the γ-PGA configuration on the hydrolysis rate of the degradation enzyme was successfully alleviated, and the molecular weight of γ-PGA was further reduced to 3-5kDa, and the yield was as high as 34.27±0.25g / L, realizing the one-step synthesis of ultra-low molecular weight polyglutamic acid. This process further broadens the molecular weight distribution range of biosynthesized γ-PGA and simplifies the production process of low-molecular-weight polyglutamic acid, which is beneficial to promoting the application of γ-PGA in different fields.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and fermentation engineering, and specifically relates to an engineering bacterium of Bacillus amyloliquefaciens producing ultra-low molecular weight polyglutamic acid and application thereof. Background Art

[0002] With the continuous deepening of research on γ-PGA, more and more studies have shown that the biological function of γ-PGA is closely related to its molecular weight. Especially when the molecular weight is lower than 10kDa, γ-PGA exhibits unique biological activity functions. Studies have shown that γ-PGA with a molecular weight between 1-10kDa is easier to penetrate into the skin base and has the effect of transdermal moisturizing. γ-PGA with a molecular weight below 10kDa is easy to combine with drugs and improve its targeting properties, making the drug efficacy more complete, and also showing certain advantages in gene therapy. This shows that γ-PGA (1-10kDa) has shown important application prospects in the fields of cosmetics and medicine.

[0003] At present, the main ways of γ-PGA degradation are physical degradation, chemical degradation and enzymatic degradation. Previous studies have found that it is difficult to reduce the molecular weight of γ-PGA to below 10KDa by physical and chemical degradation. At the same time, these methods are prone to unstable product quality, a wide range of molecular weights and serious environmental pollution during the production process. In the process of preparing γ-PGA with different molecular weights by bioenzymatic method, endo-type γ-PGA degrading enzyme plays a major role. However, the production process of regulating the molecular weight of γ-PGA by the two-step method of enzyme production and catalysis is too complicated, and commercial γ-PGA degrading enzyme is currently unavailable. The production cost is too high, making it difficult to achieve large-scale application.

[0004] In recent years, with the continuous development of molecular biology, the research on key enzymes for γ-PGA synthesis has made further breakthroughs. One-step direct fermentation has attracted more and more attention due to its simplified process flow and low cost. Previous studies have shown that the degradation enzyme has the characteristics of substrate configuration selectivity, so the difference in substrate configuration will seriously affect the hydrolysis rate of the degradation enzyme, and adjusting the configuration is an effective measure to promote the hydrolysis efficiency. Secondly, by heterologous cloning and expression of γ-PGA synthase and glutamate racemase from different strains, it was found that the molecular weight of γ-PGA synthesized by microorganisms has the characteristics of being dependent on the differences between polymerase and racemase. The γ-PGA synthase gene cluster pgsBCA from Bacillus licheniformis NK-03 and Bacillus amyloliquefaciens LL3 was expressed in Escherichia coli JM109. The results showed that the average molecular weight of γ-PGA synthesized by γ-PGA synthase from B. licheniformis NK-03 was 3.74±0.38×10 4 Da, while the average molecular weight of γ-PGA synthesized by γ-PGA synthase from B. amyloliquefaciens LL3 was relatively low, which was 3.23±0.26×10 4 Da, further coupling expression of glutamate racemase racE will further cause changes in the molecular weight of γ-PGA.

[0005] Based on this, in order to achieve the synthesis of ultra-low molecular weight γ-PGA, alleviate the interference of γ-PGA stereochemical composition on the hydrolysis rate of γ-PGA degrading enzyme, functional analysis and verification of key enzymes regulating the configuration of related γ-PGA were carried out, and a stereochemical composition regulation model in the γ-PGA synthesis process was constructed, and the γ-PGA degrading enzyme hydrolysis process was further coupled to achieve the synthesis of ultra-low molecular weight γ-PGA. This study is of great significance for promoting the application of γ-PGA in different fields, and also provides a useful reference for the molecular weight control of other similar polymer compounds. Summary of the invention

[0006] The technical problem to be solved by the present invention is to address the deficiencies of the prior art, design experiments and verify that the combination of γ-PGA stereochemical component regulation and degradation enzyme hydrolysis process is an effective strategy for regulating the molecular weight of γ-PGA, and provide an engineered bacillus amyloliquefaciens strain producing ultra-low molecular weight polyglutamic acid and its application.

[0007] The technical problem that the present invention is to solve is the application of the above-mentioned Bacillus amyloliquefaciens engineered bacteria in the production of ultra-low molecular weight polyglutamic acid.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0009] A method for constructing an engineered Bacillus amyloliquefaciens strain producing ultra-low molecular weight polyglutamic acid is characterized in that Bacillus amyloliquefaciens NX-2S CCTCC NO:M 2016346 is used as a starting strain, endogenous plasmid p2Sip is eliminated, and the polyglutamate degradation enzyme gene pgdSA is further knocked out to obtain recombinant Bacillus amyloliquefaciens NS, and the recombinant Bacillus amyloliquefaciens NS is coupled and co-expressed with pgsA-yrpC-pgdS to obtain the engineered Bacillus amyloliquefaciens NS-LM.

[0010] Among them, in the yrpC-pgsA-pgdS, the glutamate racemase gene yrpC is derived from Bacillus amyloliquefaciens NS, the γ-PGA synthase gene pgsA is derived from Bacillus anthracis BA, and the γ-PGA degrading enzyme gene pgdS is derived from Bacillus subtilis NX-2.

[0011] Specifically, the nucleotide sequence of the glutamate racemase gene yrpC is shown in SEQ ID NO:27, the nucleotide sequence of the γ-PGA synthase gene pgsA is shown in SEQ ID NO:54, and the nucleotide sequence of the γ-PGA degradase gene pgdS is shown in SEQ ID NO:18.

[0012] The method for constructing the engineered bacterium Bacillus amyloliquefaciens comprises the following steps:

[0013] (1) The construction process of recombinant Bacillus amyloliquefaciens NS is as follows: Using Bacillus amyloliquefaciens NX-2S (CCTCC NO: M 2016346) as the starting strain, the endogenous plasmid P2Sip was eliminated, and the polyglutamate degrading enzyme gene pgdSA was further knocked out. The elimination process of the endogenous plasmid P2Sip of Bacillus amyloliquefaciens NX-2S has been described in the authorized patent CN 108624546B. In order to achieve the seamless knockout of the polyglutamate degrading enzyme gene pgdSA, the genome of Bacillus amyloliquefaciens NX-2S was used as a template, and the primers pgdSAUP / DN-F / R were used to amplify the 800bp homology arms pgdSA-UP / DN upstream and downstream of the target gene pgdSA. At the same time, the constitutive strong promoter PHpaⅡ was amplified using pDR-PHpaⅡ-F / R using the expression plasmid pMA5 as a template. After gel recovery, the promoter and upstream and downstream homologous arms were fused into the fragment PHpaⅡ-geneUP-geneDN by overlapping PCR, and then the fusion fragment was cloned into the vector pDR-pheS* double-digested with EcoR I and Xho I by one-step cloning method (see ClonExpress Multis One Step Cloning Kit for steps) to obtain the recombinant plasmid pDR-pheS*-pgdSA. After demethylation, the verified correct recombinant plasmid was electrotransformed (voltage 2.0-3.0kv, electric shock time 4ms) into Bacillus amyloliquefaciens NX-2S, and the primer pDR-F / R was used to verify whether the transformation was successful. The strain containing the recombinant plasmid was cultured in LB plate medium containing 100μg / mL spectinomycin at 30℃ for 12h, and then transferred to LB culture medium containing 100μg / mL spectinomycin at 42℃ for 24-48h to induce single exchange of plasmid. After the single exchange positive clones were relaxedly cultured for 2 generations in LB medium without resistance, they were diluted and spread on LB plate culture medium. Single colonies were picked for double exchange verification using pgdSA-OUT-F / R primers. The one that was successfully verified was the recombinant Bacillus amyloliquefaciens NS.

[0014] (2) Using the genome of Bacillus amyloliquefaciens NS as a template, PCR amplification technology was used to amplify the fragment yrpC using primers pHY-yrpC-F / R.

[0015] The nucleotide sequence of primer pHY-yrpC-F: 5'--3' is shown in SEQ ID NO:21, and the nucleotide sequence of primer pHY-yrpC-R: 5'--3' is shown in SEQ ID NO:22.

[0016] (3) Using the genome of Bacillus anthracis BA as a template, PCR amplification technology was used to amplify the fragment pgsA using primers pHY-pgsA(BA)-F / R.

[0017] The nucleotide sequence of primer pHY-pgsA(BA)-F: 5'--3' is shown in SEQ ID NO:50, and the nucleotide sequence of primer pHY-pgsA(BA)-R: 5'--3' is shown in SEQ ID NO:51.

[0018] (4) Using the genome of Bacillus subtilis NX-2 as a template, PCR amplification technology was used to amplify the fragment pgdS using primers PHY-pgdS-F / R.

[0019] The nucleotide sequence of primer PHY-pgdS-F: 5'--3' is shown in SEQ ID NO: 19, and the nucleotide sequence of primer PHY-pgdS-R: 5'--3' is shown in SEQ ID NO: 20.

[0020] (5) Construction of plasmid pHY-yrpC-pgdS:

[0021] a: using shuttle plasmid pHY300PLK as a backbone, linearizing by double restriction digestion with BamH I and Hind III to obtain vector backbone A; using plasmid PMA5 and Bacillus amyloliquefaciens NS genome as templates, amplifying promoter PHpaⅡ and terminator Tamy fragments by PCR; connecting promoter PHpaⅡ, pgdS fragment obtained in step (4) and terminator Tamy fragment by overlapping PCR to obtain fragment PHpaⅡ-pgdS; connecting the obtained fragment PHpaⅡ-pgdS and vector backbone A by one-step cloning to obtain plasmid pHY-PHpaⅡ-pgdS;

[0022] b: Plasmid pHY-PHpaⅡ-pgdS was used as the backbone, and the vector backbone B was obtained after double restriction digestion with EcoRⅠ and BamHⅠ; plasmid PMA5 and Bacillus amyloliquefaciens NS genome were used as templates to amplify the promoter PHpaⅡ and terminator Tamy fragments by PCR; the promoter PHpaⅡ, yrpC fragment and terminator Tamy fragment were connected by overlapping PCR to obtain the fragment PHpaⅡ-yrpC; the obtained fragment PHpaⅡ-yrpC was connected with the vector backbone B by one-step cloning to obtain the plasmid pHY-yrpC-pgdS;

[0023] (6) The fragments yrpC and pgsA amplified in step (2) and step (3) are subjected to fusion PCR to obtain a fusion fragment yrpC-pgsA, which is then double-digested with Sal I and Pst I to obtain a vector backbone C, and the fusion fragment is connected to the vector backbone C to obtain a recombinant plasmid pHY-yrpC-pgsA-pgdS;

[0024] (7) The recombinant plasmid pHY-yrpC-pgsA-pgdS obtained in step (6) is transformed into the recombinant Bacillus amyloliquefaciens NS to obtain a recombinant strain, namely, the engineered Bacillus amyloliquefaciens NS-LM.

[0025] The Bacillus amyloliquefaciens engineered bacteria producing ultra-low molecular weight polyglutamic acid constructed by the above construction method is also within the scope of protection of the present invention.

[0026] The use of the above-mentioned Bacillus amyloliquefaciens engineered bacteria in the production of ultra-low molecular weight polyglutamic acid is also within the scope of protection of the present invention.

[0027] The ultra-low molecular weight polyglutamic acid has a molecular weight of 3-5 kDa and a yield of up to 34.27±0.25 g / L.

[0028] The seed solution of the Bacillus amyloliquefaciens engineered bacteria is inoculated into a fermentation medium, and cultured at 30-37°C and 180-240 rpm for 48-72 hours to ferment and prepare ultra-low molecular weight polyglutamic acid. The preferred fermentation conditions are 32°C and 200 rpm for 72 hours.

[0029] Specifically, the seed solution is prepared by: taking a small amount of bacterial solution from a glycerol tube stored at -80°C, streaking on an LB plate culture medium, and culturing overnight at 32°C. Pick a single colony from the LB plate culture medium and transfer it to a 250mL shake flask containing 50mL of LB liquid culture medium, and culture it at 32°C and 200rpm for 12h.

[0030] Specifically, the inoculation amount is 6% v / v.

[0031] Specifically, the fermentation medium formula is: inulin crude extract 70g / L, (NH4)2SO4 5g / L, K2HPO4·3H2O 20g / L, KH2PO4 2g / L, MgSO4 0.45g / L, MnSO4·H2O 0.06g / L, pH 8.0.

[0032] Beneficial effects: First, the present invention identifies the positive effect of γ-PGA stereochemical configuration regulation on γ-PGA molecular weight control: by analyzing the substrate configuration of the γ-PGA degrading enzyme screened in the early stage, it is found that it is more inclined to hydrolyze γ-PGA substrates with a high proportion of D-glutamic acid monomers. On this basis, in order to achieve the regulation of the γ-PGA stereochemical configuration, the effects of the glutamate racemase gene yrpC and the γ-PGA synthase gene pgsA on the γ-PGA configuration were investigated respectively, and it was found that overexpression of both is beneficial to increase the proportion of D-glutamic acid monomers in γ-PGA. Finally, by recombinantly expressing yrpC, pgsA and pgdS, and combining and replacing the pgsA genes from different bacterial sources, the recombinant strain NS-LM was obtained, which successfully alleviated the limitation of the γ-PGA configuration on the hydrolysis rate of the degrading enzyme, further reduced the γ-PGA molecular weight to 3-5kDa, and the yield reached 34.27±0.25g / L. This process further broadens the molecular weight distribution range of biosynthesized γ-PGA, which is beneficial to promoting the application of γ-PGA in different fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0034] Figure 1 The effect of the stereochemical configuration of γ-PGA on the hydrolysis efficiency of the degradation enzyme pgdS.

[0035] Figure 2 The effects of enhanced expression of genes yrpC and pgsA on γ-PGA synthesis. (A) Cell growth; (B) γ-PGA production; (C) γ-PGA molecular weight; (D) γ-PGA stereochemical composition.

[0036] Figure 3 Effects of combined expression of genes yrpC, pgsA and pgdS on cell growth (A), γ-PGA production (B) and γ-PGA molecular weight (C).

[0037] Figure 4 To study the effect of replacing the pgsA gene from different sources on γ-PGA synthesis. DETAILED DESCRIPTION

[0038] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0039] In the following examples, the experimental materials used are from the following sources:

[0040] All primers and DNA sequences were synthesized by GenScript Biotech Co., Ltd.;

[0041] All plasmid and DNA fragment sequencing was completed by GenScript Biotech Co., Ltd.

[0042] Plasmids were extracted using the plasmid small-quantity DNA extraction kit (AP-MN-P-250G) provided by Axygen;

[0043] DNA fragments were recovered using the DNA gel recovery kit (AP-MN-P-250G) provided by Axygen;

[0044] The bacterial genome was extracted using the TIANamp Bacteria DNA Kit provided by Tiangen Biochemical Technology Co., Ltd.

[0045] DNA fragment PCR was performed using Kodaq 2X PCR MasterMix with dye high-fidelity enzyme from Ibim Biotech Co., Ltd.;

[0046] The ligase used for one-step cloning ligation was the ligase from Novezan Biotechnology Co., Ltd. (ClonExpress II One Step Cloning Kit, ClonExpress MultiS One Step Cloning Kit);

[0047] The restriction endonuclease used was the restriction endonuclease of Baoriyi Biotechnology Co., Ltd.

[0048] In the following examples, the PCR reaction system used is:

[0049] The NS genome of Bacillus amyloliquefaciens was used as a template, and the target gene was amplified using relevant primers. The reaction mixture contained 2 μL of template, 2 μL of each primer, 25 μL of KOD enzyme and 19 μL of double distilled water. The PCR reaction parameters were as follows: stage one: 95°C, 30 s, stage two: 95°C, 5 s; 60°C, 60 s; 40 cycles. The overlapping PCR reaction system was as follows: the reaction mixture contained 2 μL of each template to be overlapped, 2 μL of each first and last primer, 25 μL of KOD enzyme and 17 μL of double distilled water. The overlapping PCR reaction parameters were as follows: stage one: 95°C, 30 s, stage two: 95°C, 5 s; 60°C, 60 s; 40 cycles.

[0050] In the following examples, the recombinant strains NS(yrpC), NS(pgsA) and NS(yrpC-pgsA) described in Example 2, the recombinant strains NS(yrpC-pgdS), NS(pgsA-pgdS) and NS(yrpC-pgsA-pgdS) described in Example 3, and the recombinant strains constructed by replacing the pgsA gene from different sources described in Example 4, the fermentation process is as follows:

[0051] (1) Take a small amount of bacterial solution from the -80℃ glycerol tube, streak it on LB plate, and culture it at 32℃ overnight.

[0052] (2) Pick a single colony from the LB plate medium and transfer it to a 250 mL shake flask containing 50 mL of LB liquid medium. Cultivate at 32°C and 200 rpm for 12 h.

[0053] (3) The seed solution was inoculated into a 500 mL shake flask containing 80 mL of fermentation medium at an inoculum volume of 6% v / v and cultured at 32°C and 200 rpm for 72 h.

[0054] The LB medium formula is: yeast powder 5g / L, peptone 10g / L, sodium chloride 10g / L, pH 6.0-8.0.

[0055] The fermentation medium formula is: crude inulin extract 70 g / L, (NH4)2SO4 5 g / L, K2HPO4·3H2O 20 g / L, KH2PO4 2 g / L, MgSO4 0.45 g / L, MnSO4·H2O 0.06 g / L, pH 8.0.

[0056] In the following examples, the yield and molecular weight of γ-PGA were determined as follows:

[0057] The molecular weight of the fermentation product was determined by gel permeation chromatography (GPC). The fermentation purified product was appropriately diluted and the bacteria were removed by a 0.22 μm filter membrane, and 20 μL was directly injected. The chromatographic column was selected: Shodex Ohpak SB-806M HQ; 0.2 mol / L Na2SO4 solution was prepared as the mobile phase of the liquid phase, and the pH was adjusted to about 4.0 with acetic acid; in order to control the pressure in the liquid chromatography column not to be too high, the mobile phase flow rate was adjusted to 1.0 mL / min. The peak time of the sample was compared with the γ-PGA standard to determine the molecular weight of the product.

[0058] In the following examples, the D / L configuration of γ-PGA is determined as follows:

[0059] The D / L monomer content in γ-PGA was determined by a chiral column. The γ-PGA product was hydrolyzed in 6mol / L HCl at 110℃ for 12h. The hydrolyzate was adjusted to pH 7.0 with NaOH, filtered with a 0.22μm filter membrane, and 20μL was directly injected. The chromatographic column was selected: Sumichiral OA-5000; 1mM CuSO4 solution was prepared as the mobile phase of the liquid phase. In order to control the pressure in the liquid chromatography column not too high, the mobile phase flow rate was adjusted to 1.0mL / min. The injection temperature was controlled at 35℃. The peak time of the sample was compared with the D / L-glutamic acid standard to determine the ratio of D / L-glutamic acid monomers.

[0060] Example 1 Analysis of the configuration tendency of the substrate hydrolyzed by the γ-PGA degrading enzyme pgdS from Bacillus subtilis

[0061] In the process of characterizing the properties of γ-PGA degrading enzymes, it is necessary to examine the preference of the degrading enzymes for the stereochemical configuration of γ-PGA, because this characteristic may restrict the high efficiency of the degrading enzymes for hydrolyzing γ-PGA, thereby affecting the final molecular weight of the product. In this study, in order to study the preference of the degrading enzyme pgdS from Bacillus subtilis NX-2 for the configuration of the hydrolyzed substrate, γ-PGA containing 80%, 50% and 20% of D-glutamic acid monomers were used as substrates for hydrolysis.

[0062] Specifically, the fermentation liquid of the recombinant strain NS-pHY-PHpaⅡ-pgdS was centrifuged to obtain a crude enzyme solution containing the degradative enzyme; γ-PGA with different D-glutamate monomers was dissolved in 0.05mM phosphate buffer (pH 7.4) to obtain a 10g / L γ-PGA aqueous solution. The enzyme reaction solution contained 200μL crude enzyme solution and 800μL γ-PGA solution and was cultured at 37°C for 4h. A blank sample containing an inactivated enzyme was set up for each sample to correct the results for non-enzymatic release of amino groups. The reaction was stopped by heating in boiling water for 5min, and then 1mL of ninhydrin colorimetric solution was added, mixed evenly, heated at 100°C for 15min, cooled, and diluted with 3mL of 60% ethanol, and the OD was detected by a UV spectrophotometer. 570 , compare the hydrolysis rates of enzyme solutions on different substrates.

[0063] The results are as follows Figure 1As shown, the degradation enzyme pgdS has the highest hydrolysis rate for γ-PGA containing 80% D-glutamic acid monomers, and the molecular weight of γ-PGA drops sharply from 1500kDa to 150kDa after 24h of hydrolysis. When γ-PGA containing 50% D-glutamic acid monomers is used as the hydrolysis substrate, the hydrolysis rate of the degradation enzyme PgdS decreases, and the molecular weight of γ-PGA drops from 1300kDa to about 500kDa. However, for γ-PGA containing 20% ​​D-glutamic acid monomers, the degradation enzyme PgdS has no obvious hydrolysis effect on it, and the molecular weight of γ-PGA does not change significantly during the enzymatic hydrolysis process. These results show that the degradation enzyme pgdS derived from Bacillus subtilis NX-2 has a good hydrolysis effect on γ-PGA containing a high proportion of D-glutamic acid monomers.

[0064] The construction process of the recombinant strain NS-pHY-PHpaⅡ-pgdS is as follows:

[0065] Bacillus amyloliquefaciens NX-2S (CCTCC NO: M 2016346) was used as the starting strain to eliminate the endogenous plasmid P2Sip and further knock out the polyglutamate degrading enzyme gene pgdSA. The elimination process of the endogenous plasmid P2Sip of Bacillus amyloliquefaciens NX-2S has been described in the authorized patent CN 108624546 B. In order to achieve the seamless knockout of the polyglutamate degrading enzyme gene pgdSA, the genome of Bacillus amyloliquefaciens NX-2S was used as a template, and the primers pgdSAUP / DN-F / R were used to amplify the 800bp homology arms pgdSA-UP / DN upstream and downstream of the target gene pgdSA. At the same time, the expression plasmid pMA5 was used as a template, and the constitutive strong promoter PHpaⅡ was amplified using pDR-PHpaⅡ-F / R. After gel recovery, the promoter and upstream and downstream homologous arms were fused into the fragment PHpaⅡ-geneUP-geneDN by overlapping PCR, and then the fusion fragment was cloned into the vector pDR-pheS* double-digested with EcoR I and Xho I by one-step cloning method (see ClonExpressMultis One Step Cloning Kit for steps) to obtain the recombinant plasmid pDR-pheS*-pgdSA. After demethylation, the verified correct recombinant plasmid was electrotransformed (voltage 2.0-3.0kv, electric shock time 4ms) into Bacillus amyloliquefaciens NX-2S, and the primer pDR-F / R was used to verify whether the transformation was successful. The strain containing the recombinant plasmid was cultured in LB plate medium containing 100μg / mL spectinomycin at 30℃ for 12h, and then transferred to LB culture medium containing 100μg / mL spectinomycin at 42℃ for 24-48h to induce single exchange of plasmid. After the single exchange positive clones were relaxedly cultured for 2 generations in LB medium without resistance, they were diluted and spread on LB plate culture medium, and single colonies were picked for double exchange verification using pgdSA-OUT-F / R primers. The one that was successfully verified was the recombinant Bacillus amyloliquefaciens NS (starting strain) constructed.

[0066] Among them, the nucleotide sequence of primer pgdSAUP-F: 5'--3' is shown in SEQ ID NO:1, the nucleotide sequence of primer pgdSAUP-R: 5'--3' is shown in SEQ ID NO:2, the nucleotide sequence of primer pgdSADN-F: 5'--3' is shown in SEQ ID NO:3, the nucleotide sequence of primer pgdSADN-R: 5'--3' is shown in SEQ ID NO:4, the nucleotide sequence of primer pDR-PHpaⅡ-F: 5'--3' is shown in SEQ ID NO:5, the nucleotide sequence of primer pDR-PHpaⅡ-R: 5'--3' is shown in SEQ ID NO:6, the nucleotide sequence of primer pDR-F: 5'--3' is shown in SEQ ID NO:7, the nucleotide sequence of primer pDR-R: 5'--3' is shown in SEQ ID NO:8, and the nucleotide sequence of primer pgdSA-OUT-F: 5'--3' is shown in SEQ ID NO:9. NO:9, and the nucleotide sequence of primer pgdSA-OUT-R:5'--3' is shown in SEQ ID NO:10.

[0067] The promoter PHpaⅡ and terminator Tamy fragments were amplified from plasmid PMA5 and Bacillus amyloliquefaciens NS genome using primers pHY-PHpaⅡ-F / R and pHY-Tamy-F / R, respectively. In order to facilitate the replacement of overexpressed genes in the later stage, the restriction site KpnⅠ was introduced after the promoter and the restriction site NdeⅠ was introduced before the terminator. The polyglutamate degrading enzyme gene pgdS was amplified using primers PHY-pgdS-F / R with the genome of Bacillus subtilis NX-2 as a template. Subsequently, the promoter PHpaⅡ, pgdS and terminator Tamy fragments were fused by overlapping PCR using primers pHY-PHpaⅡ-F and pHY-Tamy-R. After gel recovery and purification, the fusion fragment was connected with the recombinant expression vector, Escherichia coli-Bacillus subtilis shuttle plasmid pHY300PLK, which was linearized and recovered by double restriction digestion of BamH I and HindⅢ, by one-step cloning to obtain the gene overexpression recombinant plasmid pHY-PHpaⅡ-pgdS. The successfully verified recombinant plasmid was demethylated and then electrotransformed into the constructed recombinant Bacillus amyloliquefaciens NS (starting strain) to obtain the recombinant strain NS-pHY-PHpaⅡ-pgdS.

[0068] Among them, the nucleotide sequence of the recombinant expression vector is shown in SEQ ID NO:11, the nucleotide sequence of the promoter PHpaⅡ is shown in SEQ ID NO:12, the nucleotide sequence of the terminator Tamy is shown in SEQ ID NO:13, the nucleotide sequence of the primer pHY-PHpaⅡ-F: 5'--3' is shown in SEQ ID NO:14, the nucleotide sequence of the primer pHY-PHpaⅡ-R: 5'--3' is shown in SEQ ID NO:15, the nucleotide sequence of the primer pHY-Tamy-F: 5'--3' is shown in SEQ ID NO:16, the nucleotide sequence of the primer pHY-Tamy-R: 5'--3' is shown in SEQ ID NO:17, the nucleotide sequence of the pgdS is shown in SEQ ID NO:18, the nucleotide sequence of the primer PHY-pgdS-F: 5'--3' is shown in SEQ ID NO:19, and the nucleotide sequence of the primer PHY-pgdS-R: 5'--3' is shown in SEQ ID NO:20.

[0069] Example 2 Effects of glutamate racemase yrpC and γ-PGA synthase pgsA on the stereochemical configuration of γ-PGA

[0070] In order to evaluate the effects of glutamate racemase yrpC and γ-PGA synthase pgsA on γ-PGA synthesis, the glutamate racemase gene yrpC and γ-PGA synthase gene pgsA were expressed separately and coupled in the recombinant Bacillus amyloliquefaciens NS (starting strain) to obtain the recombinant strains NS (yrpC), NS (pgsA) and NS (yrpC-pgsA). The specific steps are as follows:

[0071] The genome of recombinant Bacillus amyloliquefaciens NS (starting strain) was used as a template, and the fragments yrpC and pgsA were amplified using primers pHY-yrpC-F / R and pHY-pgsA-F / R, respectively. The previously constructed pHY-PHpaⅡ-pgdS plasmid was used as a backbone, and the linearized cloning vector was obtained after enzyme digestion and recovery using KpnⅠ and NdeⅠ. The yrpC and pgsA fragments were connected to the linearized cloning vector using one-step cloning and transformed into E.coli GM2163. Single colonies were picked to extract plasmids and enzyme digestion was performed to verify the correctness of the recombinant plasmids, and the recombinant plasmids pHY-yrpC and pHY-pgsA were obtained. In order to co-express yrpC and pgsA, two gene fragments were amplified from the genome using primers pHY-yrpC-F / pHY-yrpCA-R and pHY-pgsA(BAY)-F / pHY-pgsA-R, respectively. The two fragments were fused and overlapped using primers pHY-yrpC-F / pHY-pgsA-R, and the fragment yrpC-pgsA was obtained by gel recovery. The previously constructed pHY-PHpaⅡ-pgdS plasmid was used as the backbone, and the linearized cloning vector was obtained after enzyme digestion and recovery using KpnⅠ and NdeⅠ. The yrpC-pgsA fragment and the above-mentioned linearized cloning vector were connected by one-step cloning and transformed into E.coli GM2163. A single colony was picked to extract the plasmid and enzyme digestion was used to verify the correctness of the recombinant plasmid to obtain the recombinant plasmid pHY-yrpC-pgsA.

[0072] The successfully verified plasmids pHY-yrpC, pHY-pgsA and pHY-yrpC-pgsA were transformed into host bacteria to obtain recombinant strains NS(yrpC), NS(pgsA) and NS(yrpC-pgsA);

[0073] Among them, the nucleotide sequence of primer pHY-yrpC-F: 5'--3' is shown in SEQ ID NO:21, the nucleotide sequence of primer pHY-yrpC-R: 5'--3' is shown in SEQ ID NO:22, the nucleotide sequence of primer pHY-pgsA-F: 5'--3' is shown in SEQ ID NO:23, the nucleotide sequence of primer pHY-pgsA-R: 5'--3' is shown in SEQ ID NO:24, the nucleotide sequence of primer pHY-yrpCA-R: 5'--3' is shown in SEQ ID NO:25, the nucleotide sequence of primer pHY-pgsA(BAY)-F: 5'--3' is shown in SEQ ID NO:26, and the nucleotide sequence of yrpC is shown in SEQ ID NO:27.

[0074] The fermentation results are as follows Figure 2As shown, the cell dry weights of the recombinant strains NS(yrpC), NS(pgsA) and NS(yrpC-pgsA) were all higher than that of the control strain NS. In addition, the yield of γ-PGA synthesized by the recombinant strains NS(yrpC), NS(pgsA) and NS(yrpC-pgsA) increased from 24.26±0.58g / L to 26.47±0.25g / L, 28.79±0.43g / L and 30.14±0.56g / L, respectively. Figure 2 B) These results indicate that overexpression of yrpC and pgsA is beneficial to cell growth and γ-PGA synthesis. At the same time, we also studied the effects of different recombinant strains on the molecular weight and stereochemical composition of synthesized γ-PGA. Figure 2 As shown in Figure C, the molecular weight of γ-PGA produced by strains NS(yrpC), NS(pgsA) and NS(yrpC-pgsA) increased from 1300-1400 kDa to 1400-1550 kDa, 1600-1750 kDa and 1800-1900 kDa, respectively, and the proportion of D-glutamic acid monomer in γ-PGA synthesized by the corresponding strains increased from 57% to 61%, 71% and 80%, respectively ( Figure 2 D). These results indicate that enhancing the expression of yrpC and pgsA is an effective method to regulate the molecular weight and stereochemical composition of γ-PGA. In particular, the molecular weight and D-glutamic acid monomer ratio of γ-PGA obtained by regulating the expression of pgsA were higher than those of γ-PGA obtained by controlling the expression level of yrpC, that is, regulating the composition of γ-PGA by regulating the expression of pgsA is more effective than regulating the expression of yrpC.

[0075] To further investigate the effects of yrpC and pgsA on γ-PGA synthesis, knockout experiments were performed on the yrpC and pgsA genes, respectively.

[0076] In order to achieve the seamless knockout of yrpC and pgsA, referring to the knockout process of pgdSA, the genome of Bacillus amyloliquefaciens NX-2S was used as a template, and the upstream 800bp and downstream 800bp were selected to form homology repair templates according to the yrpC and pgsA gene sequences, respectively. The upstream and downstream 800bp homology arms yrpC-UP / DN and pgsA-UP / DN of the target genes yrpC and pgsA were amplified using primers yrpCUP / DN-F / R and pgsAUP / DN-F / R. After gel recovery, the promoter PHpaⅡ and the upstream and downstream homology arms were fused into the fragment PHpaⅡ-geneUP-geneDN by overlapping PCR, and then the fusion fragment was cloned into the vector pDR-pheS* double-digested with EcoR I and Xho I by one-step cloning to obtain the recombinant plasmid pDR-pheS*-yrpC / pgsA. After demethylation, the verified recombinant plasmid was electrotransformed into Bacillus amyloliquefaciens, and the primers pDR-F / R were used to verify whether the transformation was successful. After the strain containing the recombinant plasmid was cultured in LB plate medium containing 100μg / mL spectinomycin at 30°C for 12h, it was transferred to LB culture medium containing 100μg / mL spectinomycin at 42°C for 24-48h to induce single exchange of the plasmid. After the single exchange positive clones were relaxed cultured in LB culture medium without resistance for 2 generations, they were diluted and spread on LB plate culture medium, and single colonies were picked for double exchange verification using yrpC / pgsA-OUT-F / R primers. The successful verification was the mutant strains NS(ΔyrpC) and NS(ΔpgsA) that successfully knocked out yrpC and pgsA.

[0077] Wherein, the nucleotide sequence of primer yrpCUP-F: 5'--3' is shown in SEQ ID NO:28, the nucleotide sequence of primer yrpCUP-R: 5'--3' is shown in SEQ ID NO:29, the nucleotide sequence of primer yrpCDN-F: 5'--3' is shown in SEQ ID NO:30, the nucleotide sequence of primer yrpCDN-R: 5'--3' is shown in SEQ ID NO:31, the nucleotide sequence of primer pgsAUP-F: 5'--3' is shown in SEQ ID NO:32, the nucleotide sequence of primer pgsAUP-R: 5'--3' is shown in SEQ ID NO:33, the nucleotide sequence of primer pgsADN-F: 5'--3' is shown in SEQ ID NO:34, the nucleotide sequence of primer pgsADN-R: 5'--3' is shown in SEQ ID NO:35, the nucleotide sequence of primer yrpC-OUT-F: 5'--3' is shown in SEQ ID NO:36, the nucleotide sequence of primer yrpC-OUT-R: 5'--3' is shown in SEQ ID NO:37, the nucleotide sequence of primer pgsA-OUT-F: 5'--3' is shown in SEQ ID NO:38, and the nucleotide sequence of primer pgsA-OUT-R: 5'--3' is shown in SEQ ID NO:39.

[0078] The results are shown in Table 1. The deletion of the glutamate racemase gene yrpC had a negative impact on bacterial growth and γ-PGA synthesis. The molecular weight of γ-PGA synthesized by NS (ΔyrpC) decreased from 1300-1400 kDa to 1000-1100 kDa. The content of D-glutamic acid monomer in γ-PGA also decreased from 57% to 52%. When the pgsA gene was deleted, no γ-PGA was detected in the fermentation broth. This result indicates that pgsA is necessary for the synthesis of γ-PGA by Bacillus amyloliquefaciens. The complementation of the yrpC and pgsA genes in the mutant strain restored the normal growth of the cells and the synthesis of γ-PGA, indicating that the genes yrpC and pgsA are important for maintaining cell growth and γ-PGA synthesis.

[0079] Table 1 Effects of knockout and complementation of genes yrpC and pgsA on γ-PGA synthesis

[0080]

[0081] Example 3 Effect of coupled expression of genes yrpC, pgsA and pgdS on the molecular weight of γ-PGA

[0082] Although the ratio of D-glutamate monomers in γ-PGA has been optimized by enhanced expression of yrpC and pgsA, the specific distribution of D-glutamate monomers and L-glutamate monomers on the chain is still unclear. Therefore, in order to further verify whether the increased ratio of D-glutamate monomers will promote the hydrolysis rate of the degradation enzyme pgdS, we coupled expressed yrpC-pgdS, pgsA-pgdS and yrpC-pgsA-pgdS in Bacillus amyloliquefaciens NS. The specific steps are as follows:

[0083] Using the genome of Bacillus amyloliquefaciens NS as a template, the primers pHY-yrpC'-F / R and pHY-pgsA'-F / R were used to amplify the fragments yrpC and pgsA, respectively. At the same time, the promoter pHpaⅡ and terminator Tamy fragments were amplified from the plasmid pMA5 and the genome of Bacillus amyloliquefaciens NS using primers pHY-PHpaⅡ-F / R and pHY-Tamy-F / R, respectively. In order to facilitate the replacement of the co-expression fragments in the later stage, the restriction site SalⅠ was introduced after the promoter, and the restriction site PstⅠ was introduced before the terminator. The promoter PHpaⅡ, yrpC fragment and terminator Tamy fragment were fused by overlapping PCR using primers pHY-PHpaⅡ-F / pHY-Tamy-R, and the fragment PHpaⅡ-yrpC-Tamy was obtained by gel recovery and purification. The fusion fragment PHpaⅡ-pgsA-Tamy was obtained in the same way. The plasmid pHY-PHpaⅡ-pgdS was digested with EcoRⅠ and BamHⅠ restriction endonucleases to obtain a linearized digestion vector. The PHpaⅡ-yrpC-Tamy and PHpaⅡ-pgsA-Tamy fusion fragments were respectively connected to the above-mentioned linearized digestion vector and transformed into E. coli GM2163 to obtain recombinant plasmids pHY-yrpC-pgdS and pHY-pgsA-pgdS.

[0084] The fragments yrpC and pgsA were amplified using primers pHY-yrpC'-F / pHY-yrpCA-R and pHY-pgsA(BAY)-F / pHY-pgsA'-R, respectively. The two fragments were then fused by PCR using primers pHY-yrpC-F / pHY-pgsA-R to obtain the fusion fragment yrpC-pgsA. The previously constructed plasmid pHY-yrpC-pgdS was double-digested with SalⅠ and PstⅠ to obtain a linearized restriction vector. The fusion fragment was connected to the above-mentioned restriction vector to obtain the recombinant plasmid pHY-yrpC-pgsA-pgdS. The plasmids that were successfully verified by sequencing were transformed into strain NS, respectively, to obtain recombinant strains NS(yrpC-pgdS), NS(pgsA-pgdS) and NS(yrpC-pgsA-pgdS).

[0085] Among them, the nucleotide sequence of primer pHY-yrpC'-F: 5'--3' is shown as SEQ ID NO:40, the nucleotide sequence of primer pHY-yrpC'-R: 5'--3' is shown as SEQ ID NO:41, the nucleotide sequence of primer pHY-pgsA'-F: 5'--3' is shown as SEQ ID NO:42, the nucleotide sequence of primer pHY-pgsA'-R: 5'--3' is shown as SEQ ID NO:43, the nucleotide sequence of primer pHY-pgsA(BAY)-F: 5'--3' is shown as SEQ ID NO:26, and the nucleotide sequence of primer pHY-yrpCA-R: 5'--3' is shown as SEQ ID NO:25.

[0086] The fermentation results are as follows Figure 3 As shown, the biomass of the recombinant strains NS(yrpC-pgdS), NS(pgsA-pgdS) and NS(yrpC-pgsA-pgdS) was similar to that of the control strain. In addition, the γ-PGA production of the three recombinant strains increased to 27.2±0.35g / L, 30.3±0.33g / L and 30.8±0.28g / L, respectively. Finally, the molecular weight of γ-PGA produced by NS(yrpC-pgdS), NS(pgsA-pgdS) and NS(yrpC-pgsA-pgdS) was successfully reduced to 17-25kDa, 13-16kDa and 9-11kDa, respectively, which was much lower than that of the control strain NS(PHpaⅡ)(21-30kDa). Compared with the expression of single gene yrpC or pgsA, the molecular weight of γ-PGA obtained by co-expression of the two genes was lower. These results indicate that regulating the stereochemical configuration of γ-PGA is an effective measure for Bacillus amyloliquefaciens to synthesize low molecular weight γ-PGA. It is worth noting that the molecular weight of γ-PGA produced by the recombinant strain NS (pgsA-pgdS) is lower than that synthesized by the recombinant strain NS (yrpC-pgdS), which further confirms that pgsA has a more significant promoting effect on the regulation of the stereochemical configuration of γ-PGA than yrpC.

[0087] Example 4 Effect of expression of pgsA genes from different sources on the molecular weight of γ-PGA

[0088] Based on the experiment of Example 3, it was found that coupling regulation of the stereochemical configuration of γ-PGA and the expression of the degradation enzyme is beneficial to reducing the molecular weight of γ-PGA. In order to further reduce the molecular weight of γ-PGA, the combined expression of yrpC, pgsA and pgdS was optimized. In view of the fact that pgsA has a significantly better regulatory effect on the stereochemical composition of γ-PGA than yrpC, optimizing the source of pgsA is more conducive to the synthesis of low molecular weight γ-PGA. Therefore, we studied the effect of pgsA genes from different Bacillus sources on the synthesis of γ-PGA, including Bacillus anthracis B. anthracis, Bacillus subtilis B. subtilis, Bacillus licheniformis B. licheniformis, Bacillus megaterium B. megaterium and Bacillus pumilus B.

[0089] The pgsA in Example 3 was replaced with the pgsA gene derived from Bacillus anthracis, Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium and Bacillus pumilus, respectively, and the other conditions remained unchanged, and the recombinant strains were constructed respectively.

[0090] Wherein, the nucleotide sequence of primer pHY-pgsA(BA)-F: 5'--3' is shown in SEQ ID NO:44, wherein, the nucleotide sequence of primer pHY-pgsA(BA)-R: 5'--3' is shown in SEQ ID NO:45, the nucleotide sequence of primer pHY-pgsA(BS)-F: 5'--3' is shown in SEQ ID NO:46, the nucleotide sequence of primer pHY-pgsA(BS)-R: 5'--3' is shown in SEQ ID NO:47, the nucleotide sequence of primer pHY-pgsA(BL)-F: 5'--3' is shown in SEQ ID NO:48, the nucleotide sequence of primer pHY-pgsA(BL)-R: 5'--3' is shown in SEQ ID NO:49, the nucleotide sequence of primer pHY-pgsA(BM)-F: 5'--3' is shown in SEQ ID NO: NO:50, the nucleotide sequence of primer pHY-pgsA(BM)-R: 5'--3' is shown in SEQ ID NO:51, the nucleotide sequence of primer pHY-pgsA(BP)-F: 5'--3' is shown in SEQ ID NO:52, the nucleotide sequence of primer pHY-pgsA(BP)-R: 5'--3' is shown in SEQ ID NO:53, and the nucleotide sequence of pgsA derived from Bacillus anthracis is shown in SEQ ID NO:54.

[0091] The fermentation results are as follows Figure 4As shown, replacing the pgsA gene from different Bacillus sources had a significant effect on the molecular weight of γ-PGA. When pgsA was replaced with genes from Bacillus subtilis and Bacillus licheniformis, the cell dry weight and γ-PGA yield of the recombinant strain did not show significant differences compared with the recombinant strain NS (yrpC-pgsA-pgdS) in Example 3. However, when pgsA from Bacillus megaterium and Bacillus brevis was overexpressed, the cell dry weight and γ-PGA yield decreased slightly. Overexpression of pgsA from Bacillus anthracis increased the cell dry weight and γ-PGA yield of the bacteria to 6.1±0.32 g / L and 34.27±0.25 g / L, respectively. In addition, we further determined the effect of the expression of pgsA from different sources on the molecular weight of γ-PGA. The results are shown in Figure 4 As shown, the molecular weights of γ-PGA produced by strains expressing pgsA from Bacillus subtilis and Bacillus licheniformis were 14-15 kDa and 14-17 kDa, respectively, which did not further reduce the molecular weight compared to the molecular weight of γ-PGA synthesized by the recombinant strain NS (yrpC-pgsA-pgdS) (9-11 kDa). When the pgsA genes from Bacillus megaterium and Bacillus pumilus were overexpressed, the molecular weight of γ-PGA synthesized by the recombinant strain was significantly higher than that of γ-PGA produced by the recombinant strain NS (yrpC-pgsA-pgdS). When the pgsA gene from Bacillus anthracis was expressed, the molecular weight of the synthesized γ-PGA was the lowest, reaching 3-5 kDa. These results indicate that replacing pgsA from different sources is effective for Bacillus amyloliquefaciens to synthesize γ-PGA of different molecular weights, and the Bacillus amyloliquefaciens NS-LM constructed by coupling and co-expressing the pgsA gene from Bacillus anthracis, the yrpC gene from Bacillus amyloliquefaciens and the pgdS gene from Bacillus subtilis can effectively synthesize ultra-low molecular weight γ-PGA (Mw: 3-5kDa), and the yield of γ-PGA is the highest, reaching 34.27±0.25g / L.

[0092] In summary, the results of Examples 1-4 show that:

[0093] (1) The substrate configuration preference of the degradation enzyme pgdS from Bacillus subtilis NX-2 was identified. Enzymatic hydrolysis reactions were carried out using γ-PGA containing 80%, 50% and 20% D-glutamic acid monomers as substrates. It was found that the degradation enzyme had a higher hydrolysis efficiency for γ-PGA containing 80% D-glutamic acid monomers, indicating that the pgdS gene from B. subtilis NX-2 has a better hydrolysis effect on γ-PGA containing a higher proportion of D-glutamic acid monomers.

[0094] (2) The effects of glutamate racemase yrpC and γ-PGA polymerase pgsA on the stereochemical configuration of γ-PGA were investigated. Overexpression and knockout experiments were performed on the two genes. The results showed that when yrpC, pgsA and yrpC-pgsA were overexpressed, the proportion of D-glutamate monomers in γ-PGA synthesized by the corresponding strains increased from 57% to 61%, 71% and 80%, respectively. This indicates that the enhanced expression of genes yrpC and pgsA led to an increase in the proportion of D-glutamate monomers in γ-PGA.

[0095] (3) Synergistic regulation of γ-PGA stereochemical configuration and degradation process. By combining the expression of yrpC-pgdS, pgsA-pgdS and yrpC-pgsA-pgdS, the molecular weight was successfully reduced from 21-30 kDa to 17-25 kDa, 13-16 kDa and 9-11 kDa, respectively, successfully alleviating the limitation of configuration on degradation. The source of pgsA gene was further optimized, the molecular weight of γ-PGA was reduced to 3-5 kDa, and the yield was increased to 34.27±0.25 g / L.

[0096] The present invention provides an engineering bacterium for amyloliquefaciens producing ultra-low molecular weight polyglutamic acid and its application ideas and methods. There are many methods and approaches to implement the technical solution. 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 principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for constructing an engineered bacterium of Bacillus amyloliquefaciens that produces ultra-low molecular weight polyglutamic acid, characterized in that: Bacillus amyloliquefaciens NX-2S CCTCC NO:M 2016346 was used as the starting strain, the endogenous plasmid p2Sip was eliminated, and the polyglutamate degrading enzyme gene pgdSA was further knocked out to obtain the recombinant Bacillus amyloliquefaciens NS. The recombinant Bacillus amyloliquefaciens NS was coupled with co-expression of pgsA-yrpC-pgdS to obtain the engineered Bacillus amyloliquefaciens NS-LM.

2. The construction method according to claim 1, characterized in that: In the yrpC-pgsA-pgdS, the glutamate racemase gene yrpC is derived from Bacillus amyloliquefaciens NS, the γ-PGA synthase gene pgsA is derived from Bacillus anthracis BA, and the γ-PGA degrading enzyme gene pgdS is derived from Bacillus subtilis NX-2.

3. The construction method according to claim 1, characterized in that: The steps include: (1) Using Bacillus amyloliquefaciens NX-2S as the starting strain, eliminating the endogenous plasmid P2Sip and knocking out the polyglutamate degradation enzyme gene pgdSA to construct a recombinant Bacillus amyloliquefaciens NS; (2) Using the B. amyloliquefaciens NS genome as a template, the fragment yrpC was amplified by PCR; (3) Using the Bacillus anthracis BA genome as a template, the fragment pgsA was amplified by PCR; (4) Using the genome of Bacillus subtilis NX-2 as a template, the fragment pgdS was amplified by PCR; (5) Construction of plasmid pHY-yrpC-pgdS: a: using shuttle plasmid pHY300PLK as a backbone, linearizing by double restriction digestion with BamH I and Hind III to obtain vector backbone A; using plasmid PMA5 and Bacillus amyloliquefaciens NS genome as templates, amplifying promoter PHpaⅡ and terminator Tamy fragments by PCR; connecting promoter PHpaⅡ, pgdS fragment obtained in step (4) and terminator Tamy fragment by overlapping PCR to obtain fragment pHpaⅡ-pgdS; connecting the obtained fragment PHpaⅡ-pgdS and vector backbone A by one-step cloning to obtain plasmid pHY-PHpaⅡ-pgdS; b: Plasmid pHY-PHpaⅡ-pgdS was used as the backbone, and the vector backbone B was obtained after double restriction digestion with EcoRⅠ and BamHⅠ; plasmid PMA5 and Bacillus amyloliquefaciens NS genome were used as templates to amplify the promoter PHpaⅡ and terminator Tamy fragments by PCR; the promoter PHpaⅡ, yrpC fragment and terminator Tamy fragment were connected by overlapping PCR to obtain the fragment PHpaⅡ-yrpC; the obtained fragment PHpaⅡ-yrpC was connected with the vector backbone B by one-step cloning to obtain the plasmid pHY-yrpC-pgdS; (6) The fragments yrpC and pgsA amplified in step (2) and step (3) are subjected to fusion PCR to obtain a fusion fragment yrpC-pgsA, which is then double-digested with Sal I and Pst I to obtain a vector backbone C, and the fusion fragment is connected to the vector backbone C to obtain a recombinant plasmid pHY-yrpC-pgsA-pgdS; (7) The recombinant plasmid pHY-yrpC-pgsA-pgdS obtained in step (6) is transformed into the recombinant Bacillus amyloliquefaciens NS to obtain a recombinant strain, namely, the engineered Bacillus amyloliquefaciens NS-LM.

4. The engineered Bacillus amyloliquefaciens producing ultra-low molecular weight polyglutamic acid constructed by the construction method according to any one of claims 1 to 3.

5. Use of the Bacillus amyloliquefaciens engineered bacteria according to claim 4 in producing ultra-low molecular weight polyglutamic acid.

6. The use according to claim 5, characterized in that: The ultra-low molecular weight polyglutamic acid has a molecular weight of 3-5 kDa.

7. The use according to claim 5, characterized in that: The seed liquid of the Bacillus amyloliquefaciens engineered bacteria is inoculated into a fermentation medium, the culture temperature is 30-37° C., the shaking speed is 180-240 r / min, the culture is carried out for 48-72 hours, and ultra-low molecular weight polyglutamic acid is fermented and prepared.

8. The use according to claim 7, characterized in that: The inoculation amount is 6% v / v.

9. The use according to claim 7, characterized in that: The fermentation medium formula is: inulin crude extract 70g / L, (NH4)2SO4 5g / L, K2HPO4·3H2O 20g / L, KH2PO4 2g / L, MgSO4 0.45g / L, MnSO4·H2O0.06g / L, pH 8.0.

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