CN116004497A

CN116004497A - Bacillus amyloliquefaciens engineering bacterium for producing ultra-low molecular weight polyglutamic acid and application thereof

Info

Publication number: CN116004497A
Application number: CN202211445858.XA
Authority: CN
Inventors: 王延斌; 冯小海; 许宗奇; 金如昌; 孟恒君
Original assignee: Nanjing Xuankai Biotechnology Co ltd; Xuan Kai Biotechnology Chuzhou Co ltd
Current assignee: Nanjing Xuankai Biotechnology Co ltd; Xuan Kai Biotechnology Chuzhou Co ltd
Priority date: 2022-11-18
Filing date: 2022-11-18
Publication date: 2023-04-25

Abstract

The invention discloses a biosynthesis method of ultra-low molecular weight polyglutamic acid. To eliminate endogenous plasmid P2sip, knock out polyglutamic acid hydrolase pgdSA to obtain bacillus amyloliquefaciens NS, and further coupling co-expression glutamate racemase genes yrpC, gamma-PGA polymerase genes pgsA and gamma-PGA degrading enzyme genes pgdS to obtain host bacteria NS-LM. The positive effect of gamma-PGA stereochemical configuration regulation on gamma-PGA molecular weight control was determined by substrate configuration analysis of the Bacillus subtilis NX-2 source pgdS. Further, by recombinant expression of the yrpC, pgsA and pgdS and combined substitution of pgsA genes from different bacterial sources, the restriction of gamma-PGA configuration on the hydrolysis rate of degrading enzymes is successfully relieved, the molecular weight of gamma-PGA is further reduced to 3-5kDa, the yield is up to 34.27 +/-0.25 g/L, and the ultra-low molecular weight polyglutamic acid is synthesized in one step. The process further widens the molecular weight distribution range of the biosynthesis of the gamma-PGA, simplifies the production process of the low molecular weight polyglutamic acid, and is beneficial to promoting the application of the gamma-PGA in different fields.

Description

Bacillus amyloliquefaciens engineering bacterium for producing ultra-low molecular weight polyglutamic acid and application thereof

Technical Field

The invention belongs to the fields of genetic engineering and fermentation engineering, and in particular relates to bacillus amyloliquefaciens for producing ultra-low molecular weight polyglutamic acid and application thereof.

Background

With the continued depth of research into γ -PGA, more and more studies have shown that the biological function of γ -PGA has a close relationship with its molecular weight. Especially when the molecular weight is below 10kDa, γ -PGA exhibits a unique biologically active function. Research shows that gamma-PGA with molecular weight of 1-10kDa is easier to permeate into skin and muscle sole, and has transdermal moisturizing effect. The gamma-PGA with the molecular weight lower than 10kDa is easy to combine with the drug, improves the targeting property, promotes the drug effect to be more complete, and also shows certain advantages in the aspect of gene therapy. This shows that gamma-PGA (1-10 kDa) has an important application prospect in the fields of cosmetics and medicines.

The current gamma-PGA degradation mode is mainly physical degradation, chemical degradation and enzymatic degradation. Earlier studies have found that it is difficult to reduce the molecular weight of γ -PGA to less than 10kDa by means of physical and chemical degradation, and that unstable product quality, a wide molecular weight range, serious environmental pollution, etc. are easily caused during the reproduction process of these means. In the preparation process of realizing different molecular weight gamma-PGA by the biological enzyme method, the endo gamma-PGA degrading enzyme plays a main role. However, the production process for regulating and controlling the molecular weight of the gamma-PGA by the enzyme-producing and catalytic two-step method is too complex, and the commercial gamma-PGA degrading enzyme cannot be obtained at present, so that the production cost is too high, and the large-scale application is difficult to realize.

In recent years, with the continuous development of molecular biology, research on Guan-PGA synthesis key enzyme has made a further breakthrough, and direct fermentation in one step is receiving more and more attention due to its simplified process flow and low cost. Earlier studies have shown that degrading enzymes have the characteristic of selectivity of substrate configuration, so that the difference of substrate configuration can seriously affect the hydrolysis rate of degrading enzymes, and the regulation of configuration is an effective measure for promoting the hydrolysis efficiency. Secondly, by carrying out heterologous clone expression on gamma-PGA synthetase and glutamic acid racemase which are derived from different strains, the molecular weight of the gamma-PGA synthesized by microorganisms is found to have the characteristic of dependence on the difference between polymerase and racemase. As a result of plasmid free expression of the Bacillus licheniformis B.lichenifermis NK-03 and Bacillus amyloliquefaciens B.amyloliquefaciens LL 3-derived gamma-PGA synthase gene cluster pgsBCA using E.coli Escherichia coli JM109 as an expression host, it was found that the average molecular weight of gamma-PGA synthesized by B.lichenifermis NK-03-derived gamma-PGA synthase was 3.74.+ -. 0.38X10 ⁴ Da, whereas the average molecular weight of gamma-PGA synthesized by the B.amyloquefaciens LL 3-derived gamma-PGA synthetase is relatively low, 3.23.+ -. 0.26X 10 ⁴ Da, further coupled expression of the glutamate racemase racE will further cause a change in the molecular weight of gamma-PGA.

Based on the above, in order to realize the synthesis of the ultra-low molecular weight gamma-PGA, the interference of gamma-PGA stereochemical composition on the hydrolysis rate of gamma-PGA degrading enzyme is relieved, functional analysis and verification are carried out on key enzymes for regulating and controlling related gamma-PGA configuration, a stereochemical composition regulating and controlling model in the gamma-PGA synthesis process is constructed, the gamma-PGA degrading enzyme hydrolysis process is further coupled, and the synthesis of the ultra-low molecular weight gamma-PGA is realized. The research has important significance for promoting the application of the gamma-PGA in different fields, and provides a beneficial reference for the control of the molecular weight of other similar high molecular compounds.

Disclosure of Invention

Aiming at the defects of the prior art, the invention designs experiments and verifies that the combination of gamma-PGA stereochemical component regulation and degradation enzyme hydrolysis process is an effective strategy for regulating the molecular weight of gamma-PGA, and provides a bacillus amyloliquefaciens engineering bacterium for producing ultra-low molecular weight polyglutamic acid and application thereof.

The invention also solves the technical problem that the bacillus amyloliquefaciens engineering bacteria are applied to producing ultra-low molecular weight polyglutamic acid.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a construction method of bacillus amyloliquefaciens engineering bacteria for producing ultra-low molecular weight polyglutamic acid is characterized in that bacillus amyloliquefaciens NX-2S CCTCC NO:M 2016346 is taken as an original strain, an endogenous plasmid p2Sip is eliminated, a polyglutamic acid degrading enzyme gene pgdSA is further knocked out to obtain recombinant bacillus amyloliquefaciens NS, and the recombinant bacillus amyloliquefaciens NS is coupled to co-express pgsA-yrpC-pgdS to obtain bacillus amyloliquefaciens engineering bacteria NS-LM.

Wherein, in the yrpC-pgsA-pgdS, the glutamate racemase gene yrpC is derived from bacillus amyloliquefaciens NS, the gamma-PGA synthetase gene pgsA is derived from bacillus anthracis BA, and the gamma-PGA degrading enzyme gene pgdS is derived from bacillus subtilis NX-2.

Specifically, the nucleotide sequence of the glutamate racemase gene yrpC is shown in SEQ ID NO. 27, the nucleotide sequence of the gamma-PGA synthetase gene pgsA is shown in SEQ ID NO. 54, and the nucleotide sequence of the gamma-PGA degrading enzyme gene pgdS is shown in SEQ ID NO. 18.

The construction method of the bacillus amyloliquefaciens engineering bacteria comprises the following steps:

(1) The construction process of the recombinant bacillus amyloliquefaciens NS is as follows: bacillus amyloliquefaciens NX-2S (CCTCC NO: M2016146) is taken as an initial strain, the endogenous plasmid P2Sip is eliminated, and the polyglutamic acid degrading enzyme gene pgdSA is further knocked out. The process of elimination of the Bacillus amyloliquefaciens NX-2S endogenous plasmid P2Sip has been described in patent CN 108624546B. In order to realize the traceless knockout of the polyglutamic acid degrading enzyme gene pgdSA, the genome of bacillus amyloliquefaciens NX-2S is taken as a template, and the primers pgdSAUP/DN-F/R are used for amplifying the homologous arms pgdSA-UP/DN of 800bp at the upstream and downstream of the target gene pgdSA. Meanwhile, the expression plasmid pMA5 is used as a template, and the pDR-PHpaII-F/R is utilized to amplify the constitutive strong promoter PHpaII. After gel recovery, fusing the promoter and upstream and downstream homology arms into a fragment PHpa II-geneUP-geneDN through overlapping PCR, and cloning the fusion fragment to a vector pDR-pheS of EcoR I and Xho I double enzyme digestion by a one-step cloning method (see ClonExpress Multis One Step Cloning Kit kit for steps) to obtain a recombinant plasmid pDR-pheS of pgdSA. After demethylation, the recombinant plasmid with correct verification is electrically transformed (voltage 2.0-3.0kv, electric shock time 4 ms) into bacillus amyloliquefaciens NX-2S, and whether the transformation is successful is verified by using a primer pDR-F/R. The strain containing the recombinant plasmid is cultured in LB plate medium containing 100 mug/mL spectinomycin at 30 ℃ for 12 hours, and then transferred to LB culture solution containing 100 mug/mL spectinomycin at 42 ℃ for 24-48 hours to induce plasmid single exchange. After the single-exchange positive clone is subjected to loose culture for 2 generations in an LB culture solution without resistance, the single-exchange positive clone is diluted and coated on an LB plate culture medium, and a single colony is selected to carry OUT double-exchange verification by using a pgdSA-OUT-F/R primer, so that the constructed recombinant bacillus amyloliquefaciens NS is successfully verified.

(2) The bacillus amyloliquefaciens NS genome is used as a template, a PCR amplification technology is adopted, and a primer pHY-yrpC-F/R is used for amplifying a fragment yrpC.

Wherein, the primer pHY-yrpC-F: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 21, and the primer pHY-yrpC-R: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 22.

(3) The bacillus anthracis BA genome is used as a template, a PCR amplification technology is adopted, and a primer pHY-pgsA (BA) -F/R is utilized to amplify the fragment pgsA.

Wherein, the primer pHY-pgsA (BA) -F: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO:50, wherein the primer pHY-pgsA (BA) -R: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 51.

(4) The bacillus subtilis NX-2 genome is used as a template, a PCR amplification technology is adopted, and a primer PHY-pgdS-F/R is used for amplifying a fragment pgdS.

Wherein, primer PHY-pgdS-F: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 19, wherein the primer PHY-pgdS-R: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 20.

(5) Construction of plasmid pHY-yrpC-pgdS:

taking shuttle plasmid pHY300PLK as a framework, and carrying out double-enzyme tangential linearization on BamH I and HindIII to obtain a carrier framework A; the plasmid PMA5 and the bacillus amyloliquefaciens NS genome are used as templates, and a promoter PHpa II and a terminator Tamy fragment are amplified through PCR; connecting the promoter PHpa II, the pgdS fragment obtained in the step (4) and the terminator Tamy fragment by using an overlapping PCR (polymerase chain reaction) mode to obtain a fragment PHpa II-pgdS; connecting the obtained fragment PHpa II-pgdS with a vector skeleton A by a one-step cloning method to obtain a plasmid pHY-PHpa II-pgdS;

b, taking plasmid pHY-PHpaII-pgdS as a framework, and carrying out double-enzyme tangential linearization on EcoRI and BamHI to obtain a carrier framework B; the plasmid PMA5 and the bacillus amyloliquefaciens NS genome are used as templates, and a promoter PHpa II and a terminator Tamy fragment are amplified through PCR; connecting the promoter PHpa II, the yrpC fragment and the terminator Tamy fragment by using an overlapping PCR mode to obtain a fragment PHpa II-yrpC; connecting the obtained fragment PHpa II-yrpC with a vector skeleton B by a one-step cloning method to obtain plasmid pHY-yrpC-pgdS;

(6) Obtaining fusion fragments yrpC-pgsA by fusion PCR of fragments yrpC and pgsA obtained by amplifying in the step (2) and the step (3), then obtaining a carrier skeleton C by double digestion and recovery of the fusion fragments with Sal I and Pst I of the plasmid pHY-yrpC-pgdS constructed in the step (5), and connecting the fusion fragments with the carrier skeleton C to obtain recombinant plasmid pHY-yrpC-pgsA-pgdS;

(7) And (3) converting the recombinant plasmid pHY-yrpC-pgsA-pgdS obtained in the step (6) into recombinant bacillus amyloliquefaciens NS to obtain a recombinant strain, namely bacillus amyloliquefaciens engineering bacteria NS-LM.

The bacillus amyloliquefaciens which is constructed by the construction method and produces the ultra-low molecular weight polyglutamic acid is also within the scope of the invention.

The application of the bacillus amyloliquefaciens engineering bacteria in producing ultra-low molecular weight polyglutamic acid is also within the scope of the invention.

Wherein the molecular weight of the ultra-low molecular weight polyglutamic acid is 3-5kDa, and the yield is up to 34.27 +/-0.25 g/L.

Wherein, the bacillus amyloliquefaciens seed liquid is inoculated into a fermentation culture medium to be cultured for 48 to 72 hours at the temperature of 30 to 37 ℃ and the rpm of 180 to 240rpm, and the ultra-low molecular weight polyglutamic acid is prepared by fermentation. The preferred fermentation conditions are 32℃and 200rpm for 72 hours.

Specifically, the seed liquid is prepared by the following steps: sucking a small amount of bacterial liquid from a glycerol pipe preserved at-80 ℃, streaking and separating on an LB plate medium, and standing and culturing at 32 ℃ for overnight. Single colonies were picked from LB plate medium and transferred to 250mL shake flasks containing 50mL of LB liquid medium, and cultured at 32℃with shaking at 200rpm for 12h.

Specifically, the inoculation amount is 6% v/v.

Specifically, the formula of the fermentation medium is as follows: 70g/L of inulin crude extract (NH) ₄ ) ₂ SO ₄ 5g/L，K ₂ HPO ₄ ·3H ₂ O 20g/L，KH ₂ PO ₄ 2g/L，MgSO ₄ 0.45g/L，MnSO ₄ ·H ₂ O 0.06g/L，pH 8.0。

The beneficial effects are that: firstly, positive effects of gamma-PGA stereochemical configuration regulation on gamma-PGA molecular weight control are identified: analysis of the substrate configuration of the pre-selected gamma-PGA degrading enzyme revealed that it was more prone to hydrolyze gamma-PGA substrates containing a high proportion of D-glutamic acid monomers. On the basis, in order to realize the regulation and control of the gamma-PGA stereochemical configuration, the influence of the glutamate racemase gene yrpC and the gamma-PGA synthetase gene pgsA on the gamma-PGA configuration is examined respectively, and the over-expression of the glutamate racemase gene yrpC and the gamma-PGA synthetase gene pgsA is found to be beneficial to the improvement of the D-glutamate monomer proportion in the gamma-PGA. Finally, recombinant strain NS-LM is obtained by recombinant expression of the yrpC, pgsA and pgdS and combined substitution of pgsA genes from different bacterial sources, so that the limit of gamma-PGA configuration on the hydrolysis rate of degrading enzyme is successfully relieved, the molecular weight of gamma-PGA is further reduced to 3-5kDa, and the yield reaches 34.27 +/-0.25 g/L. The process further widens the molecular weight distribution range of the biosynthesis of the gamma-PGA, and is beneficial to promoting the application of the gamma-PGA in different fields.

Drawings

The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.

FIG. 1 shows the effect of the stereochemical configuration of gamma-PGA on the hydrolytic efficiency of the degrading enzyme pgdS.

FIG. 2 shows the effect of enhanced expression of gene yrpC and pgsA on gamma-PGA synthesis. (A) cell growth amount; (B) γ -PGA yield; (C) gamma-PGA molecular weight; (D) gamma-PGA stereochemistry.

FIG. 3 shows the effect of the combined expression of genes yrpC, pgsA and pgdS on cell growth (A), gamma-PGA yield (B) and gamma-PGA molecular weight (C).

FIG. 4 shows the effect of the replacement of the pgsA gene of different origin on the synthesis of gamma-PGA.

Detailed Description

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

In the examples below, the experimental materials used were derived as follows:

all primers and DNA sequences were synthesized by gold sri biotechnology limited;

all plasmid and DNA fragment sequencing work was done by golden sri biosciences, inc.;

the plasmid is extracted by using a plasmid small-quantity DNA extraction kit (AP-MN-P-250G) provided by Axygen company;

the recovery of the DNA fragment is carried out by using a DNA gel recovery kit (AP-MN-P-250G) provided by Axygen company;

the extraction of the bacterial genome is carried out by using a bacterial genome DNA extraction kit (TIANamp Bacteria DNA Kit) provided by Tiangen biochemical technology Co., ltd;

the DNA fragment PCR is performed by using Kodaq 2X PCR MasterMix with dye high-fidelity enzyme of Aiping Biotechnology Co., ltd;

the ligase used for the one-step cloning ligation was selected from the ligases of Norvezan Biotechnology Co., ltd (ClonExpress II One Step Cloning Kit, clonExpress MultiS One Step Cloning Kit);

the restriction enzyme is selected from restriction enzymes of Takiosks, inc.

In the following examples, the PCR reaction system used was:

respectively amplifying target genes by using bacillus amyloliquefaciens NS genome as a template and using related primers, wherein a reaction mixture contains 2 mu L of the template, 2 mu L of each primer, 25 mu L of KOD enzyme and 19 mu L of double distilled water; the PCR reaction parameters are as follows: stage one: 95 ℃,30s, stage two: 95 ℃ for 5s;60 ℃ for 60s;40 cycles. The overlapping PCR reaction system is as follows: the reaction mixture contained 2. Mu.L of each template to be overlapped, 2. Mu.L of each head-to-tail primer, 25. Mu.L of KOD enzyme and 17. Mu.L of double distilled water; the overlapping PCR reaction parameters were: stage one: 95 ℃,30s, stage two: 95 ℃ for 5s;60 ℃ for 60s;40 cycles.

In the following examples, recombinant strains NS (yrpC), NS (pgsA) and NS (yrpC-pgsA) described in example 2, recombinant strains NS (yrpC-pgdS), NS (pgsA-pgdS) and NS (yrpC-pgsA-pgdS) described in example 3, and recombinant strains constructed by substituting the pgsA gene from different sources described in example 4 were fermented as follows:

(1) Sucking a small amount of bacterial liquid from a glycerol pipe preserved at-80 ℃, streaking and separating on an LB plate medium, and standing and culturing at 32 ℃ for overnight.

(2) Single colonies were picked from LB plate medium and transferred to 250mL shake flasks containing 50mL of LB liquid medium, and cultured at 32℃with shaking at 200rpm for 12h.

(3) The seed solution was inoculated at 6% v/v into 500mL shake flasks containing 80mL of fermentation medium and incubated at 32℃for 72h at 200 rpm.

Wherein, the formula of the LB culture medium is as follows: 5g/L yeast powder, 10g/L peptone, 10g/L sodium chloride and pH 6.0-8.0.

Wherein, the formula of the fermentation medium is as follows: 70g/L of inulin crude extract (NH) ₄ ) ₂ SO ₄ 5g/L，K ₂ HPO ₄ ·3H ₂ O 20g/L，KH ₂ PO ₄ 2g/L，MgSO ₄ 0.45g/L，MnSO ₄ ·H ₂ O 0.06g/L，pH 8.0。

In the following examples, the production and molecular weight measurement methods of γ -PGA were as follows:

the molecular weight of the fermentation product was measured by Gel Permeation Chromatography (GPC), and after the fermentation purified product was diluted appropriately, the cell was removed by using a 0.22 μm filter, and 20. Mu.L of the sample was directly introduced. Selecting a chromatographic column: shodex Ohpak SB-806M HQ; preparing 0.2mol/L Na ₂ SO ₄ The solution is taken as a mobile phase of a liquid phase, and the pH value is adjusted to be about 4.0 by 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 1.0mL/min. The peak time of the sample was compared with the gamma-PGA standard to determine the molecular weight of the product.

In the following examples, the D/L configuration of gamma-PGA was determined as follows:

the D/L monomer content in the gamma-PGA was determined using a chiral column and the gamma-PGA product was subjected to hydrolysis at 110℃for 12h with 6mol/L HCl. The hydrolysate was adjusted to pH 7.0 with NaOH, filtered with a 0.22 μm filter, and 20. Mu.L was directly fed. Selecting a chromatographic column: sumichiral OA-5000; preparation of 1mM CuSO ₄ The solution was used as a mobile phase of the liquid phase, and the flow rate of the mobile phase was adjusted to 1.0mL/min in order to control the pressure in the liquid chromatography column not to be too high. The sample injection temperature is controlled at 35 ℃. And comparing the peak-out time of the sample with a D/L-glutamic acid standard substance, and determining the proportion of the D/L-glutamic acid monomer.

EXAMPLE 1 analysis of propensity to hydrolysis of substrate configuration by the Bacillus subtilis-derived gamma-PGA degrading enzyme pgdS

In characterizing the properties of γ -PGA degrading enzymes, it is necessary to examine the propensity of degrading enzymes to the stereochemical configuration of γ -PGA, as this property may limit the effectiveness of degrading enzymes in hydrolysis of γ -PGA, thereby affecting the final molecular weight of the product. In this study, in order to investigate the tendency of the degrading enzyme pgdS derived from B.subtilis NX-2 to hydrolyze the substrate configuration, gamma-PGA containing D-glutamic acid monomers in a proportion of 80%, 50% and 20%, respectively, was hydrolyzed as a substrate.

Specifically, the recombinant strain NS-pHY-PHpa II-pgdS fermentation broth is centrifuged to obtain a crude enzyme solution containing degrading enzymeThe method comprises the steps of carrying out a first treatment on the surface of the Gamma-PGA of different D-glutamic acid monomers was dissolved in 0.05mM phosphate buffer (pH 7.4) to obtain 10g/L of an aqueous gamma-PGA solution. The enzyme reaction solution contained 200. Mu.L of the crude enzyme solution and 800. Mu.L of the gamma-PGA solution, and was incubated at 37℃for 4 hours. A blank sample containing inactivated enzyme was set for each sample to correct the results for non-enzymatic release of amino groups. Heating in boiling water for 5min to stop reaction, adding 1mL ninhydrin color developing solution, mixing, heating at 100deg.C for 15min, cooling, adding 3mL 60% ethanol for dilution, and detecting OD with ultraviolet spectrophotometer ₅₇₀ The hydrolysis rates of the enzyme solutions on the different substrates were compared.

As a result, as shown in FIG. 1, the degradation enzyme pgdS had the highest hydrolysis rate of gamma-PGA containing 80% of D-glutamic acid monomer, and the molecular weight of gamma-PGA was drastically reduced from 1500kDa to 150kDa after 24 hours of hydrolysis. When gamma-PGA containing 50% of D-glutamic acid monomer is used as hydrolysis substrate, the hydrolysis rate of degradation enzyme PgdS is reduced, and the molecular weight of gamma-PGA is reduced from 1300kDa to about 500 kDa. In the case of gamma-PGA containing 20% D-glutamic acid monomer, the degradation enzyme PgdS has no obvious hydrolysis effect, and the molecular weight of gamma-PGA is not changed obviously in the enzymolysis process. These results indicate that the degrading enzyme pgdS derived from Bacillus subtilis NX-2 has a good hydrolysis effect on gamma-PGA containing a higher proportion of D-glutamic acid monomers.

Wherein, the construction process of the recombinant strain NS-pHY-PHpaII-pgdS is as follows:

bacillus amyloliquefaciens NX-2S (CCTCC NO: M2016146) is taken as an initial strain, the endogenous plasmid P2Sip is eliminated, and the polyglutamic acid degrading enzyme gene pgdSA is further knocked out. The process of elimination of the Bacillus amyloliquefaciens NX-2S endogenous plasmid P2Sip has been described in patent CN 108624546B. In order to realize the traceless knockout of the polyglutamic acid degrading enzyme gene pgdSA, the genome of bacillus amyloliquefaciens NX-2S is taken as a template, and the primers pgdSAUP/DN-F/R are used for amplifying the homologous arms pgdSA-UP/DN of 800bp at the upstream and downstream of the target gene pgdSA. Meanwhile, the expression plasmid pMA5 is used as a template, and the pDR-PHpaII-F/R is utilized to amplify the constitutive strong promoter PHpaII. After gel recovery, fusing the promoter and upstream and downstream homology arms into a fragment PHpa II-geneUP-geneDN through overlapping PCR, and cloning the fusion fragment to a vector pDR-pheS of EcoR I and Xho I double enzyme digestion by a one-step cloning method (see ClonExpress Multis One Step Cloning Kit kit for steps) to obtain a recombinant plasmid pDR-pheS of pgdSA. After demethylation, the recombinant plasmid with correct verification is electrically transformed (voltage 2.0-3.0kv, electric shock time 4 ms) into bacillus amyloliquefaciens NX-2S, and whether the transformation is successful is verified by using a primer pDR-F/R. The strain containing the recombinant plasmid is cultured in LB plate medium containing 100 mug/mL spectinomycin at 30 ℃ for 12 hours, and then transferred to LB culture solution containing 100 mug/mL spectinomycin at 42 ℃ for 24-48 hours to induce plasmid single exchange. After the single-exchange positive clone is subjected to loose culture for 2 generations in an LB culture solution without resistance, the single-exchange positive clone is diluted and coated on an LB plate culture medium, and a single colony is selected to carry OUT double-exchange verification by using a pgdSA-OUT-F/R primer, so that the constructed recombinant bacillus amyloliquefaciens NS (starting strain) is successfully verified.

Wherein, the primer pgdSAUP-F: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 1, and the primer pgdSAUP-R: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 2, and the primer pgdSADN-F: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO:3, and the primer pgdSADN-R: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 4, and the primer pDR-PHpa II-F: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 5, and the primer pDR-PHpa II-R: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 6, and the primer pDR-F: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 7, and the primer pDR-R: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 8, and the primer pgdSA-OUT-F: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO:9, and the primer pgdSA-OUT-R: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 10.

The primers pHY-PHpa II-F/R and pHY-Tamy-F/R are used for amplifying the promoter PHpa II and the terminator Tamy fragment from the genome of the plasmid PMA5 and the Bacillus amyloliquefaciens NS respectively, and in order to facilitate the replacement of the post-overexpression genes, a cleavage site Kpn I is introduced behind the promoter, and a cleavage site NdeI is introduced in front of the terminator. The bacillus subtilis NX-2 genome is used as a template, and a primer PHY-pgdS-F/R is used for amplifying the polyglutamic acid degrading enzyme gene pgdS. And then, fusing the promoter PHpa II, pgdS and the terminator Tamy fragment by using primers pHY-PHpa II-F and pHY-Tamy-R through overlapping PCR, recovering and purifying the fused fragment by using a gel, and connecting the fused fragment with a recombinant expression vector obtained by carrying out double-enzyme tangential recovery on BamH I and HindIII by using a one-step cloning method, namely an escherichia coli-bacillus subtilis shuttle plasmid pHY300PLK, so as to obtain a gene overexpression recombinant plasmid pHY-PHpa II-pgdS. And (3) carrying out demethylation on the recombinant plasmid which is verified to be successful, and then electrically converting the recombinant plasmid into the constructed recombinant bacillus amyloliquefaciens NS (original strain) to obtain the recombinant strain NS-pHY-PHpa II-pgdS.

The nucleotide sequence of the recombinant expression vector is shown as SEQ ID NO. 11, the nucleotide sequence of the promoter PHpa II is shown as SEQ ID NO. 12, the nucleotide sequence of the terminator Tamy is shown as SEQ ID NO. 13, and the primer pHY-PHpa II-F: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 14, and the primer pHY-PHpa II-R: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 15, and the primer pHY-Tamy-F: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 16, and the primer pHY-Tamy-R: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 17, the nucleotide sequence of pgdS is shown as SEQ ID NO. 18, and the primer PHY-pgdS-F: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 19, and the primer PHY-pgdS-R: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 20.

EXAMPLE 2 Effect of glutamate racemase yrpC and gamma-PGA synthetase pgsA on the stereochemical configuration of gamma-PGA

To evaluate the effect of glutamate racemase yrpC and γ -PGA synthase pgsA on γ -PGA synthesis, recombinant strains NS (yrpC), NS (pgsA) and NS (yrpC-pgsA) were obtained by expressing glutamate racemase gene yrpC and γ -PGA synthase gene pgsA, respectively, alone and in a coupled manner in recombinant bacillus amyloliquefaciens NS (starting strain). The method comprises the following specific steps:

the genome of recombinant Bacillus amyloliquefaciens NS (starting strain) is used as a template, and primers pHY-pyroC-F/R and pHY-pgsA-F/R are used for respectively amplifying fragments yrpC and pgsA. And taking the pHY-PHpa II-pgdS plasmid constructed before as a framework, carrying out enzyme digestion and recovery by using KpnI and NdeI to obtain a linearization cloning vector, connecting the yrpC and pgsA fragments with the linearization cloning vector by adopting one-step cloning, converting the connected with the linearization cloning vector into E.coll GM2163, picking a single colony to carry out plasmid enzyme digestion to verify the correctness of the recombinant plasmid, and obtaining the recombinant plasmids pHY-yrpC and pHY-pgsA. For co-expression of the 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, and the two fragments were fused and overlapped using primers pHY-yrpC-F/pHY-pgsA-R, and the fragment yrpC-pgsA was recovered by gel. The pHY-PHpa II-pgdS plasmid constructed before is taken as a framework, kpnI and NdeI are used for carrying out enzyme digestion and recovery to obtain a linearization cloning vector, one-step cloning is adopted to connect the yrpC-pgsA fragment with the linearization cloning vector and convert the connected pyroC-pgsA fragment into E.colli GM2163, single colony plasmid extraction is selected for enzyme digestion to verify the correctness of the recombinant plasmid, and the recombinant plasmid pHY-yrpC-pgsA is obtained.

The plasmids pHY-pyroC, pHY-pgsA and pHY-pyroC-pgsA which were confirmed to be successful were transformed into host bacteria, respectively, to obtain recombinant strains NS (pyroC), NS (pgsA) and NS (pyroC-pgsA);

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

As shown in FIG. 2, the cell dry weights of recombinant strains NS (yrpC), NS (pgsA) and NS (yrpC-pgsA) were higher than those of the control strain NS. In addition, the production of γ -PGA synthesized by 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 (FIG. 2B). These results indicate that overexpression of yrpC and pgsA is beneficial for cell growth and synthesis of γ -PGA. Meanwhile, the influence of different recombinant strains on the molecular weight and stereochemical composition of the synthesized gamma-PGA is also studied. As shown in FIG. 2C, the molecular weights of γ -PGA produced by strains NS (yrpC), NS (pgsA) and NS (yrpC-pgsA) increased from 1300-1400kDa to 1400-1550kDa, 1600-1750kDa and 1800-1900kDa, respectively, and the D-glutamic acid monomer ratios in the corresponding strains synthesized γ -PGA increased from 57% to 61%, 71% and 80%, respectively (FIG. 2D). These results indicate that enhancing the expression of yrpC and pgsA is an effective method for modulating the molecular weight and stereochemistry of γ -PGA. In particular, γ -PGA obtained by regulating pgsA expression has a higher molecular weight and a higher D-glutamic acid monomer ratio than γ -PGA obtained by controlling the level of expression of yrpC, i.e., it is more effective to regulate the composition of γ -PGA by regulating pgsA expression than to regulate the expression of yrpC.

To further investigate the effect of yrpC and pgsA on γ -PGA synthesis, knock-out experiments were performed on the genes yrpC and pgsA, respectively.

In order to realize the traceless knockout of the yrpC and the pgsA, referring to the knockout process of pgdSA, the bacillus amyloliquefaciens NX-2S genome is taken as a template, the homologous repair template consisting of 800bp upstream and 800bp downstream is selected according to the gene sequences of the yrpC and the pgsA, and the primers yrpCUP/DN-F/R and pgsapup/DN-F/R are used for amplifying the homologous arms yrpC-UP/DN and pgsA-UP/DN of the target genes yrpC and pgsA of 800bp upstream and downstream respectively. After glue recovery, fusing the promoter PHpa II and upstream and downstream homology arms into a fragment PHpa II-geneUP-geneDN through overlapping PCR, and cloning the fusion fragment to EcoR I and Xho I double-enzyme-cut vector pDR-pheS by a one-step cloning method to obtain recombinant plasmid pDR-pheS-yrpC/pgsA. After demethylation, the recombinant plasmid with correct verification is electrically transformed into bacillus amyloliquefaciens, and whether the transformation is successful is verified by using a primer pDR-F/R. The strain containing the recombinant plasmid is cultured in LB plate medium containing 100 mug/mL spectinomycin at 30 ℃ for 12 hours, and then transferred to LB culture solution containing 100 mug/mL spectinomycin at 42 ℃ for 24-48 hours to induce plasmid single exchange. After the single exchange positive clones are subjected to loose culture for 2 generations in an LB culture solution without resistance, the single exchange positive clones are diluted and coated on an LB plate culture medium, single colonies are picked up and subjected to double exchange verification by using a yrpC/pgsA-OUT-F/R primer, and the verification success is that mutant strains NS (delta-yrpC) and NS (delta-pgsA) of the yrpC and pgsA are successfully knocked OUT.

Wherein, primer yrpCUP-F: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 28, and the primer is the pyroCUP-R: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 29, and the primer is pyroCDN-F: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO:30, and the primer is pyroCDN-R: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 31, and the primer pgsADP-F: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 32, and the primer pgsADP-R: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 33, and the primer pgsADN-F: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO:34, and the primer pgsADN-R: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO:35, and the primer is pyroC-OUT-F: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO:36, and the primer is pyroC-OUT-R: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO:37, and the primer pgsA-OUT-F: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO:38, and the primer pgsA-OUT-R: the nucleotide sequence of 5 '-3' is shown as SEQ ID NO. 39.

As shown in Table 1, deletion of the glutamate racemase gene yrpC adversely affects the amount of bacterial growth and the synthesis of γ -PGA. The molecular weight of gamma-PGA synthesized by NS (Δyrpc) decreases from 1300-1400kDa to 1000-1100kDa. The D-glutamic acid monomer content in the gamma-PGA was also reduced from 57% to 52%. When pgsA gene was deleted, no gamma-PGA was detected in the fermentation broth. This result indicates that pgsA is necessary for the synthesis of Bacillus amyloliquefaciens gamma-PGA. The restoration of the yrpC and pgsA genes in the mutant restored normal cell growth and γ -PGA synthesis, indicating that the genes yrpC and pgsA are significant for maintaining cell growth and γ -PGA synthesis.

TABLE 1 influence of knockout and anaplerosis of the gene yrpC and pgsA on the synthesis of gamma-PGA

Example 3 influence of coupled expressed genes yrpC, pgsA and pgdS on the molecular weight of γ -PGA

Although the D-glutamic acid monomer ratio in gamma-PGA has been optimized by enhanced expression of yrpC and pgsA, the specific distribution of D-glutamic acid monomer and L-glutamic acid monomer on the chain has not been clarified. Therefore, to further verify whether up-regulation of the D-glutamic acid monomer ratio promotes the hydrolysis rate of the degrading enzyme pgdS, we expressed in Bacillus amyloliquefaciens NS in a coupled manner, respectively, yrpC-pgdS, pgsA-pgdS and yrpC-pgsA-pgdS. The method comprises the following specific steps:

the primers pHY-pyroC '-F/R and pHY-pgsA' -F/R were used to amplify fragments yrpC and pgsA, respectively, using Bacillus amyloliquefaciens genome NS as template. Meanwhile, primers pHY-PHpa II-F/R and pHY-Tamy-F/R are used for amplifying a promoter pHpa II and a terminator Tamy fragment from the genome of the Bacillus amyloliquefaciens NS respectively. To facilitate the replacement of the late co-expression fragment, the cleavage site SalI was introduced after the promoter and the cleavage site PstI was introduced before the terminator. The primers pHY-PHpa II-F/pHY-Tamy-R are used for fusing the promoter PHpa II, the yrpC fragment and the terminator Tamy fragment through overlapping PCR, and the fragment PHpa II-yrpC-Tamy is obtained through glue recovery and purification. The fusion fragment PHpa II-pgsA-Tamy was obtained in the same manner. And simultaneously, carrying out enzyme digestion and recovery on plasmid pHY-PHpa II-pgdS by using EcoRI and BamHI restriction enzymes to obtain a linearized digestion vector, respectively connecting PHpa II-yrpC-Tamy and PHpa II-pgsA-Tamy fusion fragments with the linearized digestion vector and transforming into E.coli GM2163 to obtain recombinant plasmids pHY-yrpC-pgdS and pHY-pgsA-pgdS.

The primers pHY-pyroC '-F/pHY-pyroCA-R and pHY-pgsA (BAY) -F/pHY-pgsA' -R are used for amplifying fragments yrpC and pgsA respectively, and the primers pHY-pyroC-F/pHY-pgsA-R are used for carrying out fusion PCR on the two fragments to obtain a fusion fragment yrpC-pgsA. And (3) carrying out double digestion and recovery on the plasmid pHY-yrpC-pgdS constructed in the previous step through SalI and PstI to obtain a linearization digestion vector, and connecting the fusion fragment with the digestion vector to obtain the recombinant plasmid pHY-yrpC-pgsA-pgdS. Plasmids which were successfully sequenced were transformed into strain NS, respectively, to give recombinant strains NS (yrpC-pgdS), NS (pgsA-pgdS) and NS (yrpC-pgsA-pgdS).

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

As a result of fermentation, as shown in FIG. 3, 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 gamma-PGA yields 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 weights of γ -PGA produced by NS (yrpC-pgdS), NS (pgsA-pgdS) and NS (yrpC-pgsA-pgdS) were successfully reduced to 17-25kDa, 13-16kDa and 9-11kDa, respectively, which were significantly lower than that of the control strain NS (PHpa II) (21-30 kDa). Co-expression of the two genes gives a lower molecular weight of gamma-PGA than expression of the single gene, yrpC or pgsA. These results indicate that modulating the stereochemical configuration of γ -PGA is an effective measure for synthesizing low molecular weight γ -PGA by Bacillus amyloliquefaciens. Notably, recombinant strain NS (pgsA-pgdS) produced a lower molecular weight of γ -PGA than that synthesized by recombinant strain NS (yrpC-pgdS), further confirming that pgsA significantly promotes the regulation of γ -PGA stereochemical configuration than yrpC.

EXAMPLE 4 Effect of expression of pgsA Gene from different sources on the molecular weight of gamma-PGA

Based on the experiments of example 3, it was found that coupling modulation of the stereochemical configuration of γ -PGA with the expression of degrading enzymes is beneficial for reducing the molecular weight of γ -PGA. 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 effect on the regulation of the stereochemical composition of gamma-PGA than that of yrpC, optimizing the source of pgsA is more advantageous for the synthesis of gamma-PGA of low molecular weight. Thus, we studied the effect of pgsA genes from different Bacillus sources on gamma-PGA synthesis, including B.anthracis, B.subtilis, B.licheniformis, B.megaterium and B.pumilus.

The pgsA gene of example 3 was replaced with pgsA gene derived from Bacillus anthracis B.anthracis, bacillus subtilis B.subtilis, bacillus licheniformis B.lichenifermis, bacillus megaterium B.megaterium and Bacillus pumilus, respectively, and the other conditions were unchanged, to construct recombinant strains, respectively.

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

As shown in FIG. 4, the substitution of pgsA gene from different Bacillus sources has a large effect on the molecular weight of gamma-PGA. When pgsA was replaced with genes derived from Bacillus subtilis and Bacillus licheniformis, the cell dry weight and the γ -PGA yield of the recombinant strain did not show a significant difference compared to the recombinant strain NS (yrpC-pgsA-pgdS) in example 3, whereas when pgsA of Bacillus megaterium and Bacillus pumilus was overexpressed, the cell dry weight and the γ -PGA yield were slightly decreased. Overexpression of pgsA from B.anthracis resulted in an increase in cell dry weight and gamma-PGA production to 6.1.+ -. 0.32g/L and 34.27.+ -. 0.25g/L, respectively. Furthermore, we further examined the effect of expression of pgsA from different sources on the molecular weight of gamma-PGA. As a result, as shown in FIG. 4, the strains expressing pgsA derived from Bacillus subtilis and Bacillus licheniformis produced gamma-PGA having molecular weights of 14-15kDa and 14-17kDa, respectively, and had no further effect of reducing the molecular weight compared with the molecular weight (9-11 kDa) of gamma-PGA synthesized by recombinant strain NS (yrpC-pgsA-pgdS). And when the pgsA genes derived from bacillus megaterium and bacillus pumilus are overexpressed, the molecular weight of the gamma-PGA synthesized by the recombinant strain is obviously higher than that of the gamma-PGA generated by the recombinant strain NS (yrpC-pgsA-pgdS). When expressing the pgsA gene from B.anthracis, the molecular weight of the synthesized gamma-PGA is the lowest, reaching 3-5kDa. These results indicate that substitution of pgsA from different sources is effective for synthesizing gamma-PGA of different molecular weights from Bacillus amyloliquefaciens, while coupling the pgsA gene from Bacillus anthracis, the yrpC gene from Bacillus amyloliquefaciens and the pgdS gene from Bacillus subtilis construct to obtain Bacillus amyloliquefaciens B.amyloliquefaciens NS-LM can effectively synthesize gamma-PGA of ultra-low molecular weight (Mw: 3-5 kDa) with the highest yield of gamma-PGA reaching 34.27.+ -. 0.25g/L.

Taken together, the results of examples 1-4 demonstrate that:

(1) Identifying the substrate configuration tendency of the degrading enzyme pgdS from bacillus subtilis B.subilis NX-2, and respectively taking gamma-PGA containing 80%, 50% and 20% of D-glutamic acid monomers as a substrate for enzymolysis reaction, wherein the degrading enzyme has higher hydrolysis efficiency on gamma-PGA containing 80% of D-glutamic acid monomers, which shows that the pgdS gene from B.subilis NX-2 has better hydrolysis effect on gamma-PGA containing higher D-glutamic acid monomers.

(2) The influence of glutamate racemase yrpC and gamma-PGA polymerase pgsA on gamma-PGA stereochemical configuration was examined, and overexpression and knockout experiments were performed on the two genes, respectively, and as a result, it was found that when the pyroc, pgsA and yrpC-pgsA were overexpressed, the proportion of D-glutamate monomer in the gamma-PGA synthesized by the corresponding strain was increased from 57% to 61%, 71% and 80%, respectively, indicating that the enhanced expression of both genes yrpC and pgsA resulted in an increase in the proportion of D-glutamate monomer in gamma-PGA.

(3) By combining and expressing the yrpC-pgdS, pgsA-pgdS and yrpC-pgsA-pgdS, the molecular weight is successfully reduced from 21 kDa to 30kDa to 17kDa to 25kDa, 13 kDa to 16kDa and 9 kDa to 11kDa respectively, and the restriction of the configuration on degradation is successfully relieved. And further optimize pgsA gene source, the molecular weight of gamma-PGA is reduced to 3-5kDa, and the yield is increased to 34.27 + -0.25 g/L.

The invention provides a bacillus amyloliquefaciens engineering bacterium for producing ultra-low molecular weight polyglutamic acid and an application thought and a method thereof, and particularly the method and the way for realizing the technical scheme are numerous, the above is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made by a person of ordinary skill in the art without departing from the principle of the invention, and the improvements and the modifications are also regarded as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.

Claims

1. A construction method of bacillus amyloliquefaciens engineering bacteria for producing ultra-low molecular weight polyglutamic acid is characterized in that bacillus amyloliquefaciens NX-2S CCTCC NO:M 2016346 is taken as an original strain, an endogenous plasmid p2Sip is eliminated, a polyglutamic acid degrading enzyme gene pgdSA is further knocked out to obtain recombinant bacillus amyloliquefaciens NS, and the recombinant bacillus amyloliquefaciens NS is coupled to co-express pgsA-yrpC-pgdS to obtain bacillus amyloliquefaciens engineering bacteria NS-LM.

2. The method according to claim 1, wherein the gene for glutamate racemase, yrpC, is derived from bacillus amyloliquefaciens NS, the gene for γ -PGA synthase, pgsA is derived from bacillus anthracis BA, and the gene for γ -PGA degrading enzyme, pgdS, is derived from bacillus subtilis NX-2.

3. The construction method as claimed in claim 1, comprising the steps of:

(1) Taking bacillus amyloliquefaciens NX-2S as an initial strain, eliminating an endogenous plasmid P2Sip, knocking out a polyglutamic acid degrading enzyme gene pgdSA, and constructing recombinant bacillus amyloliquefaciens NS;

(2) Using a bacillus amyloliquefaciens NS genome as a template, and amplifying a fragment yrpC by using PCR;

(3) Using bacillus anthracis BA genome as a template, and amplifying a fragment pgsA by using PCR;

(4) Using bacillus subtilis NX-2 genome as a template, and amplifying a fragment pgdS by using PCR;

(5) Construction of plasmid pHY-yrpC-pgdS:

taking shuttle plasmid pHY300PLK as a framework, and carrying out double-enzyme tangential linearization on BamH I and HindIII to obtain a carrier framework A; the plasmid PMA5 and the bacillus amyloliquefaciens NS genome are used as templates, and a promoter PHpa II and a terminator Tamy fragment are amplified through PCR; connecting the promoter PHpa II, the pgdS fragment obtained in the step (4) and the terminator Tamy fragment by using an overlapping PCR mode to obtain a fragment pHpa II-pgdS; connecting the obtained fragment PHpa II-pgdS with a vector skeleton A by a one-step cloning method to obtain a plasmid pHY-PHpa II-pgdS;

4. The bacillus amyloliquefaciens which is constructed by the construction method according to any one of claims 1-3 and produces ultra-low molecular weight polyglutamic acid.

5. The bacillus amyloliquefaciens of claim 4, wherein the bacillus amyloliquefaciens is used for producing ultra-low molecular weight polyglutamic acid.

6. The use according to claim 5, wherein the ultra-low molecular weight polyglutamic acid has a molecular weight of 3-5kDa.

7. The use according to claim 5, wherein the seed solution of the bacillus amyloliquefaciens is inoculated into a fermentation medium, the culture temperature is 30-37 ℃, the rotation speed of a shaking table is 180-240r/min, the culture is carried out for 48-72h, and the ultra-low molecular weight polyglutamic acid is prepared by fermentation.

8. The use according to claim 7, wherein the inoculation is performed in an amount of 6% v/v.

9. The use according to claim 7, wherein the fermentation medium is formulated as follows: 70g/L of inulin crude extract (NH) ₄ ) ₂ SO ₄ 5g/L，K ₂ HPO ₄ ·3H ₂ O 20g/L，KH ₂ PO ₄ 2g/L，MgSO ₄ 0.45g/L，MnSO ₄ ·H ₂ O 0.06g/L，pH 8.0。