WO2023124062A1 - METHOD FOR PREPARING γ-PGA COUPLED WITH ATP REGENERATION ENZYME AND POLY(GLUTAMIC ACID) SYNTHETASE - Google Patents

Abstract

Provided is a method for preparing γ-PGA coupled with an ATP regeneration enzyme and a poly(glutamic acid) synthetase, specifically a method for preparing a poly(glutamic acid) (γ-PGA) using glutamic acid or sodium glutamate as a substrate by means of an enzymic method, belonging to the technical field of biological engineering. The method comprises biological enzymes used in combination, or an expression vector or a cloning vector expressing the biological enzymes used in combination, or a transgenic cell line expressing the biological enzymes used in combination, or a genetically engineered bacterium expressing the biological enzymes used in combination, preferably, poly(glutamic acid) synthetase PgsB and polyphosphate kinase PPK used in combination.

Description

A method for preparing γ-PGA coupled with ATP regenerating enzyme and polyglutamic acid synthetase technical field

The invention belongs to the field of biology, and in particular relates to a method for preparing gamma-PGA coupled with ATP regenerating enzyme and polyglutamic acid synthetase.

Background technique

γ-polyglutamic acid (γ-PGA) is a natural polymer formed by condensation of glutamic acid monomers through γ-amide bonds in microorganisms. It has excellent film-forming, fibroblast, plasticity, moisture retention, Biodegradability and other advantages can be used as hydrogels, moisturizers, film-forming agents, thickeners, dispersants, drug-controlled release carriers, gene carriers, nano-wound dressings, etc. in cosmetics, food additives, environmental governance, biological Medicine and other fields have excellent commercial value.

Microbial fermentation is currently the main method for preparing γ-PGA. However, the production cycle of poly-γ-glutamic acid prepared by fermentation is long (generally more than 70 hours), and the conversion rate of substrate glutamic acid is low (30-50%), and with different strains and fermentation processes, the output of γ-PGA ( 10g/L-120g/L) and molecular weight (10kDa-1000kDa) vary widely. It is of great significance to develop efficient, low-cost, high-yield γ-PGA production process.

The static cell (or enzyme) conversion method uses the whole cell (or enzyme) of microorganisms as a reaction catalyst. On the basis of keeping the conditions of the fermentation method mild and low in cost, the production cycle is shorter than the fermentation method, and the conversion rate of the substrate is higher. The reaction system It is simple, has fewer by-products, is easier to extract and purify later products, and is more conducive to industrial production. However, so far there are few reports on the preparation of γ-PGA by resting cell (or enzyme) conversion method.

With the in-depth study of γ-PGA metabolic pathway, it is found that γ-PGA synthesis involves at least 3-4 genes (pgsBCA or pgsBCAE) in the pgs gene cluster. The introduction of the gene cluster into the host cell can greatly enhance the ability of the host cell to produce γ-PGA. The inventor's previous research found that engineering bacteria expressing only one gene of pgsB or its enzyme can convert glutamic acid to prepare γ-PGA, and the substrate glutamic acid conversion rate exceeds 70% after 6 hours of reaction, but adenosine triphosphate (ATP) must be added during the preparation process. ATP is the most important high-energy phosphate compound in living organisms. In industry, the enzymatic synthesis of glutathione, theanine, 7-aminocephalosporanic acid, glutamyl-taurine and other important bioactive substances or biochemical drugs requires the participation of ATP. ATP is expensive, and adding a large amount will inevitably increase the production cost of the product. ATP recycling is an important issue in the development of enzyme engineering, and it is also a key factor that restricts whether industrial production is economical.

Contents of the invention

The purpose of the present invention is to provide a method for preparing γ-PGA coupled with ATP regeneration enzyme and polyglutamic acid synthetase, the coupling of enzyme reaction and ATP regeneration effectively increases the output of γ-PGA, the reaction rate is fast, and the conversion efficiency is high. The production cost of γ-PGA is comparable to the existing fermentation preparation method, which provides a new path for the synthesis of γ-PGA. Specifically: first construct the No. 1 recombinant bacterium that overexpresses the pgsB gene of polyglutamic acid synthase, and the No. 2 recombinant bacterium that overexpresses the ppk2 gene of polyphosphate kinase; The mixed bacteria are obtained by mixing; the gamma-polyglutamic acid is prepared by using the mixed bacteria and the optimized reaction system.

In the present invention, pgsB is used to represent a gene, wherein "pgs" is in italics, and PgsB is used to represent a protein, and other proteins or genes are expressed in the same way.

The object of the present invention is achieved through the following technical solutions:

The invention provides a method for preparing γ-PGA coupled with ATP regenerating enzyme and polyglutamic acid synthetase, using glutamic acid, sodium glutamate or glutamine as a substrate, and enzymatically preparing γ-polyglutamic acid,

The method for enzymatically preparing γ-polyglutamic acid includes: using a combination of biological enzymes, or a recombinant vector expressing the combination of biological enzymes, or a transgenic cell line expressing the combination of biological enzymes, or expressing the combination of biological enzymes. Genetically engineered bacteria with biological enzymes;

The amino acid sequence of the combined biological enzyme contains:

1) the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2; or,

2) On the basis of the amino acid sequence defined by SEQ ID NO.1, it is formed by base deletion, substitution, insertion or mutation, and has the activity of catalyzing the polymerization of glutamic acid or sodium glutamate into γ-PGA through γ-amide bonds The amino acid sequence of polyglutamate synthase; or the amino acid sequence of glutaminase; or,

3) On the basis of the amino acid sequence defined by SEQ ID NO.2, it is formed by base deletion, substitution, insertion or mutation, and has a polyphosphokinase PPK that catalyzes ATP regeneration activity; or,

4) Amino acid sequences of acetate kinase, pyruvate kinase, and creatine kinase that can be used to catalyze ATP regeneration, wherein the amino acid sequence of the acetate kinase protein is shown in SEQ ID NO.5, and the amino acid sequence of the creatine kinase protein is shown in SEQ ID NO. .6 shown.

In one embodiment of the present invention, the substrate for synthesizing γ-PGA by the method is glutamic acid, sodium glutamate or glutamine;

The biological enzyme that described method utilizes is the biological enzyme of any one or more groups in following (1) to (8):

(1) polyglutamic acid synthetase and polyphosphate kinase PPK;

(2) polyglutamic acid synthetase and acetate kinase;

(3) polyglutamic acid synthetase and pyruvate kinase;

(4) polyglutamic acid synthetase and creatine kinase;

(5) Transglutaminase and polyphosphate kinase PPK;

(6) Transglutaminase and acetate kinase;

(7) Transglutaminase and pyruvate kinase;

(8) Transglutaminase and creatine kinase.

In one embodiment of the present invention, the presence and utilization of the enzyme in the preparation method includes any one of the following (1) to (6):

(1) any combination of the biological enzymes;

(2) Genes encoding the following biological enzymes: polyglutamic acid synthase, polyphosphate kinase, acetate kinase, pyruvate kinase, creatine kinase;

(3) Genes containing the following biological enzymes: polyglutamic acid synthase, polyphosphate kinase, acetate kinase, pyruvate kinase, creatine kinase;

(4) Expression vectors or cloning vectors of genes containing the following biological enzymes: polyglutamic acid synthetase, polyphosphate kinase, acetate kinase, pyruvate kinase, creatine kinase;

(5) Transgenic cell lines containing the genes for the following biological enzymes: polyglutamic acid synthetase, polyphosphate kinase, acetate kinase, pyruvate kinase, creatine kinase;

(6) Genetically engineered bacteria containing the genes of the following biological enzymes: (polyglutamic acid synthase, polyphosphate kinase, acetate kinase, pyruvate kinase, creatine kinase.

In one embodiment of the present invention, the combined biological enzyme is selected as polyglutamic acid synthetase PgsB and polyphosphate kinase PPK, wherein polyglutamic acid synthetase PgsB has the expression as shown in SEQ ID No.1 Protein sequence, polyphosphate kinase PPK2 has a protein sequence as shown in SEQ ID No.2.

In one embodiment of the present invention, the preparation method of γ-PGA coupling ATP regeneration enzyme and polyglutamic acid synthetase specifically comprises the following steps:

(a) obtain the No. 1 recombinant bacterium of overexpressing pgsB gene, described pgsB gene expresses polyglutamic acid synthetase PgsB, and this polyglutamic acid synthetase PgsB has the protein sequence as shown in SEQ ID No.1;

(b) Obtain the No. 2 recombinant bacterium of overexpressing ppk2 gene, said ppk2 gene expresses polyphosphate kinase PPK2, and this polyphosphate kinase is ATP regeneration enzyme, and this polyphosphate kinase PPK2 has as shown in SEQ ID No.2 the protein sequence;

(c) Mix the No. 1 recombinant bacterium obtained in step (a) with the No. 2 recombinant bacterium obtained in step (b) to obtain mixed bacterial cells, and then catalyze the synthesis of γ-containing substrates containing glutamic acid and/or glutamate - polyglutamic acid. Gene and protein names are case-sensitive.

In step (a), the preparation steps of the No. 1 recombinant bacterium overexpressing the pgsB gene are as follows:

Construct the No. 1 recombinant plasmid expressing the pgsB gene of polyglutamic acid synthetase, and then transform the No. 1 recombinant plasmid into microorganisms to obtain No. 1 recombinant bacteria;

Or use genome editing technology to integrate the pgsB gene expressing polyglutamic acid synthetase into the genome of the microorganism to obtain No. 1 recombinant bacteria.

Preferably, the pgsB gene is derived from the pgsB gene in Bacillus subtilis having a nucleotide sequence as shown in SEQ ID No. 3 or an amino acid sequence having more than 90% homology with the sequence.

In step (a), the No. 1 recombinant bacterium after activation is induced and cultivated separately to the mid-log phase and above to collect the No. 1 bacterium. The specific induction process is: place the No. 1 recombinant bacterium in the activation medium of a shaker for activation No. 1 liquid seed was obtained overnight, the shaker speed was 200rpm, the temperature was 37°C, the formula of the activation medium was NaCl 10g/L, yeast extract 5g/L and tryptone 10g/L;

Move the activated No. 1 liquid seed to the shake flask proliferation medium, and cultivate it in a shaker with a temperature of 37°C and a rotation speed of 200 rpm until the cell concentration OD ₆₀₀ =0.6-0.8, and add a final concentration of 0.4-0.8mM The isopropyl thiogalactoside and 0.5-5mM magnesium sulfate were induced to express at 25°C for 14-16 hours, and the No. 1 bacterial cells were collected by centrifugation.

In step (b), the preparation steps of No. 2 recombinant bacteria overexpressing the ppk2 gene are:

Construct the No. 2 recombinant plasmid expressing the ppk2 gene of polyphosphate kinase, and then transform the No. 2 recombinant plasmid into microorganisms to obtain No. 2 recombinant bacteria;

Or use the genome editing technology to integrate the ppk2 gene expressing polyphosphate kinase into the genome of the microorganism to obtain No. 2 recombinant bacteria.

Preferably, the ppk2 gene is derived from Rhodopseudomonas or Deinococcus having a nucleotide sequence as shown in SEQ ID No.4 or an amino acid sequence with a homology of more than 90% with this sequence ppk2 gene.

In step (b), the No. 2 recombinant bacterium after activation is induced and cultured separately to the mid-log phase and above to collect the bacterium to obtain the No. 2 bacterium. The specific induction process is: place the No. 2 recombinant bacterium on the activation medium of the shaker No. 2 liquid seeds were obtained by activating in medium overnight, the shaker speed was 200rpm, the temperature was 37°C, and the formula of the activation medium was NaCl 10g/L, yeast extract 5g/L and tryptone 10g/L;

Move the activated No. 2 liquid seed to the shake flask proliferation medium, and cultivate it in a shaker with a temperature of 37°C and a rotation speed of 200 rpm until the cell concentration OD ₆₀₀ =0.6-0.8, and add a final concentration of 0.4-0.8mM The isopropyl thiogalactoside and 0.5-5mM magnesium sulfate were induced to express at 25°C for 14-16 hours, and the No. 2 bacteria were collected by centrifugation.

In step (a) and step (b), the microorganism is Escherichia coli.

In step (c), the No. 1 recombinant bacterium and the No. 2 recombinant bacterium can be mixed first and then cultivated for a period of time, or can be cultivated to a desired level before mixing.

In step (c), the weight ratio of No. 1 recombinant bacterium to No. 2 recombinant bacterium is 8:1-2:1, preferably 3:1-2:1.

In step (c), the mixed bacteria cells are incubated at 16-37° C. and pH 6-8 for 3-12 hours to directly catalyze the synthesis of γ-polyglutamic acid.

In step (c), the substrate of the catalyzed synthesis is a mixture of glutamic acid and/or glutamate, magnesium ions (magnesium ions are cofactors of the two enzymes), adenosine monophosphate and polyphosphate . The cooperation of No. 2 recombinant bacteria, adenosine monophosphate and polyphosphate can convert adenosine monophosphate into adenosine triphosphate, and adenosine triphosphate provides energy for No. 1 recombinant bacterium producing γ-PGA and converts it into adenosine monophosphate, making the whole The mixed bacteria system can continue to produce γ-PGA under the condition of continuous addition of glutamic acid or sodium glutamate, and the catalytic conversion rate of this system to glutamic acid/sodium glutamate is much higher than that containing No. 1 recombinant bacteria and Catalytic conversion rate of ATP system to glutamate/sodium glutamate.

Preferably, the concentration ratio of magnesium ions, adenosine monophosphate and polyphosphate is 1:1:0.5. These substrates are dissolved in a phosphate solution in order to carry out the catalytic reaction.

Preferably, the polyphosphate comprises hexametaphosphate.

Preferably, the magnesium ions are provided by magnesium sulfate heptahydrate.

The method integrates the target gene into the genome of host cells such as Escherichia coli, solves the problem that plasmids are easily lost during the culture of recombinant bacteria, and saves the use of antibiotics during the culture of bacteria and the subsequent treatment of antibiotic-containing wastewater. The present invention utilizes the optimized system, reacts at 25°C for 12 hours, converts 50 g/L sodium glutamate to obtain 44.25 g/L γ-PGA, the conversion rate of sodium glutamate is 88.5%, and the highest space-time conversion rate reaches 4.34 g/L L/h. The space-time conversion rate (obtained by dividing the yield and fermentation time) is 2.2-2.5 times higher than the static conversion method of single bacteria (enzyme) and the Bacillus subtilis fermentation method reported so far to prepare γ-PGA. The production cost of γ-PGA is equivalent to the existing fermentation preparation method. In a word, the present invention provides a new path for low-cost, fast and efficient preparation of γ-PGA.

Compared with the prior art, the present invention has the following advantages:

1) The enzyme preparation method is energy-saving and environmentally friendly.

2) The multi-enzyme system synergistically prepares γ-polyglutamic acid to obtain the product γ-polyglutamic acid and regenerates ATP in situ at the same time. The consumption and regeneration of ATP are carried out at the same time, so that only a very low concentration of ATP needs to be input into the reaction system at the initial stage. Avoid the investment of a large amount of ATP.

3) The reaction conditions for the production of γ-polyglutamic acid are optimized, the reaction speed is fast, and the substrate conversion rate is high.

4) The cost of raw materials, equipment and synthesis reaction process is saved to the greatest extent.

Description of drawings

Fig. 1 is the PCR amplification of pgsB gene;

Fig. 2 is the enzyme digestion identification figure of recombinant plasmid pET-22b-pgsB;

Figure 3 shows the expression of recombinant PgsB analyzed by SDS-PAGE, and the thick band in the box is the location of the target recombinant protein.

Figure 4 shows the expression of recombinant PPK2 analyzed by SDS-PAGE, and the thick band in the box is the position of the target recombinant protein;

Figure 5 is a comparison of the γ-PGA production ability of the single recombinant bacteria and the coupled double-group bacteria;

Fig. 6 is the influence of the weight ratio of PgsB and PPK2 on the conversion rate of glutamic acid;

Fig. 7 is the situation of producing γ-PGA under the coupled double-group bacteria amplification system and feeding in Example 11.

Detailed ways

The present invention (a method for preparing γ-PGA coupled with ATP regenerating enzyme and polyglutamic acid synthetase) will be described in detail below with reference to the accompanying drawings and specific examples.

A method for preparing γ-PGA coupled with ATP regeneration enzyme and polyglutamic acid synthetase, the preparation method specifically comprising the following steps:

(a) Construct the No. 1 recombinant plasmid expressing the pgsB gene of polyglutamic acid synthetase, and then transform the No. 1 recombinant plasmid into the microorganism to obtain the No. 1 recombinant bacterium that overexpresses the pgsB gene, or use genome editing technology to express polyglutamic acid The pgsB gene of amino acid synthase was integrated into the genome of the microorganism to obtain the No. 1 recombinant bacterium that overexpressed the pgsB gene, and then the activated No. The induction process is as follows: Place No. 1 recombinant bacteria in the activation medium of a shaker to activate overnight to obtain No. 1 liquid seed. The shaker speed is 200rpm, the temperature is 37°C, the formula of the activation medium is NaCl 10g/L, yeast extract 5 g/L and tryptone 10 g/L, then the activated No. 1 liquid seeds were transferred to the shake flask proliferation medium, and cultivated in a shaker with a temperature of 37 ° C and a rotation speed of 200 rpm until the cell concentration OD ₆₀₀ = 0.6 At -0.8, add 0.4-0.8mM isopropylthiogalactoside and 0.5-5mM magnesium sulfate at a final concentration of 0.5-5mM to induce expression at 25°C for 14-16h, and collect by centrifugation to obtain No. 1 bacterial cell. The pgsB gene expresses polyglutamic acid synthetase, and the polyglutamic acid synthetase PgsB has a protein sequence as shown in SEQ ID No.1, wherein the pgsB gene is derived from Bacillus subtilis and has a protein sequence such as SEQ ID No.3 The nucleotide sequence shown or the pgsB gene having an amino acid sequence whose homology is more than 90% with the sequence.

(b) Construct the No. 2 recombinant plasmid expressing the ppk2 gene of polyphosphate kinase, and then transform the No. 2 recombinant plasmid into the microorganism to obtain the No. 2 recombinant bacterium that overexpresses the ppk2 gene, or use genome editing technology to express polyphosphate kinase The ppk2 gene was integrated into the genome of the microorganism to obtain the No. 2 recombinant bacterium overexpressing the ppk2 gene, and then the activated No. 2 recombinant bacterium was induced and cultured to the mid-log phase and above to collect the bacteria to obtain the No. 2 bacteria. The specific induction process For: put the No. 2 recombinant bacteria in the activation medium of the shaker to activate overnight to obtain the No. 2 liquid seed, the shaker speed is 200rpm, the temperature is 37°C, the formula of the activation medium is NaCl 10g/L, yeast extract 5g /L and tryptone 10g/L, then the activated No. 2 liquid seeds were transferred to the shake flask proliferation medium, and cultivated in a shaker with a temperature of 37°C and a rotation speed of 200rpm until the cell concentration OD ₆₀₀ =0.6-0.8 At 25°C, the expression was induced by adding isopropylthiogalactopyranoside at a final concentration of 0.4-0.8mM and magnesium sulfate at a final concentration of 0.5-5mM for 14-16h, and collected by centrifugation to obtain No. 2 bacterial cells. The ppk2 gene expresses polyphosphate kinase PPK2, and the polyphosphate kinase PPK2 has a protein sequence as shown in SEQ ID No.2, wherein the ppk2 gene is derived from Rhodopseudomonas or Deinococcus and has a protein sequence as shown in SEQ ID No.2. The nucleotide sequence shown in ID No.4 or the ppk2 gene having an amino acid sequence whose homology is more than 90% with the sequence.

(c) Mix No. 1 recombinant bacteria obtained in step (a) and No. 2 recombinant bacteria obtained in step (b) at a weight ratio of 8:1 to 2:1 to obtain mixed bacterial cells, and then at 16-37°C, Under the condition of pH 6-8, the substrate containing glutamic acid and/or glutamate is catalyzed to synthesize γ-polyglutamic acid, the substrate is glutamic acid and/or glutamate, magnesium ion, mono A mixture of adenosine phosphate and polyphosphate.

Embodiment 1: the establishment of recombinant bacterial strain

According to the Bacillus licheniformis ATCC 14580, complete genome (NC_006270.3) in the NCBI database, use SnapGene to design primers pgsB-F and pgsB-R (as shown in Table 1), and use the non-glutamate-dependent Bacillus subtilis screened by our laboratory The genomic DNA of the bacillus was used as a template, and the pgsB gene was amplified by PCR (as shown in Figure 1, see SEQ ID No 3 for the sequence), the M swimming lane was the DNA marker, and the sizes of each band were 100, 250, 500, 750, 1000, 1500, 2000, 3000, 5000bp, 1 lane is pgsB PCR product. The target gene pgsB and the plasmid vector pET-22b were digested with restriction endonucleases BamH1 and Nco1 respectively, separated by electrophoresis, and recovered by tapping gel to obtain a gene fragment and a linearized plasmid fragment containing the same sticky end. - DNA ligase connection to obtain No. 1 recombinant plasmid, which was transformed into E.coli BL21 (DE3) competent cells (Escherichia coli), and positive single clones were screened (as shown in Figure 2) to obtain the recombinant strain pET -22b-pgsB-BL21, the No. 1 recombinant bacterium, was stored in 50% glycerol. The M lane in Figure 2 is a DNA marker, and the sizes of each band were 100, 250, 500, 750, and 1000 from bottom to top. , 1500, 2000bp, Lane 1, Lane 2, Lane 3 and Lane 4 are the results of BamH1 and Nco1 double digestion of plasmids picked from four single clones. It can be seen from the figure that the single clones in Lane 3 and Lane 4 are positive clones.

According to the protein sequence (WP_013615652, SEQ ID No 2) of Deinococcus proteolyticus polyphosphate kinase in the NCBI database, its ppk2 gene was synthesized according to the codon bias of Escherichia coli, and primers PPK2-F and PPK2-R (as shown in Table 1) were designed to The synthesized ppk2 gene was used as a template (see SEQ ID No 4 for the sequence), the Deipr-ppk2 gene was amplified, and the recombinant plasmid pET-Duet-Deipr-ppk2 was constructed by means of cutting, splicing, transfection, amplification, and detection, namely the No. 2 recombinant plasmid , transform the plasmid into E.coli BL21 (DE3) competent cells (Escherichia coli), and obtain the recombinant strain pETDuet-Deipr-ppk2-BL21, namely No. 2 recombinant bacteria, which are stored in 50% glycerol.

Embodiment 2: Preparation of recombinant free cells

Escherichia coli strains pET-22b-pgsB-BL21 and pET-Duet-Deipr-ppk2-BL21 preserved in 50% glycerol were inoculated according to 1% volume ratio (volume ratio is the volume ratio of the added bacterial solution to the volume ratio of the new culture solution) The amount was placed in the activation medium of the shaker to activate overnight, the shaker speed was 200rpm, the temperature was 37°C, the formula of the activation medium was NaCl 10g/L, yeast extract 5g/L, tryptone 10g/L.

The above-mentioned activated seeds (obtained after the above-mentioned activation culture in the shaker) are moved to the shake flask proliferation medium (the formula of this proliferation medium is K ₂ HPO _9.4g /L according to the inoculum size of 2% volume ratio) , KH ₂ PO ₄ 2.2g/L, yeast extract 23.6g/L, tryptone 11.8g/L, glycerol 4mL/L), cultivated in a shaker at 37°C and 200rpm until the cell concentration OD ₆₀₀ = about 0.6 , add 0.4mM isopropylthiogalactopyranoside and 0.5mM magnesium sulfate at a final concentration, induce expression at 25°C for 14 hours, and collect the bacteria by centrifugation, including pET-22b-pgsB-BL21 recombinant free cells and pET-22b-pgsB-BL21 - Duet-Deipr-ppk2-BL21 recombinant free cells were analyzed by SDS-PAGE (Figure 3 shows the expression of recombinant PgsB analyzed by SDS-PAGE, and Figure 4 shows the expression of recombinant PPK2 analyzed by SDS-PAGE), and then stored at -20°C , in Figure 3 and Figure 4 (the boxes in the two figures indicate the expression position of the target protein), lane M is the protein marker, lane 1 is before induction, lane 2 is after induction, lane 3 is the supernatant after induction, lane 4 Precipitation after induction; the size of each standard protein band in the protein marker lane is 14.4, 18.4, 25, 35, 45, 66.2, 116kDa from small to large.

Example 3: Preparation of γ-PGA by No. 1 Recombinant Bacteria Resting Cells Example 1

The No. 1 recombinant plasmid and the No. 1 recombinant bacterium were sequentially constructed using the method described in Example 1; the resting cells of the No. 1 recombinant bacterium were obtained by culturing and inducing expression using the method described in Example 2.

Resuspend the resting cells of No. 1 recombinant bacteria in phosphate buffer with a concentration of 0.1 mol/L and a pH of 8; add the resting cells of No. , 1mM MgSO ₄ in 0.1mol/L phosphate reaction solution with a pH of 8 (phosphate acts as a buffer); in a shaker at 25°C and 200rpm for 8h, take a sample every 1-2h; 12000rpm The supernatant was obtained by centrifugation at a rotating speed of 5°C and a temperature of 4° C. to obtain a reaction solution containing γ-PGA; the output of γ-PGA was measured by the CTAB method, and the conversion rate was as shown in Figure 5 (the conversion rate formula is conversion rate=(initial valley Concentration of Acid/Glutamate - Concentration of Glutamate/Glutamate at a certain point in time)/Concentration of Initial Glutamate/Glutamate, Concentration of Glutamate/Glutamate at a certain point in time The concentration can be calculated from the production of γ-PGA). It can be seen that within 2 hours before the reaction, the content of γ-PGA and the conversion rate of glutamic acid gradually increased with the prolongation of time; after 2 hours, the conversion rate of glutamic acid tended to a stable state at about 30%, and almost no longer increased. It shows that No. 1 recombinant bacterium can convert glutamic acid to prepare γ-PGA under the synergistic action of ATP.

Example 4: Preparation of γ-PGA by No. 1 Recombinant Bacteria Resting Cells Example 2

The No. 1 recombinant plasmid and the No. 1 recombinant bacterium were sequentially constructed using the method described in Example 1; the resting cells of the No. 1 recombinant bacterium were obtained by culturing and inducing expression using the method described in Example 2.

Resuspend the resting cells of No. 1 recombinant bacteria in phosphate buffer with a concentration of 0.1mol/L and a pH of 8; add the resting cells of No. 1 recombinant bacteria to the solution containing 1.7g/L sodium glutamate, 1mM AMP, 0.1mol/L of 1mM MgSO ₄ in a phosphate reaction solution with a pH of 8; shake the reaction at a constant temperature at 25°C and 200rpm for 8h, and take samples every 1-2h; rotate at 12000rpm and 4°C The supernatant was collected by centrifugation at a temperature above 100°C to obtain a reaction solution containing γ-PGA; the yield of γ-PGA was measured by the CTAB method, and the conversion rate was shown in Figure 5. It can be seen that there is almost no production of γ-PGA or conversion of glutamate in the entire reaction time range of the assay. It shows that when the No. 1 recombinant strain lacks ATP in the system, the No. 1 recombinant strain cannot synthesize γ-PGA even though there is AMP, the substrate for PPK2 regeneration, without the ATP regeneration enzyme PPK2.

Example 5: Preparation of γ-PGA by No. 1 Recombinant Bacteria Resting Cells Example 3

The No. 1 recombinant plasmid and the No. 1 recombinant bacterium were sequentially constructed using the method described in Example 1; the resting cells of the No. 1 recombinant bacterium were obtained by culturing and inducing expression using the method described in Example 2.

Resuspend the resting cells of No. 1 recombinant bacteria in phosphate buffer with a concentration of 0.1mol/L and a pH of 8; add the resting cells of No. 1 recombinant bacteria to the solution containing 1.7g/L sodium glutamate, 1mM AMP, 1mM MgSO ₄ , 0.5mM polyphosphate (polyP) in 0.1mol/L phosphate reaction solution with a pH of 8; shake the reaction at a constant temperature at 25°C and 200rpm for 8h, every 1-2h Take a sample; centrifuge at 12000rpm and 4°C to get the supernatant to obtain a reaction solution containing γ-PGA; use the CTAB method to measure the yield of γ-PGA, and the conversion rate is shown in Figure 5. It can be seen that there is almost no production of γ-PGA or conversion of glutamate in the entire reaction time range of the assay. It shows that when the No. 1 recombinant strain lacks ATP, the No. 1 recombinant strain cannot synthesize γ-PGA although there are substrates AMP and polyphosphate for PPK2 regeneration, but there is no ATP regeneration enzyme PPK2.

Example 6: Preparation of γ-PGA by mixing resting cells of No. 1 recombinant bacteria and No. 2 recombinant bacteria Example 1

The method described in Example 1 was used to sequentially construct No. 1 recombinant plasmid and No. 2 recombinant plasmid, No. 1 recombinant bacterium and No. 2 recombinant bacterium; the method described in Example 2 was used to cultivate and induce expression to obtain No. 1 recombinant bacterium and No. 2 recombinant bacterium resting cells.

Resuspend the resting cells of the No. 1 recombinant bacteria and the resting cells of the No. The resting cells of the bacteria were added into the phosphate reaction solution containing 1.7g/L sodium glutamate, 1mM AMP, and 1mM MgSO ₄ with a pH of 8 at a mass ratio of 3:1; at 25°C and 200rpm Shake the reaction at a constant temperature for 8 hours, and take a sample every 1-2 hours; centrifuge at a speed of 12000 rpm and a temperature of 4°C to obtain the supernatant to obtain a reaction solution containing γ-PGA; use the CTAB method to measure the output of γ-PGA, The conversion rate is shown in Figure 5. It can be seen that there is almost no γ-PGA production or glutamic acid conversion within the entire measured reaction time range (that is, the curves of Examples 4, 5, and 6 in Fig. 5 basically overlap). It shows that when there is recombinant PgsB and PPK2 but lacks ATP, there are only substrates AMP and polyphosphate for PPK2 regeneration, and because ATP cannot be regenerated, recombinant quiescent cells cannot synthesize γ-PGA.

Example 7: Preparation of γ-PGA Example 2 by Mixing Resting Cells of No. 1 Recombinant Bacteria and No. 2 Recombinant Bacteria

The method described in Example 1 was used to sequentially construct No. 1 recombinant plasmid and No. 2 recombinant plasmid, No. 1 recombinant bacterium and No. 2 recombinant bacterium; the method described in Example 2 was used to cultivate and induce expression to obtain No. 1 recombinant bacterium and No. 2 recombinant bacterium resting cells.

Resuspend the resting cells of the No. 1 recombinant bacteria and the resting cells of the No. The resting cells of the bacteria were added to 0.1mol/L pH 8 containing 1.7g/L sodium glutamate, 1mM AMP, 1mM MgSO ₄ , 0.5mM polyphosphate (polyP) at a mass ratio of 3:1. In the phosphate reaction solution; shake the reaction at a constant temperature at 25°C and 200rpm for 8h, and take a sample every 1-2h; centrifuge at 12000rpm and 4°C to get the supernatant to obtain the reaction containing γ-PGA solution; the output of γ-PGA was measured by CTAB method, and the conversion rate was shown in Figure 5. It can be seen that within the reaction time range of the entire measurement, with the prolongation of the reaction time, the production of γ-PGA or the conversion rate of glutamic acid increased rapidly in the first 3 hours, then leveled off, and then hardly changed, and the conversion rate of glutamic acid in the end up to 30%. It shows that when the system has recombinant PgsB and PPK2 but lacks ATP, PPK2 can use substrate AMP and polyphosphate to regenerate ATP, and restore the ability of No. 1 recombinant strain to produce γ-PGA. It can also be seen from Figure 5 that compared with the γ-PGA-producing ability of No. 1 recombinant strain alone in the presence of ATP, the γ-PGA-producing ability of the mixed strain was inferior to that of No. 1 recombinant strain in the first 3.5 hours, and gradually surpassed the former after 3.5 hours. The 8h substrate glutamate conversion rate (51%) increased by 70% compared with the former (30%).

Example 8: Preparation of γ-PGA by mixing resting cells of No. 1 recombinant bacteria and No. 2 recombinant bacteria Example 3

The method described in Example 1 was used to sequentially construct No. 1 recombinant plasmid and No. 2 recombinant plasmid, No. 1 recombinant bacterium and No. 2 recombinant bacterium; the method described in Example 2 was used to cultivate and induce expression to obtain No. 1 recombinant bacterium and No. 2 recombinant bacterium resting cells.

Resuspend the resting cells of the No. 1 recombinant bacteria and the resting cells of the No. The resting cells of the bacteria were added to the 0.1mol/L phosphate reaction solution with a pH of 8 and containing 1.7g/L sodium glutamate, 1mM AMP, 1mM MgSO ₄ , and 0.5mM polyP at a mass ratio of 8:1; Shake the reaction at a constant temperature at 25°C and 200rpm for 8 hours, and take samples every 1-2 hours; centrifuge at a speed of 12,000rpm and a temperature of 4°C to obtain a reaction solution containing γ-PGA; use CTAB method to determine The yield and conversion rate of γ-PGA are shown in Figure 6. It can be seen that within the reaction time range of the entire measurement, as the reaction time prolongs, the production of γ-PGA or the conversion rate of glutamic acid rises rapidly in the first 4 hours, then tends to be flat, and then hardly changes, and finally the conversion rate of glutamic acid up to 16%. Compared with the situation in which the resting cells of No. 1 recombinant bacteria and No. 2 recombinant bacteria had a mass ratio of 3:1, the latter (i.e. Example 7) had a stronger ability to produce γ-PGA, indicating that recombinant PgsB and PPK2 The ratio of cells will affect the level of γ-PGA produced by mixed cells.

Example 9: Preparation of γ-PGA by mixing resting cells of No. 1 recombinant bacteria and No. 2 recombinant bacteria Example 4

The method described in Example 1 was used to sequentially construct No. 1 recombinant plasmid and No. 2 recombinant plasmid, No. 1 recombinant bacterium and No. 2 recombinant bacterium; the method described in Example 2 was used to cultivate and induce expression to obtain No. 1 recombinant bacterium and No. 2 recombinant bacterium resting cells.

Resuspend the resting cells of the No. 1 recombinant bacteria and the resting cells of the No. The resting cells of the bacteria were added to the 0.1mol/L pH 8 phosphate reaction solution containing 1.7g/L sodium glutamate, 1mM AMP, 1mM MgSO ₄ , 0.5mM polyP at a mass ratio of 4:1; Shake the reaction at a constant temperature at 25°C and 200rpm for 8 hours, and take samples every 1-2 hours; centrifuge at a speed of 12,000rpm and a temperature of 4°C to obtain a reaction solution containing γ-PGA; use CTAB method to determine The yield and conversion rate of γ-PGA are shown in Figure 6. It can be seen that within the reaction time range of the entire measurement, as the reaction time prolongs, the production of γ-PGA or the conversion rate of glutamic acid rises rapidly in the first 4 hours, then tends to be flat, and then hardly changes, and finally the conversion rate of glutamic acid up to 37%. In this case, the ability of the mixed bacteria to produce γ-PGA is better than that of the resting cells of the No. 1 recombinant strain and the resting cells of the No. 2 recombinant strain at a mass ratio of 8:1, but lower than that of the resting cells of the No. 1 recombinant strain The resting cell mass ratio of No. 2 recombinant bacteria was 3:1, indicating that the ratio of recombinant PgsB and PPK2 cells would affect the level of γ-PGA produced by mixed cells.

Example 10: Preparation of γ-PGA by mixing resting cells of No. 1 recombinant bacteria and No. 2 recombinant bacteria Example 5

The method described in Example 1 was used to sequentially construct No. 1 recombinant plasmid and No. 2 recombinant plasmid, No. 1 recombinant bacterium and No. 2 recombinant bacterium; the method described in Example 2 was used to cultivate and induce expression to obtain No. 1 recombinant bacterium and No. 2 recombinant bacterium resting cells.

Resuspend the resting cells of the No. 1 recombinant bacteria and the resting cells of the No. The resting cells of the bacteria were added to the 0.1mol/L pH 8 phosphate reaction solution containing 1.7g/L sodium glutamate, 1mM AMP, 1mM MgSO ₄ , 0.5mM polyP at a mass ratio of 2:1; Shake the reaction at a constant temperature at 25°C and 200rpm for 8 hours, and take samples every 1-2 hours; centrifuge at a speed of 12,000rpm and a temperature of 4°C to obtain a reaction solution containing γ-PGA; use CTAB method to determine The yield and conversion rate of γ-PGA are shown in Figure 6. It can be seen that within the reaction time range of the entire measurement, as the reaction time prolongs, the production of γ-PGA or the conversion rate of glutamic acid rises rapidly in the first 4 hours, then tends to be flat, and then hardly changes, and finally the conversion rate of glutamic acid up to 65.4%. In this case, the ability of the mixed bacteria to produce γ-PGA is better than that of the resting cells of the No. 1 recombinant strain and the resting cells of the No. 2 recombinant strain at a mass ratio of 8:1 to 3:1, indicating that the ratio of recombinant PgsB and PPK2 cells will increase. It affects the level of γ-PGA produced by mixed cells, and the amount of recombinant PPK2 plays a dominant role in the process of converting glutamic acid to γ-PGA.

Example 11: Preparation of γ-PGA by mixing resting cells of No. 1 recombinant bacteria and No. 2 recombinant bacteria Example 6

The method described in Example 1 was used to sequentially construct No. 1 recombinant plasmid and No. 2 recombinant plasmid, No. 1 recombinant bacterium and No. 2 recombinant bacterium; the method described in Example 2 was used to cultivate and induce expression to obtain No. 1 recombinant bacterium and No. 2 recombinant bacterium resting cells.

Resuspend the resting cells of the No. 1 recombinant bacteria and the resting cells of the No. The resting cells of the bacteria were added into the 0.1mol/L phosphate reaction solution with a pH of 8 and containing 30g/L sodium glutamate, 30mM AMP, 30mM MgSO ₄ , and 15mM polyP at a mass ratio of 2:1; ℃, 200rpm in a shaking table at a constant temperature for 12 hours, and samples were taken every 1-2 hours; the supernatant was collected by centrifugation at a speed of 12000rpm and a temperature of 4℃ to obtain a reaction solution containing γ-PGA; the CTAB method was used to determine γ- PGA output. Because the production of γ-PGA leveled off at 4h and 8h, in order to verify that the flattening of the reaction was caused by the depletion of the substrate, 15g/L of sodium glutamate and 10mM polyP were added to the reaction at 6h, and the reaction At 10 h, 5 g/L sodium glutamate and 5 mM polyP were added again. The production of γ-PGA over time is shown in Figure 7. It can be seen that after substrate supplementation, the production of γ-PGA further increased. In the first 6 hours of the reaction, 30g/L of the substrate sodium glutamate was efficiently converted into 26.02g/L of the product γ-PGA, the conversion rate of glutamic acid was 86.7%, and the conversion rate was 4.34g/L/h; the entire 12h conversion process, 50g/L sodium glutamate was converted into 44.25g/L product γ-PGA, the conversion rate of glutamic acid was 88.5%, and the conversion rate was 3.69g/L/h.

Example 12: Construction of No. 1 Recombinant Bacteria Using Gene Editing Technology

In this example, CRISPR gene editing technology was used to knock in the pgsB gene. The steps of gene editing are described in detail below.

1) According to the insertion position of the target gene pgsB sequence and the surrounding sequence characteristics, design and prepare gRNA, clone the gRNA and Donor sequence into the gene editing vector Donor plasmid, and verify by sequencing to ensure that the gRNA and Donor sequences in the constructed vector are consistent with the target Sequence is consistent;

2) Prepare Escherichia coli BL21(DE3) electroporation competent, transform the Cas9 plasmid into BL21(DE3) competent, pick a single clone to prepare BL21(DE3)-Cas9 electroporation competent, and transform the Donor plasmid into BL21(DE3)-Cas9 electroporation Competent, add arabinose to induce and spread the plate, and carry out the gene editing strain screening experiment;

3) PCR amplification verification of the edited BL21(DE3) monoclonal, the band size of the successful knock-in of pgsB gene was 1193bp, and no band of successful knock-in. The results of agarose gel electrophoresis showed that the knock-in of pgsB gene was successfully screened The monoclonal pETDuet-1-pgsB-BL21(DE3), the No. 1 recombinant bacterium, was stored in 50% glycerol.

Example 13: Construction of No. 2 Recombinant Bacteria Using Gene Editing Technology

In this example, CRISPER gene editing technology was used to knock in the ppk2 gene. The steps of gene editing are described in detail below.

1) According to the insertion position of the target gene ppk2 sequence and the surrounding sequence characteristics, design and prepare gRNA, clone the gRNA and Donor sequence into the gene editing vector Donor plasmid, and verify by sequencing to ensure that the gRNA and Donor sequence in the constructed vector are consistent with the target Sequence is consistent;

2) Prepare Escherichia coli BL21(DE3) electrotransfer competent, transform Cas9 plasmid into BL21(DE3) competent, pick a single clone to prepare BL21(DE3)-Cas9 electrotransfer competent, and transform Donor plasmid into BL21(DE3)-Cas9 electrotransfer Competent, add arabinose to induce and spread the plate, and carry out the gene editing strain screening experiment;

3) PCR amplification verified the edited BL21(DE3) monoclonal, the band size of ppk2 gene knock-in was 858bp, and there was no band if knock-in was not successful. The results of agarose gel electrophoresis showed that ppk2 gene knock-in was successfully screened The monoclonal pETDuet-1-Deipr-ppk2-BL21(DE3), namely No. 2 recombinant bacteria, was stored in 50% glycerol.

Table 1 Primer names and sequences

SEQ ID No.1 PgsB protein sequence

SEQ ID No.2 PPK2 protein sequence:

Nucleic acid sequence of SEQ ID No.3 pgsB

The nucleic acid sequence of SEQ ID No.4 ppk2

SEQ ID No.5 acetate kinase protein sequence

SEQ ID No.6 creatine kinase protein sequence

The above descriptions of the embodiments are for those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described here to other embodiments without creative efforts. Therefore, the present invention is not limited to the above-mentioned embodiments. Improvements and modifications made by those skilled in the art according to the disclosure of the present invention without departing from the scope of the present invention should fall within the protection scope of the present invention.

Claims (10)

1. A method for preparing γ-PGA coupled with ATP regenerating enzyme and polyglutamic acid synthetase, characterized in that,

   Using glutamic acid, sodium glutamate or glutamine as substrates, enzymatically prepares γ-polyglutamic acid,

   The method for enzymatically preparing γ-polyglutamic acid includes: using a combination of biological enzymes, or a recombinant vector expressing the combination of biological enzymes, or a transgenic cell line expressing the combination of biological enzymes, or expressing the combination of biological enzymes. Genetically engineered bacteria with biological enzymes;

   The amino acid sequence of the combined biological enzyme contains:

   1) the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2; or,

   2) On the basis of the amino acid sequence defined by SEQ ID NO.1, it is formed by base deletion, substitution, insertion or mutation, and has the activity of catalyzing the polymerization of glutamic acid or sodium glutamate into γ-PGA through γ-amide bonds The amino acid sequence of polyglutamate synthase; or the amino acid sequence of glutaminase; or,

   3) On the basis of the amino acid sequence defined by SEQ ID NO.2, it is formed by base deletion, substitution, insertion or mutation, and has a polyphosphokinase PPK that catalyzes ATP regeneration activity; or,

   4) Amino acid sequences of acetate kinase, pyruvate kinase, and creatine kinase that can be used to catalyze ATP regeneration, wherein the amino acid sequence of acetate kinase protein is shown in SEQ ID NO.5, and the amino acid sequence of creatine kinase protein is shown in SEQ ID Shown in NO.6.
2. A kind of γ-PGA preparation method coupling ATP regeneration enzyme and polyglutamic acid synthetase according to claim 1, is characterized in that, the substrate of described method synthesis γ-PGA is glutamic acid, sodium glutamate or glutamine;

   The biological enzyme that described method utilizes is the biological enzyme of any one or more groups in following (1) to (8):

   (1) polyglutamic acid synthetase and polyphosphate kinase PPK;

   (2) polyglutamic acid synthetase and acetate kinase;

   (3) polyglutamic acid synthetase and pyruvate kinase;

   (4) polyglutamic acid synthetase and creatine kinase;

   (5) Transglutaminase and polyphosphate kinase PPK;

   (6) Transglutaminase and acetate kinase;

   (7) Transglutaminase and pyruvate kinase;

   (8) Transglutaminase and creatine kinase.
3. A method for preparing γ-PGA coupling ATP regenerating enzyme and polyglutamic acid synthetase according to claim 1 or 2, characterized in that the existence and utilization of enzymes in the preparation method include the following (1) to Any one of (6):

   (1) any combination of biological enzymes described in claim 2;

   (2) genes encoding the following biological enzymes in claim 2: polyglutamic acid synthase, polyphosphate kinase, acetate kinase, pyruvate kinase, creatine kinase;

   (3) genes containing the following biological enzymes in claim 2: polyglutamic acid synthase, polyphosphate kinase, acetate kinase, pyruvate kinase, creatine kinase;

   (4) expression vectors or cloning vectors containing the genes of the following biological enzymes in claim 2: polyglutamic acid synthetase, polyphosphate kinase, acetate kinase, pyruvate kinase, creatine kinase;

   (5) Transgenic cell lines containing the genes of the following biological enzymes in claim 2: polyglutamic acid synthase, polyphosphate kinase, acetate kinase, pyruvate kinase, creatine kinase;

   (6) genetically engineered bacteria containing the gene of the following biological enzymes in claim 2: (polyglutamic acid synthase, polyphosphate kinase, acetate kinase, pyruvate kinase, creatine kinase.
4. A method for preparing γ-PGA coupled with ATP regenerating enzyme and polyglutamic acid synthetase according to claim 1, characterized in that, the combined biological enzyme is selected as polyglutamic acid synthase PgsB and polyphosphoric acid Kinase PPK, wherein polyglutamic acid synthase PgsB has a protein sequence as shown in SEQ ID No.1, and polyphosphate kinase PPK2 has a protein sequence as shown in SEQ ID No.2.
5. A method for preparing γ-PGA coupling ATP regeneration enzyme and polyglutamic acid synthetase according to claim 1, wherein the preparation method specifically comprises the following steps:

   (a) obtaining the No. 1 recombinant bacterium that overexpresses the pgsB gene, and the pgsB gene expresses polyglutamic acid synthetase;

   (b) obtaining the No. 2 recombinant bacterium that overexpresses the ppk2 gene, and the ppk2 gene expresses polyphosphate kinase;

   (c) Mix the No. 1 recombinant bacterium obtained in step (a) with the No. 2 recombinant bacterium obtained in step (b) to obtain mixed bacterial cells, and then catalyze the synthesis of γ-containing substrates containing glutamic acid and/or glutamate - polyglutamic acid.
6. A method for preparing γ-PGA coupling ATP regeneration enzyme and polyglutamic acid synthetase according to claim 5, characterized in that, the gene pgsB of polyglutamic acid synthetase described in step (a) is derived from Subtilis subtilis Bacillus, its amino acid sequence is:

   an amino acid sequence as shown in SEQ ID No.1; or,

   Based on the amino acid sequence defined by SEQ ID NO.1, it is formed by base deletion, substitution, insertion or mutation, and has the activity of catalyzing the polymerization of glutamic acid or sodium glutamate into γ-PGA through γ-amide bonds. Amino acid sequence of glutamate synthase.
7. A method for preparing γ-PGA coupling ATP regeneration enzyme and polyglutamic acid synthetase according to claim 5, wherein the ppk2 gene encoding polyphosphokinase in step (b) is derived from Rhodopseudomonas genus or Deinococcus;

   Its amino acid sequence is:

   an amino acid sequence as shown in SEQ ID No.2; or,

   Based on the amino acid sequence defined by SEQ ID NO.2, it is formed by base deletion, substitution, insertion or mutation, and is a polyphosphokinase PPK that catalyzes ATP regeneration.
8. A kind of γ-PGA preparation method coupling ATP regeneration enzyme and polyglutamic acid synthetase according to claim 5, it is characterized in that, in step (c), the weight ratio of No. 1 recombinant bacterium and No. 2 recombinant bacterium is 8:1～2:1.
9. A method for preparing γ-PGA coupling ATP regenerating enzyme and polyglutamic acid synthetase according to claim 5, characterized in that, in step (c), the mixed bacteria cells are at 16-37°C and the pH is 6- Under the condition of 8, incubate for 3-12h to directly catalyze the synthesis of γ-polyglutamic acid.
10. A method for preparing γ-PGA coupling ATP regeneration enzyme and polyglutamic acid synthetase according to claim 5, characterized in that, in step (c), the substrate of the catalyzed synthesis is glutamic acid and/or Or a mixture of glutamate, magnesium ions, adenosine monophosphate and polyphosphate.

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