CN116694658A - Method for improving gamma-polyglutamic acid yield - Google Patents

Abstract

The invention discloses a method for increasing the output of γ-polyglutamic acid. In the method, the rapA gene in the gamma-polyglutamic acid producing strain is replaced by the mutant gene rapA, so that the yield of the gamma-polyglutamic acid of the producing strain is significantly increased. The nucleotide sequence of the mutant gene rapA is shown in SEQ ID NO.1, and the 575th base is mutated from A to G. Bacillus Velez PGA‑224 contains the mutant gene rapA, and its preservation number is GDMCCNo.62295. The present invention replaces the rapA gene in the gamma-polyglutamic acid producing strain with the mutated rapA gene, and significantly increases the gamma-polyglutamic acid production.

Description

A method for increasing the output of gamma-polyglutamic acid

technical field

The invention belongs to the technical field of microorganisms, and in particular relates to a method for increasing the yield of gamma-polyglutamic acid.

Background technique

γ-polyglutamic acid is an extracellular polymer synthesized by microorganisms. It is a type of homopolyamino acid polymerized from glutamic acid monomers through γ-glutamine bonds. It has excellent characteristics such as strong water absorption, degradability, strong biocompatibility, and non-toxicity. It is used in industry, food, and medicine. , environment, agriculture, daily chemical and other industries have broad application prospects.

γ-polyglutamic acid is mainly produced by bacteria belonging to the genus Bacillus. The industrial production of γ-polyglutamic acid is mainly carried out by Bacillus Velez, Bacillus subtilis and Bacillus licheniformis. The current methods to increase the production of γ-polyglutamic acid mainly screen high-yielding strains in natural environments, adjust medium formulations and gene editing. The improvement of the method of changing the fermentation medium is limited, and the method of screening new strains is time-consuming and laborious. Gene editing is mainly to knock out the gene of the enzyme that degrades γ-polyglutamic acid, and replace the promoter of glutamic acid and γ-polyglutamic acid synthetase. But editing multiple genes takes a long time. The rapA gene mainly encodes the dephosphorylated SpoOF~P protein. SpoOF~P protein can promote the phosphorylation of SpoOA, and SpoOA~P allows the bacteria to enter the spore cycle. However, the present invention finds another way to replace the rapA gene in the γ-polyglutamic acid producing strain with the mutated rapA gene, so that the γ-polyglutamic acid yield of the producing bacterium is significantly increased.

Contents of the invention

The purpose of the present invention is to provide a method for replacing the rapA gene in the gamma-polyglutamic acid producing strain with a mutated gene so as to increase the yield of gamma-polyglutamic acid.

The first object of the present invention is to provide a mutant gene rapA that increases the production of γ-polyglutamic acid, the nucleotide sequence of which is shown in SEQ ID NO.1.

Specifically, the protein expressed by the gene is a dephosphorylase (rapA). The 575th base in the rapA gene is mutated from A to G, and a missense mutation occurs. Partic acid becomes glycine.

The second object of the present invention is to provide a strain containing the mutant gene rapA.

Preferably, the bacterial strain is Bacillus Velez PGA-224, which is preserved in the Guangdong Provincial Microbial Culture Collection Center (GDMCC); accession number GDMCC No.62295; preservation date: March 16, 2022; preservation address : 5th Floor, Building 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, China, 510070.

The third object of the present invention is to provide a method for improving the yield of γ-polyglutamic acid, which is characterized in that it comprises the following steps: replacing the rapA gene in the γ-polyglutamic acid production strain by the mutant gene rapA, so that The yield of gamma-polyglutamic acid of the producing bacteria is significantly increased.

Preferably, the method comprises the steps of:

(1) Synthesizing the sgRNA sequence of the rapA gene in the genome of the γ-polyglutamic acid producing bacteria;

(2) Insert the sgRNA sequence into the pJOE8999 plasmid to obtain pJOE8999-sgRNA;

(3) Digest pJOE8999-sgRNA;

(4) amplifying the upstream and downstream homologous sequences of the rapA gene of the γ-polyglutamic acid producing bacteria;

(5) amplifying the mutant gene rapA sequence;

(6) Perform homologous recombination on the digested pJOE8999-sgRNA plasmid, the amplified upstream and downstream homologous sequence fragments of the rapA gene of the γ-polyglutamic acid producing bacteria, and the mutant gene rapA sequence fragments to obtain pJOE8999-sgRNA-rapA224 plasmid;

(7) The pJOE8999-sgRNA-rapA224 plasmid was transformed into γ-polyglutamic acid producing bacteria, and fermented to produce γ-polyglutamic acid.

Preferably, the specific steps are as follows:

(1) In the rapA gene sequence in the gamma-polyglutamic acid producing bacteria genome, tacg is added in front of the 5' end of the first 20 bases of the 5'-NGG PAM motif, and tacg is added in front of the 5' end of the complementary sequence. Go to aaac to get the sgRNA sequence and synthesize the sgRNA sequence;

(2) Digest the pJOE8999 plasmid with BsaI, and recover it by tapping the rubber; link the digested plasmid with the sgRNA sequence to obtain pJOE8999-sgRNA;

(3) Carry out SmaI and XbaI double enzyme digestion to the pJOE8999-sgRNA plasmid;

(4) using the genomic DNA of the gamma-polyglutamic acid-producing bacteria as a template, using PCR-specific primers to amplify the upstream and downstream homologous sequences of the rapA gene;

(5) Using the genomic DNA of Bacillus Velez PGA-224 as a template, amplify the mutant gene rapA gene sequence using PCR-specific primers;

(6) The digested pJOE8999-sgRNA plasmid, amplified to obtain the upstream and downstream homologous sequence fragments of the rapA gene of the gamma-polyglutamic acid-producing bacterium, and the amplified sequence of the mutant gene rapA of Bacillus Velez PGA-224 The fragment was subjected to homologous recombination to obtain the pJOE8999-sgRNA-rapA224 plasmid;

(7) Transform the pJOE8999-sgRNA-rapA224 plasmid into γ-polyglutamic acid producing bacteria, and select transformants on LB plates containing 20 μg/mL kanamycin and 1.0 g/L mannose at 30°C; The positive mutants grown on the plate were transferred to common LB plates to eliminate the plasmid at 45°C; then the positive clones were screened by colony PCR using the verified primers; the positive clones were fermented to produce γ-polyglutamic acid.

Preferably, the sequences of the verification primers are: rapA-y-F: ATGAAGCAGACTATTCCGTCCT and rapA-y-R: TAGACAATCTCCCTCTCTGGATT.

Preferably, the γ-polyglutamic acid-producing bacteria are Bacillus subtilis, Bacillus licheniformis or Bacillus Velez.

The fourth object of the present invention is to provide the application of the mutant gene rapA and the bacterial strain Bacillus velesi PGA-224 in improving the yield of γ-polyglutamic acid.

The beneficial effects of the invention are: the rapA gene in the gamma-polyglutamic acid producing strain is replaced by the mutated rapA gene, and the yield of the gamma-polyglutamic acid of the producing strain is significantly increased. The γ-polyglutamic acid of Bacillus licheniformis ATCC9945a reached 29.54g/L, and the γ-polyglutamic acid of Bacillus licheniformis ATCC9945a-224rapA after gene editing reached 35.54g/L, and the yield of γ-polyglutamic acid was significantly increased .

Bacillus velezensis PGA-224 was deposited in Guangdong Microbial Culture Collection Center (GDMCC); accession number GDMCC No.62295; preservation date: March 16, 2022; preservation address: No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, China 5th Floor, Building 59, Zip Code: 510070.

Detailed ways

The following examples are to further illustrate the present invention, rather than limit the present invention.

The Bacillus licheniformis in the following examples is Bacillus licheniformis ATCC9945a, its deposit number: ATCC No: 9945a. Bacillus subtilis is Bacillus subtilis (Bacillus subtilis) PGA-7, its preservation number is: CCTCC NO: M206102, the strain is disclosed in the patent CN200610122640.5, the invention name is γ-polyglutamic acid producing bacteria and using the strain to prepare γ - In the patent of the method of polyglutamic acid.

Example 1:

The starting strain Bacillus veleisi PGA-7.1 was inoculated on a solid medium plate and cultured at 37°C for 24h. The bacteria on the plate were eluted with 2 mL of sterile water. Then mix with skimmed milk so that the final concentration of skimmed milk is 10% by volume. Aliquot 1.5mL bacterial solution into 2mL cryovials, then place at 4°C for 30min, then place at -20°C for 90min, then place at -80°C overnight. Finally, freeze-dry and transfer to Beijing Aerospace Corporation after drying and packaging. Another tube of freeze-dried bacterial powder was used as the ground control strain. The samples of the control group were placed in a 4°C refrigerator before launch, and the control samples were placed in the natural environment after the spacecraft lifted off; The bacterial culture of the sample after loading was eluted into 10mL sterile water, diluted and gradiently coated on a solid medium plate, and after culturing for 24-48 hours, a single colony was picked, and the orifice plate was initially screened. The composition of the solid medium: peptone 10g/L, beef extract 3g/L, sodium chloride 5g/L, agar 20g/L, the balance is water, pH 7.0-7.2; the preparation method is to mix the above ingredients evenly Finally, adjust the pH value, and then sterilize for later use.

The semi-solid fermentation medium was melted, and 100 μL of the melted medium was added to a 96-well culture plate, and the distance between the liquid surface of the melted medium and the lid of the well plate was 5 mm. The composition of the semi-solid fermentation medium: the formula of the medium contains per liter: glycerol 30g/L, peptone 50g/L, citric acid 15g/L, magnesium sulfate heptahydrate 0.6g/L, dipotassium hydrogen phosphate 1g/L L, ammonium chloride 7g/L, manganese sulfate monohydrate 0.104g/L and agar 2g/L, the balance is water, pH 6.5.

After the culture medium in the orifice plate is condensed, use a toothpick to pick a single colony on the nutrient agar plate into the well of a 96-well culture plate containing a semi-solid fermentation medium, and cover the orifice plate well. The orifice plate was then inverted and placed in an incubator with a relative humidity above 80% for 96 hours at 37°C. γ-polyglutamic acid is a product that is secreted extracellularly, and γ-polyglutamic acid has relatively high viscosity. When the orifice plate is cultured upside down, if the output of γ-polyglutamic acid is large and accumulates more, when its own gravity is greater than its viscosity, the product of gravity will drop on the cover of the orifice plate; if the output of γ-polyglutamic acid is small , when its own gravity is less than its adhesive force, the product can only stick in the orifice plate. Therefore, visually observe whether there is a liquid product on the orifice plate cover, and the strain inserted into the hole corresponding to the position of the orifice plate cover with the liquid product is a high-yielding γ-polyglutamic acid bacteria. Thus, Bacillus velezensis PGA-224 with high yield of γ-polyglutamic acid was screened out. Bacillus Velez PGA-224 was deposited in Guangdong Microbial Culture Collection Center (GDMCC); accession number GDMCC No.62295; date of preservation: March 16, 2022; address of preservation: martyrs, Yuexiu District, Guangzhou City, Guangdong Province, China 5th Floor, Building 59, No. 100 Middle Road, Zip Code: 510070.

Example 2:

Inoculate the screened Bacillus Velez PGA-224 strain on a solid medium slant, and culture at 37°C for 16-24 hours to obtain activated Bacillus Velez PGA-224 strain. The composition of the solid medium: the formula of the medium contains per liter: peptone 10g/L, beef extract 3g/L, sodium chloride 5g/L, agar 20g/L, the balance is water, pH 7.0-7.2; The preparation method is to mix the above ingredients evenly, adjust the pH value, and then sterilize for future use.

Preparation of seed solution: Take 2 rings of the above-mentioned activated Bacillus Velez PGA-224 strain, insert it into a 300mL Erlenmeyer flask containing 50mL of fermentation medium, and culture it with shaking at 100r/min at 37°C for 18h to obtain Velez Bacillus PGA-224 seed solution. The composition of the fermentation medium: the formula of the medium contains per liter: 30 g of limonine glycerin, 50 g of peptone, 15 g of citric acid, 0.6 g of magnesium sulfate heptahydrate, 1 g of dipotassium hydrogen phosphate, 7 g of ammonium chloride and manganese sulfate monohydrate 0.104g, the balance is water, pH 6.5; the preparation method is to mix the above ingredients evenly, adjust the pH value, and then sterilize for later use.

Liquid shake flask fermentation: Prepare 50 mL of the fermentation medium according to the formula above and put it into a 300 mL Erlenmeyer flask, inoculate the seed solution of Bacillus Velesi PGA-224 into the fermentation medium according to the inoculum volume fraction of 10%, and the fermentation temperature is 37 ℃. The culture was shaken on a rotary shaker for 7 days, and the speed of the shaker was 250r/min. Fermentation completes, detection gamma-polyglutamic acid (detection method is prior art, such as patent CN200610122640.5, invention name gamma-polyglutamic acid producing bacterium and the testing method of the method utilizing this bacterial strain to prepare gamma-polyglutamic acid ) yield of 9.543g/L.

Example 3:

The 1-ring activated Bacillus velesi PGA-224 and the starting bacterium Bacillus velesii PGA-7.1 were respectively inserted into 300mL Erlenmeyer flasks containing 50mL nutrient broth medium, and cultured at 37°C with shaking at 100r/min for 18h , the composition of described nutrient broth medium: peptone 10g/L, beef extract 3g/L, sodium chloride 5g/L, agar 20g/L, surplus is water, pH 7.0～7.2; After the ingredients are mixed evenly, the pH value is adjusted, and then sterilized for later use. Centrifuge at 8000r/min for 10min (4°C), discard the supernatant, wash the precipitate twice with sterile water, freeze in liquid nitrogen, and send the sample on dry ice. The starting bacterium, Bacillus Velesi PGA-7.1, is used for whole-genome sequencing and mutagenesis Genome resequencing of Bacillus Velez PGA-224 to analyze the mutation site of Bacillus Velez PGA-224. The mutation site is that the 575th base in the rapA gene is mutated from A to G, and a missense mutation occurs, and the coding gene sequence after the mutation is shown in SEQ ID NO.1.

The nucleotide sequence of the mutant rapA gene (1137bp) of Bacillus Velez PGA-224 is shown in SEQ ID NO.1.

Example 4:

Gene editing of Bacillus licheniformis ATCC No: 9945a:

(1) Synthesize the sgRNA sequence of the rapA gene of Bacillus licheniformis ATCC No: 9945a, sgRNA-F: tacgTACGGACGCCGAAATGCTGA, sgRNA-R: aaacTCAGCATTTCGGCGTCCGTA.

(2) The pJOE8999 plasmid (purchased from Shanghai Haijihaoge Biotechnology Co., Ltd.) was single-digested with BsaI, and recovered by tapping rubber; the digested plasmid was linked with the sgRNA sequence to obtain pJOE8999-sgRNA.

(3) The pJOE8999-sgRNA plasmid was double digested with SmaI and XbaI, and recovered by tapping rubber.

(4) Using the genomic DNA of Bacillus licheniformis ATCC No: 9945a as a template, PCR-specific primers were used to amplify the upstream and downstream homologous sequences of the rapA gene, each with a length of 800 bp, and the amplified sequences were recovered from the gel. The primers are:

Primers for amplification of upstream homologous sequences are:

rapA-U-F: gtcgacggccaacgaggcccgggCGGCGCCGGGAACTGTCGCCAA

rapA-U-R: gacggaatagtctgcttcatCCCTTACGCCCCTTCCTTTTTAT

The primers for amplification of downstream homologous sequences are:

rapA-D-F: tccagagaggagattgtctaTATGAAATCTAAACTGTTGTTA

rap A-D-R: gatgaagattatttcttaatctagaCGGGTGACTGATGGATCAGCCT

(5) Using the genomic DNA of Bacillus Velez PGA-224 as a template, the rapA gene sequence was amplified using PCR-specific primers, and the amplified sequence was recovered from the gel. The primers are:

224rapA-F: aaaaggaaggggcgtaagggATGAAGCAGACTATTCCGTCCT

224 rap A-R: acaacagtttagatttcataTAGACAATCTCCCTCTCTGGATT

(6) The pJOE8999-sgRNA plasmid after digestion, the amplified upstream and downstream homologous sequence fragments of the rapA gene of Bacillus licheniformis and the amplified sequence fragments of the rapA gene of Bacillus Velez PGA-224 were added to the recombinase, 50 Incubate for 15 minutes at °C. Then the recombinant product was transformed into Escherichia coli, and the transformants were selected on LB plates with 50 μg/mL kanamycin. Then use rapA-U-F and rapA-D-R to carry out bacterial PCR verification, and the PCR product is 2600bp, which is a correct positive recombinant transformant. Correct positive recombinant transformants were cultured overnight at 37°C in 50 μg/mL kanamycin LB liquid. Finally, the bacteria were collected, and the plasmid was extracted to obtain the pJOE8999-sgRNA-rapA224 plasmid.

(7) The pJOE8999-sgRNA-rapA224 plasmid was transformed into Bacillus licheniformis. Transformants were selected on LB plates containing 20 μg/mL kanamycin and 1.0 g/L mannose at 30°C. Positive mutants grown on the plate were transferred to common LB plates to eliminate plasmids at 45 °C. Several clones were then screened by colony PCR using validated primers. The gene-edited Bacillus licheniformis ATCC9945a-224rapA strain was obtained. The sequence of the verification primer is:

rapA-y-F: ATGAAGCAGACTATTCCGTCCT

rapA-y-R: TAGACAATCTCCCTCTCTGGATT

Example 5:

The preparation steps for the activation of the Bacillus licheniformis ATCC9945a-224rapA strain are the same as in Example 2.

Preparation of Bacillus licheniformis ATCC9945a-224rapA seed solution: Take 2 rings of the above-mentioned activated Bacillus licheniformis ATCC9945a-224rapA strain, insert them into the fermentation medium, shake and cultivate at 37°C for 18 hours, and the shaker speed is 200r/min. for the seed liquid. The composition of the fermentation medium: citric acid 12g/L, glycerol 80g/L, L-glutamic acid 20g/L, ammonium chloride 7g/L, dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate heptahydrate 0.5g /L, calcium chloride dihydrate 0.15g/L, manganese sulfate monohydrate 0.104g/L, ferric chloride hexahydrate 0.04g/L, the balance is water, adjust the pH to 6.5; the preparation method is to mix the above ingredients evenly Finally, adjust the pH value, and then sterilize for later use.

Liquid shake flask fermentation: divide 50mL of the fermentation medium into a 300mL Erlenmeyer flask, insert the seed solution of Bacillus licheniformis ATCC9945a-224rapA into the fermentation medium according to the inoculum volume fraction of 10%, the fermentation temperature is 37°C, and shake on the shaker Cultivate for 96 hours, the shaker rotates at 200r/min. The output of gamma-polyglutamic acid was detected to be 35.54g/L.

Embodiment 6:

Gene editing of Bacillus subtilis PGA-7:

(1) Synthesize the sgRNA sequence of the rapA gene of Bacillus subtilis, sgRNA-F: tacgAAAGGGGGGATATATAATCT, sgRNA-R: aaacAGATTATATATCCCCCTTTA.

(2) The pJOE8999 plasmid was single-digested with BsaI, and recovered by tapping the rubber; the digested plasmid was linked with the sgRNA sequence to obtain pJOE8999-sgRNA.

(3) The pJOE8999-sgRNA plasmid was double digested with SmaI and XbaI, and recovered by tapping rubber.

(4) Using the genomic DNA of Bacillus subtilis PGA-7 as a template, PCR-specific primers were used to amplify the upstream and downstream homologous sequences of the rapA gene, each with a length of 800 bp, and the amplified sequences were recovered from the gel. The primers are:

Primers for amplification of upstream homologous sequences are:

rapA-U-F: gtcgacggccaacgaggcccgggGCCGCTCCCCCCCACACCCGTCA

rapA-U-R: aatagtctgcttcatCCTCAATAAATCCCCCCTTTTG

The primers for amplification of downstream homologous sequences are:

rapA-D-F: ctatatgaaatctaaACTGTTGTCAGGATTGCTGCTA

rapA-D-R: gatgaagattatttcttaatctagaTCGCTGTTTCCCGTCATATAGC

(5) Using the genomic DNA of Bacillus Velez PGA-224 as a template, the rapA gene sequence was amplified using PCR-specific primers, and the amplified sequence was recovered from the gel. The primers are:

224rapA-F: ggggatttattgaggATGAAGCAGACTATTCCGTCCT

224rapA-R: aatcctgacaacagtTTAGATTTCATATAGACAATCT

(6) The pJOE8999-sgRNA plasmid digested, the amplified upstream and downstream homologous sequence fragments of the rapA gene of Bacillus subtilis PGA-7, and the amplified rapA gene sequence fragments of Bacillus Velez PGA-224 were subjected to homologation. source recombination to obtain the pJOE8999-sgRNA-rapA224 plasmid.

(7) The pJOE8999-sgRNA-rapA224 plasmid was transformed into Bacillus subtilis PGA-7. Transformants were selected on LB plates containing 20 μg/mL kanamycin and 1.0 g/L mannose at 30°C. Positive mutants grown on the plate were transferred to common LB plates to eliminate plasmids at 45 °C. Several clones were then screened by colony PCR using validated primers. The gene-edited Bacillus subtilis PGA-7-224rapA strain was obtained. The sequence of the verification primer is:

rapA-y-F: ATGAAGCAGACTATTCCGTCCT

rapA-y-R: TAGACAATCTCCCTCTCTGGATT

Embodiment 7:

The preparation steps for the activation of the Bacillus subtilis PGA-7-224rapA strain are the same as in Example 2.

Preparation of Bacillus subtilis PGA-7-224rapA seed solution: Take 2 rings of the above-mentioned activated Bacillus subtilis PGA-7-224rapA strain, insert it into the fermentation medium, shake and cultivate at 37°C for 18 hours, and the shaker speed is 200r /min, this is the seed solution. The composition of the fermentation medium: sucrose 90g/L, ammonium sulfate 20g/L, dipotassium hydrogen phosphate 1g/L, magnesium sulfate heptahydrate 0.06g/L, calcium chloride dihydrate 0.15g/L, manganese sulfate monohydrate 0.05g/L, ferric chloride hexahydrate 0.01g/L, the balance is water, and the pH is adjusted to 6.5; the preparation method is to mix the above ingredients evenly, adjust the pH value, and then sterilize for later use.

Liquid shake flask fermentation: 50mL of the fermentation medium was divided into 300mL Erlenmeyer flasks, and the seed solution of Bacillus subtilis PGA-7-224rapA was inserted into the fermentation medium according to the inoculation amount as 10% by volume fraction, and the fermentation temperature was 37°C. Bed shaking culture 96h, shaker speed 200r/min. The output of gamma-polyglutamic acid was detected to be 25.24g/L.

Comparative example 1:

The steps for the activation of the Bacillus Velez PGA-7.1 strain and the preparation of the seed solution are the same as in Example 2.

Liquid shake flask fermentation: fermentation medium (the fermentation medium of formula with embodiment 2) subpackage 50mL enters 300mL Erlenmeyer flask, the seed liquid of fermented strain Bacillus Velesi PGA-7.1 is inoculated into 10% by volume fraction according to the inoculum size In the fermentation medium, the fermentation temperature is 37°C. The culture was shaken on a rotary shaker for 7 days, and the speed of the shaker was 250r/min. After the fermentation was completed, the output of gamma-polyglutamic acid was detected to be 2.647g/L.

Comparative example 2:

The preparation steps for the activation of the Bacillus licheniformis ATCC No: 9945a strain are the same as in Example 2. The preparation steps of Bacillus licheniformis ATCC No: 9945a seed solution are the same as in Example 5.

Liquid shake flask fermentation: 50mL of the fermentation medium (the same formulation as the fermentation medium of Example 5) is subpackaged into a 300mL Erlenmeyer flask, and the Bacillus licheniformis ATCC No: 9945a seed solution is inoculated into the fermentation medium according to the inoculum size of 10% by volume fraction Medium, the fermentation temperature is 37°C. The culture was shaken on a shaker for 96 hours, and the speed of the shaker was 200r/min. After the fermentation was completed, the output of gamma-polyglutamic acid was detected to be 29.54g/L.

Comparative example 3:

The preparation steps for the activation of the Bacillus subtilis PGA-7 strain are the same as in Example 2. The preparation steps of the Bacillus subtilis PGA-7 seed solution are the same as in Example 7.

Liquid shake flask fermentation: fermentation medium (the fermentation medium of formula with embodiment 7) subpackage 50mL enters 300mL Erlenmeyer flask, the seed liquid of Bacillus subtilis is inoculated in the fermentation medium according to inoculum size as volume fraction 10%, fermentation The temperature was 37°C, and the culture was shaken on a shaker for 96 hours, and the speed of the shaker was 200r/min. After the fermentation, the output of gamma-polyglutamic acid was detected to be 16.23g/L. SEQ ID NO.1 (nucleotide sequence of mutant gene rapA)

ATGAAGCAGACTATTCCGTCCTCTTTTGTCGGGCTCAAAATTAATGAATGGTATACCCATATCCGGCAGTTTCACGTCCTTGAGGCGGAGCGCGTCAAACGTGAAGTAGAGAGAGAGATTGAGGATATGGAAGAAGATCAGGATCTGCTGCTGTATTATTCATTAATGGAATTCAGACACCGCGTCATGCTGGATTACATCAAGCCCTTAAAGGAGGA CCCTTTCAGCCTGAGTTTTCAGAATTATTGGAAGACATTGAAGGCAACCAGTATAAACTGACAGGACTGCTTGATTACTACTTTAATTTTTTTCGAGGAATGTACGAATTTAAACAGAAAATGTTCCTAAACGCCATGATGTATTACAAACGGGCTGAAAAAAACCTTGCACTCGTTTCTGATGACATCGAGAAAAGCTGAGTTTGCTTTTAAAATGGCCGAGATTT TTTACAACCTGAAACAAACCTACGTCTCCATGAGTTATGCCGTTCAAGCCTTGAAACGTACCAATCGTATGAGACGTACAACGTCCGCAGAATCCAATGTGAATTCGTTATTGCAGGGAATTATGATGGCATGCAGTATCCAAGAAAGAGCATTGCCCCACTTAGAACTTGCTTTAGATCTTGCAAAACAAGAAGGCAATCCCCGTCTTATCAGTTCAGCCTTATAATACT CGGAAACTGTTACGAAAAAATGGGAGACCTCTCTAAA

GCAGCCGAATATTTTGAGACAGCCGTTTCCATTTGCAGGTCAGAAAAGTTCGATAATCTT

CCGCATTCTATTACTCATTAACACAGGTTCTTTATAAACAGAACACTACAGCCGAAGCA

GAGAAGCAATATCGCCTCGGGCTCAGTATCGCCCGCGAATACAATGATGAATTGTTTGTC

AATCTGTTCCAATTTTTGCATGCGTTATACGGCAAGGAAATGGATAACGCATCCGTCAGG

CGCACGTTTGATTTTCTGGAAGAACACATGCTGTATCCGTATGTGGAAGAATTGGCGCAT

GACGCTGCCAAATTTTACATGAAACACGGACAGCCTGAAAAAGCGCTCACGTTTTATGA

AAAAATGGTGCACGCCCAAAAACAAATCCAGAGAGGAGATTGTCTATATGAAATCTAA.

Claims (10)

1. A mutant gene rapA that improves the yield of γ-polyglutamic acid, characterized in that its nucleotide sequence is as shown in SEQ ID NO.1. 2. the bacterial strain containing the mutant gene rapA described in claim 1. 3. Bacillus Velez PGA-224, the preservation number of which is GDMCC No.62295. 4. A method for increasing the yield of gamma-polyglutamic acid, comprising the following steps: replacing the rapA gene in the gamma-polyglutamic acid-producing strain with the mutant gene rapA of claim 1. 5 . The method according to claim 4 , characterized in that, the γ-polyglutamic acid-producing bacteria are Bacillus Veles, Bacillus subtilis or Bacillus licheniformis. 6. the method according to claim 5 is characterized in that, described Bacillus licheniformis is Bacillus licheniformisATCC9945a, and its deposit number: ATCCNo: 9945a; Described Bacillus subtilis is Bacillus subtilis (Bacillus subtilis) PGA- 7. Its deposit number is: CCTCCNO: M206102. 7. The method according to claim 4, characterized in that, comprising the steps of: (1) Synthesizing the sgRNA sequence of the rapA gene in the genome of the γ-polyglutamic acid producing bacteria; (2) Insert the sgRNA sequence into the pJOE8999 plasmid to obtain pJOE8999-sgRNA; (3) Digest pJOE8999-sgRNA; (4) amplifying the upstream and downstream homologous sequences of the rapA gene of the γ-polyglutamic acid producing bacteria; (5) amplifying the mutant gene rapA sequence described in claim 1; (6) Perform homologous recombination on the digested pJOE8999-sgRNA plasmid, the amplified upstream and downstream homologous sequence fragments of the rapA gene of the γ-polyglutamic acid producing bacteria, and the mutant gene rapA sequence fragments to obtain pJOE8999-sgRNA-rapA224 plasmid; (7) The pJOE8999-sgRNA-rapA224 plasmid was transformed into γ-polyglutamic acid producing bacteria, and fermented to produce γ-polyglutamic acid. 8. The method according to claim 7, wherein the specific steps are as follows: (1) add tacg in front of the 5' end of the first 20 bases of the rapA gene in the gamma-polyglutamic acid producing bacteria genome, and add aaac in front of the 5' end of the complementary sequence, thereby obtaining the sgRNA sequence and synthesizing the sgRNA sequence; (2) Digest the pJOE8999 plasmid with BsaI, and recover it by tapping the rubber; link the digested plasmid with the sgRNA sequence to obtain pJOE8999-sgRNA; (3) Carry out SmaI and XbaI double enzyme digestion to the pJOE8999-sgRNA plasmid; (4) using the genomic DNA of the gamma-polyglutamic acid-producing bacteria as a template, using PCR-specific primers to amplify the upstream and downstream homologous sequences of the rapA gene; (5) using the genomic DNA of the Bacillus bacillus PGA-224 according to claim 3 as a template, use PCR specific primer amplification to obtain the mutant gene rapA gene sequence; (6) The digested pJOE8999-sgRNA plasmid, amplified to obtain the upstream and downstream homologous sequence fragments of the rapA gene of the gamma-polyglutamic acid-producing bacterium, and the amplified sequence of the mutant gene rapA of Bacillus Velez PGA-224 The fragment was subjected to homologous recombination to obtain the pJOE8999-sgRNA-rapA224 plasmid; (7) Transform the pJOE8999-sgRNA-rapA224 plasmid into γ-polyglutamic acid producing bacteria, and select transformants on LB plates containing 20 μg/mL kanamycin and 1.0 g/L mannose at 30°C; The positive mutants grown on the plate were transferred to common LB plates to eliminate the plasmid at 45°C; then the positive clones were screened by colony PCR using the verified primers; the positive clones were fermented to produce γ-polyglutamic acid. 9 . The method according to claim 8 , wherein the sequences of the verification primers are: rapA-y-F: ATGAAGCAGACTATTCCGTCCT and rapA-y-R: TAGACAATCTCCCTCTCTGGATT. 10. The application of the mutated gene rapA as claimed in claim 1 and the bacterial strain as claimed in claim 3 or 4 in increasing the output of γ-polyglutamic acid.