Application of glutamate decarboxylase mutant in production of gamma-aminobutyric acid

Abstract

The invention provides an application of a glutamic acid decarboxylase mutant in production of gamma-aminobutyric acid, and the amino acid sequence of the glutamic acid decarboxylase mutant is shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7. The invention further provides a preparation method of the glutamic acid decarboxylase mutant. The optimum pH and temperature range of glutamate decarboxylase is widened, and the yield of gamma-aminobutyric acid produced through fermentation of corynebacterium glutamicum is further increased.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to application of a glutamate decarboxylase mutant in producing gamma-aminobutyric acid. Background Art

[0002] γ-aminobutyric acid (GABA), also known as 4-aminobutyric acid, is a natural non-protein amino acid widely distributed in microorganisms, animals, humans and plants. At present, microbial fermentation is mainly used in industry to produce GABA, and glutamate decarboxylase is used for whole-cell catalysis in the production process. When using microbial fermentation to produce γ-aminobutyric acid, the glutamate decarboxylase gene is introduced into Corynebacterium glutamicum to obtain recombinant bacteria as fermentation strains for fermentation. Since the optimal pH range of glutamate decarboxylase is between 4-5.5, the enzyme activity decreases seriously when the pH is lower than 4 or higher than 5.5. In addition, the optimal catalytic temperature range of glutamate decarboxylase is between 35-45°C. When the temperature is lower than 35°C or higher than 45°C, the catalytic activity of the enzyme decreases seriously. In the actual production process, glutamate decarboxylase is not conducive to large-scale whole-cell catalytic production and application within the current pH and temperature range. Summary of the invention

[0003] In view of the shortcomings of the prior art, the present invention provides an application of a glutamate decarboxylase mutant in the production of γ-aminobutyric acid, which broadens the optimal pH and temperature range of glutamate decarboxylase, thereby increasing the yield of γ-aminobutyric acid produced by fermentation with Corynebacterium glutamicum.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] In a first aspect, the present invention provides an application of a glutamate decarboxylase mutant in producing γ-aminobutyric acid, wherein the amino acid sequence of the glutamate decarboxylase mutant is shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQID NO.6 or SEQ ID NO.7.

[0006] It should be noted that the present application is obtained by mutating the glutamate decarboxylase having an amino acid sequence as shown in SEQ ID NO.2 in the following manner: the tyrosine at position 10 is mutated to valine (Y10V), and the amino acid sequence is SEQ ID NO.3; or, the lysine at position 22 is mutated to aspartic acid (K22D), and the amino acid sequence is SEQ ID NO.4; or, the tyrosine at position 39 is mutated to threonine (Y39T), and the amino acid sequence is SEQ ID NO.5; or, the asparagine at position 41 is mutated to serine (N41S), and the amino acid sequence is SEQ ID NO.6; or, the lysine at position 56 is mutated to glutamic acid (K56E), and the amino acid sequence is SEQ ID NO.7.

[0007] In a second aspect, the present invention provides a gene encoding the glutamate decarboxylase mutant.

[0008] Preferably, when the amino acid sequence of the glutamate decarboxylase mutant is SEQ ID NO.3, the nucleotide sequence encoding SEQ ID NO.3 is shown as SEQ ID NO.8; when the amino acid sequence of the glutamate decarboxylase mutant is SEQ ID NO.4, the nucleotide sequence encoding SEQ ID NO.4 is shown as SEQ ID NO.9; when the amino acid sequence of the glutamate decarboxylase mutant is SEQ ID NO.5, the nucleotide sequence encoding SEQ ID NO.5 is shown as SEQ ID NO.10; when the amino acid sequence of the glutamate decarboxylase mutant is SEQ ID NO.6, the nucleotide sequence encoding SEQ ID NO.6 is shown as SEQ ID NO.11; when the amino acid sequence of the glutamate decarboxylase mutant is SEQ ID NO.7, the nucleotide sequence encoding SEQ ID NO.7 is shown as SEQ ID NO.12.

[0009] In a third aspect, the present invention provides a recombinant expression vector carrying the gene.

[0010] Preferably, pET-28a is used as the original expression vector.

[0011] In a fourth aspect, the present invention provides a recombinant bacterium containing the recombinant expression vector.

[0012] Preferably, the recombinant bacteria uses Corynebacterium glutamicum or Escherichia coli as a host bacterium.

[0013] In a fifth aspect, the present invention provides a use of the gene, the recombinant expression vector or the recombinant bacteria in synthesizing γ-aminobutyric acid.

[0014] Preferably, glutamate or sodium glutamate is used as a substrate, and the recombinant bacteria is a fermentation strain to produce γ-aminobutyric acid.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) The present invention broadens the optimal pH and temperature range of glutamate decarboxylase by mutating glutamate decarboxylase, and glutamate decarboxylase can also be well expressed when the pH value is close to neutral and the temperature is close to 50°C, so that the optimal pH range of glutamate decarboxylase is close to that of Corynebacterium glutamicum. This promotes Corynebacterium glutamicum to better ferment and produce γ-aminobutyric acid, thereby increasing the production yield of γ-aminobutyric acid.

[0017] (2) The mutants of glutamate decarboxylase, Y10V, K22D, Y39T, N41S, and K56E, were recombinantly expressed in Escherichia coli as a host, and the expression effect was better than that of the wild type. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0019] Sources of key experimental materials: PLP (pyridoxal phosphate) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., and sodium glutamate was purchased from Shanghai Bioengineering Co., Ltd.

[0020] LB solid medium: yeast powder 5g / L, peptone 10g / L, NacL 10g / L, agar powder 20g / L.

[0021] Example 1 Construction of an engineered Escherichia coli containing the G9 gene

[0022] The decarboxylase G9 gene used in the present invention is derived from Enterococcus faecalis, and the gene sequence is synthesized on the plasmid pEC-XK99E by Wuhan Jinkairui Bioengineering Co., Ltd., and the inserted restriction sites are BamH I (5' end) and SalI (3' end), thereby obtaining the plasmid pEC-XK99E-G9. The nucleotide sequence of the G9 gene is shown in SEQ ID NO: 1, as follows, and the amino acid sequence of the enzyme expressed by the G9 gene is shown in SEQ ID NO: 2, as follows. PCR was used to obtain the target gene G9, with upstream primer G9-F (TGGTGCCGCGCGGCA GCCATATGACCAAGAAGAACGATCTGAACGAATACC) and downstream primer G9-R (TCGAGTGCGGCCGCAAGCTTTTAGATGATGGACT GGTTTGGGCGC) as primers. The PCR amplification reaction system for the target gene G9 was as follows: upstream primer G9-F 2μL, downstream G9-R 2μL, template pEC-XK99E-G9 (100ng / μL) 0.5μL, DNA polymerase 2×Prime STAR Max DNA 25μL, and sterile water to 50μL. The PCR reaction program was as follows: PCR amplification program: 98℃ pre-denaturation for 3min, 98℃ denaturation for 10s, 58℃ annealing for 5sec, 72℃ extension for 20s, 30 cycles, and finally 72℃ extension for 5min, and the PCR product was stored at 12℃.

[0023] PCR was used to obtain the linear plasmid pET28a, the upstream primer pET28a-F (AAGCTTG CGGCCGCACTCGAGCACC), and the downstream primer pET28a-R (ATGGCTGC CGCGCGGCACCAGGCCGCTG). The reaction system for PCR amplification to obtain the target fragment was as follows: 2 μL of upstream primer pET28a-F, 2 μL of downstream pET28a-R, 0.5 μL of template pET28a (100 ng / μL), 25 μL of DNA polymerase 2× Prime STAR Max DNA, and sterile water to 50 μL. The PCR reaction program was as follows: PCR amplification program: 98°C pre-denaturation for 3 min, 98°C denaturation for 10 s, 58°C annealing for 5 sec, 72°C extension for 80 s, 30 cycles, and finally 72°C extension for 5 min, and the PCR product was stored at 12°C.

[0024] The above-obtained PCR products, G9 gene fragments and pET28a linear fragments were detected by 1% agarose gel nucleic acid electrophoresis and recovered by gel cutting with an OMEGA gel recovery kit. The two recovered PCR products were digested by T5 exonuclease for 5 minutes to obtain the recombinant expression vector pET28a-G9. Then it was added to DH5α competent cells, heat-shocked at 42°C for 60 seconds, 1mL LB liquid culture medium was added and cultured in a shaker at 37°C and 200rpm for 45 minutes, and then evenly coated on LB solid culture medium containing 50μg / mL kanamycin, and cultured inverted overnight in a 37°C incubator. Single clones were picked, verified by colony PCR and sent to the company for sequencing. The strains with correct sequencing were cultured, plasmids were extracted, and re-transformed into E. coli BL21 (DE3) competent cells. Culture medium was added and cultured at 37°C and 200rpm for 45 minutes. Then, the cells were spread on LB solid culture medium containing 50 μg / mL kanamycin and cultured inverted at 37°C overnight for about 10 hours to obtain the recombinant transformant E. coli BL21 (DE3) / pET28a-G9 monoclonal strain for expressing the G9 gene.

[0025] Example 2 Construction of recombinant expression vector of G9 mutant

[0026] In this example, the mutants Y10V, K22D, Y39T, N41S, and K56E of G9 were constructed for recombinant expression. The primers of the mutants are shown in Table 1:

[0027] Table 1: Primers for G9 enzyme mutant genes

[0028] Primer Sequence (5-3) Y10V-F GAACGATCTGAACGAAGTCCAGGAATACCTGG Y10V-R CCAGGTATTCCTGGACTTCGTTCAGATCGTTC K22D-F CTTCCTGGGCTCCGACGGCTCCCAGCGC K22D-R GCGCTGGGAGCCGTCGGAGCCCAGGAAG Y39T-F GAAACCCCAATGACCGCAAACCTGGC Y39T-R GCCAGGTTTGCGGTCATTGGGGTTTC N41S-F CCAATGTACGCATCCCTGGCCCGCGA N41S-R TCGCGGGCCAGGGATGCGTACATTGG K56E-F CCTGAACGAAGCAGAGGCAAACCAGAACC K56E-R GGTTCTGGTTTGCCTCTGCTTCGTTCAGG

[0029] The recombinant expression vector pET28a-10 for constructing mutant Y10V is specifically described. PCR site-directed mutagenesis, 1) the reaction system is as follows: primer Y10V-F 2μL, Y10V-F 2μL, template pET28a-G9 (100ng / μL) 0.5μL, DNA polymerase 2× Prime STAR Max DNA 10μL, and sterile water to make up to 20μL. PCR reaction program is as follows: PCR amplification program: 98℃ pre-denaturation for 3min, 98℃ denaturation for 10s, 58℃ annealing for 5sec, 72℃ extension for 80s, 30 cycles, and finally 72℃ extension for 5min, and PCR product preservation at 12℃; 2) Dpn I eliminates the template, and the reaction system is as follows: Dpn I 1μL, 10×buffer 1μL, PCR product 8μL, react at 37℃ for 3h; 4) The product treated with Dpn I is added to DH5α competent cells, heat-shocked at 42℃ for 60s, and 1mL LB liquid culture medium is added to the shaker at 37℃ and 200rpm. After culturing for 45min, it is evenly coated on LB solid culture medium containing 50μg / mL kanamycin, and inverted overnight in a 37℃ incubator. Pick a single clone, verify the colony PCR and send it to the company for sequencing, determine the correct mutant expression vector pET28a-10, culture the corresponding single clone strain, and extract the expression vector.

[0030] The remaining mutants K22D, Y39T, N41S and K56E corresponding expression vectors pET28a-22, pET28a-39 and pET28a-56 used the primers in Table X, and the remaining steps were consistent with the steps for constructing the pET28a-10 vector.

[0031] Example 3 Construction of a recombinant expression host for the G9 mutant

[0032] The mutant recombinant expression pET28a-10, pET28a-22, pET28a-39, pET28a-41 and pET28a-56 in Example 2 were transformed into the E. coli BL21 (DE3) host to construct the expression host. Taking the construction of E. coli BL21 (DE3) / pET28a-10 as an example, the specific process is as follows: 1) Take 10 μL of the expression vector pET28a-10 into the competent E. coli BL21 (DE3), heat shock at 42°C for 60s, add 1mL LB, culture at 37°C and 200rpm for 45min, apply it on the LB solid culture medium containing 50 μg / mL kanamycin, and culture it at 37°C overnight for about 10h to obtain the recombinant expression host E. coli BL21 (DE3) / pET28a-10 for expressing the mutant Y10V.

[0033] The expression hosts of the remaining mutants were E. coli BL21(DE3) / pET28a-22, E. coli BL21(DE3) / pET28a-39, E. coli BL21(DE3) / pET28a-41 and E. coli BL21(DE3) / pET28a-56, and the construction process was consistent with that of E. coli BL21(DE3) / pET28a-10.

[0034] Example 4 Purification of G9 mutants

[0035] The present embodiment purifies wild-type G9 and its mutants, and now takes mutant Y10V recombinant expression strain E.coli BL21 (DE3) / pET28a-10 as an example, and is specifically described as follows: 50 μL E.coli BL21 (DE3) / pET28a-10 is taken into a 50mL conical flask containing 10mL LB liquid culture medium, and cultured on a shaker set at 37°C and 200 rpm for 11 hours. Afterwards, 5mL of seed culture solution is taken out, transferred to a 2L conical flask containing 500mL LB liquid culture medium, and continued to be cultured on a shaker at the same temperature and speed for 2 hours. Subsequently, an IPTG inducer with a final concentration of 0.1mM is added thereto, and the fermentation temperature is lowered to 25°C to promote the recombinant expression of glutamate decarboxylase, and this process lasts for 24 hours.

[0036] Glutamate decarboxylase was purified by nickel-NTA (Ni-NTA) affinity chromatography column. First, the bacterial solution at the end of fermentation was collected, centrifuged at 8000g for 20min at 4°C, the supernatant was discarded, and the bacteria were collected. The bacteria were resuspended in 0.2M, pH7.0 phosphate buffer, and then the cells were lysed by high-pressure crushing. Centrifuged again at 8000g for 20min at 4°C to separate the cell-free fraction containing glutamate decarboxylase and its mutants. The cell-free extract was added to the pre-cooled Ni-NTA chromatography column. The purification process was divided into two steps of elution: the first step was to wash with buffer A containing 50mM phosphate buffer (PBS) and 100mM imidazole (pH7.4) to remove non-specifically bound impurities; the second step was to use buffer B containing 50mM PBS and 300mM imidazole (pH7.4) for elution to effectively recover glutamate decarboxylase and its mutants. Finally, the purified enzyme and mutant were dialyzed in 0.2M, pH 7.0 phosphate buffer, and stored in a -80°C refrigerator after aliquoting. The buffer used in the entire purification process must be precooled to 4°C before use. In this way, purified glutamate decarboxylase was successfully obtained.

[0037] The purification process of the enzymes from the recombinant expression strains of the remaining mutants K22D, Y39T, N41S, and K56E was consistent with that of the mutant Y10V.

[0038] Example 5 Optimization of Glutamate Decarboxylase Mutant Conversion to γ-Aminobutyric Acid in Vitro

[0039] The ability of wild-type glutamate decarboxylase and mutants Y10V, K22D, Y39T, N41S and K56E pure enzymes to convert sodium glutamate into γ-aminobutyric acid in vitro was verified. In a 250 mL conical flask, the reaction system was as follows: 10 mg pure enzyme, final concentration 0.1 mM PLP (pyridoxal phosphate), final concentration 100 g / L sodium glutamate, and phosphate buffer in the system was supplemented to 50 mL. When the reaction temperature setting value was 40 ° C, the optimal pH in the catalytic reaction system was optimized, and 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7 and 7.5 were selected. The optimal pH was selected, and then the optimal temperature in the reaction system was optimized, and 20 ° C, 30 ° C, 40 ° C, 50 ° C, 60 ° C and 70 ° C were selected. At different pH and temperature, the reaction was carried out for 5 hours, and then the content of glutamate decarboxylase and mutants in converting glutamate to synthesize γ-aminobutyric acid under different conditions was detected, and the maximum synthesis of γ-aminobutyric acid by wild-type glutamate decarboxylase under different conditions was set to 100g / L. The experimental results are shown in Tables 2 and 3. The experimental results show that compared with wild-type glutamate decarboxylase, the ability of mutants Y10V, K22D, Y39T, N41S and K56E to catalyze the synthesis of γ-aminobutyric acid from substrate glutamate is significantly improved in the pH range of 5-7.5 and the temperature range of 20-70°C.

[0040] Table 2: Effect of different pH on the synthesis of γ-aminobutyric acid (g / L) by glutamate decarboxylase

[0041] Enzyme pH 3.5 pH 4 pH 4.5 pH 5 pH 5.5 pH6 pH6.5 pH7 pH7.5 Wild type 91.0 100.0 99.9 92.0 65.3 64.6 73.3 28.7 16.7 Y10V 76.7 91.4 101.0 106.2 113.3 121.9 118.6 67.6 58.6 K22D 87.1 96.2 91.0 103.8 105.7 110.0 118.1 101.9 44.3 Y39T 93.7 100.8 109.1 110.7 123.5 124.8 124.3 104.3 90.0 N41S 62.9 86.2 91.9 109.0 102.9 119.5 114.3 92.9 71.9 K56E 50.5 91.4 95.7 108.6 100.5 109.0 121.4 69.5 42.4

[0042] Table 3: Effect of different temperatures on the synthesis of γ-aminobutyric acid (g / L) by glutamate decarboxylase

[0043] Enzyme 20℃ 30℃ 40℃ 50℃ 60℃ 70℃ Wild type 23.4 32.9 55.4 100.0 78.7 34.4 Y10V 65.6 62.9 104.9 106.8 85.1 81.0 K22D 66.5 73.3 94.6 107.5 81.9 85.5 Y39T 68.9 92.9 107.4 108.3 91.0 82.8 N41S 68.8 85.5 102.8 113.6 93.7 86.9 K56E 64.3 75.6 93.8 105.6 90.5 84.6

[0044] Gene sequence and amino acid sequence

[0045] SEQ ID NO.1 (G9: gene sequence)

[0046] ATGACCAAGAAGAACGATCTGAACGAATACCAGGAATACCTGGAAGATTCC

[0047] TTCCTGGGCTCCAAGGGCTCCCAGCGCCTGCTGCCAAACACCAAGATGCCA

[0048] GAAACCCCAATGTACGCAAACCTGGCCCGCGATCTGATCGAACACTTCCGC

[0049] CTGAACGAAGCAAAGGCAAACCAGAACCTGGCCACCTTCTGCACCACCCAG

[0050] ATGGAAAAGCAGGCCGATGAACTGATCCTGAACTCCCTGAACACCAACGCA

[0051] ATCGACAAGTCCGAATACCCAAAGACCTCCGCAATGGAAAACTCCTGCATC

[0052] TCCATGATCGCCCACCTGTGGAACGTGGGCGACGATAAGGAAATCTTCAAG

[0053] GACTTCATCGGCACCTCCACCGTGGGCTCCTCCGAAGGCTGCATGCTGGGCG

[0054] GCCTGGCCCTGTTGAAGTCCTGGCGCAAGCGCGCCGCAGATGTGGGCATCG

[0055] ACGTGGACAACATGAAGGATCACAAGCCAAACCTGGTGATCATGTCCGGCT

[0056] ACCAGGTGGTGTGGGAAAAGTTCTGCGTGTACTGGGACGTGGAACTGCGCT

[0057] CCATCCCAGTGTCCAAGGATCACATGTCCCTGGATACCGATTCCGTGATGTC

[0058] CTACGTGGACGAAAACACCATCGGCATCGTGGGCGTGCACGGCATCACCTA

[0059] CACCGGCGCAGTGGACGACATCCAGAAGCTGGATGAACTGGTGTCCGAATA

[0060] CAACAAGAACTCCAAGCTGGACCTGCGCATCCACGTGGACGCAGCATTCGG

[0061] CGGCCTGTTCTCCCCATTCATGGATGGCTTCAAGCCATGGGACTTCCGCCTG

[0062] AAGAACGTGGTGTCCATCAACGTGTCCGGCCACAAGTACGGCATGGTGTAC

[0063] CCAGGCATCGGCTGGGTGATCTGGCGCGAAAACAACCTGGACCTGCTGCCA

[0064] GAATCCATGCGCTTCGAAGTGGCCTACCTGGGCGATAAGGTGGATTCCATC

[0065] GCCATCAACTTCTCCCACTCCGGCGCACACATCGTGGCACAGTACTACAACT

[0066] TCATCCGCTTCGGCTTCTACGGCTACAAGAAGATCATGGAAGGCGTGCGCA

[0067] AGGTGTCCATCGCACTGGCAGATGAAATCTCCTCCACCGGCTTCTTCGAAAT

[0068] CATCAACGATGGCTCCCAGCTGCCAATCGTGTGCTGGAAGCTGAACGATGA

[0069] AATCAAGACCGATTGGTCCCTGTACGATCTGGAATCCGTGCTGGCCAAGCA

[0070] CGGCTGGCAGGTGCCAGCCTACCCACTGCCAAAGAACCGCGAAAACACCAC

[0071] CATCTCCCGCATCGTGGTGCGCCCATCCATGACCATGACCGTGTGCGGCGAC

[0072] TTCATCGACGATCTGAAGCTGTCCATCAAGGAACTGGAAACCTCCCGCCTG

[0073] GTGCGCCCAAACCAGTCCATCATCTAASEQ ID NO.2 (G9: Amino acid sequence)

[0074] MTKKNDLNEYQEYLEDSFLGSKGSQRLLPNTKMPETPMYANLARDLIEHFRLN

[0075] EAKANQNLATFCTTQMEKQADELILNSLNTNAIDKSEYPKTSAMENSCISMIAH

[0076] LWNVGDDKEIFKDFIGTSTVGSSEGCMLGGLALLKSWRKRAADVGIDVDNMK

[0077] DHKPNLVIMSGYQVVWEKFCVYWDVELRSIPVSKDHMSLDTDSVMSYVDENT

[0078] IGIVGVHGITYTGAVDDIQKLDELVSEYNKNSKLDLRIHVDAAFGGLFSPFMDG

[0079] FKPWDFRLKNVVSINVSGHKYGMVYPGIGWVIWRENNLDLLPESMRFEVAYL

[0080] GDKVDSIAINFSHSGAHIVAQYYNFIRFGFYGYKKIMEGVRKVSIALADEISSTG

[0081] FFEIINDGSQLPIVCWKLNDEIKTDWSLYDLESVLAKHGWQVPAYPLPKNRENT

[0082] TISRIVVRPSMTMTVCGDFIDDLKLSIKELETSRLVRPNQSII

[0083] SEQ ID NO.3 (Y10V: Amino Acid Sequence)

[0084] MTKKNDLNEVQEYLEDSFLGSKGSQRLLPNTKMPETPMYANLARDLIEHFRLN

[0085] EAKANQNLATFCTTQMEKQADELILNSLNTNAIDKSEYPKTSAMENSCISMIAH

[0086] LWNVGDDKEIFKDFIGTSTVGSSEGCMLGGLALLKSWRKRAADVGIDVDNMK

[0087] DHKPNLVIMSGYQVVWEKFCVYWDVELRSIPVSKDHMSLDTDSVMSYVDENT

[0088] IGIVGVHGITYTGAVDDIQKLDELVSEYNKNSKLDLRIHVDAAFGGLFSPFMDG

[0089] FKPWDFRLKNVVSINVSGHKYGMVYPGIGWVIWRENNLDLLPESMRFEVAYL

[0090] GDKVDSIAINFSHSGAHIVAQYYNFIRFGFYGYKKIMEGVRKVSIALADEISSTG

[0091] FFEIINDGSQLPIVCWKLNDEIKTDWSLYDLESVLAKHGWQVPAYPLPKNRENT

[0092] TISRIVVRPSMTMTVCGDFIDDLKLSIKELETSRLVRPNQSII

[0093] SEQ ID NO.4 (K22D: Amino Acid Sequence)

[0094] MTKKNDLNEYQEYLEDSFLGSDGSQRLLPNTKMPETPMYANLARDLIEHFRLN

[0095] EAKANQNLATFCTTQMEKQADELILNSLNTNAIDKSEYPKTSAMENSCISMIAH

[0096] LWNVGDDKEIFKDFIGTSTVGSSEGCMLGGLALLKSWRKRAADVGIDVDNMK

[0097] DHKPNLVIMSGYQVVWEKFCVYWDVELRSIPVSKDHMSLDTDSVMSYVDENT

[0098] IGIVGVHGITYTGAVDDIQKLDELVSEYNKNSKLDLRIHVDAAFGGLFSPFMDG

[0099] FKPWDFRLKNVVSINVSGHKYGMVYPGIGWVIWRENNLDLLPESMRFEVAYL

[0100] GDKVDSIAINFSHSGAHIVAQYYNFIRFGFYGYKKIMEGVRKVSIALADEISSTG

[0101] FFEIINDGSQLPIVCWKLNDEIKTDWSLYDLESVLAKHGWQVPAYPLPKNRENT

[0102] TISRIVVRPSMTMTVCGDFIDDLKLSIKELETSRLVRPNQSII

[0103] SEQ ID NO.5 (Y39T: Amino Acid Sequence)

[0104] MTKKNDLNEYQEYLEDSFLGSKGSQRLLPNTKMPETPMTANLARDLIEHFRLN

[0105] EAKANQNLATFCTTQMEKQADELILNSLNTNAIDKSEYPKTSAMENSCISMIAH

[0106] LWNVGDDKEIFKDFIGTSTVGSSEGCMLGGLALLKSWRKRAADVGIDVDNMK

[0107] DHKPNLVIMSGYQVVWEKFCVYWDVELRSIPVSKDHMSLDTDSVMSYVDENT

[0108] IGIVGVHGITYTGAVDDIQKLDELVSEYNKNSKLDLRIHVDAAFGGLFSPFMDG

[0109] FKPWDFRLKNVVSINVSGHKYGMVYPGIGWVIWRENNLDLLPESMRFEVAYL

[0110] GDKVDSIAINFSHSGAHIVAQYYNFIRFGFYGYKKIMEGVRKVSIALADEISSTG

[0111] FFEIINDGSQLPIVCWKLNDEIKTDWSLYDLESVLAKHGWQVPAYPLPKNRENT

[0112] TISRIVVRPSMTMTVCGDFIDDLKLSIKELETSRLVRPNQSII

[0113] SEQ ID NO.6 (N41S: Amino Acid Sequence)

[0114] MTKKNDLNEYQEYLEDSFLGSKGSQRLLPNTKMPETPMYASLARDLIEHFRLN

[0115] EAKANQNLATFCTTQMEKQADELILNSLNTNAIDKSEYPKTSAMENSCISMIAH

[0116] LWNVGDDKEIFKDFIGTSTVGSSEGCMLGGLALLKSWRKRAADVGIDVDNMK

[0117] DHKPNLVIMSGYQVVWEKFCVYWDVELRSIPVSKDHMSLDTDSVMSYVDENT

[0118] IGIVGVHGITYTGAVDDIQKLDELVSEYNKNSKLDLRIHVDAAFGGLFSPFMDG

[0119] FKPWDFRLKNVVSINVSGHKYGMVYPGIGWVIWRENNLDLLPESMRFEVAYL

[0120] GDKVDSIAINFSHSGAHIVAQYYNFIRFGFYGYKKIMEGVRKVSIALADEISSTG

[0121] FFEIINDGSQLPIVCWKLNDEIKTDWSLYDLESVLAKHGWQVPAYPLPKNRENT

[0122] TISRIVVRPSMTMTVCGDFIDDLKLSIKELETSRLVRPNQSIISEQ ID NO.7 (K56E: Amino Acid Sequence)

[0123] MTKKNDLNEYQEYLEDSFLGSKGSQRLLPNTKMPETPMYANLARDLIEHFRLN

[0124] EAEANQNLATFCTTQMEKQADELILNSLNTNAIDKSEYPKTSAMENSCISMIAH

[0125] LWNVGDDKEIFKDFIGTSTVGSSEGCMLGGLALLKSWRKRAADVGIDVDNMK

[0126] DHKPNLVIMSGYQVVWEKFCVYWDVELRSIPVSKDHMSLDTDSVMSYVDENT

[0127] IGIVGVHGITYTGAVDDIQKLDELVSEYNKNSKLDLRIHVDAAFGGLFSPFMDG

[0128] FKPWDFRLKNVVSINVSGHKYGMVYPGIGWVIWRENNLDLLPESMRFEVAYL

[0129] GDKVDSIAINFSHSGAHIVAQYYNFIRFGFYGYKKIMEGVRKVSIALADEISSTG

[0130] FFEIINDGSQLPIVCWKLNDEIKTDWSLYDLESVLAKHGWQVPAYPLPKNRENT

[0131] TISRIVVRPSMTMTVCGDFIDDLKLSIKELETSRLVRPNQSIISEQ ID NO.8(Y10V: Gene sequence)

[0132] ATGACCAAGAAGAACGATCTGAACGAAGTCCAGGAATACCTGGAAGATTCC

[0133] TTCCTGGGCTCCAAGGGCTCCCAGCGCCTGCTGCCAAACACCAAGATGCCA

[0134] GAAACCCCAATGTACGCAAACCTGGCCCGCGATCTGATCGAACACTTCCGC

[0135] CTGAACGAAGCAAAGGCAAACCAGAACCTGGCCACCTTCTGCACCACCCAG

[0136] ATGGAAAAGCAGGCCGATGAACTGATCCTGAACTCCCTGAACACCAACGCA

[0137] ATCGACAAGTCCGAATACCCAAAGACCTCCGCAATGGAAAACTCCTGCATC

[0138] TCCATGATCGCCCACCTGTGGAACGTGGGCGACGATAAGGAAATCTTCAAG

[0139] GACTTCATCGGCACCTCCACCGTGGGCTCCTCCGAAGGCTGCATGCTGGGCG

[0140] GCCTGGCCCTGTTGAAGTCCTGGCGCAAGCGCGCCGCAGATGTGGGCATCG

[0141] ACGTGGACAACATGAAGGATCACAAGCCAAACCTGGTGATCATGTCCGGCT

[0142] ACCAGGTGGTGTGGGAAAAGTTCTGCGTGTACTGGGACGTGGAACTGCGCT

[0143] CCATCCCAGTGTCCAAGGATCACATGTCCCTGGATACCGATTCCGTGATGTC

[0144] CTACGTGGACGAAAACACCATCGGCATCGTGGGCGTGCACGGCATCACCTA

[0145] CACCGGCGCAGTGGACGACATCCAGAAGCTGGATGAACTGGTGTCCGAATA

[0146] CAACAAGAACTCCAAGCTGGACCTGCGCATCCACGTGGACGCAGCATTCGG

[0147] CGGCCTGTTCTCCCCATTCATGGATGGCTTCAAGCCATGGGACTTCCGCCTG

[0148] AAGAACGTGGTGTCCATCAACGTGTCCGGCCACAAGTACGGCATGGTGTAC

[0149] CCAGGCATCGGCTGGGTGATCTGGCGCGAAAACAACCTGGACCTGCTGCCA

[0150] GAATCCATGCGCTTCGAAGTGGCCTACCTGGGCGATAAGGTGGATTCCATC

[0151] GCCATCAACTTCTCCCACTCCGGCGCACACATCGTGGCACAGTACTACAACT

[0152] TCATCCGCTTCGGCTTCTACGGCTACAAGAAGATCATGGAAGGCGTGCGCA

[0153] AGGTGTCCATCGCACTGGCAGATGAAATCTCCTCCACCGGCTTCTTCGAAAT

[0154] CATCAACGATGGCTCCCAGCTGCCAATCGTGTGCTGGAAGCTGAACGATGA

[0155] AATCAAGACCGATTGGTCCCTGTACGATCTGGAATCCGTGCTGGCCAAGCA

[0156] CGGCTGGCAGGTGCCAGCCTACCCACTGCCAAAGAACCGCGAAAACACCAC

[0157] CATCTCCCGCATCGTGGTGCGCCCATCCATGACCATGACCGTGTGCGGCGAC

[0158] TTCATCGACGATCTGAAGCTGTCCATCAAGGAACTGGAAACCTCCCGCCTGGTGCGCCCAAACCAGTCCATCATCTAA。

[0159] SEQ ID NO.9 (K22D: Gene Sequence)

[0160] ATGACCAAGAAGAACGATCTGAACGAATACCAGGAATACCTGGAAGATTCC

[0161] TTCCTGGGCTCCGACGGCTCCCAGCGCCTGCTGCCAAACACCAAGATGCCA

[0162] GAAACCCCAATGTACGCAAACCTGGCCCGCGATCTGATCGAACACTTCCGC

[0163] CTGAACGAAGCAAAGGCAAACCAGAACCTGGCCACCTTCTGCACCACCCAG

[0164] ATGGAAAAGCAGGCCGATGAACTGATCCTGAACTCCCTGAACACCAACGCA

[0165] ATCGACAAGTCCGAATACCCAAAGACCTCCGCAATGGAAAACTCCTGCATC

[0166] TCCATGATCGCCCACCTGTGGAACGTGGGCGACGATAAGGAAATCTTCAAG

[0167] GACTTCATCGGCACCTCCACCGTGGGCTCCTCCGAAGGCTGCATGCTGGGCG

[0168] GCCTGGCCCTGTTGAAGTCCTGGCGCAAGCGCGCCGCAGATGTGGGCATCG

[0169] ACGTGGACAACATGAAGGATCACAAGCCAAACCTGGTGATCATGTCCGGCT

[0170] ACCAGGTGGTGTGGGAAAAGTTCTGCGTGTACTGGGACGTGGAACTGCGCT

[0171] CCATCCCAGTGTCCAAGGATCACATGTCCCTGGATACCGATTCCGTGATGTC

[0172] CTACGTGGACGAAAACACCATCGGCATCGTGGGCGTGCACGGCATCACCTA

[0173] CACCGGCGCAGTGGACGACATCCAGAAGCTGGATGAACTGGTGTCCGAATA

[0174] CAACAAGAACTCCAAGCTGGACCTGCGCATCCACGTGGACGCAGCATTCGG

[0175] CGGCCTGTTCTCCCCATTCATGGATGGCTTCAAGCCATGGGACTTCCGCCTG

[0176] AAGAACGTGGTGTCCATCAACGTGTCCGGCCACAAGTACGGCATGGTGTAC

[0177] CCAGGCATCGGCTGGGTGATCTGGCGCGAAAACAACCTGGACCTGCTGCCA

[0178] GAATCCATGCGCTTCGAAGTGGCCTACCTGGGCGATAAGGTGGATTCCATC

[0179] GCCATCAACTTCTCCCACTCCGGCGCACACATCGTGGCACAGTACTACAACT

[0180] TCATCCGCTTCGGCTTCTACGGCTACAAGAAGATCATGGAAGGCGTGCGCA

[0181] AGGTGTCCATCGCACTGGCAGATGAAATCTCCTCCACCGGCTTCTTCGAAAT

[0182] CATCAACGATGGCTCCCAGCTGCCAATCGTGTGCTGGAAGCTGAACGATGA

[0183] AATCAAGACCGATTGGTCCCTGTACGATCTGGAATCCGTGCTGGCCAAGCA

[0184] CGGCTGGCAGGTGCCAGCCTACCCACTGCCAAAGAACCGCGAAAACACCAC

[0185] CATCTCCCGCATCGTGGTGCGCCCATCCATGACCATGACCGTGTGCGGCGAC

[0186] TTCATCGACGATCTGAAGCTGTCCATCAAGGAACTGGAAACCTCCCGCCTG

[0187] GTGCGCCCAAACCAGTCCATCATCTAA

[0188] SEQ ID NO.10 (Y39T: Gene Sequence)

[0189] ATGACCAAGAAGAACGATCTGAACGAATACCAGGAATACCTGGAAGATTCC

[0190] TTCCTGGGCTCCAAGGGCTCCCAGCGCCTGCTGCCAAACACCAAGATGCCA

[0191] GAAACCCCAATGACCGCAAACCTGGCCCGCGATCTGATCGAACACTTCCGC

[0192] CTGAACGAAGCAAAGGCAAACCAGAACCTGGCCACCTTCTGCACCACCCAG

[0193] ATGGAAAAGCAGGCCGATGAACTGATCCTGAACTCCCTGAACACCAACGCA

[0194] ATCGACAAGTCCGAATACCCAAAGACCTCCGCAATGGAAAACTCCTGCATC

[0195] TCCATGATCGCCCACCTGTGGAACGTGGGCGACGATAAGGAAATCTTCAAG

[0196] GACTTCATCGGCACCTCCACCGTGGGCTCCTCCGAAGGCTGCATGCTGGGCG

[0197] GCCTGGCCCTGTTGAAGTCCTGGCGCAAGCGCGCCGCAGATGTGGGCATCG

[0198] ACGTGGACAACATGAAGGATCACAAGCCAAACCTGGTGATCATGTCCGGCT

[0199] ACCAGGTGGTGTGGGAAAAGTTCTGCGTGTACTGGGACGTGGAACTGCGCT

[0200] CCATCCCAGTGTCCAAGGATCACATGTCCCTGGATACCGATTCCGTGATGTC

[0201] CTACGTGGACGAAAACACCATCGGCATCGTGGGCGTGCACGGCATCACCTA

[0202] CACCGGCGCAGTGGACGACATCCAGAAGCTGGATGAACTGGTGTCCGAATA

[0203] CAACAAGAACTCCAAGCTGGACCTGCGCATCCACGTGGACGCAGCATTCGG

[0204] CGGCCTGTTCTCCCCATTCATGGATGGCTTCAAGCCATGGGACTTCCGCCTG

[0205] AAGAACGTGGTGTCCATCAACGTGTCCGGCCACAAGTACGGCATGGTGTAC

[0206] CCAGGCATCGGCTGGGTGATCTGGCGCGAAAACAACCTGGACCTGCTGCCA

[0207] GAATCCATGCGCTTCGAAGTGGCCTACCTGGGCGATAAGGTGGATTCCATC

[0208] GCCATCAACTTCTCCCACTCCGGCGCACACATCGTGGCACAGTACTACAACT

[0209] TCATCCGCTTCGGCTTCTACGGCTACAAGAAGATCATGGAAGGCGTGCGCA

[0210] AGGTGTCCATCGCACTGGCAGATGAAATCTCCTCCACCGGCTTCTTCGAAAT

[0211] CATCAACGATGGCTCCCAGCTGCCAATCGTGTGCTGGAAGCTGAACGATGA

[0212] AATCAAGACCGATTGGTCCCTGTACGATCTGGAATCCGTGCTGGCCAAGCA

[0213] CGGCTGGCAGGTGCCAGCCTACCCACTGCCAAAGAACCGCGAAAACACCAC

[0214] CATCTCCCGCATCGTGGTGCGCCCATCCATGACCATGACCGTGTGCGGCGAC

[0215] TTCATCGACGATCTGAAGCTGTCCATCAAGGAACTGGAAACCTCCCGCCTG

[0216] GTGCGCCCAAACCAGTCCATCATCTAA

[0217] SEQ ID NO.11 (N41S: Gene Sequence)

[0218] ATGACCAAGAAGAACGATCTGAACGAATACCAGGAATACCTGGAAGATTCC

[0219] TTCCTGGGCTCCAAGGGCTCCCAGCGCCTGCTGCCAAACACCAAGATGCCA

[0220] GAAACCCCAATGTACGCATCCCTGGCCCGCGATCTGATCGAACACTTCCGC

[0221] CTGAACGAAGCAAAGGCAAACCAGAACCTGGCCACCTTCTGCACCACCCAG

[0222] ATGGAAAAGCAGGCCGATGAACTGATCCTGAACTCCCTGAACACCAACGCA

[0223] ATCGACAAGTCCGAATACCCAAAGACCTCCGCAATGGAAAACTCCTGCATC

[0224] TCCATGATCGCCCACCTGTGGAACGTGGGCGACGATAAGGAAATCTTCAAG

[0225] GACTTCATCGGCACCTCCACCGTGGGCTCCTCCGAAGGCTGCATGCTGGGCG

[0226] GCCTGGCCCTGTTGAAGTCCTGGCGCAAGCGCGCCGCAGATGTGGGCATCG

[0227] ACGTGGACAACATGAAGGATCACAAGCCAAACCTGGTGATCATGTCCGGCT

[0228] ACCAGGTGGTGTGGGAAAAGTTCTGCGTGTACTGGGACGTGGAACTGCGCT

[0229] CCATCCCAGTGTCCAAGGATCACATGTCCCTGGATACCGATTCCGTGATGTC

[0230] CTACGTGGACGAAAACACCATCGGCATCGTGGGCGTGCACGGCATCACCTA

[0231] CACCGGCGCAGTGGACGACATCCAGAAGCTGGATGAACTGGTGTCCGAATA

[0232] CAACAAGAACTCCAAGCTGGACCTGCGCATCCACGTGGACGCAGCATTCGG

[0233] CGGCCTGTTCTCCCCATTCATGGATGGCTTCAAGCCATGGGACTTCCGCCTG

[0234] AAGAACGTGGTGTCCATCAACGTGTCCGGCCACAAGTACGGCATGGTGTAC

[0235] CCAGGCATCGGCTGGGTGATCTGGCGCGAAAACAACCTGGACCTGCTGCCA

[0236] GAATCCATGCGCTTCGAAGTGGCCTACCTGGGCGATAAGGTGGATTCCATC

[0237] GCCATCAACTTCTCCCACTCCGGCGCACACATCGTGGCACAGTACTACAACT

[0238] TCATCCGCTTCGGCTTCTACGGCTACAAGAAGATCATGGAAGGCGTGCGCA

[0239] AGGTGTCCATCGCACTGGCAGATGAAATCTCCTCCACCGGCTTCTTCGAAAT

[0240] CATCAACGATGGCTCCCAGCTGCCAATCGTGTGCTGGAAGCTGAACGATGA

[0241] AATCAAGACCGATTGGTCCCTGTACGATCTGGAATCCGTGCTGGCCAAGCA

[0242] CGGCTGGCAGGTGCCAGCCTACCCACTGCCAAAGAACCGCGAAAACACCAC

[0243] CATCTCCCGCATCGTGGTGCGCCCATCCATGACCATGACCGTGTGCGGCGAC

[0244] TTCATCGACGATCTGAAGCTGTCCATCAAGGAACTGGAAACCTCCCGCCTG

[0245] GTGCGCCCAAACCAGTCCATCATCTAASEQ ID NO.12(K56E: Gene Sequence)

[0246] ATGACCAAGAAGAACGATCTGAACGAATACCAGGAATACCTGGAAGATTCC

[0247] TTCCTGGGCTCCAAGGGCTCCCAGCGCCTGCTGCCAAACACCAAGATGCCA

[0248] GAAACCCCAATGTACGCAAACCTGGCCCGCGATCTGATCGAACACTTCCGC

[0249] CTGAACGAAGCAGAGGCAAACCAGAACCTGGCCACCTTCTGCACCACCCAG

[0250] ATGGAAAAGCAGGCCGATGAACTGATCCTGAACTCCCTGAACACCAACGCA

[0251] ATCGACAAGTCCGAATACCCAAAGACCTCCGCAATGGAAAACTCCTGCATC

[0252] TCCATGATCGCCCACCTGTGGAACGTGGGCGACGATAAGGAAATCTTCAAG

[0253] GACTTCATCGGCACCTCCACCGTGGGCTCCTCCGAAGGCTGCATGCTGGGCG

[0254] GCCTGGCCCTGTTGAAGTCCTGGCGCAAGCGCGCCGCAGATGTGGGCATCG

[0255] ACGTGGACAACATGAAGGATCACAAGCCAAACCTGGTGATCATGTCCGGCT

[0256] ACCAGGTGGTGTGGGAAAAGTTCTGCGTGTACTGGGACGTGGAACTGCGCT

[0257] CCATCCCAGTGTCCAAGGATCACATGTCCCTGGATACCGATTCCGTGATGTC

[0258] CTACGTGGACGAAAACACCATCGGCATCGTGGGCGTGCACGGCATCACCTA

[0259] CACCGGCGCAGTGGACGACATCCAGAAGCTGGATGAACTGGTGTCCGAATA

[0260] CAACAAGAACTCCAAGCTGGACCTGCGCATCCACGTGGACGCAGCATTCGG

[0261] CGGCCTGTTCTCCCCATTCATGGATGGCTTCAAGCCATGGGACTTCCGCCTG

[0262] AAGAACGTGGTGTCCATCAACGTGTCCGGCCACAAGTACGGCATGGTGTAC

[0263] CCAGGCATCGGCTGGGTGATCTGGCGCGAAAACAACCTGGACCTGCTGCCA

[0264] GAATCCATGCGCTTCGAAGTGGCCTACCTGGGCGATAAGGTGGATTCCATC

[0265] GCCATCAACTTCTCCCACTCCGGCGCACACATCGTGGCACAGTACTACAACT

[0266] TCATCCGCTTCGGCTTCTACGGCTACAAGAAGATCATGGAAGGCGTGCGCA

[0267] AGGTGTCCATCGCACTGGCAGATGAAATCTCCTCCACCGGCTTCTTCGAAAT

[0268] CATCAACGATGGCTCCCAGCTGCCAATCGTGTGCTGGAAGCTGAACGATGA

[0269] AATCAAGACCGATTGGTCCCTGTACGATCTGGAATCCGTGCTGGCCAAGCA

[0270] CGGCTGGCAGGTGCCAGCCTACCCACTGCCAAAGAACCGCGAAACACCAC

[0271] CATCTCCCGCATCGTGGTGCGCCCATCCATGACCATGACCGTGTGCGGCGAC

[0272] TTCATCGACGATCTGAAGCTGTCCATCAAGGAACTGGAAACCTCCCGCCTG

[0273] GTGCGCCCAAACCAGTCCATCATCTAA

[0274] Other raw materials or structures not specifically described in the present invention already exist in the prior art and can be directly purchased from the market.

[0275] The above are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a glutamate decarboxylase mutant in the production of γ-aminobutyric acid, characterized in that: The amino acid sequence of the glutamate decarboxylase mutant is shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.

7.

2. A gene encoding the glutamate decarboxylase mutant according to claim 1.

3. The gene according to claim 2, characterized in that When the amino acid sequence of the glutamate decarboxylase mutant is SEQ ID NO.3, the nucleotide sequence encoding SEQ ID NO.3 is shown in SEQ ID NO.8; When the amino acid sequence of the glutamate decarboxylase mutant is SEQ ID NO.4, the nucleotide sequence encoding SEQ ID NO.4 is shown in SEQ ID NO.9; When the amino acid sequence of the glutamate decarboxylase mutant is SEQ ID NO.5, the nucleotide sequence encoding SEQ ID NO.5 is shown in SEQ ID NO.10; When the amino acid sequence of the glutamate decarboxylase mutant is SEQ ID NO.6, the nucleotide sequence encoding SEQ ID NO.6 is shown in SEQ ID NO.11; When the amino acid sequence of the glutamate decarboxylase mutant is SEQ ID NO.7, the nucleotide sequence encoding SEQ ID NO.7 is shown as SEQ ID NO.

12.

4. A recombinant expression vector carrying the gene according to claim 2 or 3.

5. The recombinant expression vector according to claim 4, characterized in that pET-28a was used as the original expression vector.

6. A recombinant bacterium containing the recombinant expression vector according to claim 4 or 5.

7. The recombinant bacterium according to claim 6, characterized in that The recombinant bacteria uses Corynebacterium glutamicum or Escherichia coli as host bacteria.

8. Use of the gene according to claim 2 or 3, the recombinant expression vector according to claim 4 or 5, or the recombinant bacterium according to claim 6 or 7 in synthesizing γ-aminobutyric acid.

9. The use according to claim 8, characterized in that Using glutamate or sodium glutamate as a substrate, the recombinant bacteria described in claim 7 are fermented to produce gamma-aminobutyric acid.