L-2-aminobutyric acid producing strain, construction method and application thereof

Abstract

This invention provides an L-2-aminobutyric acid (GABA) producing strain, its construction method, and its application. E. coli W3110, as a chassis strain, was knocked out. tdh , rhtA, ilvE Genes, upregulated ppc, aspC , thrA fbr BC , ilvA fbr , tdcB , yeaS , pntAB The transcriptional level of genes weakens ilvI Genes, heterologously introduced from Bacillus subtilis B. subtilis 168 bcd, gdh Genes, and those derived from Corynebacterium glutamicum K051 cysK Genes; the strain does not contain plasmids, is genetically stable, requires no induction or addition of resistance substances, and efficiently synthesizes L-2-aminobutyric acid de novo using glucose as a substrate. It has good L-2-aminobutyric acid synthesis ability, stable performance, and high acid production efficiency.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and fermentation engineering technology, and in particular to an L-2-aminobutyric acid (GABA) producing strain, its construction method, and its application. Background Technology

[0002] L-2-Aminobutyric acid (ABA) is a non-protein chiral α-amino acid that can inhibit the transmission of nerve signals in the human body, enhance glucose phosphodiesterase activity, and promote brain cell metabolism. At the same time, ABA is an important chemical raw material and pharmaceutical precursor, and has been used as a key intermediate in the synthesis of various chiral drugs, such as the antiepileptic drugs levetiracetam and briracetam, and the antituberculosis drug ethambutol.

[0003] Currently, the main methods for producing L-2-aminobutyric acid (L-2-aminobutyric acid) include chemical synthesis, enzymatic catalysis, and microbial fermentation. Chemical synthesis suffers from drawbacks such as high cost, severe pollution, demanding reaction conditions, and easy generation of byproducts. Enzymatic catalysis faces challenges such as low coenzyme recycling efficiency and enzyme stability, as well as difficulties in scale-up. Microbial fermentation, on the other hand, offers advantages such as high specificity, mild reaction conditions, environmental friendliness, and sustainable production, and is widely used in the industrial production of L-2-aminobutyric acid. However, in the industrial production of L-2-aminobutyric acid, engineered bacteria carrying plasmids exhibit poor genetic stability and a heavy metabolic burden. Therefore, there is an urgent need to develop an efficient, convenient, and stable engineered bacterial strain. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a strain for producing L-2-aminobutyric acid.

[0005] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned L-2-aminobutyric acid producing strain.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned L-2-aminobutyric acid producing strain.

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0008] A strain for producing L-2-aminobutyric acid, said strain as E.coli W3110, as a chassis strain, was knocked out. tdh , rhtA, ilvE Genes, upregulated ppc, aspC , thrA fbr BC , ilvA fbr , tdcB , yeaS , pntAB The transcriptional level of genes weakensilvI The gene, heterologously introduced from Bacillus subtilis B.subtilis 168 bcd, gdh Genes, and those derived from Corynebacterium glutamicum K051 cysK Gene.

[0009] Preferably, the above-mentioned L-2-aminobutyric acid producing strain, in order to E. coli W3110, as a chassis strain, had the gene knocked out from the strain's genome. tdh , rhtA, ilvE Genes; using artificial promoter P BBa_j23114 Replacement of acetylhydroxy acid synthase ilvI The natural promoter of the gene; enhanced using the trc promoter. ppc Gene transcription and integration into the genome. yjiV Pseudogene loci; enhanced using the TRC promoter. aspC Gene transcription and integration into the genome. ilvG Pseudogene sites; the threonine operon was enhanced using the TRC promoter. thrA fbr BC Gene transcription and integration into the genome. ycgH Pseudogene locus and double copy to yeeP Pseudogene loci; enhanced using the TRC promoter. ilvA fbr Gene transcription and integration into the genome. yeeL Pseudogene locus and double copy to ylbE Pseudogene loci; enhanced using the TRC promoter. tdcB Gene transcription and integration into the genome. yjgX Pseudogene loci; enhanced using the TRC promoter. yeaS Gene transcription and integration into the genome. yciQ Pseudogene loci; enhanced using the TRC promoter. pntAB Gene transcription and integration into the genome. yghE Pseudogene sites; enhanced from TRC promoters. B. subtilis 168 gdh Gene transcription and integration into the genome. rhtA Pseudogene sites; enhanced from TRC promoters. ​ 168 ​ Gene transcription, followed by three copies of integration into the genome. ​ , ​ , ​ Pseudogene sites; enhanced from TRC promoters. ​ K051 ​ Gene transcription and integration into the genome.​ Pseudogene loci.

[0010] The above-mentioned L-2-aminobutyric acid producing strain, wherein ​ fbr ​ The gene underwent a point mutation, specifically changing the base at position 1034 from C to T, resulting in the amino acid residue at position 345 changing from serine to phenylalanine; [The above...] ​ fbr The gene underwent point mutations, specifically changing the base at position 1054 from T to G, the base at position 1055 from T to C, the base at position 1084 from C to T, the base at position 1085 from G to T, and the base at position 1086 from T to C. This resulted in the amino acid residue at position 352 changing from phenylalanine to alanine, and the amino acid residue at position 362 changing from arginine to phenylalanine.

[0011] Preferably, the above-mentioned L-2-aminobutyric acid producing strain, wherein ​ W3110 is ​ W3110 ATCC27325.

[0012] Preferably, the above-mentioned L-2-aminobutyric acid producing strain, wherein ​ The nucleotide sequence is shown in SEQ ID NO.2 of the sequence listing; ​ The nucleotide sequence is shown in SEQ ID NO.4 of the sequence listing; ​ The nucleotide sequence is shown in SEQ ID NO.6 of the sequence listing.

[0013] Preferably, the above-mentioned L-2-aminobutyric acid producing strain, wherein ​ The nucleotide sequence is shown in SEQ ID NO.3 of the sequence listing; ​ The nucleotide sequence is shown in SEQ ID NO.5 of the sequence listing; ​ The nucleotide sequence is shown in SEQ ID NO.7 of the sequence listing; ​ The nucleotide sequence is shown in SEQ ID NO.8 of the sequence listing; ​ The nucleotide sequence is shown in SEQ ID NO.9 of the sequence listing; ​ fbr ​ The nucleotide sequence is shown in SEQ ID NO.10 of the sequence listing; ​ fbr The nucleotide sequence is shown in SEQ ID NO.11 of the sequence listing; ​ The nucleotide sequence is shown in SEQ ID NO.12 of the sequence listing; ​ The nucleotide sequence is shown in SEQ ID NO.13 of the sequence listing; ​The nucleotide sequence is shown in SEQ ID NO.14 of the sequence listing; ​ The nucleotide sequence is shown in the sequence listing SEQ ID NO.15.

[0014] Preferably, in the above-mentioned L-2-aminobutyric acid producing strain, the nucleotide sequence of the trc promoter is shown in SEQ ID NO.1 of the sequence listing; the promoter P BBa_j23114 The nucleotide sequence is shown in the sequence listing SEQ ID NO.16.

[0015] The above method for constructing L-2-aminobutyric acid producing strains involves starting with the strain... ​ The targeted modification based on W3110 involves the following three modules:

[0016] (1) Increase the production of the precursor threonine: including increasing the phosphoenolpyruvate carboxylase gene ​ Transcription level; increase aspartate transaminase gene ​ Transcription level; increase threonine operon ​ fbr ​ Transcriptional levels; knockout of L-threonine efflux transporter genes ​ Knockout of threonine catabolism gene ​ Increase the supply of precursors for the L-2-aminobutyric acid (GABA) production pathway;

[0017] (2) Enhance reducing power: Enhance the pyridine nucleotide transhydrogenase gene ​ The transcriptional level increased the supply of NADPH, introducing substances derived from [the source]. ​ 168 glucose 1-dehydrogenase gene ​ This increased NADPH supply and knocked out branched-chain amino acid aminotransferase genes. ​ This reduces NADPH consumption;

[0018] (3) Increase L-2-aminobutyric acid production: enhance threonine dehydrogenase gene ​ fbr threonine dehydrogenase gene ​ The transcriptional level, increased from ​ 168 leucine dehydrogenase ​ Gene transcription level; improved from ​ K051 cysteine ​​synthase gene ​ At the transcriptional level, increase the transcriptional levels of key enzymes in the L-2-aminobutyric acid (GABA) synthesis pathway; use the artificial promoter P BBa_j23114 Replacement of acetylhydroxy acid synthase ​The natural promoter of the gene weakens the branching pathway of the precursor 2-ketobutyrate towards isoleucine; enhances the L-2-aminobutyrate efflux transport protein gene. ​ It increases the transcriptional level and the production of L-2-aminobutyric acid.

[0019] Application of the above-mentioned L-2-aminobutyric acid producing strains in the fermentation production of L-2-aminobutyric acid.

[0020] The aforementioned L-2-aminobutyric acid (GABA) producing strains were fermented in a culture medium under suitable fermentation conditions to produce GABA. The culture medium included, but was not limited to, carbon sources, nitrogen sources, inorganic salts, and vitamins. Fermentation conditions included fermentation temperature, pH, dissolved oxygen levels, pressure, and time. All the culture media could be obtained using conventional methods and used for GABA production. The fermentation conditions could be adjusted to suit the production characteristics of the strains.

[0021] Preferably, the above application follows these steps:

[0022] ① Strain activation: The L-2-aminobutyric acid producing strain was streaked onto an activation slant, incubated at 37℃ for 12 h, and passaged once;

[0023] ② Seed culture: Use an inoculation loop to scrape a loop of slanted seeds and inoculate them into an Erlenmeyer flask containing seed culture medium. Seal the flask with gauze and incubate at 36℃ and 200r / min for 10h.

[0024] ③ Fermentation culture: Inoculate the seed liquid prepared after activation of the strain at an inoculation rate of 10%-15% into an Erlenmeyer flask containing fermentation culture medium, seal the flask with gauze, maintain the temperature at 37±0.2℃, and shake culture at 220 r / min. During the fermentation process, ammonia water is added to maintain the pH at 7.0-7.2; glucose solution is added to supplement the carbon source required by the cells.

[0025] Preferably, in the application of the above-mentioned L-2-aminobutyric acid producing strain, the slant culture medium used in step ① is the general-purpose LB solid medium.

[0026] Preferably, in the application of the above-mentioned L-2-aminobutyric acid producing strain, the seed culture medium used in step ② is: glucose 25 g / L, yeast extract 5 g / L, citric acid 1 g / L, MgSO4·7H2O 0.6 g / L, KH2PO4·H2O 3 g / L, peptone 3 g / L, V B Mixture 0.5 mg / L, V H 1 mg / L, glutamic acid 2 g / L, methionine 0.2 g / L, lysine 0.5 g / L, the remainder being water.

[0027] Preferably, in the application of the above-mentioned L-2-aminobutyric acid producing strain, the fermentation medium used in step ③ is: citric acid 2 g / L, MgSO4·7H2O 1.6 g / L, FeSO4·7H2O 10 mg / L, MnSO4·7H2O 10 mg / L, KH2PO4 4 g / L, V B Mixture 0.5 mg / L, V H 0.5 mg / L, yeast powder 5 g / L, glutamic acid 2 g / L, lysine 3 g / L, methionine 2 g / L, corn steep liquor 20 ml / L, the remainder being water.

[0028] All of the above-mentioned culture media can be prepared using standard methods.

[0029] Beneficial effects:

[0030] The aforementioned L-2-aminobutyric acid (GABA) producing strain is plasmid-free, genetically stable, requires no induction or the addition of antibiotics, and efficiently synthesizes L-2-aminobutyric acid de novo using glucose as a substrate. It exhibits excellent L-2-aminobutyric acid synthesis capability, low production cost, stable performance, and high acid production efficiency, demonstrating good economic benefits and broad industrial application prospects. Specifically:

[0031] (1) By improving ​ , ​ The transcriptional level of the gene increased the supply of NADPH, and the knockout of the ilvE gene reduced the consumption of NADPH, providing a large amount of reducing power for the synthesis of L-2-aminobutyric acid.

[0032] (2) Introduction from ​ 168 leucine dehydrogenase ​ Gene substitution knockout of branched-chain amino acid aminotransferase gene ​ Its function is to convert 2-ketobutyric acid into L-2-aminobutyric acid;

[0033] (3) Using the artificial promoter P BBa_j23114 Replacement of acetylhydroxyl synthase gene ​ The natural promoter of the precursor 2-ketobutyrate weakens the branching pathway towards isoleucine, thus reducing the competitive metabolic pathway.

[0034] (4) Introduction from ​ K051 cysteine ​​synthase gene ​ This improved the key enzyme genes in the L-2-aminobutyric acid synthesis pathway. ​ fbr ​ , ​ fbr The transcriptional level. Attached Figure Description

[0035] ​ This diagram illustrates the de novo synthesis pathway modification process of L-2-aminobutyric acid (GABA) genetically engineered bacteria. Detailed Implementation

[0036] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0037] Unless otherwise specified, the percentage sign "%" used in the examples refers to the mass percentage. The percentage of a solution refers to the number of grams of solute contained in 100 mL. The percentage between liquids refers to the volume ratio of the solution at 25°C.

[0038] The gene editing method used is referenced in the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21.). Unless otherwise specified, all technical terms used in this invention are explained in this article. In this invention, "knockout" refers to the inactivation of the target gene, and "introduction" refers to the insertion of a foreign gene into the engineered bacterial genome after linking it with a promoter and terminator.

[0039] The examples used ​ W3110 is ​ W3110 ATCC 27325, the corresponding promoter and gene are shown in the sequence listing, and the primers used in the strain construction process are shown in Table 1.

[0040] Table 1

[0041] ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ GTGTGGAATTGTGAGCGGATAACAATTTCACAGGAAACAGACCATGTATCCGGATTTAAAAGGAAAAGTCGTC gdh-a SEQ ID NO.24 GACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAACCGCGGCCTGCCTG pGRB-tdh-s SEQ ID NO.25 AGTCCTAGGTATAATACTAGTGGCGAAGATGTGCTGGTTTCGTTTTAGAGCTAGAA pGRB-tdh-a SEQ ID NO.26 TTCTAGCTCTAAAACGAAACCAGCACATCTTCGCCACTAGTATTATACCTAGGACT tdh-US SEQ ID NO.27 GTCAGTTCCGTGAGCAAATGTCG tdh-UA SEQ ID NO.28 GTATCCGCTCAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGCACCGGGATGGTTTTTTG tdh-DS SEQ ID NO.29 CTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATAAGGCTTGTTCATTAAAGGTATTTACGG tdh-DA SEQ ID NO.30 GGTGCTGTCATCATAAATTACCGAC cysKs SEQ ID NO.31 GTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGATTGGAGCACCACCCGA cysK-a SEQ ID NO.32 CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAGTCGCGGATGTCTTCGTAAAGAA in pGRB-ilvE SEQ ID NO.33 AGTCCTAGGTATATACTAGTGATTATCGCTGCTTTCCCGTGTTTTAGAGCTAGAA pGRB-ilvE-a SEQ ID NO.34 TTCTAGCTCTAAAACACGGGAAAGCAGCGATAATCACTAGTATTATACCTAGGACT ilvE-US SEQ ID NO.35 GGAGAAATTATCATGATGCAACATCAGG ilvE-UA SEQ ID NO.36 GAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAATGGCGGAATACAACCGGTC ilvE-DS SEQ ID NO.37 GGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCGCAGCGTAGACGGTATTCAG ilvE-DA SEQ ID NO.38 TGGCCCATGGCAATCCCATC bcd-s SEQ ID NO.39 GTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGAACTTTTTAAATATATGGAGAAATACG bcd-a SEQ ID NO.40 CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAACGTCTGCTTAATACACTGTGGC pGRB-yjiV-s SEQ ID NO.41 GAACCAACTATCTACCAGCGTTAACAGATCGTTTAGATCCGA pGRB-yjiV-a SEQ ID NO.42 GTAGGTCCACTTGAGACGGTTATATAATCGCCATCACTTTCC yjiV-US SEQ ID NO.43 GACTGTGGAAGCCCTGTATACG yjiV-UA SEQ ID NO.44 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACTGTCCCTTGTCGACTGTCTGT yjiV-DS SEQ ID NO.45 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTGAACGAGTTTATTCTGCCGG yjiV-DA SEQ ID NO.46 TGGCGACATTCCCTTCCTT ppc-s SEQ ID NO.47 CTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAACGAACAATATTCCGCATTGC ppc-a SEQ ID NO.48 AACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAGCCGGTATTACGCATACCTGC pGRB-ilvG-s SEQ ID NO.49 AGTCCTAGGTATAATACTAGTTATCGGCACTGACGCATTTCGTTTTAGAGCTAGAA pGRB-ilvG-a SEQ ID NO.50 AGTCCTAGGTATAATACTAGTGGAAGAGTTGCCGCGCATCAGTTTTAGAGCTAGAA ilvG-US SEQ ID NO.51 ACCGAGGAGCAGACAATGAATAA ilvG-UA SEQ ID NO.52 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGTGATGGCAACAACAGGGA ilvG-DS SEQ ID NO.53 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCTATCTACGCGCCGTTGTTGTT ilvG-DA SEQ ID NO.54 GCGCTGGCTAACATGAGGAA aspC-s SEQ ID NO.55 ATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGTTTGAGAACATTACCGCCGCTCCT aspC-a SEQ ID NO.56 ACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACAGCACTGCCACAATCGC pGRB-ycgH-s SEQ ID NO.57 AGTCCTAGGTATAATACTAGTTATGCGTCTGAACGACCGTGGTTTTAGAGCTAGAA pGRB-ycgH-a SEQ ID NO.58 TTCTAGCTCTAAAACCACGGTCGTTCAGACGCATAACTAGTATTATACCTAGGACT pGRB-4#-s SEQ ID NO.59 ATGCACAGGAGACTTTCTGATGCGCTGGTTGATTTCTTCTAGGGTCATAGTAATCCAGCAACT pGRB-4#-a SEQ ID NO.60 AGTTGCTGGATTACTATGACCCTAGAAGAAATCAACCAGCGCATCAGAAAGTCTCCTGTGCAT pGRB-1#-s SEQ ID NO.61 CTGAACAACATCATATTTAAATGAACATAACTCAATTTGTAGGCTAGCATAACCCCTTGGGGC pGRB-1#-a SEQ ID NO.62 GCCCCAAGGGGTTATGCTAGCCTACAAATTGAGTTATGTTCATTTAAATATGATGTTGTTCAG ycgH-US SEQ ID NO.63 TAAACTCGTCAGCGGCACAA ycgH-UA SEQ ID NO.64 GAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGTAGGCGTTTCTGTTGATTCTG ycgH-DS-4# SEQ ID NO.65 ATGCACAGGAGACTTTCTGATGCGCTGGTTGATTTCTTCTAGGGTCATAGTAATCCAGCAACTGCGTGTCGGATTATCGTTCG ycgH-DS-1# SEQ ID NO.66 CTGAACAACATCATATTTAAATGAACATAACTCAATTTGTAGGCTAGCATAACCCCTTGGGGCGCGTGTCGGATTATCGTTCG ycgH-DS SEQ ID NO.67 GACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGCGTGTCGGATTATCGTTCG ycgH-DA SEQ ID NO.68 GATTCAGGTTGCCATTTACGC thr1-s SEQ ID NO.69 GTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCGAGTGTTGAAGTTCGG thr1-a-4# SEQ ID NO.70 AGTTGCTGGATTACTATGACCCTAGAAGAAATCAACCAGCGCATCAGAAAGTCTCCTGTGCATGTTTTCCAGATTAAGGCCATGTACATTG thr2-S SEQ ID NO.71 CTTACGTGTCTGCGGTGTTG thr2-A-1# SEQ ID NO.72 GCCCCAAGGGGTTATGCTAGCCTACAAATTGAGTTATGTTCATTTAAATATGATGTTGTTCAGGCCTGCCAGATGTCGCC thr3-S SEQ ID NO.73 TCGACGGCAGAAGCCAGG thr3-A SEQ ID NO.74 GATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACTGATGATTCATCATCAATTTACGCAACG pGRB-yeeP-s SEQ ID NO.75 AGTCCTAGGTATAATACTAGTTGAACAGTTTACCGGTGCGGGTTTTAGAGCTAGAA pGRB-yeeP-a SEQ ID NO.76 TTCTAGCTCTAAAACCCGCACCGGTAAACTGTTCAACTAGTATTATACCTAGGACT yeeP-US SEQ ID NO.77 GGTCAGGAGGTAACTTATCAGCG yeeP-UA SEQ ID NO.78 GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAATGGCAGGGCTCCGTTTT yeeP-DS-4# SEQ ID NO.79 ATGCACAGGAGACTTTCTGATGCGCTGGTTGATTTCTTCTAGGGTCATAGTAATCCAGCAACTGAACTGGATTTTCTTCTGAACCTGT yeeP-DS-1# SEQ ID NO.80 CTGAACAACATCATATTTAAATGAACATAACTCAATTTGTAGGCTAGCATAACCCCTTGGGGCGAACTGGATTTTCTTCTGAACCTGT yeeP-DS SEQ ID NO.81 GGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGAACTGGATTTTCTTCTGAACCTGT yeeP-DA SEQ ID NO.82 ACGATGTCAGCAGCCAGC pGRB-yeeL-s SEQ ID NO.83 AGTCCTAGGTATAATACTAGTAACACAGCAATACGGTACGCGTTTTAGAGCTAGAA pGRB-yeeL-a SEQ ID NO.84 TTCTAGCTCTAAAACGCGTACCGTATTGCTGTGTTACTAGTATTATACCTAGGACT eeL-US SEQ ID NO.85 TTCATCGGGACGAGTGGAGA eeL-UA SEQ ID NO.86 TCCACACATTATACGAGCCGGATGATTAATTGTCAACCATAGCATCGCCAATCTGATCGGG eeL-DS SEQ ID NO.87 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATACCCAAAGGTGAAGATA yesL-YES SEQ ID NO.88 CATTCCCTCTACAGAACTAG <![CDATA[ilvA fbr -s]]> SEQ ID NO.89 CATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACAGGAAACAGACCATGGCTGACTCGCAACCC <![CDATA[ilvA fbr -a]]> SEQ ID NO.90 AACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGCTAACCCGCCAAAAAGAACCTGA ilvA-TB SEQ ID NO.91 aaggcagcgccctcaaattctgccaactgcttggcgggctctcggtcac ilvA-TB-a SEQ ID NO.92 gtgaccgagagcccgccaagcagttggcagaatttgagggcgctgcctt pGRB-ylbE-s SEQ ID NO.93 AGTCCTAGGTATACTAGTACACTGGCTGGATGTGCAACGTTTTAGAGCTAGAA pGRB-ylbE-a SEQ ID NO.94 TTCTAGCTCTAAAACGTTGCACATCCAGCCAGTGTACTAGTATTATACCTAGGACT ylbe-US SEQ ID NO.95 ACCCAACCTTACGCAACCAG ylbE-UA SEQ ID NO.96 GTGAAATTGTTATCCGCTCAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATTGTTCGATAACCGCAGCAT ylbE-DS SEQ ID NO.97 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCGCTGGCGTGCTTTGAA ylbe-DA SEQ ID NO.98 GGCGTAACTCAGCAGGCAG pGRB-yjgX-s SEQ ID NO.99 AGTCCTAGGTATAATACTAGTTCGCGACCACCGTAACTGGCGTTTTAGAGCTAGAA pGRB-yjgX-a SEQ ID NO.100 TTCTAGCTCTAAAACGCCAGTTACGGTGGTCGCGAACTAGTATTATACCTAGGACT yjgX-US SEQ ID NO.101 GGAAGTCAACGGGTTATGCG yjgX-UA SEQ ID NO.102 GTATCCGCTCAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAAAAATCACCACGAATACCAGAATCGC yjgX-DS SEQ ID NO.103 GACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATACAGTGTCTTCCCTGAGCCG yjgX-DA SEQ ID NO.104 GGCGAAGGATACCATCAAGC with tdcB SEQ ID NO.105 CTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCATATTACATACGATCTGCCGG tdcB-a SEQ ID NO.106 ACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAAGCGTCAACGAAACCGGTG pGRB-yciQ-s SEQ ID NO.107 AGTCCTAGGTATACTAGTAAACAACGTTTCTTGCCTCAGTTTTAGAGCTAGAA pGRB-yciQ-a SEQ ID NO.108 TTCTAGCTCTAAACTGAGGCAAGAAACGTTGTTTACTAGTATTATACCTAGGACT yciQ-US SEQ ID NO.109 TTACTTGAAGCATTGGGCGAAC yciQ-UA SEQ ID NO.110 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCAGTCAAGATGCCAGGGTTC yciQ-DS SEQ ID NO.111 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGTCTGACAAGAACCAGCAAATCCT yciQ-DA SEQ ID NO.112 ATAGCTTCACCGTGGGCATAAC yeaS-s SEQ ID NO.113 GCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCGTGTTCGCTGAATACGGGGTTC yeaS-a SEQ ID NO.114 acaaacaacagataaaacgaaaggcccagtctttcgactgagcctttcgttttatttgtcaggattgcagcgtcgc pGRB-yghE-s SEQ ID NO.115 AGTCCTAGGTATAATACTAGTGCTGAAAAAATATCGCCCACGTTTTAGAGCTAGAA pGRB-yghE-a SEQ ID NO.116 TTCTAGCTCTAAAACGTGGGCGATATTTTTTCAGCACTAGTATTATACCTAGGACT yghE-US SEQ ID NO.117 GTCAGGCACTGGCGAAAGAT yghE-UA SEQ ID NO.118 TTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACGCAAGCCATAAACCCACAAG yghE-DS SEQ ID NO.119 GCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTTCCGACATCGAAATGCGTGG yghE-DA SEQ ID NO.120 AGGCGTTGTTGTGGCAGAT pntAB-s SEQ ID NO.121 CCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCGAATTGGCATACCAAGAG pntAB-a SEQ ID NO.122 CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACAGAGCTTTCAGGATTGCATCCAC pGRB-mbhA-s SEQ ID NO.123 AGTCCTAGGTATAATACTAGTGCGTGATGTGAATGAGAAAAGTTTTAGAGCTAGAA pGRB-mbhA-a SEQ ID NO.124 TTCTAGCTCTAAAACTTTTCTCATTCACATCACGCACTAGTATTATACCTAGGACT mbhA-US SEQ ID NO.125 GCCAGCACGAACATAATCCC mbhA-UA SEQ ID NO.126 GGTCTGTTTCCTGCTAGCACTATACCTAGGACTGAGCTAGCCGTAAACACGGTGGCAGGTTTTGG mbhA-DS SEQ ID NO.127 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGACCAAAAGTGCGTCCGATAC mbhA-DA SEQ ID NO.128 CGGCGTAATCACAAACTGGC pGRB-ygaY-s SEQ ID NO.129 AGTCCTAGGTATAATACTAGTCACTGATGGCGCTGGCATTAGTTTTAGAGCTAGAA pGRB-ygaY-a SEQ ID NO.130 TTCTAGCTCTAAAACTAATGCCAGCGCCATCAGTGACTAGTATTATACCTAGGACT ygaY-US SEQ ID NO.131 ACCCAACCTTACGCAACCAG ygaY-UA SEQ ID NO.132 GTGAAATTGTTATCCGCTCAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATTGTTCGATAACCCGCAGCAT ygaY-DS SEQ ID NO.133 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCGCTGGCGTGCTTTGAA ygaY-DA SEQ ID NO.134 GGCGTAACTCAGCAGGCAG pGRB-ilvI-s SEQ ID NO.135 AGTCCTAGGTATAATACTAGTATTGCTTAAGCAAGATCGGAGTTTTAGAGCTAGAA pGRB-ilvI-a SEQ ID NO.136 TTCTAGCTCTAAAACTCCGATCTTGCTTAAGCAATACTAGTATTATACCTAGGACT ilvI-js114-JD-S SEQ ID NO.137 gctcagtcctaggtacaatgctagc UP-ilvI-S SEQ ID NO.138 TCTCTCCTGGCATGAAGCTGC UP-ilvI-js114-A SEQ ID NO.139 TTTCCTgctagcattgtacctaggactgagctagccataaaGGGGTAATAAGAATAAACAGGAGGAAAGGT DW-ilvI-js114-S SEQ ID NO.140 ctaggtacaatgctagcAGGAAACAGACCAGGCCATGGAGATGTTGTCTGGAGCCG DW-ilvI-A SEQ ID NO.141 CCTATCAACGAGGTCGCTACCT

[0042] Example 1

[0043] like Figure 1 As shown, this embodiment aims to illustrate the specific construction steps of strain ABA-15. In particular, if there are similar gene manipulation methods in the embodiment, they will only be provided once and annotated, without further elaboration.

[0044] (1) Knockout rhtA Genes are integrated at this site from B.subtilis 168 gdh Genes: E. coliUsing the W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification of rhtA-US, rhtA-UA, rhtA-DS, and rhtA-DA, respectively. B.subtilis Using the 168 genome as a template, and with gdh-s and gdh-a as primers, the target gene fragment was amplified by PCR. Then, using upstream and downstream homologous arms and the target gene fragment as templates, and with rhtA-US and rhtA-DA as primers, overlapping fragments were amplified by overlapping PCR. Using pGRB-rhtA-s and pGRB-rhtA-a as primers, the gRNA fragment was annealed and ligated to the pGRB vector to obtain rhtA-pGRB. Preparation... E.coli W3110 electroporated competent cells, and the target fragment and rhtA-pGRB were electroporated into competent cells together. Positive transformants were then screened to obtain strain ABA-01.

[0045] (2) Knockout tdh Genes are integrated at this site from Corynebacterium glutamicum K051 cysK Gene: It has the same operation as in (1), except that it uses Corynebacterium glutamicum Gene fragments were obtained from the K051 genome using primers tdh-US, tdh-UA, tdh-DS, tdh-DA, pGRB-tdh-s, pGRB-tdh-a, cysK-s, and cysK-a. Competent cells were ABA-01, and strain ABA-02 was obtained.

[0046] (3) Knockout ilvE The gene is integrated at this site from B. subtilis 168. bcd Gene: It has the same operation as in (1), except that it uses B.subtilis Gene fragments were obtained from the 168 genome using primers ilvE-US, ilvE-UA, ilvE-DS, ilvE-DA, pGRB-ilvE-s, pGRB-ilvE-a, bcd-s, and bcd-a. Competent cells were used, and strain ABA-03 was obtained.

[0047] (4) In yghE Pseudogene sites are controlled using the TRC promoter. pntAB Gene overexpression: E.coliUsing the W3110 genome as a template, upstream and downstream homologous arms and the target gene fragment were obtained by PCR amplification using yghE-US, yghE-UA, yghE-DS, yghE-DA, pntAB-s, and pntAB-a, respectively. Then, using the upstream and downstream homologous arms and the target gene fragment as templates, overlapping fragments were obtained by overlapping PCR amplification using yghE-US and yghE-DA primers. Using pGRB-yghE-s and pGRB-yghE-a primers, gRNA fragments were annealed to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain yghE-pGRB. ABA-03 electroporation competent cells were prepared, and the target fragment and yghE-pGRB were electroporated into competent cells together. Positive transformants were screened to obtain ABA-03 competent cells, and strain ABA-04 was obtained.

[0048] (5) In yjiV Pseudogene sites are controlled using the TRC promoter. ppc Gene overexpression: It has the same operation as (4), except that it uses E.coli The W3110 genome fragment was obtained, and the primers were as follows: yjiV -US、 yjiV -UA、 yjiV -DS、 yjiV -DA、 ppc -s、 ppc -a、pGRB- yjiV -s and pGRB- yjiV -a. Competent cells were ABA-04, and strain ABA-05 was obtained.

[0049] (6) In ilvG Pseudogene sites are controlled using the TRC promoter. aspC Gene overexpression: It has the same operation as (4), except that it uses E.coli The W3110 genome fragment was obtained, and the primers were as follows: ilvG -US、 ilvG -UA、 ilvG -DS、 ilvG -DA、 aspC -s、 aspC -a、pGRB- ilvG -s and pGRB- ilvG -a. Competent cells were ABA-05, and strain ABA-06 was obtained.

[0050] (7) In ycgH Pseudogene sites are controlled using the TRC promoter. thrA fbr BC Gene overexpression:

[0051] (a) with E.coli Using the W3110 genome as a template, upstream and downstream homologous arms and the target gene fragment were amplified by PCR using ycgH-US, ycgH-UA, ycgH-DS-4#, ycgH-DA, thr1-s, and thr1-a-4#, respectively. Then, using the upstream and downstream homologous arms and the target gene fragment as templates, overlapping fragments were amplified by overlapping PCR using ycgH-US and ycgH-DA primers. Using pGRB-ycgH-s and pGRB-ycgH-a primers, gRNA fragments were annealed to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain ycgH-pGRB. ABA-06 electroporation competent cells were prepared, and the target fragments and ycgH-pGRB were electroporated into competent cells together. Positive transformants were screened to obtain ABA-06 competent cells, and strain ABA-07-1 was obtained.

[0052] (b) Upstream and downstream homologous arms were obtained by PCR amplification using thr2-S, thr2-A-1# and ycgH-DS-1#, ycgH-DA, respectively. Then, using the upstream and downstream homologous arms as templates, overlapping fragments were obtained by overlapping PCR amplification using thr2-S and ycgH-DA as primers. The gRNA fragment was obtained by annealing using pGRB-4#-S and pGRB-4#-A as primers and ligated to the pGRB vector to obtain 4#-pGRB. ABA-07-1 electroporation competent cells were prepared, and the target fragment and 4#-pGRB were electroporated into competent cells together. Positive transformants were obtained by screening, and the competent cells were ABA-07-1, resulting in strain ABA-07-2.

[0053] (c) Upstream and downstream homologous arms were obtained by PCR amplification using thr3-S, thr3-A and ycgH-DS, ycgH-DA, respectively. Then, using the upstream and downstream homologous arms as templates, overlapping fragments were obtained by overlapping PCR amplification using thr3-S and ycgH-DA as primers. The gRNA fragment was obtained by annealing using pGRB-1#-S and pGRB-1#-A primers and ligated to the pGRB vector to obtain 1#-pGRB. ABA-07-2 electroporation competent cells were prepared, and the target fragment and 1#-pGRB were electroporated into competent cells. Positive transformants were obtained by screening, and the competent cells were ABA-07-2, thus obtaining strain ABA-07-3.

[0054] (8) In yeeP Pseudogene sites are controlled using the TRC promoter. thrA fbr BCGene overexpression: The same operation method as in (7) is used, except that the primers ycgH-US are replaced with yeeP-US, ycgH-UA are replaced with yeeP-UA, ycgH-DS-4# is replaced with yeeP-DS-4#, ycgH-DS-1# is replaced with yeeP-DS-1#, ycgH-DS is replaced with yeeP-DS, ycgH-DA is replaced with yeeP-DA, pGRB-ycgH-s is replaced with pGRB-yeeP-s, pGRB-ycgH-a is replaced with pGRB-yeeP-a, and the competent cells are ABA-07-3, ABA-08-1, and ABA-08-2 respectively, and strain ABA-08-3 is obtained.

[0055] (9) In yeeL Pseudogene sites are controlled using the TRC promoter. ilvA fbr Gene overexpression: It has the same operation as (4), except that it uses E.coli The W3110 genome fragment was obtained, and the primers were as follows: yeeL -US、 yeeL -UA、 yeeL -DS、 yeeL -DA、 ilvA fbr -s、 ilvA - TB -a、 ilvA-TB-s、ilvA fbr -a、pGRB- yeeL -s and pGRB- yeeL -a. Competent cells were ABA-08-3, and strain ABA-09 was obtained.

[0056] (10) In ylbE Pseudogene sites are controlled using the TRC promoter. ilvA fbr Gene overexpression: It has the same operation as (4), except that it uses E.coli The W3110 genome fragment was obtained, and the primers were as follows: ylbE -US、 ylbE -UA、 ylbE -DS、 ylbE -DA、 ilvA fbr -s、 ilvA fbr -TB-a、 fbr ilvA fbr -a、pGRB- -s and pGRB- -TB-s、ilvA-a. Competent cells were ABA-09, and strain ABA-10 was obtained.

[0057] (11) In Pseudogene sites are controlled using the TRC promoter. ylbE Gene overexpression: It has the same operation as (4), except that it uses The W3110 genome fragment was obtained, and the primers were as follows: ylbE -US、 -UA、 ygay -DS、 -DA、 bcd -s、 -a、pGRB- E.coli -s and pGRB- -a. Competent cells were ABA-10, and strain ABA-11 was obtained.

[0058] (12) In ygay Pseudogene sites are controlled using the TRC promoter. Gene overexpression: It has the same operation as (4), except that it uses ygay The W3110 genome fragment was obtained, and the primers were as follows: -US、 ygay -UA、 -DS、 ygay -DA、 -s、 bcd -a、pGRB- -s and pGRB- bcd -a. Competent cells were ABA-11, and strain ABA-12 was obtained.

[0059] (13) In Pseudogene sites are controlled using the TRC promoter. ygay Gene overexpression: It has the same operation as (4), except that it uses The W3110 genome fragment was obtained, and the primers were as follows: ygay -US、 -UA、 mbhA -DS、 -DA、 bcd -s、 -a、pGRB- E.coli -s and pGRB- -a. Competent cells were ABA-12, and strain ABA-13 was obtained.

[0060] (14) In mbhA Pseudogene sites are controlled using the TRC promoter. Gene overexpression: It has the same operation as (4), except that it uses mbhA The W3110 genome fragment was obtained, and the primers were as follows: -US、 mbhA -UA、 -DS、 mbhA -DA、 -s、 bcd bcd mbhA mbhA yjgX tdcB E.coli yjgX yjgX yjgX yjgX tdcB tdcB yjgX yjgX yciQ yeaS E.coli yciQ yciQ yciQ yciQ yeaS yeaS -a、pGRB- yciQ -s and pGRB- yciQ -a. Competent cells were ABA-13, and strain ABA-14 was obtained.

[0061] (15) Replace the starter (P) ilvI ::P BBa_j23114 Weakening ilvI The gene disrupted the in situ promoter and integrated a weakened promoter P. BBa_j23114 :by E.coli Using the W3110 genome as a template, PCR amplification was performed using primers UP-ilvI-S and UP-ilvI-js114-A, DW-ilvI-A and DW-ilvI-js114-S to obtain the upper homologous arm UP-ilvI-js114 and the lower homologous arm DW-ilvI-js114. Using the recovered upper and lower homologous arms as templates, the target fragment ilvI-js114 required for integration was obtained by overlap PCR using primers UP-ilvI-S and DW-ilvI-A. Subsequently, the DNA fragment obtained by annealing primers pGRB-ilvI-s and pGRB-ilvI-a was ligated to plasmid pGRB to construct the pGRB-ilvI plasmid. Finally, the purified ilvI-js114 integrated fragment and plasmid pGRB-ilvI were simultaneously transformed into competent ABA-14 cells by electroporation. Positive transformants were then screened using primers ilvI-js114-JD-S and DW-ilvI-A. The competent cells were ABA-14, and strain ABA-15 was finally obtained.

[0062] Example 2

[0063] This embodiment aims to illustrate the application of strain ABA-15 obtained in Example 1 in shake-flask fermentation. The specific steps are as follows:

[0064] ① Strain activation: Genetically engineered bacteria ABA-15 were streaked onto activation slant, cultured at 37℃ for 12 h, and passaged once. The general LB solid medium was used as the slant medium.

[0065] ② Seed Culture: Using an inoculation loop, scrape a loopful of seeds from the slant and inoculate it into a 500 mL Erlenmeyer flask containing 30 mL of seed culture medium. Seal the flask with nine layers of gauze and incubate at 36℃ and 200 r / min for 10 h. The seed culture medium used was: glucose 25 g / L, yeast extract 5 g / L, citric acid 1 g / L, MgSO4·7H2O 0.6 g / L, KH2PO4·H2O 3 g / L, peptone 3 g / L, V B Mixture 0.5 mg / L, V H 1 mg / L, glutamic acid 2 g / L, methionine 0.2 g / L, lysine 0.5 g / L, the remainder being water.

[0066] ③ Fermentation Culture: Inoculate the seed culture prepared after activation of the inoculum at a rate of 10%-15% into an Erlenmeyer flask containing fermentation medium. Seal the flask with nine layers of gauze, maintain the temperature at 37±0.2℃, and culture with shaking at 220 r / min. During fermentation, maintain the pH at 7.0-7.2 by adding 25% ammonia water; add 60% glucose solution to supplement the carbon source required by the cells. The fermentation cycle is 30 h. The fermentation medium used is: citric acid 2 g / L, MgSO4·7H2O 1.6 g / L, FeSO4·7H2O 10 mg / L, MnSO4·7H2O 10 mg / L, KH2PO4 4 g / L, V B Mixture 0.5 mg / L, V H 0.5 mg / L, yeast powder 5 g / L, glutamic acid 2 g / L, lysine 3 g / L, methionine 2 g / L, corn steep liquor 20 ml / L, the remainder being water.

[0067] The L-2-aminobutyric acid producing strain ABA-15 was verified to produce 8.1 g / L of L-2-aminobutyric acid through a 500 mL shake flask fermentation experiment.

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

Claims

1. A strain for producing L-2-aminobutyric acid, characterized in that: The strain E. coli W3110, as a chassis strain, was knocked out. tdh , rhtA and ilvE Gene, overexpressed ppc, aspC , thrA fbr BC , ilvA fbr , tdcB , yeaS and pntAB Genes, using the artificial promoter P BBa_j23114 Replacement of acetylhydroxy acid synthase ilvI The natural promoter of a gene, to weaken ilvI Gene expression, heterologous expression originates from Bacillus subtilis B. subtilis 168 bcd and gdh Genes, and heterologous expression originate from Corynebacterium glutamicum K051 cysK Genes; the stated gdh The nucleotide sequence is shown in SEQ ID NO.3 of the sequence listing; cysK The nucleotide sequence is shown in SEQ ID NO.5 of the sequence listing; bcd The nucleotide sequence is shown in SEQ ID NO.7 of the sequence listing; PPC The nucleotide sequence is shown in SEQ ID NO.8 of the sequence listing; aspC The nucleotide sequence is shown in SEQ ID NO.9 of the sequence listing; thrA fbr BC The nucleotide sequence is shown in SEQ ID NO.10 of the sequence listing; ilvA fbr The nucleotide sequence is shown in SEQ ID NO.11 of the sequence listing; tdcB The nucleotide sequence is shown in SEQ ID NO.12 of the sequence listing; yeaS The nucleotide sequence is shown in SEQ ID NO.13 of the sequence listing; pntAB The nucleotide sequence is shown in SEQ ID NO.14 of the sequence listing; ilvI The nucleotide sequence is shown in SEQ ID NO.15 of the sequence listing, and the promoter P... BBa_j23114 The nucleotide sequence is shown in the sequence listing SEQ ID NO.

16.

2. The L-2-aminobutyric acid producing strain according to claim 1, characterized in that: by E. coli W3110, as a chassis strain, had the gene knocked out from the strain's genome. tdh , rhtA and ilvE Gene; using artificial promoter P BBa_j23114 Replacement of acetylhydroxy acid synthase ilvI The natural promoter of the gene; enhanced using the trc promoter. PPC Gene transcription and integration into the genome. yjiV Pseudogene loci; use TRC promoter enhancement aspC Gene transcription and integration into the genome. ilvG Pseudogene loci; The threonine operon was enhanced using the TRC promoter. thrA fbr BC Gene transcription and integration into the genome. ycgH Pseudogene locus and double copy to yeeP Pseudogene loci; Enhanced using the TRC promoter ilvA fbr Gene transcription and integration into the genome. yeeL Pseudogene locus and double copy to ylbE Pseudogene loci; Enhanced using the TRC promoter tdcB Gene transcription and integration into the genome. yjgX Pseudogene loci; Enhanced using the TRC promoter yeaS Gene transcription and integration into the genome. yciQ Pseudogene loci; Enhanced using the TRC promoter pntAB Gene transcription and integration into the genome. yghE Pseudogene loci; The TRC promoter was used to enhance the source B. subtilis 168 gdh Gene transcription and integration into the genome. rhtA Pseudogene loci; The TRC promoter was used to enhance the source B. subtilis 168 bcd Gene transcription, followed by three copies of integration into the genome. ygaY , ilvE and mbhA Pseudogene sites; enhanced from TRC promoters. Corynebacterium glutamicum K051 cysK Gene transcription and integration into the genome. tdh Pseudogene loci.

3. The L-2-aminobutyric acid producing strain according to claim 1 or 2, characterized in that: The E. coli W3110 is E. coli W3110 ATCC 27325.

4. The L-2-aminobutyric acid producing strain according to claim 1 or 2, characterized in that: The rhtA The nucleotide sequence is shown in SEQ ID NO.2 of the sequence listing; tdh The nucleotide sequence is shown in SEQ ID NO.4 of the sequence listing; ilvE The nucleotide sequence is shown in the sequence listing SEQ ID NO.

6.

5. The L-2-aminobutyric acid producing strain according to claim 2, characterized in that: The nucleotide sequence of the trc promoter is shown in the sequence listing SEQ ID NO.

1.

6. A method for constructing the L-2-aminobutyric acid producing strain according to any one of claims 1-5, characterized in that: In the originating strain E. coli The targeted modification based on W3110 involves the following three modules: (1) Increase the production of the precursor threonine: enhance the phosphoenolpyruvate carboxylase gene PPC Transcription level; increase aspartate transaminase gene aspC Transcription level; increase threonine operon thrA fbr BC Transcriptional levels; knockout of L-threonine efflux transporter genes rhtA Knockout of threonine catabolism gene tdh Increase the supply of precursors for the L-2-aminobutyric acid (GABA) production pathway; (2) Enhance reducing power: Enhance the pyridine nucleotide transhydrogenase gene pntAB The transcriptional level increased the supply of NADPH, introducing substances derived from [the source]. B. subtilis 168 glucose 1-dehydrogenase gene gdh This increased NADPH supply and knocked out branched-chain amino acid aminotransferase genes. ilvE This reduces NADPH consumption; (3) Increase L-2-aminobutyric acid production: enhance threonine dehydrogenase gene ilvA fbr and threonine dehydrogenase gene tdcB The transcriptional level, increased from B. subtilis 168 leucine dehydrogenase bcd Gene transcription level; improved from Corynebacterium glutamicum K051 cysteine ​​synthase gene cysK Transcriptional level; using artificial promoter P BBa_j23114 Replacement of acetylhydroxy acid synthase ilvI The natural promoter of the gene; gene that enhances L-2-aminobutyric acid efflux transport. yeaS The transcriptional level.

7. The use of the L-2-aminobutyric acid producing strain according to any one of claims 1-5 in the fermentation production of L-2-aminobutyric acid.

8. The application according to claim 7, characterized in that: The specific steps are as follows: ① Strain activation: The L-2-aminobutyric acid producing strain was streaked onto an activation slant and passaged once; ② Seed culture: Use an inoculation loop to scrape a loop of slanted seeds and inoculate them into an Erlenmeyer flask containing seed culture medium. Seal the flask with gauze and incubate at 36℃ and 200r / min for 10h. ③ Fermentation culture: Inoculate the seed liquid prepared after activation of the strain at an inoculation rate of 10%-15% into an Erlenmeyer flask containing fermentation culture medium, seal the flask with gauze, maintain the temperature at 37±0.2℃, and shake at 220 r / min. During the fermentation process, ammonia water is added to maintain the pH at 7.0-7.

2. Add glucose solution to supplement the carbon source required by the bacteria.

9. The application according to claim 8, characterized in that: The slant culture medium used in step ① is general-purpose LB solid medium; the seed culture medium used in step ② is: glucose 25 g / L, yeast extract 5 g / L, citric acid 1 g / L, MgSO4·7H2O 0.6 g / L, KH2PO4·H2O 3 g / L, peptone 3 g / L, V B Mixture 0.5 mg / L, V H 1 mg / L, glutamic acid 2 g / L, methionine 0.2 g / L, lysine 0.5 g / L, the remainder being water; the fermentation medium used in step ③ is: citric acid 2 g / L, MgSO4·7H2O 1.6 g / L, FeSO4·7H2O 10 mg / L, MnSO4·7H2O 10 mg / L, KH2PO4 4 g / L, V B Mixture 0.5 mg / L, V H 0.5 mg / L, yeast powder 5 g / L, glutamic acid 2 g / L, lysine 3 g / L, methionine 2 g / L, corn steep liquor 20 ml / L, the remainder being water.

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