L-lysine producing strain as well as construction method and application thereof

Abstract

The invention provides an L-lysine production strain and a construction method and application thereof.E.coliW3110 serves as an original strain, pflB, adhE, ldhA, ackA, frdBC, poxB, lacI, cadA and ldcC genes are knocked out, pntAB, ppc, aspC, lysCV339A, asd, dapAH56K, dapB, lysA and lysE genes are up-regulated, ddh genes derived from Corynebacterium glutamicum K051 are heterologously expressed, part of by-products and lysine decomposition pathway enzymes are knocked out of the strain, the diaminopimelic acid pathway of the strain and supply of NADPH are enhanced, and the L-lysine production strain has the advantages that the L L-lysine can be efficiently synthesized by taking glucose as a substrate, and the method has a relatively high production level.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering and fermentation engineering, in particular to an L-lysine production strain and a construction method and application thereof. Background Art

[0002] L-lysine is an α-basic amino acid, one of the essential amino acids for the human body. It has multiple physiological functions and is widely used in animal feed, medicine and food industries. L-lysine has become the world's second largest amino acid, with an annual global output of more than 5 million tons, and is still growing.

[0003] The production of L-lysine by microbial fermentation has broad prospects due to its mild reaction conditions, good specificity, high yield, and environmental friendliness. At present, the main strains for L-lysine fermentation production are Escherichia coli and Corynebacterium glutamicum, among which Corynebacterium glutamicum has certain advantages, but has problems such as low growth rate, high nutritional requirements, and high downstream recovery costs. In contrast, Escherichia coli has a short production cycle and low culture cost, and is more advantageous in rapid and efficient production.

[0004] At present, lysine fermentation still has many problems to be solved, including: the fermentation liquid is complex, containing impurities such as lactic acid, acetic acid, and amino acid residues, which requires high technical requirements for separating and extracting high-purity L-lysine; the synthesis process requires a large amount of NADPH, and blindly improving the HMP pathway will lead to imbalanced cell metabolism and reduced conversion rate; the pathway is long, and the conversion rate of L-lysine produced by microbial fermentation is often low, and by-products are easily accumulated, etc. Therefore, it is urgent to optimize and improve the production strain. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an L-lysine producing strain.

[0006] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned L-lysine producing strain.

[0007] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned L-lysine production strain.

[0008] In order to solve the above technical problems, the technical solution of the present invention is: An L-lysine production strain, strain WLY-32, is a starting strain E.coli Knockout pflB, adhE, ldhA, ackA, frdBC, poxB, lacI, cadA, ldcC Gene, upregulated pntAB, ppc, aspC, lysC V339A , asd, dapA H56K , dapB, lysA, lysEGene, heterologous expression from Corynebacterium glutamicum K051 ddh Gene.

[0009] Preferably, the above-mentioned L-lysine production strain uses CRIPSR / Cas9 gene editing technology as a metabolic engineering method to E.coli W3110 was used as a chassis strain for directed transformation. E.coli W3110 is E.coli W3110 ATCC27325.

[0010] Preferably, in the above-mentioned L-lysine-producing strain, the nucleotide sequence of the pflB gene is shown in the sequence listing SEQ ID NO.1, the nucleotide sequence of the adhE gene is shown in the sequence listing SEQ ID NO.2, the nucleotide sequence of the ldhA gene is shown in the sequence listing SEQ ID NO.3, the nucleotide sequence of the ackA gene is shown in the sequence listing SEQ ID NO.4, the nucleotide sequence of the frdBC gene is shown in the sequence listing SEQ ID NO.5, the nucleotide sequence of the poxB gene is shown in the sequence listing SEQ ID NO.6, the nucleotide sequence of the lacI gene is shown in the sequence listing SEQ ID NO.7, the nucleotide sequence of the cadA gene is shown in the sequence listing SEQ ID NO.8, and the nucleotide sequence of the ldcC gene is shown in the sequence listing SEQ ID NO.9.

[0011] Preferably, the above-mentioned L-lysine producing strain, pntAB The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.10, ppc The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.11, aspC The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.12. lysC V339A The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.13, asd The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.14, dapA H56K The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.15, dapB The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.16, lysA The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.17. lysE The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.18, ddh The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.19.

[0012] The method for constructing the above L-lysine production strain comprises the following steps: E.coli Directional transformation is carried out based on W3110. The specific steps are as follows: (1) Transformation of chassis bacteria: knocking out some byproduct genes: pflB, adhE, ldhA, ackA, frdBC, poxB genes; knocking out transcriptional repressors lacI Gene; (2) Increase the reducing power of NADPH content: Upregulate pntAB Genes, improve pntAB Gene transcription level, increasing the supply of NADPH; (3) Strengthening the diaminopimelic acid pathway: Upregulation aspC , lysC V339A , asd , dapA H56K , dapB , lysA , lysE Genes, among which lysC and dapA genes were subjected to point mutations to release the feedback inhibition of lysine; (4) Knockout of lysine decomposition pathway genes: cadA , ldcC Gene; (5) Shortening the lysine metabolic pathway: Upregulation ppc Genes and their origin Corynebacterium glutamicum The ddh gene of K051.

[0013] Preferably, the method for constructing the above-mentioned L-lysine production strain comprises the following specific steps: (1) Knockout pflB, adhE, ldhA, ackA, frdBC, poxB, lacI, cadA, ldcC Gene; (2) Using the Trc promoter to express the pntAB gene at the yjiK pseudogene locus; (3) Using the Trc promoter to express two copies of the ppc gene at the ycgH and mbhA pseudogene loci; (4) Using the Trc promoter, multiple copies of the aspC gene were expressed at the yghX, ilvG, ydeU, and yjiV pseudogene sites; (5) Using the Trc promoter, the yciQ, ygaY, and fhiA pseudogene loci were used to express lysC V339A The gene is expressed in multiple copies; (6) Using the Trc promoter, multiple copies of the asd gene were expressed at the yjgX, gapC, and yeeP pseudogene loci; (7) Using the Trc promoter, dapA was expressed at the yjiP, yncK, and ybfL pseudogene loci. H56K The gene is expressed in multiple copies, opening up the metabolic pathway; (8) Using the Trc promoter, the dapB gene was expressed in two copies at the nmpC and ycdN pseudogene loci respectively; (9) Using the Trc promoter, two copies of the lysA gene were expressed at the ylbE and ychg pseudogene loci, respectively; (10) Using the Trc promoter to express the lysE gene at the yghE pseudogene locus; (11) The Trc promoter was used to express two copies of the ddh gene from Corynebacterium glutamicum K051 at the rph and yhjQ pseudogene loci, respectively.

[0014] Preferably, in the method for constructing the above-mentioned L-lysine production strain, the lysC V339A The gene was subjected to site-directed mutation, that is, the base at position 1016 was changed from T to C, resulting in the amino acid residue at position 339 being changed from valine (V) to alanine (A); the above dapA H56K The gene underwent site-directed mutation, where the 166th base was changed from C to A, and the 168th base was changed from T to A, resulting in the 56th amino acid residue being changed from histidine (H) to lysine (K).

[0015] Preferably, in the above-mentioned method for constructing the L-lysine producing strain, the nucleotide sequence of the Trc promoter is shown in the sequence listing SEQ ID NO.20.

[0016] Preferably, the method for constructing the above-mentioned L-lysine production strain comprises adjusting aspC, lysC V339A The copy numbers of the asd genes are 4, 3, and 3, respectively, which enables efficient expression of key enzymes in the metabolic pathway.

[0017] Application of the L-lysine producing strain in the fermentation production of lysine.

[0018] Preferably, the L-lysine producing strain is used to synthesize L-lysine by fermentation culture in a culture medium using glucose as a substrate.

[0019] Preferably, in the application of the above-mentioned L-lysine production strain, the culture medium includes but is not limited to carbon sources, nitrogen sources, inorganic salts, vitamins, etc.; the fermentation conditions include fermentation temperature, fermentation pH, fermentation dissolved oxygen conditions, fermentation pressure, fermentation time, etc.

[0020] Preferably, the application of the above-mentioned L-lysine production strain comprises the following specific steps: ① Slant culture: inoculate the L-lysine producing strain on the slant culture medium and culture at 36-37℃ for 14-18h; ② Shake flask seed culture: Take the slant strain and inoculate it into the seed culture medium in the shake flask for fermentation. The culture temperature is 36-37℃, the culture time is 12-20h, the shaker speed is 200-240r / min, and the pH is 7.2-7.4; ③ Shake flask fermentation culture: the fermentation inoculation amount is 15-20%, the culture temperature is 36-37℃, the pH is 7.2-7.4, the culture time is 24-36h, and the shaking speed is 200-240r / min.

[0021] Preferably, in the application of the above-mentioned L-lysine producing strain, the slant culture medium is a universal LB solid culture medium.

[0022] Preferably, in the application of the above-mentioned L-lysine production strain, the seed culture medium used in step ② is: glucose 20-30 g / L, yeast powder 3-5 g / L, peptone 3-5 g / L, MgSO4.7H2O 0.5-1 g / L, KH2PO4 2-3 g / L, ammonium sulfate 4-5 g / L, biotin 1-2 mg / L, and the rest is water.

[0023] Preferably, in the application of the above-mentioned L-lysine producing strain, the fermentation medium used in step ③ is: glucose 20-30 g / L, MgSO4.7H2O 1.5-2 g / L, yeast powder 4-5 g / L, peptone 2-3 g / L, ammonium sulfate 10-20 g / L, K2HPO4.3H2O 3-6 g / L, FeSO4.7H2O 20-30 mg / L, VB1, VB3, VB5 each 2-3 mg / L, and the rest is water.

[0024] The above culture media can be prepared using standard methods.

[0025] Beneficial effects: The above-mentioned L-lysine production strain has knocked out some by-products and lysine decomposition pathway enzymes, strengthened the strain's diaminopimelic acid pathway and NADPH supply, has good L-lysine synthesis ability, can efficiently synthesize L-lysine using glucose as a substrate, and has stable performance and high acid production efficiency, thus achieving efficient production of L-lysine and having excellent industrial application prospects. Specifically: (1) The pflB, adhE, ldhA, ackA, frdBC, and poxB genes were knocked out, effectively reducing impurities such as lactic acid, acetic acid, and amino acid residues during the fermentation process, thereby reducing downstream recycling costs.

[0026] (2) By increasing the transcription level of the membrane boundary inducing enzyme pntAB gene, the supply of NADPH is increased, providing a large amount of reducing power for L-lysine synthesis.

[0027] (3) Increased aspC, lysC V339A , asd, dapA H56K , dapB, lysA, lysE Gene, strengthen the strain diaminopimelate pathway, including lysC 、 Point mutations in the dapA gene relieve the feedback inhibition of lysine and adjust the aspC, lysC V339A The copy number of the asd gene is 4:3:3, which makes the key enzymes in the metabolic pathways efficiently expressed, the metabolic pathways are smooth, and at the same time strengthens ppc gene and Heterologously expressed Corynebacterium glutamicum The ddh gene of K051 catalyzes the conversion of phosphoenolpyruvate to oxaloacetate and 2,3,4,5-tetrahydrodipicolinate to meso-2,6-diaminopimelate, shortening the L-lysine metabolic pathway to increase the conversion rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Diagram of the genetic modification process for the de novo synthesis pathway of L-lysine production strains. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0030] The percentage sign "%" involved in the embodiments, unless otherwise specified, refers to mass percentage. The percentage of a solution refers to the number of grams of a solute contained in 100 mL. The percentage between liquids refers to the volume ratio of the solution at 25°C.

[0031] The starting strain used in the embodiment is a wild type E.coli W3110 ATCC 27325, the corresponding promoter and gene are shown in the sequence list. The primers used in the construction of the strains involved are shown in Table 1.

[0032] Table 1 Primers involved in strain construction Primer Name Sequence Number Primer Sequence (5'-3') pflB-US SEQ ID NO.21 GTGGTTTGTTGGTTGGGTTGACATA pflB-UA SEQ ID NO.22 GCGTTCGGAACGATAGAGAAGGGTTGTACGCTTTGCAGGAACCTT pflB-DS SEQ ID NO.23 AAGGTTCCTGCAAAGCGTACAACCCTTCTCTATCGTTCCGAACGC pflB-DA SEQ ID NO.24 TGAATGCGACCAATAACTGACATTG pGRB-pflB-s SEQ ID NO.25 AGTCCTAGGTATAATACTAGTAACGGTGCTGCAATGTCCTTGTTTTAGAGCTAGAA pGRB-pflB-a SEQ ID NO.26 TTCTAGCTCTAAAACAAGGACATTGCAGCACCGTTACTAGTATTATACCTAGGACT adhE-US SEQ ID NO.27 AGTAATCTTGCTTACGCCACCTG adhE-UA SEQ ID NO.28 GAAGCAGACTTCCTGGCGAACTCTTCGACGATACCCATGCCG adhE-DS SEQ ID NO.29 CGGCATGGGTATCGTCGAAGAGAAGCAGACTTCCTGGCGAAC adhE-DA SEQ ID NO.30 GGAAGGTGTTCTGCAAATAGTTGTG pGRB-adhE-s SEQ ID NO.31 AGTCCTAGGTATAATACTGGAAACTCACTTCGAAGAGCGTTTTAGAGCTAGAA in pGRB-adhE SEQ ID NO.32 TTCTAGCTCTAAAACGCTCTTCGAAGTGAGTTTCCACTAGTATTATACCTAGGACT ldhA-US SEQ ID NO.33 AGCAGCGTCAACGGCAC ldhA-UA SEQ ID NO.34 TCCTGGATCACGTCGTTGGATTTATGCTGCCGTCATCGTTTACGAAA ldhA-DS SEQ ID NO.35 TTTCGTAAACGATGACGGCAGCATAAATCCAACGACGTGATCCAGGA ldhA-DA SEQ ID NO.36 GCTGTTCTGGCGTAACAGCAA pGRB-ldhA-s SEQ ID NO.37 AGTCCTAGGTATAACTAGTCCGTGATGCTAACTTCTCTCGTTTTAGAGCTAGAA in pGRB-ldhA SEQ ID NO.38 TTCTGCTCTAAAACGAGAGAAGTTAGCATCACGGACTAGTATTATACCTAGGACT ackA-US SEQ ID NO.39 CATTATGCCATTGGCTGAAAATTACG ackA-UA SEQ ID NO.40 CATTTTCACCGATACCACCAGTGAAACAGTTCTGGTTTTTGTGCCAGAA ackA-DS SEQ ID NO.41 TTCTGGCACAAAAACCAGAACTGTTTCACTGGTGGTATCGGTGAAAATG ackA-DA SEQ ID NO.42 AAACGCTCAGACGAACGCC pGRB-ackA-s SEQ ID NO.43 AGTCCTAGGTATATACTAGTCACAACCCGGCTCACCTGATGTTTTAGAGCTAGAA in pGRB-ackA SEQ ID NO.44 TTCTAGCTCTAAAACATCAGGTGAGCCGGGTTGTGACTAGTATTATACCTAGGACT frdBC-US SEQ ID NO.45 CGTCAACTTCCTCAAACACACCC frdBC-UA SEQ ID NO.46 GTTTTGGTGTGCAGCAGAGCTGGACCAGCGGTAGCTCAGGT <h2 style=";text-align:left;direction:ltr">frdBC-DS <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.47 <h2 style=";text-align:left;direction:ltr"> ACCTGAGCTACCGCTGGTCCAGCTCTGCTGCACACCCAAAAC <h2 style=";text-align:left;direction:ltr"> frdBC-DA <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.48 <h2 style=";text-align:left;direction:ltr"> AGAACGCGCTCGTAGCTC <h2 style=";text-align:left;direction:ltr"> pGRB-frdBC-s <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.49 <h2 style=";text-align:left;direction:ltr"> AGTCCTAGGTATAATACTAGTTGATTACACCGACGGTATGAGTTTTAGAGCTAGAA <h2 style=";text-align:left;direction:ltr"> pGRB-frdBC-a <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.50 <h2 style=";text-align:left;direction:ltr"> TTCTAGCTCTAAAACTCATACCGTCGGTGTAATCAACTAGTATTATACCTAGGACT <h2 style=";text-align:left;direction:ltr"> poxB-US <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.51 <h2 style=";text-align:left;direction:ltr"> TCATCGGGCTATTTAACCGTTAGTG <h2 style=";text-align:left;direction:ltr"> poxB-UA <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.52 <h2 style=";text-align:left;direction:ltr"> GAGAAGCGCGTTGCAGCAGACCGCCAGTTCTCCG <h2 style=";text-align:left;direction:ltr"> poxB-DS <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.53 <h2 style=";text-align:left;direction:ltr"> CGGAGAACTGGCGGTCTGCTGCAACGCGCCTTCTC <h2 style=";text-align:left;direction:ltr"> poxB-DA <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.54 <h2 style=";text-align:left;direction:ltr"> GCGGCTTGGTCGGGTAAC <h2 style=";text-align:left;direction:ltr"> pGRB-poxB-s <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.55 <h2 style=";text-align:left;direction:ltr"> AGTCCTAGGTATAATACTAGTCAGAAGGGGCAACCATGCACGTTTTAGAGCTAGAA <h2 style=";text-align:left;direction:ltr"> pGRB-poxB-a <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.56 <h2 style=";text-align:left;direction:ltr"> TTCTAGCTCTAAAACGTGCATGGTTGCCCCTTCTGACTAGTATTATACCTAGGACT <h2 style=";text-align:left;direction:ltr"> lacI-US <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.57 <h2 style=";text-align:left;direction:ltr"> GAAGGGGTTGAATCGCAGGC <h2 style=";text-align:left;direction:ltr"> lacI-UA <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.58 <h2 style=";text-align:left;direction:ltr"> GTTGCAGCAAGCGGTCCACGCCAGACTGGAGGTGGC <h2 style=";text-align:left;direction:ltr"> lacI-DS <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.59 <h2 style=";text-align:left;direction:ltr"> GCCACCTCCAGTCTGGCGTGGACCGCTTGCTGCAAC <h2 style=";text-align:left;direction:ltr"> lacI-DA <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.60 <h2 style=";text-align:left;direction:ltr"> AATCCGTAATCATGGTCATAGCTGTT <h2 style=";text-align:left;direction:ltr"> pGRB-lacI-s <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.61 <h2 style=";text-align:left;direction:ltr"> AGTCCTAGGTATAATACTAGTCCATGAAGACGGTACGCGACGTTTTAGAGCTAGAA <h2 style=";text-align:left;direction:ltr"> pGRB-lacI-a <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.62 <h2 style=";text-align:left;direction:ltr"> TTCTAGCTCTAAAACGTCGCGTACCGTCTTCATGGACTAGTATTATACCTAGGACT <h2 style=";text-align:left;direction:ltr"> yjiK-US <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.63 <h2 style=";text-align:left;direction:ltr"> GAATCTCACCTGCGAATGCC <h2 style=";text-align:left;direction:ltr">yjiK-UA <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.64 <h2 style=";text-align:left;direction:ltr"> TGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTTAATTGTCAAACACCATCCTGCTGGCAATAAA <h2 style=";text-align:left;direction:ltr"> yjiK-DS <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.65 <h2 style=";text-align:left;direction:ltr"> AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTTGCGATGGATGCCTCTGGT <h2 style=";text-align:left;direction:ltr"> yjiK-DA <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.66 <h2 style=";text-align:left;direction:ltr"> CCTAAGGTCAGAACCAGCATC <h2 style=";text-align:left;direction:ltr"> PntAB-S <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.67 <h2 style=";text-align:left;direction:ltr"> CCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCGAATTGGCATACCAAGAG <h2 style=";text-align:left;direction:ltr"> PntAB-A <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.68 <h2 style=";text-align:left;direction:ltr"> CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACAGAGCTTTCAGGATTGCATCCAC <h2 style=";text-align:left;direction:ltr"> pGRB-yjiK-s <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.69 <h2 style=";text-align:left;direction:ltr"> AGTCCTAGGTATAATACTAGTCAAGACTCCCCAACCAATTGGTTTTAGAGCTAGAA <h2 style=";text-align:left;direction:ltr"> pGRB-yjik-a <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.70 <h2 style=";text-align:left;direction:ltr"> TTTCTAGCTCTAAAACCAATTGGTTGGGGAGTCTTGACTAGTATTATACCTAGGACT <h2 style=";text-align:left;direction:ltr"> ycgH-US <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.71 <h2 style=";text-align:left;direction:ltr"> TAAACTCGTCAGCGGCACAAC <h2 style=";text-align:left;direction:ltr"> ycgH-UA <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.72 <h2 style=";text-align:left;direction:ltr"> AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGTAGGCGTTTCTGTTGATTCTG <h2 style=";text-align:left;direction:ltr"> ycgH-DS <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.73 <h2 style=";text-align:left;direction:ltr"> AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGCGTGTCGGATTATCGTTCG <h2 style=";text-align:left;direction:ltr"> ycgH-DA <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.74 <h2 style=";text-align:left;direction:ltr"> GATTCAGGTTGCCATTTACGC <h2 style=";text-align:left;direction:ltr"> ppc-S <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.75 <h2 style=";text-align:left;direction:ltr"> CGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAACGAACAATATTCCGCATTGC <h2 style=";text-align:left;direction:ltr"> ppc-A <h2 style=";text-align:left;direction:ltr"> SEQ ID NO.76 TCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAGCCGGTATTACGCATACCTGC pGRB-ycgH-s SEQ ID NO.77 AGTCCTAGGTATAATACTAGTTATGCGTCTGAACGACCGTGGTTTTAGAGCTAGAA pGRB-ycgH-a SEQ ID NO.78 TTCTAGCTCTAAAACCACGGTCGTTCAGACGCATAACTAGTATTATACCTAGGACT mbhA-US SEQ ID NO.79 GCCAGCACGAACATAATCCC mbhA-UA SEQ ID NO.80 GGTCTGTTTCCTGCTAGCACTATACCTAGGACTGAGCTAGCCGTAAACACGGTGGCAGGTTTTGG mbhA-DS SEQ ID NO.81 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGACCAAAAGTGCGTCCGATAC mbhA-DA SEQ ID NO.82 CGGCGTAATCACAAACTGGC pGRB-mbhA-s SEQ ID NO.83 TGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACACGGTGGCAGGTTTTGG pGRB-mbhA-a SEQ ID NO.84 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGACCAAAAGTGCGTCCGATAC yghX-US SEQ ID NO.85 TCAAACGCTTTACGCAGGAT yghX-UA SEQ ID NO.86 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGCTCATCTTTGCGGGCTT yghX-DS SEQ ID NO.87 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATATCCGCAAGCGACAGGC yghX-DA SEQ ID NO.88 CGTTGATTCGGGTGTCCAG aspC-S SEQ ID NO.89 GGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGTTTGAGAACATTACCGCCGC aspC-A SEQ ID NO.90 GACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACAGCACTGCCACAATCGC pGRB-yghX-s SEQ ID NO.91 AGTCCTAGGTATAATACTAGTTGTGGCACGGCGAGTGGCGAGTTTTAGAGCTAGAA pGRB-yghX-a SEQ ID NO.92 TTCTAGCTCTAAAACTCAGGTGCCAGGGCGATATAACTAGTATTATACCTAGGACT ilvG-US SEQ ID NO.93 ACCGAGGAGCAGACAATGAATAA ilvG-UA SEQ ID NO.94 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGTGATGGCAACAACAGGGA ilvG-DS SEQ ID NO.95 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCTATCTACGCGCCGTTGTTGTT ilvG-DA SEQ ID NO.96 GCGCTGGCTAACATGAGGAA pGRB-ilvG-s SEQ ID NO.97 AGTCCTAGGTATAATACTAGTTATCGGCACTGACGCATTTCGTTTTAGAGCTAGAA pGRB-ilvG-a SEQ ID NO.98 AGTCCTAGGTATAATACTAGTGGAAGAGTTGCCGCGCATCAGTTTTAGAGCTAGAA ydeU-US SEQ ID NO.99 CAACGATTCCGCGGCGTAT ydeU-UA SEQ ID NO.100 GTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAACCGTTGACCGTTTGTGCA ydeU-DS SEQ ID NO.101 GACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAAATACTCAGGGCATGCTGGGA ydeU-DA SEQ ID NO.102 GCGTCGCATAGCGTCATTGA pGRB-ydeU-s SEQ ID NO.103 AGTCCTAGGTATACTAGTTCAGTTTATCGCCCGGAGGCGTTTTAGAGCTAGAA pGRB-ydeU-a SEQ ID NO.104 TTCTAGCTCTAAAACGCCTCCGGGCGATAAACTGAACTAGTATTATACCTAGGACT yjiV-US SEQ ID NO.105 TGTGACTGTGGAAGCCCTGT yjiV-UA SEQ ID NO.106 GTATCCGCTCAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAATTCGGGCTGTCCCTTGTC yjiV-DS SEQ ID NO.107 GGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGTGGCACCTGAATGACGAAC yjiV-DA SEQ ID NO.108 TGGCGACATTCCCTTCCTTG pGRB-yjiV SEQ ID NO.109 AGTCCTAGGTATAATACTAGTGGCTGGATCGATTTCGTTCCGTTTTAGAGCTAGAA pGRB-yjiV-a SEQ ID NO.110 TTCTAGCTCTAAAACGGAACGAAATCGATCCAGCCACTAGTATTATACCTAGGACT yciQ-US SEQ ID NO.111 TTACTTGAAGCATTGGGCGAAC yciQ-UA SEQ ID NO.112 AATGTTATCCGCTCAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCAGTCAAGATGCCAGGGTTC yciQ-DS SEQ ID NO.113 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGTCTGACAAGAACCAGCAAATCCT yciQ-DA SEQ ID NO.114 ATAGCTTCACCGTGGGCATAAC lysC-S SEQ ID NO.115 CTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGTCTGAAATTGTTGTCTCCAAATTTGG lysC-tb-A SEQ ID NO.116 CTGACGTGGTGATTAAGTCTGCCGAAATATTATGCCGCGC lysC-tb-S SEQ ID NO.117 GCGCGGCATAATATTTCGGCAGACTTAATCACCACGTCAG lysC-A SEQ ID NO.118 CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACTCAAACAAATTACTATGCAGTTTTTGCAC pGRB-yciQ-s SEQ ID NO.119 AGTCCTAGGTATACTAGTAAACAACGTTTCTTGCCTCAGTTTTAGAGCTAGAA pGRB-yciQ-a SEQ ID NO.120 TTCTAGCTCTAACTGAGGCAAGAAACGTTGTTTACTAGTATTATACCTAGGACT ygaY-US SEQ ID NO.121 CCTACAAACCACATCGCACATT ygaY-UA SEQ ID NO.122 TCCACACATTATACGAGCCGGATGATTAATTGTCAAACACCGAAGCAACCCAAAAGACGGT ygaY-DS SEQ ID NO.123 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTTGCTTGCCGCTCCACC ygaY-DA SEQ ID NO.124 GGAGTAGGGCTTTCCATAGAGTGT pGRB-ygaY-s SEQ ID NO.125 AGTCCTAGGTATAATACTAGTCACTGATGGCGCTGGCATTAGTTTTAGAGCTAGAA pGRB-ygaY-a SEQ ID NO.126 TTCTAGCTCTAAAACTAATGCCAGCGCCATCAGTGACTAGTATTATACCTAGGACT fhiA-US SEQ ID NO.127 GGGCAATGGTGTTGATACTGG fhiA-UA SEQ ID NO.128 AATTGTTATCCGCTCAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAATCGCCAGAATCATCATCCC fhiA-DS SEQ ID NO.129 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAAT CAAGCAGGAGCTGACGGTGT fhiA-DA SEQ ID NO.130 TGCACCAATGCTGGATACTTACA with pGRB-fhiA SEQ ID NO.131 AGTCCTAGGTATACTAGTTGACGTGCGTAACCAGCTGCGTTTTAGAGCTAGAA pGRB-fhiA-a SEQ ID NO.132 TTCTAGCTCTAAAACGCAGCTGGTTACGCACGTCAACTAGTATTATACCTAGGACT yjgX-US SEQ ID NO.133 GGAAGTCAACGGGTTATGCG yjgX-UA SEQ ID NO.134 TTATCCGCTCAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAAAAATCACCACGAATACCAGAATC yjgX-DS SEQ ID NO.135 GGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATACAGTGTCTTCCCTGAGCCG yjgX-DA SEQ ID NO.136 GGCGAAGGATACCATCAAGC asd-S SEQ ID NO.137 GCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAAAAATGTTGTTTTTATCGGCTGG asd-A SEQ ID NO.138 CGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACGCCAGTTGACGAAGCATC pGRB-yjgX-s SEQ ID NO.139 AGTCCTAGGTATAATACTAGTTCGCGACCACCGTAACTGGCGTTTTAGAGCTAGAA pGRB-yjgX-a SEQ ID NO.140 TTCTAGCTCTAAAACGCCAGTTACGGTGGTCGCGAACTAGTATTATACCTAGGACT gapC-US SEQ ID NO.141 TGGGAAGAAACCACGAAACTC gapC-UA SEQ ID NO.142 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATGTTTCAGCAGGTAGGCGAGA gapC-DS SEQ ID NO.143 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATAAAACGGTCGCCTGGTACG gapC-DA SEQ ID NO.144 TTATCCGCCGACATTGCTG pGRB-gapC-s SEQ ID NO.145 AGTCCTAGGTATAATACTAGTAAGTATGCCACCAATCTGGGTTAGTTTTAGAGCTAGAA pGRB-gapC-a SEQ ID NO.146 TTCTAGCTCTAAAACTAACCCAGATTGGTGGCATACTTACTAGTATTATACCTAGGACT yeeP-US SEQ ID NO.147 GGTCAGGAGGTAACTTATCAGCG yeeP-UA SEQ ID NO.148 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAATGGCAGGGCTCCGTTTTG yeeP-DS SEQ ID NO.149 CCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGAACTGGATTTTCTTCTGAACCTGTCG yeeP-DA SEQ ID NO.150 ACGATGTCAGCAGCCAGC pGRB-yeeP-s SEQ ID NO.151 AGTCCTAGGTATAATACTAGTTGAACAGTTTACCGGTGCGGGTTTTAGAGCTAGAA pGRB-yeeP-a SEQ ID NO.152 TTCTAGCTCTAAAACCCGCACCGGTAAACTGTTCAACTAGTATTATACCTAGGACT yjiP-US SEQ ID NO.153 GCCATACCGCCAGCAAGAT yjiP-UA SEQ ID NO.154 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGCAGATATTCCCCTTTCCACC yjiP-DS SEQ ID NO.155 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGACGGATGACAAACGCAAAGC yjiP-DA SEQ ID NO.156 AAAGGCGGATTTTTACTGTGGA dapA-S SEQ ID NO.157 TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGTTCACGGGAAGTATTGTCGC dapA-tb-A SEQ ID NO.158 CATCACCACATCAGCTTTTTCGTCATGATTTAAGGTAGCG dapA-tb-S SEQ ID NO.159 CGCTACCTTAAATCATGACGAAAAAGCTGATGTGGTGATG dapA-A SEQ ID NO.160 GACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACAGCAAACCGGCATGCTTAAG pGRB-yjiP-s SEQ ID NO.161 AGTCCTAGGTATAATACTAGTTGGAAAGCGCCTCGGGGAATGTTTTAGAGCTAGAA pGRB-yjiP-a SEQ ID NO.162 TTCTAGCTCTAAAACATTCCCCGAGGCGCTTTCCAACTAGTATTATACCTAGGACT yncK-US SEQ ID NO.163 CATTTTTCACAACACCCGTGACCT yncK-UA SEQ ID NO.164 TGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGCATCAGGAAAACACAATGCCTTC yncK-DS SEQ ID NO.165 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGCCCTGAAGGATGGGGTTTTAC yncK-DA SEQ ID NO.166 CGCAGCGTAAACCACTGGTTAC pGRB-yncK-s SEQ ID NO.167 AGTCCTAGGTATAATACTAGTAATTAACTATCCTCCCAAACGTTTTAGAGCTAGAA pGRB-yncK-a SEQ ID NO.168 TTCTAGCTCTAAAACGTTTGGGAGGATAGTTAATTACTAGTATTATACCTAGGACT ybfL-US SEQ ID NO.169 CAGGGAAAGATCACGTAACGCTAC ybfL-UA SEQ ID NO.170 GAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACTTATCCGTCTTGATCTGCCCG ybfL-DS SEQ ID NO.171 GAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATACGAATGATAAGGTATTCAAGGCAG ybfL-DA SEQ ID NO.172 TCGGGATGTGTTTCCCCAAA pGRB-ybfL-s SEQ ID NO.173 AGTCCTAGGTATAATACTAGTAAAGAAAGAGCCAGAAATGAGTTTTAGAGCTAGAA pGRB-ybfL-a SEQ ID NO.174 TTCTAGCTCTAAAACTCATTTCTGGCTCTTTCTTTACTAGTATTATACCTAGGACT nmpC-US SEQ ID NO.175 GCTACCGAGTATTGAAAACACCAC nmpC-UA SEQ ID NO.176 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAAGCCAAGACGGGCATAAGTAGTAT nmpC-DS SEQ ID NO.177 GACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTAAAAATGCAGAAGTTTGGGCCG nmpC-DA SEQ ID NO.178 TGATGACTCATGATGAACCCTGTTC dapB-S SEQ ID NO.179 ATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCATGATGCAAACATCCGC dapB-A SEQ ID NO.180 AACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACAAATTATTGAGATCAAGTACATCTCGCAT pGRB-nmpC-s SEQ ID NO.181 AGTCCTAGGTATAATACTAGTCAGAATTCGGTGGTGACACTGTTTTAGAGCTAGAA pGRB-nmpC-a SEQ ID NO.182 TTCTAGCTCTAAAACAGTGTCACCACCGAATTCTGACTAGTATTATACCTAGGACT ycdN-US SEQ ID NO.183 GATTTTGACGCCACCAACACC ycdN-UA SEQ ID NO.184 GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCAATCCACATCACACAATCCATC ycdN-DS SEQ ID NO.185 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGAAGGGATTTTTGGCTATCAGG ycdN-DA SEQ ID NO.186 GTATTCGCCAGGCTGTAAATTC pGRB-ycdN-s SEQ ID NO.187 AGTCCTAGGTATAATACTAGTGCGTGGAAATCATCATGGCTGTTTTAGAGCTAGAA pGRB-ycdN-a SEQ ID NO.188 TTCTAGCTCTAAAACAGCCATGATGATTTCCACGCACTAGTATTATACCTAGGACT ylbE-US SEQ ID NO.189 ACCCAACCTTACGCAACCAG ylbE-UA SEQ ID NO.190 GTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATTGTTCGATAACCGCAGCAT ylbE-DS SEQ ID NO.191 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCGCTGGCGTGCTTTGAA ylbe-DA SEQ ID NO.192 GGCGTAACTCAGCAGGCAG lysA-S SEQ ID NO.193 CGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCCACATTCACTGTTCAGGCAC lysA-A SEQ ID NO.194 AAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAAGCAATTCCAGCGCCAGTAA pGRB-ylbE-s SEQ ID NO.195 AGTCCTAGGTATACTAGTACACTGGCTGGATGTGCAACGTTTTAGAGCTAGAA pGRB-ylbE-a SEQ ID NO.196 TTCTAGCTCTAAAACGTTGCACATCCAGCCAGTGTACTAGTATTATACCTAGGACT ychg-US SEQ ID NO.197 GGGAGCCGTTTTCTTATGCCAC ychg-UA SEQ ID NO.198 GAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGCGTTCTGCCGCTTAGTG ychg-DS SEQ ID NO.199 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATATCAAAAACCCGTTATCCTGTGAAAC ychg-DA SEQ ID NO.200 CTGCGTCTTGATCAAGCAGG pGRB-ychg-s SEQ ID NO.201 GTCCTAGGTATAATACTAGTAGGGACCATCCCGAAACGACGTTTTAGAGCTAGAA pGRB-ychg-a SEQ ID NO.202 TTCTAGCTCTAAAACGTCGTTTCGGGATGGTCCCTACTAGTATTATACCTAGGAC yghE-US SEQ ID NO.203 GTCAGGCACTGGCGAAAGAT yghE-UA SEQ ID NO.204 TTGTTATCCGCTCAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACGCAAGCCATAAACCCACAAG yghE-DS SEQ ID NO.205 GCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTTCCGACATCGAAATGCGTGG yghE-DA SEQ ID NO.206 AGGCGTTGTTGTGGCAGAT lysE-S SEQ ID NO.207 GCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCGTGTTTTCTTATTACTTTCAAGGTCTTGC Lyse-A SEQ ID NO.208 ACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGCTAACTGAACAAGGCTTGTGCATG pGRB-yghE-s SEQ ID NO.209 AGTCCTAGGTATAATACTAGTGCTGAAAAAATATCGCCCACGTTTTAGAGCTAGAA pGRB-yghE-a SEQ ID NO.210 TTCTAGCTCTAAAACGTGGGCGATATTTTCAGCACTAGTATTATACCTAGGACT rph-US SEQ ID NO.211 ATAGCGCAGGGTACATTCCACT rph-UA SEQ ID NO.212 GAAATTGTTATCCGCTCAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCTTCTTCAATAGAGGCGGTACA rph-DS SEQ ID NO.213 TGCCGCAGAGACCGACATCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAAT rph-DA SEQ ID NO.214 ACAGCGGTTGTGGTGGCA ddh-S SEQ ID NO.215 TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACAGGAAACAGACCATGACCAACATCCGCGTAGC ddh-A SEQ ID NO.216 ACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAGACGTCGCGTGCGATCA pGRB-rph-s SEQ ID NO.217 AGTCCTAGGTATAATACTAGTTGCGACGTGCTTCAGGCTGAGTTTTAGAGCTAGAA pGRB-rph-a SEQ ID NO.218 TTCTAGCTCTAAAACTCAGCCTGAAGCACGTCGCAACTAGTATTATACCTAGGACT yhjQ-US SEQ ID NO.219 CGCCGTAAACAATAAAACCGGC yhjQ-UA SEQ ID NO.220 TGTGTGAAATTGTTATCCGCTCAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACAAATCGAGCTGCGAGGTGTAG yhjQ-DS SEQ ID NO.221 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCGAACTGGTGCCTGTTGAACTAC yhjQ-DA SEQ ID NO.222 CAGCGAGAAGGTGACGATGATAC pGRB-yhjQ-s SEQ ID NO.223 AGTCCTAGGTATAATACTAGTGGGCTGGGCCAGAGCGATGCGTTTTAGAGCTAGAA pGRB-yhjQ-a SEQ ID NO.224 TTCTAGCTCTAAAACGCATCGCTCTGGCCCAGCCCACTAGTATTATACCTAGGACT cadA-US SEQ ID NO.225 ACTGGGTTGCGTGTTCTGC cadA-UA SEQ ID NO.226 GTACGGAAGGATCATATTGGCGTCAGTCAAAAATAACGCCGCACA cadA-DS SEQ ID NO.227 TGTGCGGCGTTATTTTTGACTGACGCCAATATGATCCTTCCGTAC eachA-DA SEQ ID NO.228 AAACCAGAGAAGCATATGCGCT pGRB--s SEQ ID NO.229 AGTCCTAGGTATACTAGTAAGAAACACCAAACGCAACCGTTTTAGAGCTAGAA pGRB--a SEQ ID NO.230 TTCTAGCTCTAAAACGGTTGCGTTTGGTGTTTCTTACTAGTATTATACCTAGGACT ldcC-US SEQ ID NO.231 TGGGTATCATTGCTCCGCG ldcC-UA SEQ ID NO.232 GATAAGGCAGGATCATATTTGCCGCAAAAATCACGCCGCAAATTCG ldcC-DS SEQ ID NO.233 CGAATTTGCGGCGTGATTTTTGCGGCAAATATGATCCTGCCTTATC ldcC-DA SEQ ID NO.234 CGGCGGGAACGGAAATGAGAA pGRB--s SEQ ID NO.235 AGTCCTAGGTATAATACTAGTTGTACGCCGGGGCATATGGGGTTTTAGAGCTAGAA pGRB--a SEQ ID NO.236 TTCTAGCTCTAAAACCCCATATGCCCCGGCGTACAACTAGTATTATACCTAGGACT The following Example 1 constructs an L-lysine production strain through 5 modules: (1) Chassis bacteria transformation: by E.coliW3110 is the starting strain, and some byproduct genes are knocked out: pflB, adhE, ldhA, ackA, frdBC, poxB, which effectively reduces impurities such as lactic acid, acetic acid, and amino acid residues during the fermentation process; Knockout of transcriptional repressors lacI ; (2) Increase the reducing power NADPH content: Increase pntAB Genes, improve pntAB Gene transcription level, increasing the supply of NADPH; (3) Strengthening the diaminopimelic acid pathway Increase aspC, lysC V339A , asd, dapA H56K , dapB, lysA, lysE Genes, including lysC and dapA genes, were subjected to point mutations to release the feedback inhibition of lysine and adjust aspC, lysC V339A The copy numbers of , asd genes are 4, 3, and 3, respectively, which enables the efficient expression of key enzymes in metabolic pathways; (4) Knockout of lysine decomposition pathway genes: cadA , ldcC ; (5) Shorten the lysine metabolic pathway: Increase ppc and From Corynebacterium glutamicum The ddh gene of K051 catalyzes the conversion of phosphoenolpyruvate to oxaloacetate and 2,3,4,5-tetrahydrodipicolinate to meso-2,6-diaminopimelate, shortening the L-lysine metabolic pathway to increase the conversion rate.

[0033] The gene editing method used in the above gene manipulation is referred to in the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genomeediting. Metabolic Engineering, 2015, 31: 13-21.). The professional terms involved in the present invention can be explained in the article unless otherwise noted. "Knockout" as referred to in the present invention refers to the inactivation of the target gene, and "introduction" refers to the insertion of the exogenous gene into the genome of the engineered bacteria after connecting it to the promoter and terminator.

[0034] Example 1 This example is intended to illustrate the specific construction steps of strain WLY-32. The genetic modification process is as follows Figure 1If there is a method for operating a gene of the same type in the examples, it will only be provided once and annotated without further elaboration.

[0035] 1. Knockout of the pflB gene: Using the E. coli W3110 genome as a template, pflB-US, pflB-UA and pflB-DS, pflB-DA were used to obtain the upstream and downstream homology arms by PCR amplification, and then, using the upstream and downstream homology arms as templates and pflB-US and pflB-DA as primers, overlapping fragments were obtained by overlapping PCR amplification; pGRB-pflB-s and pGRB-pflB-a were used as primers to anneal the gRNA fragments, and they were connected with the pGRB vector to obtain pflB-pGRB; E. coli W3110 electroporation competent cells were prepared, the overlapping fragments and pflB-pGRB were electroporated into the competent cells, and the positive transformants were screened to obtain the strain WLY-1.

[0036] 2. Knockout adhE Gene: The same operation method as in ①, except that the primers used are adhE -US, adhE -UA, adhE -DS, adhE -DA, pGRB- adhE -s, pGRB- adhE -a. The competent cell is WLY-1, and the strain WLY-2 is obtained.

[0037] 3. Knockout ldhA Gene: The same operation method as in ①, except that the primers used are ldhA -US, ldhA -UA, ldhA -DS, ldhA -DA, pGRB- ldhA -s, pGRB- ldhA -a. The competent cell is WLY-2, and the strain WLY-,3 is obtained.

[0038] 4. Knockout ackA Gene: The same operation method as in ①, except that the primers used are ackA -US, ackA -UA, ackA -DS, ackA -DA, pGRB- ackA -s, pGRB- ackA -a. The competent cell is WLY-3, and the strain WLY-4 is obtained.

[0039] 5. Knockout frdBCGene: The same operation method as in ①, except that the primers used are frdBC -US, frdBC -UA, frdBC -DS, frdBC -DA, pGRB- frdBC -s, pGRB- frdBC -a. The competent cell is WLY-4, and the strain WLY-5 is obtained.

[0040] 6. Knockout poxB Gene: The same operation method as in ①, except that the primers used are poxB -US, poxB -UA, poxB -DS, poxB -DA, pGRB- poxB -s, pGRB- poxB -a. The competent cell is WLY-5, and the strain WLY-6 is obtained.

[0041] 7. Knockout lacI Gene: The same operation method as in ①, except that the primers used are lacI -US, lacI -UA, lacI -DS, lacI -DA, pGRB- lacI -s, pGRB- lacI -a. The competent cell is WLY-6, and the strain WLY-7 is obtained.

[0042] 8. Integration of pntAB gene at the yjiK pseudogene site: Using the E. coli W3110 genome as a template, yjiK-US, yjiK-UA, yjiK-DS, yjiK-DA and pntAB-S, pntAB-A were used to obtain the upstream and downstream homology arms and the target fragments by PCR amplification. Then, using the upstream and downstream homology arms and the target fragments as templates, yjiK-US and yjiK-DA were used as primers to obtain overlapping fragments by overlapping PCR amplification. Since the Trc promoter was already included in the primer design, The promoter was connected to the 5' segment of the primer, so the overlapping fragment carried the Trc promoter, that is, the overlapping fragment of upstream homology arm-Trc promoter-downstream homology arm of target gene; pGRB-yjiK-s and pGRB-yjiK-a were used as primers, the gRNA fragment was annealed, and it was connected to the pGRB vector to obtain yjiK-pGRB; WLY-7 electroporation competent cells were prepared, the overlapping fragments and yjiK-pGRB were electroporated into the competent cells, and positive transformants were screened to obtain strain WLY-8.

[0043] 9. Integration of ppc gene at the ycgH pseudogene site: The same operation method as in ⑧, except that the primers used are ycgH-US, ycgH-UA, ycgH-DS, ycgH-DA, ppc-S, ppc-A, pGRB-ycgH-s, pGRB-ycgH-a. The competent cell is WLY-8, and the strain WLY-9 is obtained.

[0044] 10. Integration of ppc gene at mbhA pseudogene site: The same operation method as in ⑧, except that the primers used are mbhA-US, mbhA-UA, mbhA-DS, mbhA-DA, ppc-S, ppc-A, pGRB-mbhA-s, pGRB-mbhA-a. The competent cell is WLY-9, and the strain WLY-10 is obtained.

[0045] 11. Integration of aspC gene at the yghX pseudogene site: The same operation method as in ⑧, except that the primers used are yghX-US, yghX-UA, yghX-DS, yghX-DA, aspC-S, aspC-A, pGRB-yghX-s, pGRB-yghX-a. The competent cell is WLY-10, and the strain WLY-11 is obtained.

[0046] 12. Integration of aspC gene at the ilvG pseudogene site: The same operation method as in ⑧, except that the primers used were ilvG-US, ilvG-UA, ilvG-DS, ilvG-DA, aspC-S, aspC-A, pGRB-ilvG-s, and pGRB-ilvG-a. The competent cell was WLY-11, and the strain WLY-12 was obtained.

[0047] 13. Integration of aspC gene at the ydeU pseudogene site: The same operation method as in ⑧, except that the primers used were ydeU-US, ydeU-UA, ydeU-DS, ydeU-DA, aspC-S, aspC-A, pGRB-ydeU-s, and pGRB-ydeU-a. The competent cell was WLY-12, and the strain WLY-13 was obtained.

[0048] 14. Integration of aspC gene at the yjiV pseudogene site: The same operation method as in ⑧, except that the primers used are yjiV-US, yjiV-UA, yjiV-DS, yjiV-DA, aspC-S, aspC-A, pGRB-yjiV-s, pGRB-yjiV-a. The competent cell is WLY-13, and the strain WLY-14 is obtained.

[0049] 15. Integrate the lysC gene at the yciQ pseudogene site: Using the E. coli W3110 genome as a template, yciQ-US, yciQ-UA and yciQ-DS, yciQ-DA were used to obtain the upstream and downstream homology arms by PCR amplification, and the mutant nucleotide sequence was designed on lysC-tb-A and lysC-tb-S, and there were homologous sequences. lysC-S, lysC-tb-A and lysC-tb-S, lysC-A were used to obtain fragments 1 and 2 by PCR amplification, and then fragments 1 and 2 were used as templates, lysC-S and lysC-A were used as primers, and the target fragments were obtained by overlapping PCR amplification. Then, the upstream and downstream homology arms and The target fragment was used as a template, and yciQ-US and yciQ-DS were used as primers to obtain overlapping fragments through overlapping PCR amplification; since the Trc promoter was connected to the 5' segment of the primer during primer design, the overlapping fragment carried the Trc promoter, that is, the overlapping fragment of upstream homology arm-Trc promoter-downstream homology arm of target gene; pGRB-yciQ-s and pGRB-yciQ-a were used as primers, the gRNA fragment was annealed to obtain, and it was connected to the pGRB vector to obtain yciQ-pGRB; WLY-14 electroporation competent cells were prepared, the overlapping fragments and yciQ-pGRB were electroporated into the competent cells, and positive transformants were screened to obtain strain WLY-15.

[0050] 16. Integration of lysC gene at the ygaY pseudogene site: The same operation method as in ⑮ is used, except that the primers used are ygaY-US, ygaY-UA, ygaY-DS, ygaY-DA, lysC-S, lysC-tb-A, lysC-tb-S, lysC-A, pGRB-ygaY-s, and pGRB-ygaY-a. The competent cell is WLY-15, and the strain WLY-16 is obtained.

[0051] 17. Integration of lysC gene at fhiA pseudogene site: The same operation method as in ⑮, except that the primers used were fhiA-US, fhiA-UA, fhiA-DS, fhiA-DA, lysC-S, lysC-tb-A, lysC-tb-S, lysC-A, pGRB-fhiA-s, pGRB-fhiA-a. The competent cell was WLY-16, and the strain WLY-17 was obtained.

[0052] 18. Integration of asd gene at the yjgX pseudogene site: The same operation method as in ⑧, except that the primers used were yjgX-US, yjgX-UA, yjgX-DS, yjgX-DA, asd-S, asd-A, pGRB-yjgX-s, and pGRB-yjgX-a. The competent cell was WLY-17, and the strain WLY-18 was obtained.

[0053] 19. Integration of asd gene at gapC pseudogene site: The same operation method as in ⑧, except that the primers used are gapC-US, gapC-UA, gapC-DS, gapC-DA, asd-S, asd-A, pGRB-gapC-s, pGRB-gapC-a. The competent cell is WLY-18, and the strain WLY-19 is obtained.

[0054] 20. Integration of asd gene at the yeeP pseudogene site: The same operation method as in ⑧, except that the primers used were yeeP-US, yeeP-UA, yeeP-DS, yeeP-DA, asd-S, asd-A, pGRB-yeeP-s, and pGRB-yeeP-a. The competent cell was WLY-19, and the strain WLY-20 was obtained.

[0055] 21. Integration of dapA gene at the yjiP pseudogene site: The same operation method as in ⑮, except that the primers used were yjiP-US, yjiP-UA, yjiP-DS, yjiP-DA, dapA-S, dapA-tb-A, dapA-tb-S, dapA-A, pGRB-yjiP-s, and pGRB-yjiP-a. The competent cell was WLY-20, and the strain WLY-21 was obtained.

[0056] 22. Integration of dapA gene at the yncK pseudogene site: The same operation method as in ⑮, except that the primers used were yncK-US, yncK-UA, yncK-DS, yncK-DA, dapA-S, dapA-tb-A, dapA-tb-S, dapA-A, pGRB-yncK-s, and pGRB-yncK-a. The competent cell was WLY-21, and the strain WLY-22 was obtained.

[0057] 23. Integration of dapA gene at the ybfL pseudogene site: The same operation method as in ⑮, except that the primers used were ybfL-US, ybfL-UA, ybfL-DS, ybfL-DA, dapA-S, dapA-tb-A, dapA-tb-S, dapA-A, pGRB-ybfL-s, and pGRB-ybfL-a. The competent cell was WLY-22, and the strain WLY-23 was obtained.

[0058] 24. Integration of dapB gene at nmpC pseudogene site: The same operation method as in ⑧, except that the primers used are nmpC-US, nmpC-UA, nmpC-DS, nmpC-DA, dapB-S, dapB-A, pGRB-nmpC-s, pGRB-nmpC-a. The competent cell is WLY-23, and the strain WLY-24 is obtained.

[0059] 25. Integration of asd gene at the ycdN pseudogene site: The same operation method as in ⑧, except that the primers used were ycdN-US, ycdN-UA, ycdN-DS, ycdN-DA, dapB-S, dapB-A, pGRB-ycdN-s, and pGRB-ycdN-a. The competent cell was WLY-24, and the strain WLY-25 was obtained.

[0060] 26. Integration of lysA gene at the ylbE pseudogene site: The same operation method as in ⑧, except that the primers used were ylbE-US, ylbE-UA, ylbE-DS, ylbE-DA, lysA-S, lysA-A, pGRB-ylbE-s, and pGRB-ylbE-a. The competent cell was WLY-25, and the strain WLY-26 was obtained.

[0061] 27. Integration of lysA gene at the ychg pseudogene site: The same operation method as in ⑧, except that the primers used were ychg-US, ychg-UA, ychg-DS, ychg-DA, lysA-S, lysA-A, pGRB-ychg-s, and pGRB-ychg-a. The competent cell was WLY-26, and the strain WLY-27 was obtained.

[0062] 28. Integration of lysE gene at the yghE pseudogene site: The same operation method as in ⑧, except that the primers used were yghE-US, yghE-UA, yghE-DS, yghE-DA, lysE-S, lysE-A, pGRB-yghE-s, and pGRB-yghE-a. The competent cell was WLY-27, and the strain WLY-28 was obtained.

[0063] 29. Integrate the ddh gene from Corynebacterium glutamicum K051 at the rph pseudogene site: Using the E.coliW3110 genome as a template, rph-US, rph-UA and rph-DS, rph-DA were used to obtain the upstream and downstream homology arms by PCR amplification, and using the Corynebacterium glutamicum K051 genome as a template, ddh-S and ddh-A were used to amplify the target fragments by PCR. Next, the upstream and downstream homology arms and the target fragment were used as templates, and rph-US and rph-DA were used as primers to obtain overlapping fragments through overlapping PCR amplification; since the Trc promoter was connected to the 5' segment of the primers during primer design, the overlapping fragments carried the Trc promoter, that is, the overlapping fragments of the upstream homology arm-Trc promoter-target gene downstream homology arm; pGRB-rph-s and pGRB-rph-a were used as primers, and the gRNA fragments were annealed to obtain the gRNA fragments, which were connected to the pGRB vector to obtain rph-pGRB; WLY-28 electroporation competent cells were prepared, the overlapping fragments and rph-pGRB were electroporated into the competent cells, and the positive transformants were screened to obtain the strain WLY-29.

[0064] 30. Integration of the ddh gene from Corynebacterium glutamicum K051 at the yhjQ pseudogene site: The same operation method as in ㉙ was used, except that the primers used were yhjQ-US, yhjQ-UA, yhjQ-DS, yhjQ-DA, ddh-S, ddh-A, pGRB-yhjQ-s, and pGRB-yhjQ-a. The competent cell was WLY-29, and the strain WLY-30 was obtained.

[0065] 31. Knockout cadA Gene: The same operation method as in ①, except that the primers used are cadA -US, cadA -UA, cadA -DS, cadA -DA, pGRB- cadA -s, pGRB- cadA -a. The competent cell is WLY-30, and the strain WLY-31 is obtained.

[0066] 32. Knockout ldcC Gene: The same operation method as in ①, except that the primers used are ldcC -US, ldcC -UA, ldcC -DS, ldcC -DA, pGRB-ldcC -s, pGRB- ldcC- a. The competent cell is WLY-31, and the strain WLY-32 is obtained.

[0067] Example 2 This example is intended to illustrate the application of shake flask fermentation of engineered bacteria WLY-32, and the specific steps are as follows: ① Slant culture: Take the strain WLY-32 obtained in Example 1 as the lysine production strain and inoculate it on a slant medium, and culture it at 37°C for 16 hours. The slant medium is a general LB solid medium; ② Shake flask seed culture: take the solid slant bacteria and inoculate it into the seed culture medium in the shake flask for fermentation, the culture temperature is 37℃, the culture time is 16h, the shaker speed is 220r / min, the pH is 7.2-7.4; the seed culture medium used is: glucose 30g / L, yeast powder 5g / L, peptone 5g / L, MgSO4.7H2O 0.8g / L, KH2PO43g / L, ammonium sulfate 5g / L, biotin 1mg / L, and the rest is water; ③ Shake flask fermentation culture: the fermentation inoculation amount is 20%, the culture temperature is 37℃, the pH is 7.2-7.4, the culture time is 30h, and the shaker speed is 220r / min; the fermentation medium used is: glucose 30g / L, MgSO4.7H2O 1.5g / L, yeast powder 5g / L, peptone 2g / L, ammonium sulfate 15g / L, K2HPO4.3H2O 4g / L, FeSO4.7H2O 30mg / L, VB1, VB3, VB5 each 2mg / L, and the rest is water.

[0068] Wild-type E.coli W3110 was used as the control group, and the wild type E.coli W3110 failed to accumulate lysine, while the strain WLY-32 described in Example 1 accumulated 20.3 g / L lysine, demonstrating the effectiveness of the strain.

[0069] Example 3 This example is intended to illustrate the key enzymes aspC, lysC V339A , the copy number of asd gene is optimized to 4:3:3, the specific steps are: First, the strain with high L-lysine production was obtained by directional transformation in the process of constructing the strain according to Example 1, in which, in addition to aspC, lysC V339A The copy numbers of aspC, asd and asd genes were the same as those of WLY-32. The fermentation culture method described in Example 2 was used to optimize the expression of aspC, lysC V339A, the copy number of asd gene. A total of 9 control experiments were set up below, and the data results of the 9 shake flask fermentations for 30 h are shown in Table 2.

[0070] Table 2 aspC, lysC V339A Effect of the copy number of asd gene on L-lysine production Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 Group 9 Copy number of aspC gene 2 3 4 3 4 4 4 3 3 <![CDATA[ lysC V339A Gene copy number]]> 2 3 4 4 3 4 3 4 3 Copy number of asd gene 2 3 4 4 4 3 3 3 4 <![CDATA[Bacterial biomass OD 600nm > 50.6 47.5 38.6 43.5 44.7 41.2 45.5 43.1 46.9 L-lysine production g / L 12.3 14.8 20.6 19.8 20.1 20.2 20.3 18.4 20.1 Through the control experiments of group 1 to group 3, it can be found that with the increase of aspC, lysC V339A The copy numbers of asd and asd genes increased in groups. Although the production of L-lysine showed an increasing trend, the bacterial biomass showed a decreasing trend and the trend was greater than the increasing trend of L-lysine production. The reason for this was that the overexpression of one or more genes caused a burden on bacterial metabolism. Through the control experiments of groups 4 to 9, it was found that the L-lysine production of group 5 was the highest, but its bacterial biomass was still not high, while the bacterial biomass and L-lysine production of groups 7 and 9 were similar and both were high. Comprehensive comparison showed that when adjusting aspC, lysC V339A When the copy numbers of asd genes are 4, 3, and 3 respectively, the bacteria not only have a higher L-lysine production but also maintain a higher biomass, making it a better choice.

[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. Improvements and modifications such as strain transformation carried out by technicians in this technical field based on the method of the present invention or on the basis of the method are deemed to be within the scope of protection of the present invention.

Claims

1. An L-lysine producing strain, characterized in that: In the starting strain E. coli Knockout pflB, adhE, ldhA, ackA, frdBC, poxB, lacI, cadA, ldcC Gene, upregulated pntAB, ppc, aspC、lysC V339A 、asd、dapA H56K , dapB, lysA, lysE Gene, heterologous expression from Corynebacterium glutamicum K051 dhdh Gene.

2. The L-lysine producing strain according to claim 1, characterized in that: Said E. coli W3110 is E. coli W3110 ATCC 27325.

3. The L-lysine producing strain according to claim 1, characterized in that: The nucleotide sequence of the pflB gene is shown in the sequence listing SEQ ID NO.1, the nucleotide sequence of the adhE gene is shown in the sequence listing SEQ ID NO.2, the nucleotide sequence of the ldhA gene is shown in the sequence listing SEQ ID NO.3, the nucleotide sequence of the ackA gene is shown in the sequence listing SEQ ID NO.4, the nucleotide sequence of the frdBC gene is shown in the sequence listing SEQ ID NO.5, the nucleotide sequence of the poxB gene is shown in the sequence listing SEQ ID NO.6, the nucleotide sequence of the lacI gene is shown in the sequence listing SEQ ID NO.7, the nucleotide sequence of the cadA gene is shown in the sequence listing SEQ ID NO.8, and the nucleotide sequence of the ldcC gene is shown in the sequence listing SEQ ID NO.

9.

4. The L-lysine producing strain according to claim 1, characterized in that: Said pntAB The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.

10. ppc The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.

11. aspC The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.

12. lysC V339A The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.13, asd The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.14, dapA H56K The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.

15. dapB The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.16, lysA The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.

17. lysE The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.18, dhdh The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.

19.

5. The method for constructing the L-lysine producing strain according to any one of claims 1 to 4, characterized in that: In the starting strain E. coli Directional transformation is carried out based on W3110. The specific steps are as follows: (1) Transformation of chassis bacteria: knocking out some byproduct genes: pflB, adhE, ldhA, ackA, frdBC, poxB genes; knocking out transcriptional repressors lacI Gene; (2) Increase the reducing power of NADPH content: Upregulate pntAB Gene; (3) Strengthening the diaminopimelic acid pathway: Upregulation aspC , lysC V339A , asd , dapA H56K , dapB , lysA , lysE genes, in which lysC and dapA genes were subjected to point mutations; (4) Knockout of lysine decomposition pathway genes: cadA , LqCy Gene; (5) Shortening the lysine metabolic pathway: Upregulation ppc Genes and their origin Corynebacterium glutamicum The ddh gene of K051.

6. The method for constructing an L-lysine producing strain according to claim 5, characterized in that: The specific steps are as follows: (1) Knockout pflB, adhE, ldhA, ackA, frdBC, poxB, lacI, cadA, ldcC Gene; (2) Using the Trc promoter to express the pntAB gene at the yjiK pseudogene locus; (3) Using the Trc promoter to express two copies of the ppc gene at the ycgH and mbhA pseudogene loci; (4) Using the Trc promoter, multiple copies of the aspC gene were expressed at the yghX, ilvG, ydeU, and yjiV pseudogene sites; (5) Using the Trc promoter, the yciQ, ygaY, and fhiA pseudogene loci were used to express lysC V339A The gene is expressed in multiple copies; (6) Using the Trc promoter, multiple copies of the asd gene were expressed at the yjgX, gapC, and yeeP pseudogene loci; (7) Using the Trc promoter, dapA was expressed at the yjiP, yncK, and ybfL pseudogene loci. H56K The gene is expressed in multiple copies; (8) Using the Trc promoter, the dapB gene was expressed in two copies at the nmpC and ycdN pseudogene loci respectively; (9) Using the Trc promoter, two copies of the lysA gene were expressed at the ylbE and ychg pseudogene loci, respectively; (10) Using the Trc promoter to express the lysE gene at the yghE pseudogene locus; (11) The Trc promoter was used to express two copies of the ddh gene from Corynebacterium glutamicum K051 at the rph and yhjQ pseudogene loci, respectively.

7. The method for constructing an L-lysine producing strain according to claim 6, characterized in that: Adjust aspC, lysC V339A The copy numbers of , asd genes are 4, 3, and 3, respectively.

8. Use of the L-lysine producing strain according to any one of claims 1 to 4 in the fermentation production of lysine.

9. The use according to claim 8, characterized in that: The specific steps of synthesizing L-lysine by fermentation culture in a culture medium are as follows: ① Slant culture: inoculate the L-lysine producing strain on the slant culture medium and culture at 36-37℃ for 14-18h; ② Shake flask seed culture: Take the slant strain and inoculate it into the seed culture medium in the shake flask for fermentation. The culture temperature is 36-37℃, the culture time is 12-20h, the shaker speed is 200-240r / min, and the pH is 7.2-7.4; ③ Shake flask fermentation culture: the fermentation inoculation amount is 15-20%, the culture temperature is 36-37℃, the pH is 7.2-7.4, the culture time is 24-36h, and the shaking speed is 200-240r / min.

10. The use according to claim 9, characterized in that: The slant culture medium is a universal LB solid culture medium; the seed culture medium used in step ② is: glucose 20-30g / L, yeast powder 3-5g / L, peptone 3-5g / L, MgSO4.7H2O 0.5-1g / L, KH2PO42-3g / L, ammonium sulfate 4-5g / L, biotin 1-2mg / L, and the rest is water; the fermentation culture medium used in step ③ is: glucose 20-30g / L, MgSO4.7H2O 1.5-2g / L, yeast powder 4-5g / L, peptone 2-3g / L, ammonium sulfate 10-20g / L, K2HPO4.3H2O 3-6g / L, FeSO4.7H2O 20-30mg / L, VB1, VB3, VB5 each 2-3mg / L, and the rest is water.

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