An L-lysine-producing strain, its construction method and application

Abstract

The present invention provides an L-lysine producing strain, a method for constructing the same and an application thereof. The strain uses E.coli W3110 as the starting strain, and has knocked out pflB, adhE, ldhA, ackA, frdBC, poxB, lacI, cadA, ldcC genes, up-regulated pntAB, ppc, aspC, lysC V339A , asd, dapA H56K , dapB, lysA, lysE genes, heterologously expressed the Corynebacterium glutamicum genes derived from ddh K051. The strain has knocked out some by-products and lysine degradation pathway enzymes, strengthened the diaminopimelic acid pathway and the supply of NADPH of the strain, and can efficiently synthesize L-lysine using glucose as a substrate, and has a high production level.

Description

Technical Field

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

[0002] L-lysine is an α-basic amino acid and one of the essential amino acids for the human body. It has various physiological functions and is widely used in animal feed, medicine and the food industry. L-lysine has become the second largest amino acid in the world, with an annual global production of over 5 million tons and 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. Currently, the main L-lysine fermentation production strains are Escherichia coli and Corynebacterium glutamicum. Among them, Corynebacterium glutamicum has certain advantages, but has problems such as low growth rate, high requirements for nutrient components, and high downstream recovery costs. Relatively, Escherichia coli is more advantageous in rapid and efficient production due to its short production cycle and low cultivation cost.

[0004] At present, there are still many problems to be solved in lysine fermentation, including: the composition of the fermentation broth is complex, containing impurities such as lactic acid, acetic acid, and amino acid residues, which requires high technology for separating and extracting high-purity L-lysine; a large amount of NADPH is required in the synthesis process, and blindly increasing the HMP pathway will lead to cell metabolic imbalance and reduced conversion rate; the pathway is long, and the conversion rate of microbial fermentation to produce L-lysine is often low, and by-products are easily accumulated. 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 construction method of the above L-lysine producing strain.

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

[0008] To solve the above technical problems, the technical solution of the present invention is:

[0009] An L-lysine producing strain, namely strain WLY-32, is obtained by directional transformation on the basis of the starting strain E.coli W3110: knockout pflB, adhE, ldhA, ackA, frdBC, poxB, lacI, cadA, ldcC gene, up-regulate pntAB, ppc, aspC, lysC V339A , asd, dapA H56K, dapB, lysA, lysE The gene was heterologously expressed from Corynebacterium glutamicum the ddh gene of K051.

[0010] Preferably, for the above L-lysine producing strain, the CRIPSR / Cas9 gene editing technology is used as a metabolic engineering means to E.coli directionally modify W3110 as a chassis strain, and the E.coli W3110 is E.coli W3110 ATCC27325.

[0011] Preferably, for the above L-lysine producing strain, the nucleotide sequence of the pflB gene is shown in Sequence Listing SEQ ID NO.1, the nucleotide sequence of the adhE gene is shown in Sequence Listing SEQ ID NO.2, the nucleotide sequence of the ldhA gene is shown in Sequence Listing SEQ ID NO.3, the nucleotide sequence of the ackA gene is shown in Sequence Listing SEQ ID NO.4, the nucleotide sequence of the frdBC gene is shown in Sequence Listing SEQ ID NO.5, the nucleotide sequence of the poxB gene is shown in Sequence Listing SEQ ID NO.6, the nucleotide sequence of the lacI gene is shown in Sequence Listing SEQ ID NO.7, the nucleotide sequence of the cadA gene is shown in Sequence Listing SEQ ID NO.8, and the nucleotide sequence of the ldcC gene is shown in Sequence Listing SEQ ID NO.9.

[0012] Preferably, for the above L-lysine producing strain, the pntAB nucleotide sequence of the gene is shown in Sequence Listing SEQ ID NO.10, ppc the nucleotide sequence of the gene is shown in Sequence Listing SEQ ID NO.11, aspC the nucleotide sequence of the gene is shown in Sequence Listing SEQ ID NO.12, lysC V339A the nucleotide sequence of the gene is shown in Sequence Listing SEQ ID NO.13, asd the nucleotide sequence of the gene is shown in Sequence Listing SEQ ID NO.14, dapA H56K the nucleotide sequence of the gene is shown in Sequence Listing SEQ ID NO.15, dapB the nucleotide sequence of the gene is shown in Sequence Listing SEQ ID NO.16, lysA the nucleotide sequence of the gene is shown in Sequence Listing SEQ ID NO.17, lysE the nucleotide sequence of the gene is shown in Sequence Listing SEQ ID NO.18, ddh the nucleotide sequence of the gene is shown in Sequence Listing SEQ ID NO.19.

[0013] The construction method of the above L-lysine producing strain is to carry out directional transformation on the starting strain E.coli W3110, and the specific steps are as follows:

[0014] (1) Modification of the chassis bacterium: Knock out some by-product genes: pflB, adhE, ldhA, ackA, frdBC, poxB genes; Knock out the transcriptional repressor lacI gene;

[0015] (2) Increase the content of reducing power NADPH: Up-regulate the pntAB gene, increase the pntAB transcriptional level of the gene, and increase the supply of NADPH;

[0016] (3) Strengthen the diaminopimelic acid pathway: Up-regulate aspC , lysC V339A , asd , dapA H56K , dapB , lysA , lysE genes, among which the lysC and dapA genes are point-mutated to relieve the feedback inhibition of lysine;

[0017] (4) Knock out the lysine catabolic pathway genes: cadA , ldcC genes;

[0018] (5) Shorten the lysine metabolic pathway: Up-regulate the ppc gene and the ddh gene derived from Corynebacterium glutamicum K051.

[0019] Preferably, the construction method of the above L-lysine producing strain is as follows:

[0020] (1) Knock out the pflB, adhE, ldhA, ackA, frdBC, poxB, lacI, cadA, ldcC gene;

[0021] (2) Express the pntAB gene at the yjiK pseudogene locus using the Trc promoter;

[0022] (3) Carry out double-copy expression of the ppc gene at the ycgH and mbhA pseudogene loci respectively using the Trc promoter;

[0023] (4) Carry out multi-copy expression of the aspC gene at the yghX, ilvG, ydeU, and yjiV pseudogene loci respectively using the Trc promoter;

[0024] (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;

[0025] (6) Using the Trc promoter, multiple copies of the asd gene were expressed at the yjgX, gapC, and yeeP pseudogene loci;

[0026] (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;

[0027] (8) Using the Trc promoter, the dapB gene was expressed in two copies at the nmpC and ycdN pseudogene loci respectively;

[0028] (9) Using the Trc promoter, two copies of the lysA gene were expressed at the ylbE and ychg pseudogene loci, respectively;

[0029] (10) Using the Trc promoter to express the lysE gene at the yghE pseudogene locus;

[0030] (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.

[0031] 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).

[0032] 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.

[0033] 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.

[0034] Application of the L-lysine producing strain in the fermentation production of lysine.

[0035] Preferably, for the application of the above-mentioned L-lysine producing strain, glucose is used as a substrate for fermentation culture in a medium to synthesize L-lysine.

[0036] Preferably, for the application of the above-mentioned L-lysine producing strain, the medium includes but is not limited to carbon source, nitrogen source, inorganic salts, vitamins, etc.; the fermentation conditions include fermentation temperature, fermentation pH, fermentation dissolved oxygen conditions, fermentation pressure, fermentation time, etc.

[0037] Preferably, for the application of the above-mentioned L-lysine producing strain, the specific steps are as follows:

[0038] ① Slant culture: Inoculate the L-lysine producing strain on a slant medium and culture at 36 - 37 °C for 14 - 18 h;

[0039] ② Shake flask seed culture: Inoculate the slant strain into the seed medium in a shake flask for fermentation. The culture temperature is 36 - 37 °C, the culture time is 12 - 20 h, the shaker speed is 200 - 240 r / min, and the pH is 7.2 - 7.4;

[0040] ③ Shake flask fermentation culture: The fermentation inoculation amount is 15 - 20%, the culture temperature is 36 - 37 °C, the pH is 7.2 - 7.4, the culture time is 24 - 36 h, and the shaker speed is 200 - 240 r / min.

[0041] Preferably, for the application of the above-mentioned L-lysine producing strain, the slant medium is a general LB solid medium.

[0042] Preferably, for the application of the above-mentioned L-lysine producing strain, the seed 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.

[0043] Preferably, for 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.

[0044] The above media can all be prepared by standard methods.

[0045] Beneficial effects:

[0046] The above-mentioned L-lysine producing strain has knocked out some enzymes in the by-product and lysine degradation pathways, strengthened the diaminopimelic acid pathway and the supply of NADPH in the strain, has good L-lysine synthesis ability, can efficiently synthesize L-lysine using glucose as a substrate, has stable performance and high acid production efficiency, realizes the high-efficiency production of L-lysine, and has excellent industrial application prospects. Specifically:

[0047] (1) Genes pflB, adhE, ldhA, ackA, frdBC, and poxB were knocked out, effectively reducing impurities such as lactic acid, acetic acid, and amino acid residues during fermentation and lowering the downstream recovery cost.

[0048] (2) By increasing the transcriptional level of the membrane-bound inducible enzyme pntAB gene, the supply of NADPH was increased, providing a large amount of reducing power for L-lysine synthesis.

[0049] (3) Upregulated aspC, lysC V339A , asd, dapA H56K , dapB, lysA, lysE genes to strengthen the diaminopimelic acid pathway in the strain. Among them, point mutations were made to the lysC 、 dapA gene to relieve the feedback inhibition of lysine, and the copy numbers of aspC, lysC V339A , and asd genes were adjusted to 4:3:3, enabling the highly efficient expression of key enzymes in the metabolic pathway and a smooth metabolic pathway. At the same time, ppc gene and heterologous expression of the ddh gene derived from Corynebacterium glutamicum K051 catalyzed the conversion of phosphoenolpyruvate to oxaloacetate and the conversion of 2,3,4,5-tetrahydrodipicolinate to meso-2,6-diaminopimelate, shortening the L-lysine metabolic pathway to improve the conversion rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It is a diagram of the genetic modification process of the de novo synthesis pathway of the L-lysine producing strain. DETAILED DESCRIPTION OF THE INVENTION

[0051] In order to enable those skilled in the art to better understand the technical solution of the present invention, the following further details the technical solution of the present invention in combination with specific embodiments.

[0052] In the examples, the percentage sign "%" refers to mass percentage unless otherwise specified. The percentage of a solution refers to the number of grams of solute contained in 100 mL, and the percentage between liquids refers to the volume ratio of the solution at 25°C.

[0053] The starting strain used in the examples was the wild type E.coliW3110 ATCC 27325, the corresponding promoter and gene are shown in the sequence listing. The primers used in the construction process of the strains involved are shown in Table 1.

[0054] Table 1 Primers Involved in the Construction Process of Strains

[0055] 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 AGTCCTAGGTATAATACTAGTGGAAACTCACTTCGAAGAGCGTTTTAGAGCTAGAA pGRB-adhE-a 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 AGTCCTAGGTATAATACTAGTCCGTGATGCTAACTTCTCTCGTTTTAGAGCTAGAA pGRB-ldhA-a SEQ ID NO.38 TTCTAGCTCTAAAACGAGAGAAGTTAGCATCACGGACTAGTATTATACCTAGGACT 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 AGTCCTAGGTATAATACTAGTCACAACCCGGCTCACCTGATGTTTTAGAGCTAGAA pGRB-ackA-a SEQ ID NO.44 TTCTAGCTCTAAAACATCAGGTGAGCCGGGTTGTGACTAGTATTATACCTAGGACT frdBC-US SEQ ID NO.45 CGTCAACTTCCTCAAACACACCC frdBC-UA SEQ ID NO.46 GTTTTGGTGTGCAGCAGAGCTGGACCAGCGGTAGCTCAGGT frdBC-DS SEQ ID NO.47 ACCTGAGCTACCGCTGGTCCAGCTCTGCTGCACACCAAAAC frdBC-DA SEQ ID NO.48 AGAACGCGCTCGTAGCTC pGRB-frdBC-s SEQ ID NO.49 AGTCCTAGGTATAATACTAGTTGATTACACCGACGGTATGAGTTTTAGAGCTAGAA pGRB-frdBC-a SEQ ID NO.50 TTCTAGCTCTAAAACTCATACCGTCGGTGTAATCAACTAGTATTATACCTAGGACT poxB-US SEQ ID NO.51 TCATCGGGCTATTTAACCGTTAGTG poxB-UA SEQ ID NO.52 GAGAAGGCGCGTTGCAGCAGACCGCCAGTTCTCCG poxB-DS SEQ ID NO.53 CGGAGAACTGGCGGTCTGCTGCAACGCGCCTTCTC poxB-DA SEQ ID NO.54 GCGGCTTGGTCGGGTAAC pGRB-poxB-s SEQ ID NO.55 AGTCCTAGGTATAATACTAGTCAGAAGGGGCAACCATGCACGTTTTAGAGCTAGAA pGRB-poxB-a SEQ ID NO.56 TTCTAGCTCTAAAACGTGCATGGTTGCCCCTTCTGACTAGTATTATACCTAGGACT lacI-US SEQ ID NO.57 GAAGGGGTTGAATCGCAGGC lacI-UA SEQ ID NO.58 GTTGCAGCAAGCGGTCCACGCCAGACTGGAGGTGGC lacI-DS SEQ ID NO.59 GCCACCTCCAGTCTGGCGTGGACCGCTTGCTGCAAC lacI-DA SEQ ID NO.60 AATCCGTAATCATGGTCATAGCTGTT pGRB-lacI-s SEQ ID NO.61 AGTCCTAGGTATAATACTAGTCCATGAAGACGGTACGCGACGTTTTAGAGCTAGAA pGRB-lacI-a SEQ ID NO.62 TTCTAGCTCTAAAACGTCGCGTACCGTCTTCATGGACTAGTATTATACCTAGGACT yjiK-US SEQ ID NO.63 GAATCTCACCTGCGAATGCC yjiK-UA SEQ ID NO.64 TGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAACACCATCCTGCTGGCAATAAA yjiK-DS SEQ ID NO.65 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTTGCGATGGATGCCTCTGGT yjiK-DA SEQ ID NO.66 CCTAAGGTCAGAACCAGCATC PntAB-S SEQ ID NO.67 CCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCGAATTGGCATACCAAGAG PntAB-A SEQ ID NO.68 CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACAGAGCTTTCAGGATTGCATCCAC pGRB-yjiK-s SEQ ID NO.69 AGTCCTAGGTATAATACTAGTCAAGACTCCCCAACCAATTGGTTTTAGAGCTAGAA pGRB-yjik-a SEQ ID NO.70 TTCTAGCTCTAAAACCAATTGGTTGGGGAGTCTTGACTAGTATTATACCTAGGACT ycgH-US SEQ ID NO.71 TAAACTCGTCAGCGGCACAAC ycgH-UA SEQ ID NO.72 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGTAGGCGTTTCTGTTGATTCTG ycgH-DS SEQ ID NO.73 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGCGTGTCGGATTATCGTTCG ycgH-DA SEQ ID NO.74 GATTCAGGTTGCCATTTACGC ppc-S SEQ ID NO.75 CGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAACGAACAATATTCCGCATTGC ppc-A 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 GACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATATACTCAGGGCATGCTGGGA ydeU-DA SEQ ID NO.102 GCGTCGCATAGCGTCATTGA pGRB-ydeU-s SEQ ID NO.103 AGTCCTAGGTATAATACTAGTTCAGTTTATCGCCCGGAGGCGTTTTAGAGCTAGAA pGRB-ydeU-a SEQ ID NO.104 TTCTAGCTCTAAAACGCCTCCGGGCGATAAACTGAACTAGTATTATACCTAGGACT yjiV-US SEQ ID NO.105 TGTGACTGTGGAAGCCCTGT yjiV-UA SEQ ID NO.106 GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAATTCGGGCTGTCCCTTGTC yjiV-DS SEQ ID NO.107 GGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGTGGCACCTGAATGACGAAC yjiV-DA SEQ ID NO.108 TGGCGACATTCCCTTCCTTG pGRB-yjiV-s 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 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCAGTCAAGATGCCAGGGTTC 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 AGTCCTAGGTATAATACTAGTAAACAACGTTTCTTGCCTCAGTTTTAGAGCTAGAA pGRB-yciQ-a SEQ ID NO.120 TTCTAGCTCTAAAACTGAGGCAAGAAACGTTGTTTACTAGTATTATACCTAGGACT 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 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAATCGCCAGAATCATCATCCC fhiA-DS SEQ ID NO.129 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCAAGCAGGAGCTGACGGTGT fhiA-DA SEQ ID NO.130 TGCACCAATGCTGGATACTTACA pGRB-fhiA-s SEQ ID NO.131 AGTCCTAGGTATAATACTAGTTGACGTGCGTAACCAGCTGCGTTTTAGAGCTAGAA pGRB-fhiA-a SEQ ID NO.132 TTCTAGCTCTAAAACGCAGCTGGTTACGCACGTCAACTAGTATTATACCTAGGACT yjgX-US SEQ ID NO.133 GGAAGTCAACGGGTTATGCG yjgX-UA SEQ ID NO.134 TTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAAAAATCACCACGAATACCAGAATC yjgX-DS SEQ ID NO.135 GGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATACAGTGTCTTCCCTGAGCCG yjgX-DA SEQ ID NO.136 GGCGAAGGATACCATCAAGC asd-S SEQ ID NO.137 GCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAAAAATGTTGGTTTTATCGGCTGG 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 CGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCCACATTCACTGTTCAGCAC lysA-A SEQ ID NO.194 AAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAAAGCAATTCCAGCGCCAGTAA pGRB-ylbE-s SEQ ID NO.195 AGTCCTAGGTATAATACTAGTACACTGGCTGGATGTGCAACGTTTTAGAGCTAGAA 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 TTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACGCAAGCCATAAACCCACAAG 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 TTCTAGCTCTAAAACGTGGGCGATATTTTTTCAGCACTAGTATTATACCTAGGACT rph-US SEQ ID NO.211 ATAGCGCAGGGTACATTCCACT rph-UA SEQ ID NO.212 GAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCTTCTTCAATAGAGGCGGTACA rph-DS SEQ ID NO.213 TGCCGCAGAGACCGACATCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAAT rph-DA SEQ ID NO.214 ACAGCGGTTGTGGTGGCA ddh-S SEQ ID NO.215 TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGACCAACATCCGCGTAGC 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 CGCCGTAAACAATAAACCGGC yhjQ-UA SEQ ID NO.220 TGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACAAATCGAGCTGCGAGGTGTAG 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 cadA-DA SEQ ID NO.228 AAACCAGAGAAGCATATGCGCT pGRB--s SEQ ID NO.229 AGTCCTAGGTATAATACTAGTAAGAAACACCAAACGCAACCGTTTTAGAGCTAGAA 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

[0056] The following Example 1 constructs an L-lysine-producing strain through 5 modules:

[0057] (1) Modification of the chassis bacterium:

[0058] Using E.coli W3110 as the starting strain, knock out some by-product genes: pflB, adhE, ldhA, ackA, frdBC, poxB, effectively reducing impurities such as lactic acid, acetic acid, and amino acid residues during fermentation;

[0059] Knock out the transcriptional repressor lacI ;

[0060] (2) Increase the content of reducing power NADPH:

[0061] Up-regulate the pntAB gene, increase the pntAB transcriptional level of the gene, and increase the supply of NADPH;

[0062] (3) Strengthen the diaminopimelic acid pathway

[0063] Up-regulate the aspC, lysC V339A , asd, dapA H56K , dapB, lysA, lysE gene, in which the lysC and dapA genes are point-mutated to relieve the feedback inhibition of lysine, and the copy numbers of the aspC, lysC V339A and asd genes are 4, 3, and 3 respectively, enabling the highly efficient expression of key enzymes in the metabolic pathway;

[0064] (4) Knock out the lysine degradation pathway genes: cadA , ldcC ;

[0065] (5) Shorten the lysine metabolic pathway:

[0066] Up-regulate the ppc and ddh gene derived from Corynebacterium glutamicum K051, which catalyzes the conversion of phosphoenolpyruvate to oxaloacetate and 2,3,4,5-tetrahydropyridine dicarboxylate to meso-2,6-diaminopimelate, shortening the L-lysine metabolic pathway to improve the conversion rate.

[0067] The gene editing method used in the above gene operation refers to 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.). For the professional terms involved in the present invention, without special remarks, they can all be explained in this article. "Knockout" as referred to in the present invention means inactivating the target gene, and "introduction" means inserting the foreign gene linked with a promoter and a terminator into the genome of the engineering bacterium.

[0068] Example 1

[0069] This example aims to illustrate the specific construction steps of strain WLY-32, and the gene modification process is as Figure 1 shown. For the same type of gene operation methods in the examples, only one time is provided and annotated, and no more details are given.

[0070] 1. Knock out the pflB gene: Using the E. coli W3110 genome as a template, respectively use pflB-US, pflB-UA and pflB-DS, pflB-DA, and obtain the upstream and downstream homologous arms by PCR amplification. Then, using the upstream and downstream homologous arms as templates and pflB-US, pflB-DA as primers, obtain the overlapping fragment by overlapping PCR amplification; anneal the gRNA fragment with primers pGRB-pflB-s, pGRB-pflB-a, and connect it with the pGRB vector to obtain pflB-pGRB; prepare electrocompetent cells of E. coli W3110, electrotransform the overlapping fragment and pflB-pGRB into the electrocompetent cells together, and screen to obtain positive transformants to obtain strain WLY-1.

[0071] 2. Knock out adhE gene: It has the same operation method as in ①, the difference is that the primers used are adhE -US, adhE -UA, adhE -DS, adhE -DA, pGRB- adhE -s, pGRB- adhE -a. The electrocompetent cells are WLY-1, and strain WLY-2 is obtained.

[0072] 3. Knock out ldhA gene: It has the same operation method as in ①, the difference is that the primers used are ldhA -US, ldhA -UA,ldhA -DS, ldhA -DA, pGRB- ldhA -s, pGRB- ldhA -a. The competent cells are WLY-2, and the strain WLY-3 is obtained.

[0073] 4. Knockout ackA Gene: The same operation method as in ① is used, except that the primers used are ackA -US, ackA -UA, ackA -DS, ackA -DA, pGRB- ackA -s, pGRB- ackA -a. The competent cells are WLY-3, and the strain WLY-4 is obtained.

[0074] 5. Knockout frdBC Gene: The same operation method as in ① is used, except that the primers used are frdBC -US, frdBC -UA, frdBC -DS, frdBC -DA, pGRB- frdBC -s, pGRB- frdBC -a. The competent cells are WLY-4, and the strain WLY-5 is obtained.

[0075] 6. Knockout poxB Gene: The same operation method as in ① is used, except that the primers used are poxB -US, poxB -UA, poxB -DS, poxB -DA, pGRB- poxB -s, pGRB- poxB -a. The competent cells are WLY-5, and the strain WLY-6 is obtained.

[0076] 7. Knockout lacI Gene: The same operation method as in ① is used, except that the primers used are lacI -US, lacI -UA, lacI -DS, lacI -DA, pGRB- lacI -s, pGRB- lacI -a. The competent cells are WLY-6, and the strain WLY-7 is obtained.

[0077] 8. Integrate the pntAB gene at the yjiK pseudogene locus: Using the E. coli W3110 genome as a template, amplify the upstream and downstream homologous arms and the target fragment by PCR with yjiK-US, yjiK-UA, yjiK-DS, yjiK-DA and pntAB-S, pntAB-A respectively. Then, using the upstream and downstream homologous arms and the target fragment as templates, and yjiK-US, yjiK-DA as primers, amplify the overlapping fragment by overlapping PCR. Since the Trc promoter has been linked to the 5' segment of the primers during primer design, the overlapping fragment carries the Trc promoter, that is, the upstream homologous arm - Trc promoter - overlapping fragment of the downstream homologous arm of the target gene. Anneal the gRNA fragment with pGRB-yjiK-s, pGRB-yjiK-a as primers and ligate it with the pGRB vector to obtain yjiK-pGRB. Prepare electrocompetent cells of WLY-7, electrotransform the overlapping fragment and yjiK-pGRB into the electrocompetent cells together, and screen for positive transformants to obtain strain WLY-8.

[0078] 9. Integrate the ppc gene at the ycgH pseudogene locus: The same operation method as in ⑧ is used, 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 electrocompetent cells are WLY-8, and strain WLY-9 is obtained.

[0079] 10. Integrate the ppc gene at the mbhA pseudogene locus: The same operation method as in ⑧ is used, 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 electrocompetent cells are WLY-9, and strain WLY-10 is obtained.

[0080] 11. Integrate the aspC gene at the yghX pseudogene locus: The same operation method as in ⑧ is used, 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 electrocompetent cells are WLY-10, and strain WLY-11 is obtained.

[0081] 12. Integrate the aspC gene at the ilvG pseudogene locus: The same operation method as in ⑧ is used, with the difference that the primers used are ilvG-US, ilvG-UA, ilvG-DS, ilvG-DA, aspC-S, aspC-A, pGRB-ilvG-s, and pGRB-ilvG-a. The competent cells are WLY-11, and strain WLY-12 is obtained.

[0082] 13. Integrate the aspC gene at the ydeU pseudogene locus: The same operation method as in ⑧ is used, with the difference that the primers used are ydeU-US, ydeU-UA, ydeU-DS, ydeU-DA, aspC-S, aspC-A, pGRB-ydeU-s, and pGRB-ydeU-a. The competent cells are WLY-12, and strain WLY-13 is obtained.

[0083] 14. Integrate the aspC gene at the yjiV pseudogene locus: The same operation method as in ⑧ is used, with the difference that the primers used are yjiV-US, yjiV-UA, yjiV-DS, yjiV-DA, aspC-S, aspC-A, pGRB-yjiV-s, and pGRB-yjiV-a. The competent cells are WLY-13, and strain WLY-14 is obtained.

[0084] 15. Integrate the lysC gene at the yciQ pseudogene locus: Using the E. coli W3110 genome as a template, respectively use yciQ-US, yciQ-UA and yciQ-DS, yciQ-DA to obtain the upstream and downstream homologous arms by PCR amplification. Design the mutant nucleotide sequence on lysC-tb-A and lysC-tb-S, and there are homologous sequences. Respectively use lysC-S, lysC-tb-A and lysC-tb-S, lysC-A to obtain fragment 1 and fragment 2 by PCR amplification. Then, using fragment 1 and fragment 2 as templates and lysC-S, lysC-A as primers, obtain the target fragment by overlapping PCR amplification. Next, using the upstream and downstream homologous arms and the target fragment as templates and yciQ-US, yciQ-DS as primers, obtain the overlapping fragment by overlapping PCR amplification; Since the Trc promoter has been connected to the 5' end of the primer during primer design, the overlapping fragment carries the Trc promoter, that is, the upstream homologous arm - Trc promoter - target gene downstream homologous arm overlapping fragment; Use pGRB-yciQ-s and pGRB-yciQ-a as primers, anneal to obtain the gRNA fragment, and connect it to the pGRB vector to obtain yciQ-pGRB; Prepare electrocompetent cells of WLY-14, electrotransform the overlapping fragment and yciQ-pGRB into the competent cells together, and screen for positive transformants to obtain strain WLY-15.

[0085] 16. Integrate the lysC gene at the ygaY pseudogene locus: 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 cells are WLY-15, and strain WLY-16 is obtained.

[0086] 17. Integrate the lysC gene at the fhiA pseudogene locus: The same operation method as in ⑮ is used, except that the primers used are fhiA-US, fhiA-UA, fhiA-DS, fhiA-DA, lysC-S, lysC-tb-A, lysC-tb-S, lysC-A, pGRB-fhiA-s, and pGRB-fhiA-a. The competent cells are WLY-16, and strain WLY-17 is obtained.

[0087] 18. Integrate the asd gene at the yjgX pseudogene locus: The same operation method as in ⑧ is used, except that the primers used are yjgX-US, yjgX-UA, yjgX-DS, yjgX-DA, asd-S, asd-A, pGRB-yjgX-s, and pGRB-yjgX-a. The competent cells are WLY-17, and strain WLY-18 is obtained.

[0088] 19. Integrate the asd gene at the gapC pseudogene locus: The same operation method as in ⑧ is used, except that the primers used are gapC-US, gapC-UA, gapC-DS, gapC-DA, asd-S, asd-A, pGRB-gapC-s, and pGRB-gapC-a. The competent cells are WLY-18, and strain WLY-19 is obtained.

[0089] 20. Integrate the asd gene at the yeeP pseudogene locus: The same operation method as in ⑧ is used, except that the primers used are yeeP-US, yeeP-UA, yeeP-DS, yeeP-DA, asd-S, asd-A, pGRB-yeeP-s, and pGRB-yeeP-a. The competent cells are WLY-19, and strain WLY-20 is obtained.

[0090] 21. Integrate the dapA gene at the yjiP pseudogene locus: The same operation method as in ⑮ is used, with the difference that the primers used are 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 cells are WLY-20, and strain WLY-21 is obtained.

[0091] 22. Integrate the dapA gene at the yncK pseudogene locus: The same operation method as in ⑮ is used, with the difference that the primers used are 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 cells are WLY-21, and strain WLY-22 is obtained.

[0092] 23. Integrate the dapA gene at the ybfL pseudogene locus: The same operation method as in ⑮ is used, with the difference that the primers used are 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 cells are WLY-22, and strain WLY-23 is obtained.

[0093] 24. Integrate the dapB gene at the nmpC pseudogene locus: The same operation method as in ⑧ is used, with the difference that the primers used are nmpC-US, nmpC-UA, nmpC-DS, nmpC-DA, dapB-S, dapB-A, pGRB-nmpC-s, and pGRB-nmpC-a. The competent cells are WLY-23, and strain WLY-24 is obtained.

[0094] 25. Integrate the asd gene at the ycdN pseudogene locus: The same operation method as in ⑧ is used, with the difference that the primers used are ycdN-US, ycdN-UA, ycdN-DS, ycdN-DA, dapB-S, dapB-A, pGRB-ycdN-s, and pGRB-ycdN-a. The competent cells are WLY-24, and strain WLY-25 is obtained.

[0095] 26. Integrate the lysA gene at the ylbE pseudogene locus: Using the same operation method as in ⑧, with the difference that the primers used are ylbE-US, ylbE-UA, ylbE-DS, ylbE-DA, lysA-S, lysA-A, pGRB-ylbE-s, pGRB-ylbE-a. The competent cells are WLY-25, and the strain WLY-26 is obtained.

[0096] 27. Integrate the lysA gene at the ychg pseudogene locus: Using the same operation method as in ⑧, with the difference that the primers used are ychg-US, ychg-UA, ychg-DS, ychg-DA, lysA-S, lysA-A, pGRB-ychg-s, pGRB-ychg-a. The competent cells are WLY-26, and the strain WLY-27 is obtained.

[0097] 28. Integrate the lysE gene at the yghE pseudogene locus: Using the same operation method as in ⑧, with the difference that the primers used are yghE-US, yghE-UA, yghE-DS, yghE-DA, lysE-S, lysE-A, pGRB-yghE-s, pGRB-yghE-a. The competent cells are WLY-27, and the strain WLY-28 is obtained.

[0098] 29. Integrate the ddh gene derived from Corynebacterium glutamicum K051 at the rph pseudogene locus: Using the E. coli W3110 genome as a template, and using rph-US, rph-UA and rph-DS, rph-DA respectively, the upstream and downstream homologous arms are obtained by PCR amplification. Using the Corynebacterium glutamicum K051 genome as a template, and using ddh-S, ddh-A respectively, the target fragment is obtained by PCR amplification. Then, using the upstream and downstream homologous arms and the target fragment as templates, and using rph-US, rph-DA as primers, an overlapping fragment is obtained by overlapping PCR amplification; since the Trc promoter has been connected to the 5' end of the primer during primer design, the overlapping fragment carries the Trc promoter, that is, the upstream homologous arm - Trc promoter - target gene downstream homologous arm overlapping fragment; using pGRB-rph-s, pGRB-rph-a as primers, annealing to obtain the gRNA fragment, and ligating it with the pGRB vector to obtain rph-pGRB; preparing electrocompetent cells of WLY-28, electrotransforming the overlapping fragment and rph-pGRB into the electrocompetent cells together, and screening to obtain positive transformants, and the strain WLY-29 is obtained.

[0099] 30. Integrate the ddh gene derived from Corynebacterium glutamicum K051 at the yhjQ pseudogene locus: Using the same operation method as in ㉙, the difference is that the primers used are yhjQ-US, yhjQ-UA, yhjQ-DS, yhjQ-DA, ddh-S, ddh-A, pGRB-yhjQ-s, and pGRB-yhjQ-a. The competent cells are WLY-29, and strain WLY-30 is obtained.

[0100] 31. Knock out cadA gene: Using the same operation method as in ①, the difference is that the primers used are cadA -US, cadA -UA, cadA -DS, cadA -DA, pGRB- cadA -s, pGRB- cadA -a. The competent cells are WLY-30, and strain WLY-31 is obtained.

[0101] 32. Knock out ldcC gene: Using the same operation method as in ①, the difference is that the primers used are ldcC -US, ldcC -UA, ldcC -DS, ldcC -DA, pGRB- ldcC -s, pGRB- ldcC- a. The competent cells are WLY-31, and strain WLY-32 is obtained.

[0102] Example 2

[0103] This example aims to illustrate the shake flask fermentation application of engineering bacteria WLY-32. The specific steps are as follows:

[0104] ① Slant culture: Take the strain WLY-32 obtained in Example 1 as the lysine-producing strain and inoculate it on the slant medium, and culture it at 37 °C for 16 h. The slant medium is selected as the general LB solid medium;

[0105] ② Shake flask seed culture: Inoculate the solid slant strain into the seed medium in the shake flask for fermentation. The culture temperature is 37 °C, the culture time is 16 h, the shaking speed of the shaker is 220 r / min, and the pH is 7.2 - 7.4; The seed medium used is: glucose 30 g / L, yeast powder 5 g / L, peptone 5 g / L, MgSO4·7H2O 0.8 g / L, KH2PO4 3 g / L, ammonium sulfate 5 g / L, biotin 1 mg / L, and the rest is water;

[0106] ③ Shake flask fermentation culture: The fermentation inoculation amount is 20%, the culture temperature is 37 °C, the pH is 7.2 - 7.4, the culture time is 30 h, and the shaker speed is 220 r / min; The fermentation medium used is: glucose 30 g / L, MgSO4·7H2O 1.5 g / L, yeast powder 5 g / L, peptone 2 g / L, ammonium sulfate 15 g / L, K2HPO4·3H2O 4 g / L, FeSO4·7H2O 30 mg / L, VB1, VB3, VB5 each 2 mg / L, and the rest is water.

[0107] Using the wild type E.coli W3110 as the control group, after 30 h of shake flask fermentation verification, the wild type E.coli W3110 failed to accumulate lysine, and the strain WLY-32 described in Example 1 accumulated 20.3 g / L of lysine, proving the effectiveness of this strain.

[0108] Example 3

[0109] This example aims to illustrate the optimization of the copy numbers of the key enzymes aspC, lysC V339A and asd genes of the engineered strain WLY-32 to be 4:3:3. The specific steps are as follows:

[0110] First, during the process of constructing the strain with reference to Example 1, a strain with higher L-lysine production was obtained through directional transformation. Except for the copy numbers of aspC, lysC V339A and asd genes, the rest are the same as WLY-32. According to the fermentation culture method described in Example 2, the copy numbers of aspC, lysC V339A and asd genes were optimized by the shake flask fermentation method. A total of 9 groups of control experiments were set up below, and the data results of 9 groups of shake flask fermentation for 30 h are shown in Table 2.

[0111] Table 2 Effects of the copy numbers of aspC, lysC V339A and asd genes on the L-lysine yield

[0112] 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[Cell 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

[0113] Through the control experiments of the 1st group to the 3rd group, it can be found that as the copy numbers of aspC, lysC V339A, the copy number of the asd gene increases in groups. Although the yield of L-lysine shows an upward trend, the cell biomass shows a downward trend, and the trend is greater than the upward trend of the L-lysine yield. The reason is that the overexpression of one or more genes causes a burden on cell metabolism. Through the control experiments of groups 4 to 9, it can be found that the L-lysine yield of group 5 is the highest, but its cell biomass is still not high. The cell biomass and L-lysine yield of groups 7 and 9 are similar and relatively high. After comprehensive comparison, when the copy numbers of the aspC, lysC V339A , and asd genes are 4, 3, and 3 respectively, the cells not only have a high L-lysine yield but also maintain a relatively high cell biomass, making it a better choice.

[0114] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and retouches can be made. The improvements and retouches such as strain transformation by those skilled in the art using the method of the present invention or based on this method are all regarded as the protection scope of the present invention.

Claims

1. An L-lysine producing strain, characterized in that: it is It was obtained by directed modification based on the starting strain E. coli W3110: the pflB, adhE, ldhA, ackA, frdBC, poxB, lacI, cadA, and ldcC genes were knocked out, and the pntAB, ppc, aspC, and lysC genes were upregulated. V339A 、asd, dapA H56K 、dapB, lysA, lysE genes and their expression was driven by the Trc promoter. The copy numbers of the aspC, lysC V339A 、asd genes are 4, 3, and 3 respectively, and the ddh gene from Corynebacterium glutamicum K051 was heterologously expressed; the pntAB nucleotide sequence of the gene is shown in SEQ ID NO.10 of the sequence listing, ppc the nucleotide sequence of the gene is shown in SEQ ID NO.11 of the sequence listing, aspC the nucleotide sequence of the gene is shown in SEQ ID NO.12 of the sequence listing, lysC V339A the nucleotide sequence of the gene is shown in SEQ ID NO.13 of the sequence listing, asd the nucleotide sequence of the gene is shown in SEQ ID NO.14 of the sequence listing, dapA H56K the nucleotide sequence of the gene is shown in SEQ ID NO.15 of the sequence listing, dapB the nucleotide sequence of the gene is shown in SEQ ID NO.16 of the sequence listing, lysA the nucleotide sequence of the gene is shown in SEQ ID NO.17 of the sequence listing, lysE the nucleotide sequence of the gene is shown in SEQ ID NO.18 of the sequence listing, ddh the nucleotide sequence of the gene is shown in SEQ ID NO.19 of the sequence listing.

2. The L-lysine-producing strain according to claim 1, wherein: The said E. coli W3110 is E. coli W3110 ATCC 27325.

3. The L-lysine-producing strain according to claim 1, wherein: The nucleotide sequence of the pflB gene is shown in SEQ ID NO.1 in the sequence listing; the nucleotide sequence of the adhE gene is shown in SEQ ID NO.2 in the sequence listing; the nucleotide sequence of the ldhA gene is shown in SEQ ID NO.3 in the sequence listing; the nucleotide sequence of the ackA gene is shown in SEQ ID NO.4 in the sequence listing; the nucleotide sequence of the frdBC gene is shown in SEQ ID NO.5 in the sequence listing; the nucleotide sequence of the poxB gene is shown in SEQ ID NO.6 in the sequence listing; the nucleotide sequence of the lacI gene is shown in SEQ ID NO.7 in the sequence listing; the nucleotide sequence of the cadA gene is shown in SEQ ID NO.8 in the sequence listing; the nucleotide sequence of the ldcC gene is shown in SEQ ID NO.9 in the sequence listing.

4. The method for constructing an L-lysine-producing strain according to any one of claims 1 to 3, characterized in that: Based on the starting strain E. coli W3110, the directed modification is carried out 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 content of reducing power NADPH: Express the pntAB gene at the yjiK pseudogene locus using the Trc promoter to upregulate pntAB gene; (3) Strengthen the diaminopimelic acid pathway: upregulate aspC , lysC V339A , asd , dapA H56K , dapB , lysA , lysE genes, among which the lysC and dapA genes are subjected to point mutations; among them, the aspC gene is expressed in multiple copies at the yghX, ilvG, ydeU, and yjiV pseudogene loci using the Trc promoter; the lysC V339A gene is expressed in multiple copies at the yciQ, ygaY, and fhiA pseudogene loci using the Trc promoter; the asd gene is expressed in multiple copies at the yjgX, gapC, and yeeP pseudogene loci using the Trc promoter; the dapA H56K gene is expressed in multiple copies at the yjiP, yncK, and ybfL pseudogene loci using the Trc promoter; the dapB gene is expressed in two copies at the nmpC and ycdN pseudogene loci using the Trc promoter; the lysA gene is expressed in two copies at the ylbE and ychg pseudogene loci using the Trc promoter; the lysE gene is expressed at the yghE pseudogene locus using the Trc promoter; (4) Knock out lysine catabolic pathway genes: cadA and ldcC genes; (5) Shortening the lysine metabolic pathway: upregulating ppc gene and the ddh gene derived from Corynebacterium glutamicum K051, wherein the ppc gene is expressed in a double-copy manner at the ycgH and mbhA pseudogene loci using the Trc promoter, and the ddh gene derived from Corynebacterium glutamicum K051 is expressed in a double-copy manner at the rph and yhjQ pseudogene loci using the Trc promoter; Adjust the copy numbers of aspC, lysC V339A and asd genes to 4, 3, and 3 respectively.

5. Use of the L-lysine-producing strain according to any one of claims 1-3 in the fermentation production of lysine.

6. The application according to claim 5, wherein: Fermentatively cultivate to synthesize L-lysine in a medium, and the specific steps are as follows: ① Slant culture: Inoculate the L-lysine-producing strain on a slant medium and culture at 36-37°C for 14-18 h; ② Shake flask seed culture: Inoculate the slant strain into the seed medium in a shake flask for fermentation, the culture temperature is 36-37°C, the culture time is 12-20 h, the shaker speed is 200-240 r / 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°C, the pH is 7.2-7.4, the culture time is 24-36 h, and the shaker speed is 200-240 r / min.

7. The application according to claim 6, characterized in that: The slant medium is a general LB solid medium; the seed medium used in step ② is: 20-30 g / L of glucose, 3-5 g / L of yeast powder, 3-5 g / L of peptone, 0.5-1 g / L of MgSO4·7H2O, 2-3 g / L of KH2PO4, 4-5 g / L of ammonium sulfate, 1-2 mg / L of biotin, and the rest is water; the fermentation medium used in step ③ is: 20-30 g / L of glucose, 1.5-2 g / L of MgSO4·7H2O, 4-5 g / L of yeast powder, 2-3 g / L of peptone, 10-20 g / L of ammonium sulfate, 3-6 g / L of K2HPO4·3H2O, 20-30 mg / L of FeSO4·7H2O, 2-3 mg / L each of VB1, VB3, and VB5, and the rest is water.

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