RECOMBINANT STRAIN THAT PRODUCES L-LYSINE AND METHODS OF CONSTRUCTION THEREOF AND USE THEREOF

Abstract

A method is provided for introducing point mutations into the coding sequence of the NCg12176 gene or improving its expression in Corynebacterium glutamicum, and a method for introducing point mutations into the promoter region sequence of the dapB gene in Corynebacterium glutamicum. These methods can increase the fermentation yield of L-lysine produced by a strain with the mutations.

Description

Recombinant strain that produces l-lisin and construction methods for the same and use of it This application claims priority of the Chinese Patent application No. 202010790877.0, submitted on August 7, 2020 and entitled “Recombinant Strain Producing L-Lysine and Construction Methods Therefor and use thereof; and the request for Chinese patent No. 202010514023.x, presented on June 8, 2020 and titled“ DAPB Gene Modified recombinant Strain and construction methods therefore and use thereof '; both of which requests are incorporated into the present by reference in its entirety. Technical field of the invention The invention belongs to the technical fields of genetic engineering and microorganisms, and refers to a recombinant strain with greater production capacity of L-Lisin, and construction methods for it and use of it. Background of the invention L-Lisine has physiological functions such as promoting development, improving immunity and improving the function of central nerve tissue. It is one of the eight essential amino acids that humans and animals cannot synthesize for themselves and are necessary for growth. Currently, Llisina is the second largest variety of amino acids in the world and occurs mainly through fermentation. Corynebacterium is the most important strain used in the production of amino acids, including Corynebacterium glutamicum, C. Flavum, C. crenatum, chorynebacterium pekinense, etc. Among them, Corynebacterium glutamicum is an important leisure producing strain. Approximately 90 % of L-Lisin industrial production is used as a nutrition enhancer in the feed industry, and 10 % is used as an aromatizing and sweetener agent in the food industry, as well as a pharmaceutical intermediary product in the pharmaceutical industry. Improvements in fermentation methods to produce L-Lisine can involve fermentation techniques such as oxygen agitation and supply; or involve the composition of the nutritional environment, such as the concentration of sugar during fermentation; or involve the processing of a fermentation broth in an adequate product form, such as drying and granulating the fermentation broth or by ion exchange chromatography; o You can involve inherent performances and properties of relevant microorganisms. Methods to improve the performances and properties of these microorganisms include mutagenesis, selection and screening of mutants. The strains obtained in this way are resistant to metabolites or are auxotrophic aux for regulatory importance and can produce L-Lisin. Taking Corynebacterium glutamicum as an example, 4 mol of NADPH is required to synthesize 1 mol of L-Lisin on the biosynthetic path in C. glutamicum. Therefore, in order to increase the accumulation of L-Lisin in the Biosynthetic path in C. glutamicum, it is a very important strategy I hate to increase the amount of NADPH in the metabolic pathway in C. glutamicum or reduce the amount of NADPH required in the synthetic path of L-Lisin. Dihydrodipicol¡nato reductase (DHDPR) is the second key enzyme in diamineopimelic acid and L-Lisin biosynthesis in bacteria and upper plants and catalyzes the reaction reaction of NAD-dependent dihydrodipicoline (p) h to generate hexahydrodipicoline hexahydrode. This enzyme plays a fundamental role in the formation of the cell wall. DHDPR uses either Nadh or Nadph as a cofactor. DHDPR of different bacteria have different affinities for different cofactors. For example, E. coli DHDPR prefers NADH, while DHDPR C. glutamicum mainly uses NADPH as a cofactor to participate in the synthesis of L-Lisina. Like the DHDPR discovered in other organisms, the DHDPR of C. glutamicum is encoded by the DAPB gene, and its enzymatic activity is not regulated by final products on the synthetic path, but is inhibited by the 2.6piridindicarboxylic acid (2,6-PDC). Lysine performance is related to enzymatic activity on the Biosinthetic path, which can generally be improved by amplifying one or more genes in the lysine biosynthetic pathway or applying modified promoters to genes. Brief description of the invention The invention provides a microorganism producing L -Lisine or a recombinant strain, where the expression of a polynucleotide that encodes the sequence of amino acids of the SEQ ID no: 3 is improved, and / or, the bases in the positions -49, -51 and -54 to -58 in the promoter region shown in the SEQ NO: 29 are mutated. The invention also provides a method to produce L-Lisin using the microorganism or recombinant strain. In a first aspect, the invention provides a L-Lisine producing microorganism or a recombinant strain belonging to the genus Corynebacterium, which has an improved expression of a polynucleotide that encodes the amino acid sequence of the SEQ id no: 3. According to the invention, the improved expression refers to the improvement of the expression of the polynucleotide, or the pollinucleotide. Amino acid sequence ID NO: 3 has a punctual mutation, or the polynucleotide that encodes the sequence of amino acids of the SEQ NO: 3 has a punctual mutation and improved expression. The sequence of amino acids of the SEQ NO: 3 is the amino acid sequence of the protein encoded by the NCG12176 gene. Compared to the wild or parental strain, the microorganism or the recombinant strain has a greater capacity for production of L-Lisine. Polynucleotide can encode a amino acid sequence that has approximately 90 % or more, approximately 92 % or more, approximately 95 % or more, approximately 97 % or more, approximately 98 % or more, approximately 99 % or more of homology of sequence with the amino acid sequence of the SEQ ID no: 3. As used in the present, the term "homology" refers to the percentage of identity between two modules of polynucleotes or of polypeptides. The homology of sequence between one module and another can be determined by methods known in the technique. For example, such sequence homology can be determined by the Blast algorithm. Polynucleotide expression can be improved by replacing or mutating a regulatory sequence of expression, introducing a mutation in the polynucleotide sequence, increasing the number of copies of the polynucleotide by chromosomal inclusion or the introduction of the vector, or a combination of them, and the similar ones. The polynucleotide expression regulatory sequence can be modified. The expression regulatory sequence controls the expression of the polynucleotide to which it is operationally linked, and can include a promoter, a terminator, a potentiar, a silencer, etc. Polynucleotide can have a change in the start codon. Polynucleotide can be incorporated into a chromosome into a specific place, thus increasing the number of copies. In the invention, the specific site includes a transposon site, an intergenic site, etc. In addition, polynucleotide can be incorporated into an expression vector and then the expression vector is introduced into a host cell, thus increasing the number of copies. In a modality of the invention, polynucleotide or polynucleotide with punctual mutation is incorporated into a microorganism chromosome in a specific place, thus increasing the number of copies. In a modality of the invention, the polynucleotide that carries a promoter sequence or the polynucleotide with a timely mutation that carries a promoter sequence is incorporated into a chromosome of the microorganism in a specific site, thus overexpressing, the sequence of nucleic acid. In a modality of the invention, polynucleotide or polynucleotide with punctual mutation is incorporated into an expression vector and then the expression vector is introduced into a host cell, thus increasing the number of copies. In a modality of the invention, the polynucleotide that carries a promoter sequence or the polynucleotide with a timely mutation that carries a promoter sequence is incorporated into an expression vector, and then the expression vector is introduced into a host cell, thus overexpressing the sequence of nucleic acid. In a specific modality of the invention, polynucleotide can understand the nucleotide sequence of SEQ ID no: 1. In a modality of the invention, the polynucleotide that encodes the sequence of amino acids of the SEQ ID: 3 has a punctual mutation so that the lysine residue in position 176 of the amino acid sequence of the SEQ id no: 3 is replaced with a different amino acid residue. According to the invention, it is preferred that lysine residue in position 176 will be replaced with asparagine residue. According to the invention, the amino acid sequence after the lysine residue (K) in position 176 of the amino acid sequence shown in the SEQ NO: 3 is replaced with an asparagine residue (n) that is shown in the SEQ id no: 4. In a modality of the invention, the polynucleotide sequence with punctual mutation is formed by the mutation of the base in position 528 of the polynucleotide sequence shown in SEQ ID no: 1. According to the invention, the mutation includes the adenine base mutation (a) to cytosine (c) in position 528 of the polynucleotide sequence shown in the SEQ NO: 1. In a modality of the invention, the polynucleotide sequence with punctual mutation includes the polynucleotide sequence shown in the SEQ NO: 2. As used in the present, the term linked operationally refers to a functional link between a regulatory sequence and a polynucleotide sequence, so the regulatory sequence controls the transcription and / or translation of the polynucleotide sequence. The regulatory sequence can be a strong promoter capable of increasing the level of polynucleotide expression. The regulatory sequence can be a promoter derived from microorganisms belonging to the genus Corynebacterium or can be a promoter derived from other microorganisms. For example, the promoter can be a TRC promoter, a GAP promoter, a CT promoter, a promoter T7, a LAC promoter, a TRP promoter, a Arabad promoter or a CJ7 promoter. In a specific modality of the invention, the promoter is the promoter of the polynucleotide (GEN NCG12176) that encodes the sequence of amino acids of the SEQ ID no: 3. As used in the present, the term vector refers to a polynucleotide construct that contains a gene sequence and its regulatory sequence and is configured to express the white gene in an adequate host cell. Alternatively, the vector can refer to a polynucleotide construct that includes useful sequences for homologous recombination, so the regulatory sequence of the endogenous gene into the genome of the host cell can be altered or the white gene that can be expressed is incorporated into a specific place in the host genome after introducing the vector into the host cell. In this regard, the vector used in the invention can also understand a select marker to determine the introduction of the vector into the host cell or the incorporation of the vector into the chromosome of the host cell. The selectable marker may include the score that confers a selectable phenotype such as drug resistance, auxotroph, resistance to cytotoxic agents, or surface protein expression. In the context of the use of such selective agents, transformed cells can be selected because only cells that express the selectable marker can survive or show different phenotypic features. In some specific modalities of the invention, the vector used is the PK18mobsacb plasmid or the PXMJ19 plasmid. As used in the present, the term transformation refers to the introduction of a polynucleotide in a host cell so that polynucleotide is replicable as an extragenomic element or as an element incorporated into the genome of the host cell. The vector transformation method used in the invention may include a method to introduce the nucleic acid molecule into the cell. In addition, as disseminated in the related technique, the electrical impulse method can be used according to the host cell. According to the invention, the microorganism or recombinant strain belonging to the genus oo i o Corynebacterium can be Corynebacterium glutamicum, Brevibacterium flavum, brevibacteríum lactofermentum, chorynebacterium ammonyogenes, chorynebacterium pekinense. In a modality of the invention, the microorganism that belongs to the genus Corynebacterium is Corynebacterium glutamicum YP97158, which was deposited on August 16, 2016 at the General Center for Microbiological Crops of China (Address: No.1 West Beichen Road, Chaoyang District, Beijing, Telephone: Telephone: 010-64807355) with a CGMCC Deposit number No. 12856 and has been recorded in the Chinese patent application CN106367432A (presentation date: September 1, 2016, publication date: February 1, 2017) According to the invention, the microorganism or recombinant strain can also have other improvements related to the increase in the production of L-Lisine, for example, increasing or reducing the expression of genes related to the production of NADPH (such as the gene that encodes the glucose dehydrogenase, the gene that encodes the kinase gluconate, the gene that encodes the glyceraldehyde-3-phosphate dehydrogenase, the gene that encodes the glucose-6-phosphate dehydrogenase, or the gene that encodes the 6-phosphogluconate dehydrogenase) and / or other genes involved in the biosynthesis or secretion of L-Lisine (such as the gene that encodes aminotransferase aspartate, the gene that encodes the cinase cinase SEMALDEHIDO ASPARTATO DEGIDROGENASE, the gene that encodes the synthade dihydhydopicoline, the gene that encodes the reductase dihydrodipicoline, the gene that encodes the M-Diaminepimellate dehydrogenase, the gene that encodes the diaminopimellate Discarboxylase, Lyse), or the replacement of an external gene. In a second aspect, the invention provides a polynucleotide sequence, a amino acid sequence encoded by the polynucleotide sequence, a recombinant vector that comprises the sequence of polynucleotides, and a recombinant strain that contains the polynucleotide sequence. According to the invention, the polynucleotide sequence includes a polynucleotide that encodes a peptide that comprises the sequence of amino acids of the SEQ ID no: 3, where the lysine residue in position 176 is replaced with a different amino acid residue. According to the invention, it is preferred that lysine residue in position 176 will be replaced with asparagine residue. According to the invention, the amino acid sequence after the lysine residue (K) in position 176 of the amino acid sequence shown in the SEQ NO: 3 is replaced with an asparagine residue (n) that is shown in the SEQ id no: 4. According to the invention, it is preferred that the polynucleotide sequence that encodes a polypeptide that comprises the amino acid sequence shown in the SEQ NO: 3 includes the sequence of polynucleotides shown in the SEQ ID no: 1. In a modality of the invention, the polynucleotide sequence is formed by the mutation of the nucleotide in position 528 of the polynucleotide sequence shown in the SEQ NO: 1. According to the invention, the mutation refers to the change in the site base / nucleotide, and the mutation method can be selected from at least one selected between mutagenesis methods, site-directed mutagenesis of by by and recombination homologous. In the invention, preferably, Jo I or Mutagenesis Site-Directive of PCR and / or Recombination homologous. According to the invention, the mutation includes the adenine base mutation (a) to cytosine (c) in position 528 of the polynucleotide sequence shown in the SEQ NO: 1. In a modality of the invention, the polynucleotide sequence includes the polynucleotide sequence shown in the SEQ NO: 2. According to the invention, the amino acid sequence includes the amino acid sequence shown in SEQ ID no: 4. According to the invention, the recombinant vector is constructed by introducing the polynucleotide sequence in a plasmid. In a modality of the invention, the plasmid is the PK18mobsacb plasmid. In another modality of the invention, the plasmid is PXMJ19. Specifically, the polynucleotide sequence and plasmid can be built in a recombinant vector through the Nebuider recombination system. According to the invention, the recombinant strain contains the polynucleotide sequence. As a modality of the invention, the starting strain of the recombinant strain is YP97158. In a third aspect, the invention also provides a method of building a recombinant strain of Corynebacteríum glutamicum. According to the invention, the construction method includes the passage of: Modify the polynucleotides sequence of the NCG12176 wild type as shown in the SEQ NO: 1 in a host strain to mutate the base in position 528, thus obtaining a recombinant strain of the genus Corynebacteríum that contains the encoding gene NCG12176 mutated. According to the method of building the invention, the modification method can be at least one selected from mutagenesis, Mutagenesis directed site of PCR and homologous recombination methods. According to the method of building the invention, the mutation refers to the adenine mutation (a) to cytosine (c) in position 528 of the SEQ ID no: 1; Specifically, the polynucleotide sequence includes the coding gene of NCG12176 mutated shown in SEQ ID no: 2. In addition, the construction method includes the following steps: (1) Modify the polynucleotides sequence of the NCG12176 wild type as shown in the SEQ NO: 1 to mutate the nucleotide in the 528 position, thus obtaining the sequence of polynucleotides of the NCG12176 gene mutated gene; (2) link the sequence of mutated polynucleotides with a plasmid to build a recombinant vector; (3) Enter the recombinant vector in a host strain, thus obtaining a recombinant chorynebacteríum strain containing the coding gene of NCG12176 mutated. According to the method of construction of the invention, the step (1) includes the step of building the NCG12176 mutated promptly: according to the genomic sequence of Corynebacteríum glutamicum, synthesize two pairs of primers, p1 and p2, and p3 and p4, to amplify the fragment of the gene NCG12176, introducing a specific mutation in the gen. NCG12176 Wild type as shown in SEQ ID NO: 1 by Mutagenesis Site-Directive PCR, thus obtaining the NCG12176 gene with a timely mutation that has the sequence of nucleic acid of the SEQ id no: 2, which is indicated as NCG12176A528C. In a modality of the invention, the Corynebacterium glutamicum genome can be derived from the ATCC13032 strain, whose genomic sequence is available on the NCBI website. In a modality of the invention, in step (1), primers are like below: P1: 5 ’CAGTGCCAAGCTTGCTGCTGGGGTCTCTAGCGGCGAC CGCATGGACCG 3 '(SF.Q ID NO: 5) P2: 5 'CcGGGACTGGTTTTCGGGTGTGGTGTGC 3 ’(SEQ ID NO: 6) P3: 5 'GCACCCAACCCGGAAACCAGTCCCGG 3' (SEQ ID NO: 7) P4: 5 ’CagctatgaccattacgaAtcGagCTCGTACCCGGG TCTCTCAATCGGT 3 '(SEQ ID NO: 8) In a modality of the invention, the PCR amplification procedure is as follows: 30 denaturation cycles at 94 ° C for 30 s, recounted to 52 ° C for 30 s, and extension at 72 ° C for 40 s. In a modality of the invention, the superimposed PCR amplification procedure is as follows: 30 denaturation cycles at 94 ° C for 30 s, annealing at 52 ° C for 30 s, and extension at 72 ° C for 90 s. According to the method of construction of the invention, the step (2) includes the step of building a recombinant plasmid, which includes: assemble the NCG12176A528Caislado and purified and the plasmid PK18mobsacb using the nebuider recombinant system to obtain a recombinant plasmid PK18-NCG12176A528C. According to the method of building the invention, step (3) includes the step of building a recombinant strain, which includes: transform the recombinant plasmid PK18ncg12176A528Cen a host strain, thus obtaining a recombinant strain. In a modality of the invention, the method of transformation in step (3) is an electrotransformation method. In a modality of the invention, the host strain is YP97158. In a modality of the invention, recombination is achieved by homologous recombination. In a fourth appearance, the invention also provides a method of building a recombinant corynebacterium strain. According to the invention, the construction method includes the following steps: amplify the homologous arm fragments upstairs and current down the NCG12176 gene, the coding region of the NCG12176 gene and its sequence of the promoter region, or the coding region of the NCG12176A528Cy gene its sequence of the promoter region, and introduce the NCG12176 or NCG12176A528 Recombination homologous to perform overexpression of the NCG12176 or NCG12176 A528C in the strain. In a modality of the invention, the primers to amplify the fragment of the upper -homologous arm current are like below: P7: 5'CagtgccaagtgcatgCCCGGGTCTCTAGAATGCGTT CTGGACTGAGG3 '(SEQ ID NO: 11) P8: 5'aacaccati g1ccctgti ti gggcgaaa 1ίί 1Ccggcaccgagg Aacagatg3 '(seq id no: 12) „ In a modality of the invention, the primers to amplify the fragment of the backward arm downstream are like below: P11: 5'CTACGAGACGAAGCGTTCGCTGAGGGCGCAATTAAATCAAG 3 '(SEQ ID No: 15) P12: 5'CagctatgacCatgattacg Aattcg Agtcggtaccccctat GACACCTTCAACGGATC 3 '(SEQ ID NO: 16) » In a modality of the invention, the primers to amplify the coding region of the gene and its sequence of the promoter region are as follows: P9: 5TGGGAAAATRRCOCRCAAACAGGGAATGGTGTI ATGGCATN GCAGACA TTGTGCGC3 ’(SEQ NO: 13) P10: 5'CttgattTTTGCCCTCTCGGCGAACGCTTCTCTCGTAG3 ’(SEQ id No: 14) » In a modality of the invention, using P7 and P11 previous as primers and the three mixed fragments of the upper homologous fragment, the homologous fragment downstream and the gene NCG12176 or NCG12176A528Con its own promoter such as the template, a PCR was performed to obtain a fragment of an integrative homologous arm. In a modality of the invention, the PCR system used is as follows: Ex taq 10x 5 pl, dntp mix (2.5 mm each) 4 pl, mg2+(25 mm) 4 pl, primers (10 pm) 2 pl each, ex taq (5u / pl) 0.25 pl, total volume 50 pl; The PCR amplification procedure is as follows: Initial denaturation at 94 ° C for 5 min; 30 denaturation cycles at 94 ° C for 30 s, appeal at 52 ° C for 30 s, and extension at 72 ° C for 120 s; and a final extension at 72 ° C for 10 min. In a modality of the invention, the Nebuider recombination system is used to assemble the plasmid cloning PK18Mobsacb and the fragment of the homologous integration arm to obtain an integration plasmid. In a modality of the invention, the integration plasmid is transfused into a host strain, and the NCG12176 or NCG12176A528CSE introduces into the host strain genome by homologous recombination. In a modality of the invention, the host strain is YP97158. In a modality of the invention, the host strain is a strain that carries the sequence of polynucleotides shown in the SEQ NO: 2. In a fifth aspect, the invention also provides a method of building a strain of recombinant Corynebacterium. According to the invention, the construction method includes the following steps: Amplify the coding region of the NCG12176 gene and its sequence of the promoter region, or the coding region of the NCG12176A528Cy gene its sequence of the promoter region, build a vector of overexpression plasmid and introduce the vector in a host strain to perform the overexpression of the NCG12176 or NCG12176A528 cEP. In a modality of the invention, the primers to amplify the coding region of the gene and its sequence of the promoter region are as follows: P17: 5'gcttgcatgCTGGGTCGTCTAGAGGTCCCCGGGGAAAATTTCGCCCC Aaaacag3 '(seq id no: 21), P18: 5'atcaggctgaaaatc'n ctctcatccgC'caaaactcagcgaacggcttcgt CTCGTAG 3 ’(SEQ ID NO: 22) OR In a modality of the invention, the PCR system is like then: Ex taq 10x 5 pl, mixture dntp (2.5 mm each) 4 pl, mg2+(25 mm) 4 pl, primers (10 pm) 2 pl each, ex taq (5u / pl) 0.25 pl, total volume 50 pl; The PCR amplification procedure is as follows: Initial denaturation at 94 ° C for 5 min; 30 denaturation cycles at 94 ° C for 30 s, appeal at 52 ° C for 30 s, and extension at 72 ° C for 90 s; and a final extension at 72 ° C for 10 min. In a modality of the invention, the nebuider recombination system is used to assemble the PXMJ19 cloning plasmid and the NCG12176 or NCG12176A528CON fragment with its own promoter to obtain a overexpression plasmid. In a modality of the invention, the host strain is YP97158. In a modality of the invention, the host strain is a strain that carries the sequence of polynucleotides shown in the SEQ NO: 2. The recombinant strain obtained by the invention can be used by itself to produce llisin by fermentation or can be mixed with other L-Lisine producing bacteria to produce L-Lisine by fermentation. In an additional aspect, the invention provides a nucleotide sequence of the promoter, which comprises the sequence of nucleotides obtained by mutation of the bases in the positions -49, -51 and -54 to -58 in the promoter region shown in the SEQ id no: 29. According to the invention, the nucleotide in the -49 position of the promoter region shown in the SEQ NO: 29 is mutated from cytosine (c) to adenine (a), and the nucleotide in the position -51 is mutated from guanine (g) to timina (t), and the nucleotide sequence of the positions -54 to -58 is mutated from Ctgca to Ggtgt. According to the invention, the promoter's nucleotide sequence is as follows: (a) The nucleotide sequence shown in SEQ ID no: 30; or (b) a nucleotide sequence that has at least 90 % identity, preferably at least 95 % or at least 98 % identity, with the nucleotide sequence shown in the SEQ ID no: 30 and maintaining the promoter enhancement activity in (a), in which the nucleotide in the position -49 is maintained as adenine (a), the nucleotide in the position -51 Timina (T), and the nucleotide sequence of positions -54 A -58 remains as GGTGT. The invention also provides an expression cassette that includes the previous promoter, where the expression cassette comprises the promoter and a coding sequence operationally linked to the promoter. In a modality of the invention, the coding sequence is the coding sequence of the DAPB gene. The invention also provides a recombinant vector that includes the nucleotide sequence of the promoter of the invention. According to the invention, the recombinant vector is constructed by ligating the nucleotide sequence of the promoter of the invention with a cloning plasmid; In a modality of the invention, the cloning plasmid is the PK18mobsacb plasmid. The invention also provides a recombinant strain containing the nucleotide sequence of the previous promoter or the previous recombinant vector. According to the recombinant strain of the invention, it contains the nucleotide sequence shown in SEQ ID no: 30. The nucleotide sequence shown in the SEQ NO: 30 is the promoter region of the DAPB gene. In addition, the nucleotide sequence shown in SEQ ID No: 30 is linked to the coding sequence of the DAPB gene. Specifically, the recombinant strain may contain the expression cassette or recombinant vector of the invention mentioned above, specifically, the recombinant strain of the invention is obtained by transforming an expression cassette or a recombinant vector. According to the recombinant strain of the invention, it is formed by introducing the nucleotide sequence of the mutated promoter in a host strain for recombination; The host strain can be selected from strains of L-Lisin known in the technique, for example, selected from at least one of the strains of Corynebacterium, the strain of Corynebacterium can be Corynebacterium glutamicum, Brevibacterium flavum, Corynebacterium crenatum, chorynebacterium pekinense; preferably Corynebacterium glutamicum. In a modality of the invention, the host strain is YP97158. According to the recombinant strain of the invention, the PK18Mobsacb plasmid is used as a vector. According to the recombinant strain of the invention, it can also understand other modifications. The invention also provides a method of building a recombinant strain producing L-Lisina, which includes the following steps: (1) Modify the promoter region as shown in the SEQ NO: 29 to mutate the bases in positions -49, -51 and -54 to -58, to obtain a nucleotide sequence that comprises the mutated promoting region. According to the invention, the mutation refers that the nucleotide in the position -49 of the promoter region shown in the SEQ NO: 29 is mutated from cytosine (c) to adenine (a), and the nucleotide in the position -51 is mutated from guanina (g) to timina (t), the nucleotides sequence of the positions -54 to -58 is mutated from Ctgcaa GGTGT. Specifically, the nucleotide sequence of the mutated promoting region is shown in SEQ ID No: 30. Additionally, the construction method also includes the following steps: (2) link the polynucleotide sequence of the mutated promoting region with a plasmid to build a recombinant vector; (3) Enter the recombinant vector in a host strain to obtain a recombinant strain producing L-Lisin containing the mutated promoting region. According to the invention, in the passage (1), the mutation method includes mutagenesis, site-direction mutagenesis of or recombination homologous, preferably mutagenesis site-directed site by by. According to the invention, step (1) includes: Design two pairs of primers to amplify the DAPB gene region and then perform PCR to obtain the nucleotide sequence of the mutated promoting region. In a modality of the invention, the primers used in step (1) are like below: Ργ: 5'cggaattcaccatgcggacatgggac3 '(Cor i) (seq id no: 31) P2 ’: 5 'CCTTCTGAACGGGTGTGGTA TAA TGGTGG 3' (SEQ ID NO: 32) P3 ’: 5 'CCACCATTATACCACACAACCCGTTTCAGAAGG 3’ (SEQ ID NO: 33) P4 ’: 5 'acatgcatgcgaattgacgttgaggaag v (SPH 1) (seq id no: 34) C In a modality of the invention, the step (1) includes: use Corynebacterium glutamicum atcc13032 as the template, perform amplification by PCR with the primer P1 'and P2', P3 'and P4', respectively, to obtain two DNA fragments that contain specific mutations; Using the two DNA fragments such as the template, carry out an amplification by PCR superimposed with the primer ργ and P4 ', to obtain a fragment of DNA that includes the nucleotide sequence (SEQ ID no: 30) of the promoting region of the invention. According to the invention, in passage (1), by means of amplification by superimposed PCR, the two ends of the DNA fragment obtained contain Ecori and Sphl restriction sites, respectively. According to the invention, the step (2) includes: isolating and purifying the amplified product by means of the superimposed PCR reaction, linking the doubly digested fragment (by Ecor L / SPH I) with the same doubly digested fragment (by Ecor l / SPH I) Cloning plasmid to obtain a recombinant vector by allelic substitution. According to the invention, the cloning plasmid is the PK18Mobsacb plasmid; The built recombinant vector is PK18-PDAPB (C (‘49) Ag (-51) T, CTGCA (-54-58> GGTGT) In a modality of the invention, the recombinant plasmid has a kanamycin resistance marker. In a modality of the invention, the method of transformation in step (3) is an electrotransformation method; As an example, in the passage (3), the recombinant plasmid is transformed into the yp97158 strain. The invention also provides the use of the microorganism or recombinant strain mentioned above of the invention in the preparation of L-Lisin; or a method to increase L-Lisin fermentation performance; or a method to produce L-Lisin. According to the use and method of the invention, it includes fermenting the microorganism or recombinant strain, and recovering the crop l-lisin to prepare L-Lisin. According to the use and method of invention, the recombinant strain of the invention can be used alone or mixed with other L-Lisine producing bacteria. The microorganism can be cultivated in an adequate environment under cultivation conditions known in the technique. The medium can contain: carbon sources, nitrogen sources, trace elements, and their combinations. During the cultivation process, the crop pH can be adjusted. In addition, during the cultivation process, the passage of preventing the generation of air bubbles can be included, for example, by using an antispuming agent. In addition, during the cultivation process, the step of injecting gas in the crop can be included. The gas may include any gas capable of maintaining the aerobic conditions of the crop. During the cultivation process, the temperature can be 20 ° C to 45 ° C. The resulting L-Lisine can be recovered from the crop treating cultivation with sulfuric acid or hydrochloric acid and then a combination of methods such as anionic exchange chromatography, concentration, crystallization and isoelectric precipitation can be performed. In the invention, Seq ID NO 1: sequence of NCG12176 Silvestre AtgcatttgcagacattgtgcGCGCGTCGAAACCCCCCGCGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC CTCCGGGCGTCGTGGCGTCTTGCGCGTGTCATGTCCAACCCGGAAAATTTGCC TTGTCGCGCCCCCCCCCCCCAATCAATTACCCAACAACCACCGCGGTTGGCGCCCGTTC TTCACCATCCAAGTGCCCCCTGCCAACTGTCACCGTGGGGGAAAACAGTTA CCTCATCCCCACCACACACACACACACACGCTTTTTGACCTCTGGTGGGAAGACCACACACACACAACCTTGGA CCCCGCTGGCCACCACCAGAGCCGGGTTCCCCCCCGGCCCCCCCCCCCCCCCCCTCTCTCGTGGGAAAACCCCCCGGGGTCTCTCCCGACTCGACGACCCA TCATGGTCCCTGGCTCCCCCCACTCTCGCGCATGGGAACTCGGGTTTTTTGCC ACCAAAACCCOAAACTCCCCCGGGGAACCGCTTCTACTACGAAGTCTCTCAAAG ACCCCCCGCCCCCCGTGTTCCCCCCCCGCGCCA'RGGAACCLTRGAAACC CTCCAAGOGTTCATCAACAAACACCCCCCCCCGCGCCCCTGCTGCTCCCGT GGGGACCAACCCCCACGACTATTCCGTCAGGTCAAGAGCACACAAGAAGTCCAACT GCGCAACCTGTTTTATCGAATACCCGACATGAAATGTCCTCGGGCGACGTGGCCGCCCCCCCCCCCCCCCCCCCCTCTCGGCGCGAAGGTCGAAGAACCCCCCCCCCTCTCGCGCGG GCGTRGCAATCCGTGGC'RCTCCCCCGCGGAACTGTGCCCCACGGGAACTGCC GTCACTGTCCCACCACCACCCAACGGGGCCCAAGGTCCCCGTTTCCGCCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGT TGCTGCGCGGCGGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGG CTACGAGACGAAGCGTTCG CCTGA IVIA / A / ZUZZ / U I HOB Seq Id No 2: ORF sequence of NCG12176A528C ATGGCATTTGCAGACATTGTGCGCAGCGTCGAAAACCGCACCAACGCAGCGACCC TCAACTGGTCCATCAAAAATGGCTGGAAGCCCGAAGTCACCGGATTTTCCGGGTACGG CTCCGGGCGTCGTGCGTCCCCCGCGTGTCATGTCCAACCCGGAAAATRRGC TRGTCGCCCCCCCCCCCCCAATACCCCCCAACACACCACCGGGTTGGCCCCGTTC TTCACCATCCAAGTGCCCCCTGCCAACTGTCACCGTGGGGGAAAACAGTTA CCTCATCCCCACCACACACACACACACACGCTTTTTGACCTCTGGTGGGAAGACCACACACACACAACCTTGGA CCCCGCTGGCCACCACCAGAGCCGGGTTCCCCCCCGGCCCCCCCCCCCCCCCCCTCTCTCGTGGGAAAACCCCCCGGGGTCTCTCCCGACTCGACGACCCA TCATGGTCCTGGCTTCCCCCCACTCTCGCCTGGGAACTCGGGTTTTTTTGCC ACCAACCCGGAAAACCTCCCCCGGGAACCGCTTCTACTACGAATCCTCAAAG ACCCCCCGCCCCCGTGTTCTCTCTCCCCGCGCATGGGAACCTTTTGGAAACC CTCCAAGTTTCATCAACAACAAACCCCCCCCCGCGCCCCTGCTGCTCCCGT GGGGACCAACCCCCACGACTATTCCCCTCAGGTCAAGAGCACACAAAGTCCAACT GCGCCTGTTTTTTTTTTCGAATACCCCGACTGAAATGTCCTCGTCGGCGTGGCGC CAACACGATCCCCTCATCGGCGGAAGGTCGAAGAACCCCCCTCGCAGG GCGTTGCAATCCGTGTCTCCCCCCCCGGCGGGGTGTGCTCCCCCACGGGAACTGC GTCACTGTCCACCATCACCCCAACGGGGCCAAGGTCCGTTTTCACGGCCGCCGCCGCCGCCGCGTGT TGCTGCAGCGCGCGCGCGCGCGTTCG CCTGA SEQ NO 3: Amino acid sequence of the protein encoded by the NCG12176 Silvestre type gene M a AfadivRTNAATLNWSIKNGWKPEVTGFSGSGRRVLARVLMSNPEN I.LVDAPSQSITQQAQRGWRQFFTIQVPNLPVTVTVGGKTVTSSTNSTNDNGYVDLLVEDHNLD PgwhtiqiqaegstpaarvlivariglisdidtimvtwlprallaAwnswvlhtktr kpvpgmnrfyeellkdhpdapvfyls Igawnteeilqefinkhalpdgpmllidwgp γρτ GLFRSGQEHKKVQLRNLFIEYPDMKW1LVGDGQHDPLIYGEAHPNRIGVAIRELSP GEHVLSHGTTA SLST1TTNGG QGVPVVHGRD GYELLQRYE KPFA Seq ID NO 4: amino acid sequence of the protein encoded by the NCG12176K176N gene MafadivRTNAATLNWSIKNGWKPEVTGFSGYGSGRRVLARVLMSNPEN LLVDAPSQSITQQAQRGWRQFFTIQVPNLPVTVTVGKTVTSSTNDNGYVDLLVEDHNLD PGWHT1QIQAEGSTPAEARVL1VARTAR1GL1SD1DDT1MVTWLPRALLAAWNSWVLHTNTNT KPVPGMNRFYEELKDHPDAPVFYLSTGAWNTFETLQEFINKALPDGPMLTDWGPTPT GLFRSGQEHKKVQLRNLF1EYPDMKWILVGDGQHDPLIYGEAVNRIGVA1RELSP GEHVLSHGTTA SLSTHTNGGG QGVPVVHGRD GYELLQRYET KPFA SEQ ID NO 29: SILVESTRE TYPE PROMOTER CLTAAGTCTC atatttcaaAA CATAGTCCA CCTGTGLGAT TAATCCTAG AACGGAACAAACTGATGAAC AATCGTTAAC AACACAGACC AAAACGGTCA GTTAGGTATG GATATCAGCCCTTTTTGTGGAAC GGGTCTTCT AGACTGGGG GCGTTTTGGAAA AACTCTTCGC CCCACGAAAA TGAAGGAGCA TA HATE Seq id no 29: Muta Promoting sequence CTTAAGTCLC atatttcaaa Calagtlcca Cctglgat LaalcccCTAG AACGGAACAAACTGALGAAC AatCGTTTAAC AACACAGACCC AAAACGGTCA GLLAGGGTATG GATATATCAGCACCCTLETGAAC GGGTTGGGGG A (AA (GGTGG GCGTTLGAAA AACLCTTCGCCCGAAAAA TGAAGGAGCA TA In the invention, through performing a regulation down or inactivation / elimination (Knockingout) of the NCG12176 gene, it was found that the product encoded by the gene has an impact on the production capacity of L-Lisin, and by introducing a punctual mutation in the coding sequence, or increasing the number of copies of the gene or overexpressing the gene, a recombinant strain is obtained for high concentration production of L-Lisin compared to the not modified strain. In addition, a recombinant strain is obtained by introducing specific mutations in the promoter region of the DAPB gene. Compared to the non-mutated strain, the strain obtained also greatly increased the production of L-Lisin, further improving production efficiency and the cost of production was reduced. Therefore, the invention is adequate for its promotion and application. Detailed description of the invention The technical solutions of the invention will be described in greater detail below with reference to specific examples. It should be understood that the following examples are merely an illustration and explanation of an example of the invention, and should not be interpreted as a limitation of the scope of protection of the invention. The technologies implemented based on the previous content of the invention are all covered by the scope of the planned protection of the invention. Unless otherwise indicated, raw materials and reagents used in the following examples are all commercially available, or they can be prepared through known methods; The operations carried out are also known in the technique, or are carried out according to the user manuals of commercially available products. OO I O. In the following examples, the compositions of the basic media used for the cultivation of the strains are identical, and to this basic medium you can add sucrose, kanamycin or chloramphenicol, etc. correspondingly necessary. The composition of the basic medium is like below: TAKEROUS FORMULA COMPONENT 10 g / L POLYUPP The preparation and conditions of SSCP-Page in the following examples are like below: Component Quantity (Final acrylamide concentration: 8 %) 40 %Acrylamide 8 ml DDH2O 26 ml glycerin 4 ml 10*Tbe 2 ml TeMed 40 PL 10 %Ap 600 PL Electrophoresis conditions The electrophoresis tank is placed on ice and used 1XTBE damping; Voltage: 120 V; Electrophoresis time: 10h The fermentation medium formula and the L-Lisine fermentation control process in the following examples are like below: Table 1 Fermentation medium formula Formula component Hydrolyzed sugar of starch 30 g / l (NH4) 2so4 12 g / l mgso4 0.87 g / l syrup 20 g / l Acidified corn pulp 3 ml / l h3po4 0.4 ml / l kci 0.53 g / l antispumant agent (2% enemy of bubbles) 4 ml / l feso4 120 mg / l mg / l mg / l mg / l nicotinamide 42 mg / l Calcium Pantotenato 6.3 mg / l vitamin B1 6.3 mg / l copper and zinc salt solution 0.6 g / l Biotin 0.88 mg / l Table 2 fermentation control process TEMPERATE CALIBRATION: 37 ° C, air volume: 4 l / min, speed: 1000 rpm, tank pressure: 0 MPa, calibrate after 5min amount of inoculum 10% crop temperature 37 ° C Cc) pH pH 6.9+0.05 Dissolved oxygen (OD) 10 to 30 % Initial conditions Temperature: 37 ° C, pH 6.9, tank pressure: 0 MPa, air volume: 3 l / min, speed 550 rpm Control of the complete process Control of the complete process: 1. when the OD is less than 30 %, increase the speed of 750 rpm—> 800 rpm^volume of air: 4l / min. RPM—> 950 rpm; 2. At 6 hours of fermentation, increase tank pressure to 0.01 MPA; At 12pm hours of fermentation, increase the pressure of the tank of 0.02 MPa^ 0.03 MPa—> 0.04 MPa^ 0.05 MPa Residual sugar control 0.1-0.2 % before F12H; After F12H, combined with the OD requirement, the residual sugar is controlled to be 0.1-0.05 % ammoniaconitrogen control 0.1-0.15 before F12H; 0.15-0.25 during F12-F32H; 0.1-0.15 After f32h lot materials fed 25 % ammoniacal water, 70 % concentrated sugar, 50 % ammonium sulfate, 10 % enemy of bubbles fermentation cycle approximately 48 h Example 1: Construction of the PK18-NCG12176A528Cque transformation vector includes the coding region of the NCG12176 gene with punctual mutation According to the genomic sequence of Corynebacterium glutamicum atcc13032 wild type published by the NCBI, two pairs of primers were designed and synthesized to amplify the sequence of the coding region of the gene NCG12176, and a punctual mutation was introduced in the coding region of the NCG1217 (The coded protein by which the amino acid sequence shown in the SEQ ID is: 3) of the YP97158 strain, which was deposited on August 16, 2016 at the General Center for Collection of Microbiological Crops of China (address: No. of CGMCC No. 12865 and has been recorded in the Chinese Patent application CN106367432A (Date of presentation: September 1, 2016, publication date: February 1, 2017), by allelic substitution, so that the base in position 528 of the nucleotide sequence of the NCG1217 gene changed adenine (a) to cytosine (c) No: 2: NCG12176A528C) and the amino acid residue in position 176 of the amino acid sequence of the corresponding coded protein was changed from a lysine residue to an asparagine residue (SEQ id no: 4: NCG12176K176N). The designed primers were like below (synthesized by invitrogen, Shanghai): Ρ1: 5 'Cagtgccaagctgca tgcctgcaggtcg'i agcggcgac CGCATGGACCG 3 '(SEQ ID NO: 5) P2: 5 'CCGGGGGGGTTTTCGGGTGTGGTGCC 3' (SEQ ID NO: 6) P3: 5 'GCACCCAACCCGGAAACCAGTCCCGG 3 ’(SEQ ID NO: 7) P4: 5 'CagctatgaccattacgaatTCGagCTCGTACCCCGG TCTCTCAATCGGT 3 '(SEQ ID NO: 8) Construction method: Using the genomic DNA of Corynebacterium glutamicum atcc13032 such as the template, amplifications were carried out by PCR with the primer P1 and P2, and the primer P3 and P4, respectively. PCR System EX TEQ 10X 5 PL, Mix DNTP (2.5 mm each) 4 PL, MG2+(25 mm) 4 PL, primers (10 pm) 2 pl each, ex taq (5u / pl) 0.25 pl, total volume 50 pl. PCR amplification procedure: Initial denaturation at 94 ° C for 5 min; 30 denaturation cycles at 94 ° C for 30 s, appeal at 52 ° C for 30 s, and extension at 72 ° C for 40 s; and a final extension at 72 ° C for 10 min. Two DNA fragments contained in the coding region of the NCG12176, NCG12176 UP and NCG12176 Down were obtained, with sizes of 796 PB and 786 PB, respectively. The two previous DNA fragments were isolated and purified by electrophoresis in agarose gel and then, using the two purified DNA fragments as a template, an amplification per PCR was performed superimposed with the primer P1 and P4 to obtain a fragment of approximately 1552 bp. PCR System EX TEQ 10X 5 PL, Mix DNTP (2.5 mm each) 4 PL, MG2+(25 mm) 4 PL, primers (10 pm) 2 pl each, ex taq (5u / pl) 0.25 pl, total volume 50 pl. PCR amplification procedure: Initial denaturation at 94 ° C for 5 min; 30 denaturation cycles at 94 ° C for 30 s, appeal at 52 ° C for 30 s, and extension at 72 ° C for 90 s; and a final extension at 72 ° C for 10 min. This DNA fragment (NCG12176A528C) caused the change of the base in position 528 of the coding region of the NCG12176 of YP97158 of adenine (a) to cytosine (c) and then the change of the amino acid residue in the 176 position of the coded protein of a lysine residue (K) to a waste of a gate (N). The PK18Mobsacb (Addgene) plasmid was digested with XBA I. The NCG12176A528CY The PK18Mobsacb Plsmid PK18-NCG12176A528cque contained a kanamycin resistance marker. The Vector PK18-NCG12176A528CSE sent for sequencing, and the right vector PK18-NCG12176A528cque contained the punctual mutation (A-C) was stored for later use. Jo i o Example 2: Construction of a designed strain containing the gene NCQ12176A528Ccon Punctual Mutation Construction method: The PK18-NCG12176A528C for allelic replace strain) through electroporation; The individual colonies obtained after the crop were identified by the CEBADOR P1 and the universal primer M13R respectively, and the strain from which a band of approximately 1559 bp could be amplified was a positive strain. The positive strain was cultivated in the environment containing sucrose to 15%, and the individual colonies obtained after the crop were grown in the medium containing kanamycin and without kanamycin at the same time; The strain that grew in the middle without kanamycin and did not grow in the medium containing kanamycin was also identified by PCR using the following primers (synthesized by invitrogen, Shanghai): P5: 5 'gaaacaccgccgaatc 3' (seq id no: 9) P6: 5 ’GGAGTGGTGTTTTGTGATG 3 '(SEQ ID NO: LO) The anterior PCR amplification product underwent SSCP electrophoresis after high temperature denaturation and an ice bath (the amplified fragment of the PK18NCG12176A528cse plasmid used as positive control, the amplified fragment of YP97158 was used as a negative control, and the water was used as a white control). Due to the different fragments structures, the fragments had different electrophoretic positions. The strain whose fragment had an inconsistent electrophoretic position with the negative control fragment, but the same as that of the positive control fragment had a satisfactory allelic substitution. The PCR amplification was made again with the P5 and P6 primers to amplify the white fragment of the strain with successful allelic substitution, and the amplified fragment was linked to the PMD19-T vector for the sequencing. The strain with satisfactory allelic substitution was verified by examining the sequence of mutated bases, and was appointed as YPL-4-011. Example 3: Construction of a designed strain that overexpresses the gene NCQ12176 or NCQ12176A528Cen the qenoma According to the genomic sequence of Corynebacterium glutamicum ATCC13032 Silvestre type published by the NCBI, three pairs of primers were designed and synthesized to amplify fragments of the upper and current homologous arm down and the coding region of the NCG12176 or NCG12176A528Cy the sequence of the promoter region. The NCG12176 or NCG12176A528C laid in the strain YP97158 by homologous recombination. The designed primers were like below (synthesized by invitrogen, Shanghai): HATE Ρ7: 5'Cagtgccaago l’an gccigcagg1cgaciciagaaigg γ γ CTGGACTGAGG3 ’(SEQ ID NO: 11) P8: 5'aaccattgtcctgttttgggcgaaatttcCgtgcaccgag AACAGATG3 '(SEQ NO: 12) P9: 5'CGGGAAAATTTCGCCCAAAACGGACACAATGGTTTTGGCATTGCAGACACA TTGTGCGC3 ’(SEQ NO: 13) PI0: 5'CTGATTTTTGCCTCTCTCAGGGGGGGGGTTCGTCTCGTAG3 '(SEQ ID NO: 14) P11: 5'CTACGAGACGAAGCGTTCGCTGAGGGCGCAATTAAATCAAG 3 '(SEQ ID No: 15) Pi2: 5'CagctatgacatgattacgaAtcGagcggtacccCCTAT Gaccctcaacggatc 3 ’(SEQ ID NO: 16) Construction Method: Using the genomic DNA of Corynebacterium glutamicum atcc13032 or YPL-4-011 as a template, amplifications were carried out by PCR respectively with the primers P7 and P8, P9 and P10 and P11 and P12 to obtain the fragment of the upper-homologous arm upstream of approximately 720 pb, gen ncg12176 and its promoter fragment of approximately 1092 PB, GEN NCG12176A528C and its promoter fragment of approximately 1092 bp, and the fragment of the homologous arm below approximately 653 bp. Using the mixture of the three amplified previous fragments (that is, fragment of the up -counter arm, GEN NCG12176 and its promoter fragment, fragment of the up -down -homologous arm; or fragment of the upper -homologous arm upstream, gen NCG12176A528Cy its promoter fragment, fragment of the upper -homologous arm downstairs) as template Fragment of an integral counterpart. After the PCR reaction, the amplified products were recovered by electrophoresis and the desired DNA fragments of approximately 2504 bp were recovered using a DNA recovery kit in column gel (tiangen). The fragments were linked respectively with the PK18mobsacb digested plasmid with XBA I using the nebuider recombination system to obtain the integration plasmids PK18MOBSACB-NCG12176 and PK18MOBSACB-NCG12176A528C. These two plasmids contained kanamycin resistance markers, and recombinants could be obtained with the plasmids integrated in the genomes by selecting kanamycin resistance. PCR System EX TEQ 10X 5 PL, Mix DNTP (2.5 mm each) 4 PL, MG2+(25 mm) 4 PL, primers (10 pm) 2 pl each, ex taq (5u / pl) 0.25 pl, total volume 50 pl. PCR amplification procedure: Initial denaturation at 94 ° C for 5 min; 30 denaturation cycles at 94 ° C for 30 s, appeal at 52 ° C for 30 s, and extension at 72 ° C for 120 s; and a final extension at 72 ° C for 10 min. The two integration plasmids were electrotra. A fragment of approximately 1609 bp of positive strains could be amplified, and no fragment of the original strains could be amplified. Positive strains were signed with 15 % sucrose and grown in the middle containing kanamycin and without kanamycin at the same time. The strain that grew in the middle without kanamycin and did not grow in the environment contained YPL-4-012 (without punctual mutation) and YPL-4-013 (with punctual mutation), respectively. PL3: 5 'TCCAAGGAAGATACCCCC 3 ’(SEQ ID NO: 17) P14: 5 'CCTGAGCGGAATAGTCCTGTG3 ’(SEQ ID NO: U) P15: 5 'ACGCCCGTGTTCTCT3' (SEQ ID NO: 19) P16: 5 'CGTTGGGTTGCGTTG 3 (SEQ ID NO: 20) Example 4: Construction of a designed strain that overexpresses the gene NCG12176 or NCG12176 aszscen e¡ plasmid According to the genomic sequence of Corynebacterium glutamicum ATCC13032 Silvestre type published by the NCBI, three pairs of primers were designed and synthesized to amplify the coding region of the NCG12176 or NCG12176A528CY gene. The sequence of the promoting region. The designed primers were like below (synthesized by invitrogen, Shanghai): P17: 5'GC'N GCA LGCC RGCAGGRCGACRC 1AG AGGATCCCCGGGAAA A γπ CGCCC Aaaacag3 ’(seq id no: 21) P18: 5'atcaggctgaaaatctcTCTCatCCCCCCAAACTCAGGGGGGCTTCGT CTCGTAG 3 ’(SEQ NO: 22) Construction Method: Using the genomic DNA of Corynebacterium glutamicum atcc13032 or YPL-4-011 wild type as a template, amplifications were carried out by PCR with the primer P17 and P18 respectively to obtain the gene NCG12176 or NCG12176A528Cy its promoter fragment of approximately 1140 PB. The amplified products were recovered by electrophoresis and the desired DNA fragments of approximately 1140 bp were recovered using a DNA recovery kit in column gel (tiangen). The fragments were linked to the PXMJ19 cloning plasmid with Ecor I using the Nebuider recombination system to obtain the overexpression plasmids PXMJ19-NCG12176 and PXMJ19-NCG12176A528C, respectively. These two plasmids contained chloranphenicol resistance markers, and strains transformed with plasmids could be obtained by screening chloramphenicol resistance. PCR System EX TEQ 10X 5 PL, Mix DNTP (2.5 mm each) 4 PL, MG2+(25 mm) 4 PL, primers (10 pm) 2 pl each, ex taq (5u / pl) 0.25 pl, total volume 50 pl. PCR amplification procedure: Initial denaturation at 94 ° C for 5 min; 30 denaturation cycles at 94 ° C for 30 s, appeal at 52 ° C for 30 s, and extension at 72 ° C for 90 s; and a final extension at 72 ° C for 10 min. The plasmids were electrotransformed respectively in the CEPA YP97158 Producer of L-Lisina, and the individual colonies obtained after the crop were identified by PCR with the OO I or primer M13R (-48) and P18. The strains from which a fragment of approximately 1147 bp via PCR was amplified were the strains transformed with plasmids, which were named as YPL-4-014 (without punctual mutation) and YPL-4-015 (with punctual mutation), respectively. Example 5: Construction of a designed strain that lacks one hundred NCG12176 in the genome According to the genomic sequence of Corynebacterium glutamicum ATCC13032 published by the NCBI, two pairs of primers were synthesized to amplify the fragments at the two ends of the coding region of the NCG12176 gene and the amplified fragments by PCR were used as fragments of the upper and current homologous arm. The designed primers were like below (synthesized by invitrogen, Shanghai): P19: 5'CagtgccaagctgcatgCTGGGGTCTCTAGTCAAAAGAGGGCGAGA Taat3 '(seq id no: 23) P2 (): 5'gt i catgagacccag i aggacgacc i ata P21: 5’cactgactatTTGTGGTCGTCCTGGTGTCTCATGAAC 3 '(SEQ id No: 25) P22: 5'CagctatgacCatgattacgaatTCGTCGGTACCCCCCACGATGGTT Cact3 '(SEQ NO: 26) Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, the PCR amplification was carried out with the primer P19 and P20, and P21 and P22 respectively to obtain a fragment of upper -homologous arm upstream of 852 bp and a fragment of the upper -homologous arm downstream of 787 bp; and then an superimposed PCR was carried out with the primers P19 and P22 to obtain a complete counterpart fragment of 1639 bp. After the PCR reaction, the amplified product was recovered by electrophoresis, and the desired DNA fragment of 1639 PB was recovered using a DNA recovery kit in column gel (tiangen). The fragment was linked to the PXMJ19 cloning plasmid with XBA I using the nebuider recombination system to obtain a plasmid with gene inactivation (Knock out). The plasmid contained a kanamycin resistance marker. The plasmid with gene inactivation was electrotransformed in the yp97158 yp97158 producing strain, and the individual colonies obtained after the crop were identified by PCR with the following primers (synthesized by invitrogen, Shanghai): P23: 5 'TCAAAGAGGGGAGAATAAT 3 (SEQ ID NO: 27) P24: 5 'Accgcacgatgttcact 3' (SEQ ID NO: 28) The strains that could be amplified fragments of 1521 bp and 2556 bp through the anterior PCR were positive strains, and the strains from which only a fragment of 2556 bp could be amplified were the original strains. The positive strains were signed in the midst of sucrose to 15% and were grown in the environment containing kanamycin and without kanamycin, respectively. The strain that grew in the middle without kanamycin and did not grow in the environment contained YPL-4-016. Example 6: L-Lisine fermentation experiment The strains built in examples 2 to 5 and the original strain YP97158 were fermented in the culture medium shown in Table 1 in the BLBIO-5GC-4-H model (acquired from Shanghai Bailun Biotechnology Co., Ltd.) According to the control process shown in Table 2 for fermentation experiments. The experiment for each strain was performed in triplicate. The results are shown in Table 3. Table 3 Results of the L-Lisin fermentation experiment CEPA Production of L-Lisin (G / 100 ml) OD (600 Nm) YP97158 18.8 37.3 YPL-4-011 19.0 37.2 YPL-4-012 19.3 36.5 YPL-4-013 19.8 37.0 YPL-4-014 20.1 36.8 YPL-4-015 20.3 35.9 YPL-4-016 18.2 36.7 The results are shown in Table 3. The overexpression of the NCG12176 gene, or the punctual mutation in the coding region of the NCG12176 gene, that is, NCG12176A528Cy the overexpression in Corynebacteríum glutamicum contributes to the improvement of the production of L-Lisina, while the regulation or the elimination of the gene or the elimination of the gene. lysine. Example 7: Construction of the transformation vector PK18-PDAPB <C (-49> A · Gí-57^ CTGCA (-54 ~ 58) GGTGT) that understands the promoter of the DAPB gene with punctual mutation According to the genomic sequence of Corynebacteríum glutamicum atcc13032 wild type published by the NCBI, two pairs of primers were designed and synthesized to amplify the sequence of the DAPB gene region, and specific mutations were introduced in the promoting region of the DAPB gene (seq id no: 29) By allelic substitution, so that the nucleotide in the -49 position of the nucleotide sequence of the DAPB gene GGTGT. The designed primers were like below (synthesized by invitrogen, Shanghai): P1 ’: 5 CCGGAATTCACCATGCGGACTGCGGAC3 '(ECOR I) (SEQ ID NO: 31) P2 ’: 5 'CCTTCTGAACGGGTGTGGTAATGGGGG 3' (SEQ ID NO: 32) P3 ’: 5 CCACCATTATACCACACAACCCGTTCAGAAGG 3’ (SEQ ID NO: 33) P4 ’: 5 'acatgcatgcgaattgacgttgaggaag and (SPH i) (seq id no: 34) or Construction Method: Using the genomic DNA of Corynebacterium glutamicum atcc13032 such as the template, amplifications were carried out by PCR with the PT and P2 ’primers, and the primer P3 'and P4’, respectively. PCR System EX TEQ 10X 5 PL, Mix DNTP (2.5 mm each) 4 PL, MG2+(25 mm) 4 PL, primers (10 pm) 2 pl each, ex taq (5u / pl) 0.25 pl, total volume 50 pl. PCR amplification procedure: Initial denaturation at 94 ° C for 5 min; 30 denaturation cycles at 94 ° C for 30 s, appeal to 52 ° C for 30 s, and extension at 72 ° C for 60 s; and a final extension at 72 ° C for 10 min. Two DNA fragments containing a punctual mutation, DAPB UPy DAPB Down, with 665 PB and 664 PB sizes respectively were obtained. The two previous DNA fragments were isolated and purified by electrophoresis in agarose gel and then using the two purified DNA fragments as a template, an amplification per PCR was performed superimposed with the PT and P4 ’primers to obtain a UP-Down fragment of approximately 1279 bp. Overlapping PCR system: Ex taq 10x 5 PL shock absorber, mixture dntp (2.5 mm each) 4 PL, mg2+(25 mm) 4 pl, primers (10 pm) 2 pl each, ex taq (5u / pl) 0.25 pl, total volume 50 pl. Overlapping PCR amplification procedure: Initial denaturation at 94 ° C for 5 min; 30 denaturation cycles at 94 ° C for 30 s, appeal at 52 ° C for 30 s, and extension at 72 ° C for 90 s; and a final extension at 72 ° C for 10 min. This UP-Down fragment was isolated and purified by gel electrophoresis. The fragment contained the promoter region of the DAPB gene and its sequences up and down down, and the two ends of the fragment contained Ecor I and SPH I restriction sites respectively. This DNA fragment resulted in the change of the nucleotide of C a A in the -49 position of the DAPB YP97158 gene, and the nucleotide of g to t in the position -51, and the nucleotide sequence of CTGCAA GGTGT of the positions -54 A -58. The fragment was doubly digested with the restriction enzymes Ecor I and Sph I and then linked to the plasmid cloning PK18mobsacb (Addgene) that was doubly digested with the same enzymes of restriction Ecor I and SPH I, to obtain the replacement plasmid allec CTGCA (54-58) GGTGT) that contains a kanamycin resistance marker. (C (-49) A, G (-51) T, CTGCA (-54--58) GGTGT) Que Concentrate The specific mutations was stored for later use. Example 8: Construction of a designed strain containing the vector with punctual mutation PK18PDAPB (C (-43) A, G (-51) T, CTGCA (-54-58) GGTGT) The allelic replacement plasmid PK18-PDAPB (C (-49) A, G (-51) T, CTGCA (-54-58) GGTGT> selelectrotransformed in the strain YP97158 Producer of L-Lisin (it was confirmed by sequencing that the promoter of the wild type Dapb type This strain), and the individual colonies obtained after the cultivation identified by the PT primer and the universal primer M13R, respectively. Kanamycin, respectively. The strain that grew in the middle without kanamycin and that did not grow in the environment containing kanamycin was also identified by PCR using the following primers (synthesized by invitrogen, Shanghai): P5 ’: 5 'Agatcgtcgactcattgac3' (Seq id no: 35) P6 ': 5' CaaacatagtcCCCTGTG 3 '(SEQ ID NO: 36) The anterior PCR amplification product underwent SSCP electrophoresis after high temperature denaturation and an ice bath (the amplified fragment of the PK18-PDAPB plasmid (C (-49) A, G (-51) T, CTGCA (-54-58) GGTGT) a positive control is used, the amplified control of YP97158 used as a negative control, and water was used as a blank control). Due to the different fragments structures, the fragments had different electrophoretic positions. The strain whose fragment had an inconsistent electrophoretic position with the negative control fragment, but the same as that of the positive control fragment had a satisfactory allelic substitution. The amplification by PCR was carried out again to amplify the white fragment of the positive strain, and the amplified fragment was linked to the PMD19-T vector for the sequencing. The strain with satisfactory allelic substitution was verified by examining the sequence of mutated bases, and was appointed as YPL-4-010. Example 9: L-Lisine fermentation experiment The YPL-4-010 strain built in Example 8 and the original CEPA YP97158 were fermented in the culture medium shown in Table 1 in the BLBIO-5GC-4-H (acquired from Shanghai Bailun Biotechnology Co., Ltd.) model according to the control process shown in Table 2 for fermentation experiments. The experiment for each strain was performed in triplicate. The results are shown in Table 4. HATE Table 4 Results of the L-Lisin fermentation experiment CEPA Production of L-Lisin (G / 100 ml) Conversion rate (%) YP97158 10.05 % 0.94 % Note: conversion rate = (total leisure mass / total glucose consumption)> <100 % The results are shown in Table 4, the punctual mutation of the DAPB gene promoter in C. glutamicum, pdapb ^ -49 ^ g (-51) T, CTGCA (-54-58) GGTGT), contributed to the improvement of the production of L-Lisin. The modalities of the invention have been described above. However, the invention is not limited to the previously described modalities. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the invention will be included in the scope of protection of the invention.

Claims

1. An L-lysine-producing microorganism belonging to the genus Corynebacterium, having enhanced expression of a polynucleotide encoding the amino acid sequence of SEQ ID NO: 3, and / or the bases at positions -49, -51 and -54 to -58 in the promoter region shown in SEQ ID NO: 29 are mutated; preferably, enhanced expression refers to the expression of the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 being boosted, or the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 having a point mutation, or the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 having a point mutation and boosted expression.

2. The microorganism according to claim 1, wherein the point mutation of the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 results in the substitution of the lysine residue at position 176 of the amino acid sequence of SEQ ID NO: 3 with a different amino acid residue, preferably with an asparagine residue.

3. The microorganism according to claim 1 or 2, wherein the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 comprises the nucleotide sequence of SEQ ID NO:

1.

4. The microorganism according to any of claims 1 to 3, wherein the point-mutated polynucleotide sequence is formed by the base mutation at position 528 of the polynucleotide sequence shown in SEQ ID NO: 1; preferably, the mutation includes the base mutation from adenine (A) to cytosine (C) at position 528 of the polynucleotide sequence shown in SEQ ID NO: 1; preferably, the point-mutated polynucleotide sequence comprises the polynucleotide sequence shown in SEQ ID NO:

2.

5. The microorganism according to any one of claims 1 to 4, wherein the nucleotide at position -49 of the promoter region shown in SEQ ID NO: 29 is mutated from cytosine (C) to adenine (A), and the nucleotide at position -51 is mutated from guanine (G) to thymine (T), the nucleotide sequence from positions -54 to -58 is mutated from CTGCA to GGTGT; preferably, the nucleotide sequence of the promoter is as follows: (a) the nucleotide sequence shown in SEQ ID NO: 30; or (b) a nucleotide sequence having at least 90% identity, preferably at least 95% or at least 98% identity, with the nucleotide sequence shown in SEQ ID NO: 30 and maintaining the promoter enhancer activity in (a), wherein the nucleotide at position -49 is maintained as adenine (A), the nucleotide at position -51 is maintained as thymine (T), and the nucleotide sequence from positions -54 to -58 is maintained as GGTGT.

6. The microorganism according to any of claims 1 to 5, wherein the microorganism is Corynebacterium glutamicum, preferably YP97158.

7. A polynucleotide sequence comprising a polynucleotide encoding the amino acid sequence of SEQ ID NO: 3, wherein the lysine residue at position 176 is substituted with a different amino acid residue; preferably with an asparagine residue; preferably, the polynucleotide sequence comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 4; preferably, the polynucleotide sequence is formed by the base mutation at position 528 of the polynucleotide sequence shown in SEQ ID NO: 1; preferably, the mutation includes the base mutation from adenine (A) to cytosine (C) at position 528 of the polynucleotide sequence shown in SEQ ID NO: 1; preferably, the polynucleotide sequence comprises the polynucleotide sequence shown in SEQ ID NO:

2.

8. An amino acid sequence, having the sequence shown in SEQ ID NO:

4.

9. A recombinant vector, comprising the polynucleotide sequence according to claim 7.

10. A recombinant strain, containing the polynucleotide sequence according to claim 7.

11. A promoter nucleotide sequence comprising the nucleotide sequence obtained by mutating the bases at positions -49, -51 and -54 to -58 in the promoter region shown in SEQ ID NO: 29; preferably, the nucleotide at position -49 of the promoter region shown in SEQ ID NO: 29 is mutated from cytosine (C) to adenine (A), and the nucleotide at position -51 is mutated from guanine (G) to thymine (T), and the nucleotide sequence from positions -54 to -58 is mutated from CTGCA to GGTGT.

12. The promoter nucleotide sequence according to claim 11, wherein the promoter nucleotide sequence is as follows: (a) the nucleotide sequence shown in SEQ ID NO: 30; or (b) a nucleotide sequence having at least 90% identity, preferably at least 95% or at least 98% identity, with the nucleotide sequence shown in SEQ ID NO: 30 and maintaining the promoter enhancer activity in (a), wherein the nucleotide at position -49 is maintained as adenine (A), the nucleotide at position -51 is maintained as thymine (T), and the nucleotide sequence from positions -54 to -58 is maintained as GGTGT.

13. A promoter expression cassette, wherein the expression cassette comprises the promoter nucleotide sequence according to claim 11 or 12 and a coding sequence operatively linked to the promoter; preferably, the coding sequence is the coding sequence of the dapB gene.

14. A recombinant vector, comprising the promoter nucleotide sequence according to claim 11 or 12; preferably, the recombinant vector is constructed by ligating the promoter nucleotide sequence with the pK18mobsacB plasmid.

15. A recombinant strain, containing the promoter nucleotide sequence according to claim 11 or 12 or the expression cassette according to claim 13 or the recombinant vector according to claim 14; preferably, the host strain of the recombinant strain is YP97158.

16. A method for producing L-lysine, comprising cultivating the microorganism according to any of claims 1 to 6 or the recombinant strain according to claim 10 or 15, and recovering L-lysine from the culture.