Recombinant strain producing L-lysine, method for constructing the same, and application

Abstract

The present invention provides a method for introducing a point mutation into the NCgl2176 gene coding sequence in Corynebacterium glutamicum or improving expression, and a method for performing a point mutation on the promoter region sequence of the dapB gene in Corynebacterium glutamicum. The method can increase the fermentation yield of L-lysine produced by the mutant strain.

Description

Technology Field

[0001] The present invention claims priority to patent application number 202010790877.0 filed with the National Intellectual Property Administration of China on August 7, 2020, titled "Recombinant strain producing L-lysine and method for constructing the same and application" and patent application number 202010514023.X filed with the National Intellectual Property Administration of China on June 8, 2020, titled "Recombinant strain for dapB gene modification and method for constructing the same and application". The full text of the two prior applications is incorporated into the present invention by means of citation.

[0002] The present invention belongs to the field of genetic engineering and microbial technology, and specifically relates to a recombinant strain with improved L-lysine production capacity, a method for constructing the same, and its application. Background Technology

[0003] L-lysine has physiological functions such as promoting development, strengthening immunity, and improving the function of central nervous tissue, and it is one of the eight essential amino acids required for growth that humans and animals cannot synthesize on their own. Currently, L-lysine is the second largest type of amino acid in the world, and the main production method is fermentation. Corynebacterium is the most important strain used in amino acid production, including Corynebacterium glutamicum, Corynebacterium flavum, Corynebacterium crenatum, and Corynebacterium pekinense, among which Corynebacterium glutamicum is an important lysine-producing strain. About 90% of the industrial production of L-lysine is used as a nutritional fortifier in the feed industry, and 10% is used as a flavoring agent and sweetener in the food industry and as a pharmaceutical intermediate in the pharmaceutical industry.

[0004] Improvements to the fermentation method for producing L-lysine may include fermentation techniques such as stirring and oxygen supply; or the composition of the nutrient medium, such as sugar concentration during fermentation; or the processing of the fermentation liquid into a suitable product form, such as drying and pelletizing of the fermentation liquid or ion exchange chromatography; or the unique performance and characteristics of the relevant microorganisms.

[0005] Methods to improve the performance and characteristics of these microorganisms include mutagenesis, mutant selection, and screening. Strains obtained in this manner are resistant to metabolites, have nutritional requirements for metabolites critical to regulation, and can produce L-lysine.

[0006] Taking Corynebacterium glutamicum as an example, 4 mol NADPH is required to synthesize 1 mol L-lysine in the biosynthetic pathway of C. glutamicum. Therefore, to improve the accumulation of L-lysine in the biosynthetic pathway of C. glutamicum, it is a very important strategy to increase the amount of NADPH in the metabolic pathway of C. glutamicum or to reduce the demand for NADPH in the L-lysine synthesis pathway.

[0007] Dihydrodipicolinate reductase (DHDPR) is the second key enzyme in the biosynthesis of diaminopimelane and L-lysine in bacteria and higher plants, catalyzing the NAD(P)H-dependent reduction of dihydrodipicolinate to produce hexahydrodipicolinate. This enzyme plays a pivotal role in cell wall formation. DHDPR utilizes either NADH or NADPH as a cofactor, and DHDPR in different bacteria exhibits different affinities for these cofactors. For example, DHDPR in E. coli prefers NADH, whereas DHDPR in C. glutamicum primarily uses NADPH as a cofactor to participate in L-lysine synthesis. Similar to DHDPR found in other organisms, DHDPR in C. glutamicum yes It is encoded by the B gene, and enzyme activity is not regulated by the end product of the synthetic pathway but is inhibited by 2,6-pyridindicarboxylic acid (2,6-PDC).

[0008] Lysine yield is generally related to the enzymatic activity of the biosynthetic pathway, which can be enhanced by amplifying one or more genes in the lysine biosynthetic pathway or by applying a modified promoter to the gene. The problem to be solved

[0009] The present invention provides an L-lysine-producing microorganism or recombinant strain, wherein the expression of a polynucleotide encoding an amino acid sequence indicated by SEQ ID NO:3 is improved, and / or, mutations occur at the -49th, -51st, and -54th through -58th bases of a promoter region indicated by SEQ ID NO:29. The present invention further provides a method for producing L-lysine using the said microorganism or recombinant strain. means of solving the problem

[0010] In the first aspect of the present invention, an L-lysine-producing microorganism or recombinant strain belonging to the genus Corynebacterium is provided, wherein the expression of a polynucleotide encoding an amino acid sequence represented by SEQ ID NO:3 is improved. According to the present invention, the improved expression is achieved by enhancing the expression of the polynucleotide, or by the polynucleotide encoding an amino acid sequence represented by SEQ ID NO:3 having a point mutation, or by the polynucleotide encoding an amino acid sequence represented by SEQ ID NO:3 having a point mutation and enhancing the expression.

[0011] The amino acid sequence indicated by SEQ ID NO:3 above is the protein encoded by the gene NCgl2176.

[0012] The above-mentioned microorganism or recombinant strain has an enhanced L-lysine production capacity compared to the wild type or parent strain.

[0013] The above polynucleotide may encode an amino acid sequence having about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more sequence homology with the amino acid sequence represented by SEQ ID NO:3. As used in the present invention, the term "homology" refers to the percentage of identity between a polynucleotide or two polypeptide modules. Sequence homology between one module and another module can be measured using conventional methods in the art. For example, such sequence homology can be measured using the BLAST algorithm.

[0014] The expression of polynucleotides can be enhanced through substitution or mutation of expression regulatory sequences, introduction of mutations into polynucleotide sequences, chromosomal insertion or an increase in the number of polynucleotide copies through vector introduction, or combinations thereof.

[0015] The expression regulatory sequence of the polynucleotide may be modified. The expression regulatory sequence controls the expression of the polynucleotide to which it is operably linked and may include, for example, a promoter, terminator, enhancer, silencer, etc. The polynucleotide may have a change in the start codon. The number of copies may be increased by mixing the polynucleotide into a specific location on a chromosome. In the text, the specific location may include, for example, a transposon location or an intergene location. In addition, the number of copies may be increased by mixing the polynucleotide into an expression vector and introducing the expression vector into a host cell.

[0016] In one embodiment of the present invention, a polynucleotide or a polynucleotide having a point mutation is mixed at a specific location on a microbial chromosome to increase the number of copies.

[0017] In one embodiment of the present invention, a polynucleotide having a promoter sequence or a polynucleotide having a point mutation having a promoter sequence is mixed at a specific location on a microbial chromosome to overexpress the nucleic acid sequence.

[0018] In one embodiment of the present invention, a polynucleotide or a polynucleotide having a point mutation is mixed into an expression vector, and said expression vector is introduced into a host cell to increase the number of copies.

[0019] In one embodiment of the present invention, a polynucleotide having a promoter sequence or a polynucleotide having a point mutation having a promoter sequence is mixed into an expression vector, and the expression vector is introduced into a host cell to overexpress the nucleic acid sequence.

[0020] In one specific embodiment of the present invention, the polynucleotide may include a nucleotide sequence represented by SEQ ID NO:1.

[0021] In one embodiment of the present invention, a polynucleotide encoding an amino acid sequence represented by SEQ ID NO:3 has a point mutation so that the 176th lysine of the amino acid sequence represented by SEQ ID NO:3 is substituted with a different amino acid.

[0022] According to the present invention, preferably, the 176th lysine is substituted with asparagine.

[0023] According to the present invention, the amino acid sequence represented by SEQ ID NO:3 is an amino acid sequence in which the 176th lysine (K) is substituted with asparagine (N), and is represented by SEQ ID NO:4.

[0024] In one embodiment of the present invention, the polynucleotide sequence having the point mutation is formed by a mutation in the 528th base of the polynucleotide sequence represented by SEQ ID NO:1.

[0025] According to the present invention, the mutation comprises a mutation from adenine (A) to cytosine (C) at the 528th base of a polynucleotide sequence represented by SEQ ID NO:1.

[0026] In one embodiment of the present invention, the polynucleotide sequence having the point mutation comprises the polynucleotide sequence represented by SEQ ID NO:2.

[0027] As used in the text, the term “operably linked” refers to regulating a functional link between a regulatory sequence and a polynucleotide sequence, thereby allowing the regulatory sequence to control the transcription and / or translation of the polynucleotide sequence. The regulatory sequence may be a potent promoter capable of enhancing the expression level of the polynucleotide. The regulatory sequence may be a promoter derived from a microorganism of the genus Corynebacterium or a promoter derived from another microorganism. For example, the promoter may be a trc promoter, a gap promoter, a tac promoter, a T7 promoter, a lac promoter, a trp promoter, an araBAD promoter, or a cj7 promoter.

[0028] In one specific embodiment of the present invention, the promoter is a promoter of a polynucleotide (NCgl2176 gene) encoding an amino acid sequence represented by SEQ ID NO:3.

[0029] As used in the text, the term "vector" refers to a polynucleotide construct containing a regulatory sequence of a gene and a gene sequence configured to express a target gene in a suitable host cell. Alternatively, a vector also refers to a polynucleotide construct containing a sequence for homologous recombination, wherein the vector introduced into the host cell can alter the regulatory sequence of an endogenous gene within the host cell's genome or insert a target gene that can be expressed into a specific location in the host's genome. In this regard, the vector used in the present invention may further include a selection marker to determine the introduction of the vector into the host cell or the insertion of the vector into the host cell's chromosome. The selection marker may include a marker that confers selectable phenotypes, such as drug resistance, nutrient requirements, resistance to cytotoxic agents, or the expression of surface proteins. In an environment where such a selector is used, the transformed cells can be selected because only cells expressing the selection marker may survive or exhibit different phenotypic characteristics.

[0030] In some specific embodiments of the present invention, the vectors used are pK18mobsacB plasmid and pXMJ19 plasmid.

[0031] The term "transformation" as used in the text refers to the introduction of a polynucleotide into a host cell, which can be replicated by making the polynucleotide an extragenomic element or by inserting it into the host cell's genome. The transformation method of the vector used in the present invention may include a method of introducing nucleic acids into a cell. In addition, as disclosed in related art, an electrical pulse method may be implemented depending on the host cell.

[0032] According to the present invention, microorganisms or recombinant strains belonging to the genus Corynebacterium may be Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium lactofermentum, Corynebacterium ammoniagenes, and Corynebacterium pekinense.

[0033] In one embodiment of the present invention, the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum YP97158, with deposit number CGMCC No. 12856 and deposit date August 16, 2016, and the depositing institution is the General Microbiology Center of the Chinese Microbial Species Deposit Management Committee, with an address of No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, and a contact number of 010-64807355, and has already been described in Chinese patent application CN106367432A (filing date September 1, 2016, publication date February 1, 2017).

[0034] According to the present invention, the microorganism or recombinant strain may have other improvements related to L-lysine production, for example, genes related to NADPH production (e.g., a gene encoding glucose dehydrogenase, a gene encoding gluconate kinase, a gene encoding glyceraldehyde-3-phosphate dehydrogenase, a gene encoding glucose-6-phosphate dehydrogenase, or a gene encoding 6-phosphogluconate dehydrogenase) and / or other genes involved in the biosynthesis or secretion of L-lysine (e.g., a gene encoding aspartate aminotransferase, a gene encoding aspartate kinase, a gene encoding aspartate semialdehyde dehydrogenase, a gene encoding dihydrodipicolinate synthase, a gene encoding dihydrodipicolinate reductase, a gene encoding m-diaminopimelate dehydrogenase, a gene encoding diaminopimelate decarboxylase). Increases or decreases the expression of the gene (lysE), or allows the gene to be replaced with a foreign gene.

[0035] According to a second aspect of the present invention, a polynucleotide sequence is provided, wherein the amino acid sequence encoded by the polynucleotide sequence comprises a recombinant vector of the polynucleotide sequence and contains a recombinant strain of the polynucleotide sequence.

[0036] According to the present invention, the polynucleotide sequence comprises a polynucleotide encoding a polypeptide containing an amino acid sequence represented by SEQ ID NO:3, wherein the 176th lysine is substituted with a different amino acid.

[0037] According to the present invention, preferably, the 176th lysine is substituted with asparagine.

[0038] According to the present invention, the amino acid sequence after the 176th lysine (K) in the amino acid sequence represented by SEQ ID NO:3 is substituted with asparagine (N) is represented by SEQ ID NO:4.

[0039] According to the present invention, preferably, the polynucleotide sequence of the polypeptide encoding the amino acid sequence represented by SEQ ID NO:3 comprises the polynucleotide sequence represented by SEQ ID NO:1.

[0040] In one embodiment of the present invention, the polynucleotide sequence is formed by a mutation in the 528th base of the polynucleotide sequence represented by SEQ ID NO:1.

[0041] According to the present invention, the mutation refers to a change in a base / nucleotide at the said position, and the mutation method may be selected from at least one of the following methods: mutagenic, PCR-designated mutagenic, and / or homologous recombination. In the present invention, PCR-designated mutagenic and / or homologous recombination is preferably used.

[0042] According to the present invention, the mutation comprises a mutation from adenine (A) to cytosine (C) at the 528th base of a polynucleotide sequence represented by SEQ ID NO:1.

[0043] In one embodiment of the present invention, the polynucleotide sequence comprises a polynucleotide sequence represented by SEQ ID NO:2.

[0044] According to the present invention, the amino acid sequence comprises an amino acid sequence represented by SEQ ID NO:4.

[0045] According to the present invention, the recombinant vector is constructed by introducing the polynucleotide sequence into a plasmid.

[0046] In one embodiment of the present invention, the plasmid is the pK18mobsacB plasmid.

[0047] In another embodiment of the present invention, the plasmid is the pXMJ19 plasmid.

[0048] Specifically, the polynucleotide sequence and the plasmid can be constructed into a recombinant vector through the NEBuider recombination system.

[0049] According to the present invention, the recombinant strain comprises the polynucleotide sequence.

[0050] In one embodiment of the present invention, the starting strain of the recombinant strain is YP97158.

[0051] According to a third aspect of the present invention, a method for constructing a recombinant strain of Corynebacterium glutamicum is further provided.

[0052] According to the present invention, the above construction method comprises the following steps.

[0053] The polynucleotide sequence of wild-type NCgl2176, denoted by SEQ ID NO:1 in the host strain, is modified to induce a mutation at the 528th base, thereby obtaining a recombinant Corynebacterium strain containing the mutated NCgl2176 coding gene.

[0054] According to the method of construction of the present invention, the modification comprises at least one of the following methods: mutagenesis, PCR-designated mutagenesis, and / or homologous recombination.

[0055] According to the method of construction of the present invention, the mutation refers to a mutation from adenine (A) to cytosine (C) at the 528th base of SEQ ID NO:1; specifically, the polynucleotide sequence containing the mutated NCgl2176 coding gene is denoted as SEQ ID NO:2.

[0056] Additionally, the above construction method includes the following steps.

[0057] (1) The nucleotide sequence of the wild-type NCgl2176 gene, denoted by SEQ ID NO:1, is modified so that the 528th base is mutated, thereby obtaining the mutated NCgl2176 gene polynucleotide sequence.

[0058] (2) Construct a recombinant vector by linking the above mutated polynucleotide sequence with a plasmid.

[0059] (3) The above recombinant vector is introduced into a host strain to obtain a recombinant Corynebacterium strain containing the mutant NCgl2176 coding gene.

[0060] According to the construction method of the present invention, step (1) includes the step of constructing a point-mutated NCgl2176 gene. Based on the genome sequence of Corynebacterium glutamicum, two pairs of primers P1 and P2 and P3 and P4 are synthesized to amplify the NCgl2176 gene fragment, and a point mutation is introduced into the wild-type NCgl2176 gene SEQ ID NO:1 through PCR-designated mutagenesis to obtain the point-mutated NCgl2176 gene nucleotide sequence SEQ ID NO:2, and NCgl2176 A528C It is indicated as.

[0061] In one embodiment of the present invention, the Corynebacterium glutamicum genome may be derived from the ATCC13032 strain, and its genome sequence may be obtained from the NCBI website.

[0062] In one embodiment of the present invention, in step (1), the primer is as follows.

[0063]

[0064] In one embodiment of the present invention, the PCR amplification is performed according to the following method: denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 40 seconds (30 cycles).

[0065] In one embodiment of the present invention, the nested PCR amplification is performed according to the following method: denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 90 seconds (30 cycles).

[0066] According to the method of construction of the present invention, step (2) is the separated and purified NCgl2176 through the NEBuider recombination system A528C and assembled the pK18mobsacB plasmid to form the recombinant plasmid pK18-NCgl2176 A528C It includes the step of constructing a recombinant plasmid to obtain.

[0067] According to the method of construction of the present invention, step (3) is a recombinant plasmid pK18-NCgl2176 A528C It includes a step of constructing a recombinant strain by transforming it into a host strain to obtain a recombinant strain.

[0068] In one embodiment of the present invention, the transformation of step (3) is an electric transformation method.

[0069] In one embodiment of the present invention, the host strain is YP97158.

[0070] In one embodiment of the present invention, the recombination is implemented through homologous recombination.

[0071] According to a fourth aspect of the present invention, a method for constructing a recombinant strain of Corynebacterium is further provided.

[0072] According to the present invention, the above construction method comprises the following steps.

[0073] Upstream and downstream homologous cancer fragments of the NCgl2176 gene, the sequences of the NCgl2176 gene coding region and its promoter region, or, NCgl2176 A528C Amplify the gene coding region and its promoter region sequences, and inject NCgl2176 or NCgl2176 into the host strain genome via homologous recombination. A528CBy introducing a gene, the above strain is NCgl2176 or NCgl2176 A528C Implements gene overexpression.

[0074] In one embodiment of the present invention, the primer for amplifying the upstream homologous arm fragment is as follows.

[0075]

[0076] In one embodiment of the present invention, the primer for amplifying the downstream homologous arm fragment is as follows.

[0077]

[0078] In one embodiment of the present invention, the primers for amplifying the sequences of the gene coding region and the promoter region are as follows.

[0079]

[0080] In one embodiment of the present invention, NCgl2176 or NCgl2176 having an upstream homologous fragment, a downstream homologous fragment, and a self-promoter obtained by amplification using the aforementioned P7 / P12 as primers. A528C Three fragments are mixed and amplified using a mold to obtain an integrated homologous arm fragment.

[0081] In one embodiment of the present invention, the PCR system used is as follows: 5 μL of 10×Ex Taq Buffer, 4 μL of dNTP Mixture (2.5 mM each), and Mg 2+ 4 μL of (25 mM) primer (10 pM), 2 μL each of primer (10 pM), 0.25 μL of Ex Taq (5 U / μL), total volume 50 μL; PCR amplification is performed according to the following method: initial denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 120 s (30 cycles), final extension at 72°C for 10 min.

[0082] In one embodiment of the present invention, a shuttle plasmid PK18mobsacB and an integrated homologous arm fragment are assembled using a NEBuider recombination system to obtain an integrated plasmid.

[0083] In one embodiment of the present invention, the integrated plasmid is transfected into a host strain, and NCgl2176 or NCgl2176 is transfected into the genome of the host strain in a homologous recombination manner. A528C Introduces genes.

[0084] In one embodiment of the present invention, the host strain is YP97158.

[0085] In one embodiment of the present invention, the host strain is a strain having a polynucleotide sequence indicated by SEQ ID NO:2.

[0086] According to the fifth aspect of the present invention, a method for constructing a recombinant strain of Corynebacterium is further provided.

[0087] According to the present invention, the above construction method comprises the following steps.

[0088] NCgl 2176 gene coding region and promoter region sequences, or NCgl 2176 A528C By amplifying the gene coding region and promoter region sequences, an overexpression plasmid vector is constructed, and said vector is introduced into a host strain so that said strain is NCgl 2176 or NCgl2176 A528C Implements gene overexpression.

[0089] In one embodiment of the present invention, the primers for amplifying the sequences of the gene coding region and the promoter region are as follows.

[0090]

[0091] In one embodiment of the present invention, the PCR system is as follows: 5 μL of 10×Ex Taq Buffer, 4 μL of dNTP Mixture (2.5 mM each), and Mg 2+4 μL of (25 mM), 2 μL each of primer (10 pM), 0.25 μL of Ex Taq (5 U / μL), total volume 50 μL; the PCR amplification is performed according to the following method: initial denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 90 s (30 cycles), final extension at 72°C for 10 min.

[0092] In one embodiment of the present invention, using the NEBuider recombination system, the shuttle plasmid pXMJ19 and NCgl2176 or NCgl2176 having a self-promoter A528C Assemble the fragments to obtain an overexpression plasmid.

[0093] In one embodiment of the present invention, the host strain is YP97158.

[0094] In one embodiment of the present invention, the host strain is a strain having a polynucleotide sequence indicated by SEQ ID NO:2.

[0095] The recombinant strain obtained by the present invention can be applied alone for the fermentation production of L-lysine, or it can be mixed with other L-lysine-producing bacteria for fermentation to produce L-lysine.

[0096] According to another aspect of the present invention, a promoter nucleotide sequence is provided, which comprises a nucleotide sequence formed by a mutation occurring in the -49th, -51st, -54th to -58th bases of a promoter region indicated by SEQ ID NO:29.

[0097] According to the present invention, the -49th nucleotide of the promoter region indicated by SEQ ID NO:29 is mutated from cytosine (C) to adenine (A), the -51st nucleotide is mutated from guanine (G) to thymine (T), and the -54th to -58th nucleotides are mutated from CTGCA to GGTGT.

[0098] According to the present invention, the promoter nucleotide sequence is,

[0099] (a) a nucleotide sequence denoted by SEQ ID NO:30; or,

[0100] (b) a nucleotide sequence having at least 90%, preferably at least 95%, or at least 98% identity with the nucleotide sequence represented by SEQ ID NO:30, maintaining the enhanced activity of the promoter according to (a), wherein the -49th nucleotide is maintained as adenine (A), the -51st nucleotide is maintained as thymine (T), and the -54th to -58th nucleotides are maintained as GGTGT.

[0101] The present invention further provides an expression cassette comprising the promoter, wherein the expression cassette comprises the promoter and a coding sequence operably connected to the promoter. In one embodiment of the present invention, the coding sequence is the coding sequence of the dapB gene.

[0102] The present invention further provides a recombinant vector comprising the promoter nucleotide sequence of the present invention.

[0103] According to the present invention, the promoter nucleotide sequence of the present invention is ligated with a shuttle plasmid to construct the recombinant vector; in one embodiment of the present invention, the shuttle plasmid is the pK18mobsacB plasmid.

[0104] The present invention further provides a recombinant strain comprising the promoter nucleotide sequence or the recombinant vector.

[0105] According to the recombinant strain of the present invention, it comprises a nucleotide sequence represented by SEQ ID NO:30. The nucleotide sequence represented by SEQ ID NO:30 is a promoter region of the dapB gene. Additionally, the nucleotide sequence represented by SEQ ID NO:30 is linked to the dapB gene coding sequence. Specifically, the recombinant strain may comprise the expression cassette or recombinant vector of the present invention. Specifically, the recombinant strain of the present invention is obtained by transformation using the expression cassette or recombinant vector. According to the recombinant strain of the present invention, it is formed by introducing the mutated promoter nucleotide sequence into a host strain and recombining it; The above host strain may be selected from L-lysine-producing strains known in the art, for example, selected from at least one of Corynebacterium, wherein the Corynebacterium may be Corynebacterium glutamicum, Brevibacterium flavum, Corynebacterium crenatum, or Corynebacterium pekinense; preferably, it is Corynebacterium glutamicum. In one embodiment of the present invention, the host strain is YP97158.

[0106] According to the recombinant strain of the present invention, the pK18mobsacB plasmid is used as a vector.

[0107] According to the recombinant strain of the present invention, other variations may be further included.

[0108] The present invention further provides a method for constructing a recombinant strain that produces L-lysine, comprising the following steps.

[0109] (1) Modify the promoter region indicated by SEQ ID NO:29 to mutate the -49th, -51st and -54th to -58th bases to obtain a nucleotide sequence containing the mutated promoter region.

[0110] According to the present invention, the mutation refers to the mutation in which the -49th nucleotide of the promoter region denoted by SEQ ID NO:29 is mutated from cytosine (C) to adenine (A), the -51st nucleotide is mutated from guanine (G) to thymine (T), and the -54th to -58th nucleotides are mutated from CTGCA to GGTGT. Specifically, the nucleotide sequence of the promoter region after mutation is denoted by SEQ ID NO:30. Additionally, the construction method further comprises the following steps.

[0111] (2) The above-mentioned mutated promoter region nucleotide sequence is connected to a plasmid to construct a recombinant vector.

[0112] (3) The above recombinant vector is introduced into a host strain to obtain an L-lysine-producing recombinant strain containing a mutated promoter region.

[0113] According to the present invention, in step (1), the method of mutation includes mutagenic, PCR-designated mutation, or homologous recombination, and preferably includes a PCR-designated mutation method.

[0114] According to the present invention, step (1) includes the step of designing two pairs of primers to amplify the promoter region of the dapB gene, and then obtaining a mutated promoter region nucleotide sequence through PCR technology.

[0115] In one embodiment of the present invention, the primer in step (1) is as follows.

[0116]

[0117] In one embodiment of the present invention, step (1) comprises: using Corynebacterium glutamicum ATCC13032 as a template and performing PCR amplification with primers P1' and P2' and P3' and P4', respectively, to obtain two DNA fragments containing point mutations; using the two DNA fragments as a template and performing overlap PCR amplification with P1' and P4' as primers to obtain a DNA fragment containing the promoter region nucleotide sequence (SEQ ID NO:30) of the present invention.

[0118] According to the present invention, in step (1), through overlap PCR amplification, both ends of the DNA fragment obtained each include EcoR I and Sph I restriction enzyme sites.

[0119] According to the present invention, step (2) comprises the step of isolating and purifying the product amplified by a duplicate PCR reaction, and ligating it with a shuttle plasmid that is also duplicate-restricted (EcoR I / Sph I) after the fragment double-restricted (EcoR I / Sph I) to obtain an allele-substituted recombinant vector.

[0120] According to the present invention, the shuttle plasmid is the pK18mobsacB plasmid; and the constructed recombinant vector is pK18-P dapB (C(-49)A,G(-51)T,CTGCA(-54--58)GGTGT) am.

[0121] In one embodiment of the present invention, the recombinant plasmid comprises a kanamycin resistance marker.

[0122] In one embodiment of the present invention, the transformation of step (3) is an electro-transformation method; exemplarily, in step (3), the recombinant plasmid is transformed into strain YP97158.

[0123] The present invention further provides an application of the microorganism or recombinant strain described above during L-lysine production; or provides a method for improving the amount of L-lysine fermentation; or provides a method for producing L-lysine.

[0124] According to the application and method of the present invention, the method comprises the step of culturing the microorganism or fermenting the recombinant strain, and recovering L-lysine from the culture to produce L-lysine. According to the application and method of the present invention, the recombinant strain of the present invention may be used alone or may be used in combination with other bacteria that produce L-lysine.

[0125] Microorganisms may be cultured in a suitable medium under culture conditions known in the art. The medium may include a carbon source, a nitrogen source, trace elements, and combinations thereof. During culture, the pH of the culture may be adjusted. In addition, a step of preventing the generation of bubbles during culture may be included, for example, by using an antifoaming agent. In addition, a step of injecting gas into the culture may be included during culture. The gas may include any gas capable of maintaining aerobic conditions of the culture. During culture, the temperature of the culture may be 20 to 45°C. The generated L-lysine may be recovered from the culture by treating it with sulfuric acid or hydrochloric acid and then performing a combination of methods such as anion exchange chromatography, concentration, crystallization, and isoelectric precipitation.

[0126] In the present invention,

[0127] SEQ ID NO 1: NCgl2176 Wild-type ORF sequence

[0128]

[0129] SEQ ID NO 2: NCgl2176 A528C ORF sequence

[0130]

[0131] SEQ ID NO 3: NCgl2176 Wild-type coding protein amino acid sequence

[0132]

[0133] SEQ ID NO 4: NCgl2176 K176N Coding protein amino acid sequence

[0134]

[0135] SEQ ID NO 29: Wild-type promoter sequence

[0136]

[0137] SEQ ID NO 30: Mutated promoter sequence

[0138] Effects of the invention

[0139] The beneficial effects of the present invention are as follows.

[0140] In the present invention, the NCgl2176 gene was weakened or knocked out to confirm the effect of the product encoded by the gene on the L-lysine production capacity, and a recombinant strain was obtained by introducing a point mutation into the coding sequence or increasing the number of copies of the gene or overexpressing it, and the strain obtained was advantageous for producing high concentrations of L-lysine compared to the unmodified strain.

[0141] In addition, a recombinant strain was obtained by introducing a point mutation into the promoter region of the dapB gene, and the obtained strain significantly increased the production of L-lysine compared to the non-mutated strain, further improved production efficiency, and reduced production costs, making it suitable for distribution and application. Specific details for implementing the invention

[0142] The technical means of the present invention are described in more detail in conjunction with the specific embodiments below. It must be understood that the following embodiments are merely for illustrative purposes to explain and interpret the present invention and are not to be interpreted as a limitation on the scope of protection of the present invention. All technologies implemented based on the above description of the present invention fall within the scope of protection of the present invention. Unless otherwise noted, the raw materials and prototypes used in the following embodiments may be commercially available products or can be manufactured by existing methods, and the operations performed may be known in the art or can be performed according to the instructions of commercially available products.

[0143] In the following examples, the composition of the basic medium used for culturing the strain is the same, and sucrose, kanamycin, or chloromycetin, etc., correspondingly required are added to this basic medium, and the composition of the basic medium is as follows.

[0144]

[0145] The preparation and conditions of the SSCP electrophoresis PAGE in the following examples are as follows.

[0146]

[0147] The fermentation medium formulation and fermentation control process for L-lysine in the following examples are as follows.

[0148] Table 1 Fermentation medium formulations

[0149]

[0150] Table 2 Fermentation Control Process

[0151]

[0152] Example 1: Transformation vector pK18-NCgl2176 containing a point-mutated NCgl 2176 gene-coding region A528C construction of

[0153] Based on the genome sequence of wild-type Corynebacterium glutamicum ATCC13032 released by NCBI, two pairs of primers were designed and synthesized to amplify the sequence of the NCgl2176 gene-coding region; thereby, a point mutation was introduced into the NCgl2176 gene-coding region (SEQ ID NO:1) in the background of strain YP97158 (Deposit No.: CGMCC No.12856, Deposit Date: August 16, 2016, Depository: General Microbiology Center, Board of Deposit Management of Microbial Species of China, No. 3, Block 1, Beichen West Road, Chaoyang District, Beijing, Contact: 010-6480-7355, described in Chinese Patent Application CN106367432A (Application Date: September 1, 2016, Publication Date: February 1, 2017)) via allelic substitution, and the corresponding coded protein amino acids The sequence is SEQ ID NO:3, and the 528th A in the nucleotide sequence of the NCgl2176 gene is changed to C (SEQ ID NO:2: NCgl2176 A528C ), the lysine at position 176 of the corresponding coded protein amino acid sequence is asparagine (SEQ ID NO:4: NCgl2176 K176N It changes to ).

[0154] The primers are designed as follows (synthesized by Invitrogen in Shanghai).

[0155]

[0156] Construction method: PCR amplification is performed using Corynebacterium glutamicum ATCC13032 as a template, with primers P1 and P2, and P3 and P4, respectively.

[0157] PCR System: 10×Ex Taq Buffer 5μL, dNTP Mixture (2.5mM each) 4μL, Mg 2+ (25mM) 4μL, primer (10pM) 2μL each, Ex Taq (5U / μL) 0.25μL, total volume 50μL.

[0158] The above PCR amplification is performed in the following manner: initial denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 40 s (30 cycles), final extension at 72°C for 10 min, and two DNA fragments (NCgl2176 Up and NCgl2176 Down) containing the NCgl2176 gene coding region, with sizes of 796 bp and 786 bp, respectively.

[0159] After the two DNA fragments are separated and purified by agarose gel electrophoresis, a fragment of approximately 1552 bp in length is obtained by using the two DNA fragments as a template, P1 and P4 as primers, and duplicate PCR amplification.

[0160] PCR System: 10×Ex Taq Buffer 5μL, dNTP Mixture (2.5mM each) 4μL, Mg 2+ (25mM) 4μL, primer (10pM) 2μL each, Ex Taq (5U / μL) 0.25μL, total volume 50μL.

[0161] The above PCR amplification is performed in the following manner: initial denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 90 s (30 cycles), and final extension at 72°C for 10 min.

[0162] The above DNA fragment (NCgl2176 A528C ) causes the 528th adenine (A) in the YP97158 NCgl2176 gene coding region to be changed to cytosine (C), and finally causes the 176th amino acid of the encoded protein to be changed from lysine (K) to asparagine (N).

[0163] After enzymatically restricting the pK18mobsacB plasmid (purchased from Addgene) with Xba I, NCgl2176 by agarose gel electrophoresis A528CThe and linearized pK18mobsacB plasmid was isolated and purified, and assembled via the NEBuider recombination system to vector pK18-NCgl2176 A528C Obtains, and said plasmid contains a kanamycin resistance marker. Vector pK18-NCgl2176 A528C is sent to a sequencing company for sequencing, and the vector pK18-NCgl2176 containing the exact point mutation (AC) A528C preserves.

[0164] Example 2 point-mutated NCgl2176 A528C Construction of an engineered strain including

[0165] Construction method: Plasmid pK18-NCgl2176 for allelic substitution A528C The strain is transformed into the L-lysine producing strain YP97158 via electroporation (refer to WO2014121669A1 for the construction method; sequencing confirms that the wild-type NCgl2176 gene-coding region is preserved in the strain's chromosome), and a single colony obtained by culturing is evaluated using primer P1 and general primer M13R, respectively, to amplify a band-type strain of approximately 1559 bp in size and identify it as a positive strain. The positive strain is cultured in a medium containing 15% sucrose, and the single colony obtained by culturing is cultured in media containing kanamycin and media not containing kanamycin, respectively; the strain that grows in the kanamycin-free medium and does not grow in the kanamycin-containing medium is evaluated by PCR using the following primers (synthesized by Shanghai Invitrogen).

[0166]

[0167] The above PCR amplification product was subjected to SSCP electrophoresis (plasmid pK18-NCgl2176) after high-temperature denaturation and an ice bath. A528CElectrophoresis is performed using the amplified fragment as a positive control, the YP97158 amplified fragment as a negative control, and water as a blank control. Since the fragment structures differ, their electrophoretic positions differ; therefore, strains where the electrophoretic position of the fragment does not match the position of the negative control fragment but matches the position of the positive control fragment are strains with successful allelic substitution. The target fragment of the strain with successful allelic substitution is amplified via PCR using primers P5 and P6, linked to the PMD19-T vector for sequencing, and the success of the strain's allelic substitution is verified through sequence comparison at the base sequence where the mutation occurred, and the fragment is named YPL-4-011.

[0168] Example 3 NCgl2176 or NCgl2176 in the genome A528C Construction of engineered strains that overexpress genes

[0169] According to the genome sequence of wild-type Corynebacterium glutamicum ATCC13032 released by NCBI, upstream and downstream homologous cancer fragments and NCgl2176 or NCgl 2176 A528C Three pairs of primers were designed and synthesized to amplify the gene coding region and promoter region sequences, and NCgl2176 or NCgl 2176 were injected into strain YP97158 via homologous recombination. A528C Introduces genes.

[0170] The primers are designed as follows (synthesized by Invitrogen in Shanghai).

[0171]

[0172] Construction method: PCR amplification was performed using Corynebacterium glutamicum ATCC13032 or YPL-4-011 as templates, respectively, with primers P7 / P8, P9 / P10, and P11 / P12 to obtain an upstream homologous cancer fragment of approximately 720 bp, the NCgl2176 gene and its promoter fragment of approximately 1092 bp, and NCgl2176 A528CThe gene and its promoter fragment are approximately 1092 bp, and the downstream homologous cancer fragment is approximately 653 bp. Again, using P7 / P12 as primers, the three amplified fragments (upstream homologous cancer fragment, NCgl2176 gene and its promoter fragment, downstream homologous cancer fragment; or, upstream homologous cancer fragment, NCgl2176 A528C A gene and its promoter fragment, and a downstream homologous cancer fragment) are mixed and amplified using a template to obtain an integrated homologous cancer fragment.

[0173] After the PCR reaction is complete, electrophoresis is performed on the amplified product, and the kit (TIANGEN) is recovered using a columnar DNA gel to retrieve the required DNA fragment of approximately 2504 bp. This fragment is then ligated with the recovered shuttle plasmid PK18mobsacB via the Xba I restriction enzyme using the NEBuider recombination system to form the integrated plasmid PK18mobsacB-NCgl2176 or PK18mobsacB-NCgl2176 A528C Each is obtained, and the plasmid contains a kanamycin resistance marker, and through kanamycin screening, a recombinant in which the plasmid is integrated into the genome can be obtained.

[0174] PCR System: 10×Ex Taq Buffer 5μL, dNTP Mixture (2.5mM each) 4μL, Mg 2+ (25mM) 4μL, primer (10pM) 2μL each, Ex Taq (5U / μL) 0.25μL, fill volume 50μL.

[0175] The above PCR amplification is performed in the following manner: initial denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 120 s (30 cycles), and final extension at 72°C for 10 min.

[0176] Two integrated plasmids were each electro-transformed into the L-lysine-producing strain YP97158, and single colonies obtained by culture were PCR-evaluated using P13 / P14 primers. Strains containing a fragment of approximately 1609 bp amplified by PCR were designated as positive strains, while strains without the amplified fragment were designated as original strains. Positive strains were subjected to 15% sucrose screening and cultured in kanamycin-containing and kanamycin-free media, respectively. For strains that grew in kanamycin-free media and those that did not grow in kanamycin-containing media, PCR evaluation was performed using P15 / P16 primers to amplify a strain of approximately 1123 bp to form NCgl2176 or NCgl2176 A528C The genes are incorporated into strains of the YP97158 genome and named YPL-4-012 (excluding point mutations) and YPL-4-013 (including point mutations), respectively.

[0177]

[0178] Example 4 NCgl2176 or NCgl2176 in the plasmid A528C Constructed an engineered strain that overexpresses a gene

[0179] According to the genome sequence of the wild-type Corynebacterium glutamicum ATCC13032 released by NCBI, NCgl2176 or NCgl2176 A528C A pair of primers to amplify the gene coding region and promoter region sequences are designed and synthesized, and the primers are designed as follows (synthesized by Shanghai Invitrogen).

[0180]

[0181] Construction method: PCR amplification was performed using primers P17 / P18 with wild-type Corynebacterium glutamicum ATCC13032 or YPL-4-011 as templates, respectively, to obtain NCgl2176 or NCgl2176 A528CThe gene and its promoter fragment, 1140 bp, are obtained; electrophoretic recovery is performed on the amplified product to recover the required 1140 bp DNA fragment using a column-type DNA gel recovery kit; and the fragment is ligated to the recovered shuttle plasmid pXMJ19 via EcoR I enzyme restriction using the NEBuider recombination system, thereby producing the overexpression plasmid pXMJ19-NCgl2176 or pXMJ19-NCgl2176 A528C A plasmid is obtained. The plasmid contains a chloromycetin resistance marker, and the plasmid can be transformed into a strain through chloromycetin screening.

[0182] PCR System: 10×Ex Taq Buffer 5μL, dNTP Mixture (2.5mM each) 4μL, Mg 2+ (25mM) 4μL, primer (10pM) 2μL each, Ex Taq (5U / μL) 0.25μL, fill volume 50μL.

[0183] The above PCR amplification is performed in the following manner: initial denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 90 s (30 cycles), and final extension at 72°C for 10 min.

[0184] A plasmid is electrotransformed into an L-lysine-producing strain YP97158 and cultured to obtain a single colony, which is then subjected to PCR analysis using M13R(-48) and P18 primers. The strain containing a fragment of approximately 1147 bp amplified by PCR is identified as the transformed strain and is named YPL-4-014 (without point mutation) and YPL-4-015 (with point mutation), respectively.

[0185] Example 5 Construction of an engineered strain with the NCgl2176 gene deleted in the genome

[0186] Based on the genome sequence of Corynebacterium glutamicum ATCC13032 released by NCBI, two pairs of primers were synthesized to amplify fragments at both ends of the NCgl2176 gene-coding region and used as upstream and downstream homologous cancer fragments. The primers are designed as follows (synthesized by Invitrogen Shanghai).

[0187]

[0188] PCR amplification was performed using Corynebacterium glutamicum ATCC13032 as a template with primers P19 / P20 and P21 / P22, respectively, to obtain an upstream homologous cancer fragment of 852 bp and a downstream homologous cancer fragment of 787 bp; subsequently, nested PCR was performed with primers P19 / P22 to obtain the total homologous cancer fragment of 1639. After the PCR reaction was completed, electrophoresis was performed on the amplified products, and the required 1639 bp DNA fragment was recovered using a column-type DNA gel recovery kit. The recovered fragment was then ligated to the shuttle plasmid pk18mobsacB via the NEBuider recombination system following Xba I enzyme restriction to obtain a knockout plasmid. The plasmid contains a kanamycin resistance marker.

[0189] PCR analysis was performed on single colonies obtained by electrotransforming the knockout plasmid into the lysine-producing strain YP97158 and culturing them using the following primers (synthesized by Shanghai Invitrogen).

[0190]

[0191] Band-type strains with sizes of 1521 bp and 2556 bp are amplified by the above PCR and are designated as positive strains, and the strain with only the 2556 bp band-type strain amplified is the original strain. The positive strains are screened in 15% sucrose medium and then cultured in kanamycin-containing and kanamycin-free media, respectively. PCR evaluation is performed using P23 / P24 primers on the strain that grew in kanamycin-free medium and did not grow in kanamycin-containing medium. The band-type strain amplified to a size of 1521 bp is a genetically modified strain with the NCgl2176 gene-coding region knocked out, and this is named YPL-4-016.

[0192] Example 6 L-Lysine Fermentation Experiment

[0193] The strains constructed in Examples 2 to 5 and the original strain YP97158 were placed in a BLBIO-5GC-4-H model fermenter (purchased from Shanghai Bailun Biotechnology Co., Ltd.) and fermentation experiments were performed using the media indicated in Table 1 and the control process indicated in Table 2. Each strain was repeated 3 times, and the results are as shown in Table 3.

[0194] Table 3. Results of L-lysine fermentation experiment

[0195]

[0196] The results are as shown in Table 3, where the NCgl2176 gene was overexpressed in Corynebacterium glutamicum, or point mutations were performed on the NCgl2176 gene-coding region and NCgl2176 A528C Overexpression is advantageous for improving L-lysine production, weakening or knocking out the gene, and is advantageous for the accumulation of lysine.

[0197] Example 7 Transformation vector pK18-PdapB containing a point-mutated dapB gene promoter region (C(-49)A,G(-51)T,CTGCA(-54--58)GGTGT) construction of

[0198] Based on the genome sequence of Corynebacterium glutamicum ATCC13032 released by NCBI, two pairs of primers were designed and synthesized to amplify the dapB gene promoter region sequence, and a point mutation was introduced into the dapB gene promoter region (SEQ ID NO:29) in the background of strain YP97158 by means of allelic substitution, thereby converting C at position -49bp of the dapB gene promoter region nucleotide sequence to A, converting G at position -51bp to T, and converting CTGCA at positions -54--58bp to GGTGT (SEQ ID NO:30).

[0199] The primers are designed as follows (synthesized by Invitrogen in Shanghai).

[0200]

[0201] Construction method: PCR amplification is performed using Corynebacterium glutamicum ATCC13032 as a template, with primers P1' and P2', and P3' and P4', respectively.

[0202] PCR System: 10×Ex Taq Buffer 5μL, dNTP Mixture (2.5mM each) 4μL, Mg 2+ (25mM) 4μL, primer (10pM) 2μL each, Ex Taq (5U / μL) 0.25μL, fill volume 50μL.

[0203] The above PCR amplification is performed in the following manner: initial denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 60 s, 30 cycles, and final extension at 72°C for 10 min.

[0204] Two DNA fragments (dapB Up and dapB Down fragments) with lengths of 665 bp and 644 bp, respectively, containing point mutations are obtained. The two DNA fragments are separated and purified by agarose gel electrophoresis, and then a fragment (Up-Down fragment) with a length of approximately 1279 bp is amplified by Overlap PCR using the two purified DNA fragments as templates and P1' and P4' primers.

[0205] Overlap PCR System: 10×Ex Taq Buffer 5μL, dNTP Mixture (2.5mM each) 4μL, Mg 2+ (25mM) 4μL, primer (10pM) 2μL each, Ex Taq (5U / μL) 0.25μL, fill volume 50μL.

[0206] The above Overlap PCR amplification is performed in the following manner: initial denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 90 s, 30 cycles, and final extension at 72°C for 10 min.

[0207] The above Up-Down fragment is separated and purified by agarose gel electrophoresis, and the fragment includes the dapB gene promoter region and its upstream and downstream sequences, and both ends of the fragment include EcoR I and Sph I enzyme restriction sites, respectively. This DNA fragment causes C at position -49bp of the YP97158 dapB gene promoter region to be modified to A, G at position -51bp to be modified to T, and CTGCA at positions -54--58bp to be modified to GGTGT.

[0208] After purifying the fragment with a double restriction enzyme (EcoR I / Sph I), it was ligated to the shuttle plasmid pK18mobsacB (purchased from Addgene), which had also been treated with a double restriction enzyme (EcoR I / Sph I), to obtain the plasmid pK18-PdapB for allelic substitution. (C(-49)A,G(-51)T,CTGCA(-54--58)GGTGT)Obtain the plasmid, and the plasmid contains a kanamycin resistance marker. Vector pK18-PdapB (C(-49)A,G(-51)T,CTGCA(-54--58)GGTGT) Send it to a sequencing company for sequencing, and the vector pK18-PdapB containing the exact point mutation. (C(-49)A,G(-51)T,CTGCA(-54--58)GGTGT) preserves.

[0209] Example 8 pK18-PdapB containing a point mutation (C(-49)A,G(-51)T,CTGCA(-54-58)GGTGT) Construction of manipulated strains

[0210] Plasmid pK18-PdapB for allelic substitution (C(-49)A,G(-51)T,CTGCA(-54--58)GGTGT) The L-lysine-producing strain YP97158 is transformed (sequencing confirms that the wild-type dapB gene promoter is preserved on the strain's chromosome), and a single colony obtained by culturing is evaluated using primer P1' and general primer M13F, respectively, and a 1350 bp band strain is amplified to be a positive strain. The positive strain is cultured in a medium containing 15% sucrose, and the single colony obtained by culturing is cultured in kanamycin-containing and kanamycin-free media, respectively.

[0211] PCR analysis is performed on strains grown in kanamycin-free medium and not grown in kanamycin-containing medium using the following primers (synthesized by Shanghai Invitrogen).

[0212]

[0213] The above PCR amplification product was subjected to high-temperature denaturation and an ice bath, followed by SSCP electrophoresis (plasmid pK18-PdapB (C(-49)A,G(-51)T,CTGCA(-54--58)GGTGT)Electrophoresis was performed using the amplified fragment as a positive control, the YP97158 amplified fragment as a negative control, and water as a blank control. Since the fragment structures differ, their electrophoretic positions differ; therefore, strains where the fragment electrophoretic position does not match the negative control fragment position but matches the positive control fragment position are strains with successful allelic substitution. The target fragment of the positive strain was amplified again via PCR, linked to the PMD19-T vector for sequencing, and the success of the strain's allelic substitution at the mutation-corrected sequence was verified through sequence comparison, and the result was named YPL-4-010.

[0214] Example 9 L-Lysine Fermentation Experiment

[0215] The strain YPL-4-010 constructed in Example 8 and the original strain YP97158 were placed in a BLBIO-5GC-4-H model fermenter (purchased from Shanghai Bailun Biotechnology Co., Ltd.) and fermentation experiments were performed using the media indicated in Table 1 and the control process indicated in Table 2. Each strain was repeated 3 times, and the results are as shown in Table 4.

[0216] Table 4. Results of L-lysine fermentation experiment

[0217]

[0218] Note: Transformation rate (Total mass of lysine / Total amount of glucose) × 100%

[0219] The results are as shown in Table 4. The point mutant PdapB for the dapB gene promoter in Corynebacterium glutamicum (C(-49)A,G(-51)T,CTGCA(-54--58)GGTGT) Performing this is advantageous for improving L-lysine production.

[0220] As described above, embodiments of the present invention have been explained. However, the present invention is not limited to the above embodiments. All modifications, equivalent substitutions, improvements, etc. made within the concept and principles of the present invention are all necessarily within the scope of protection of the present invention.

[0221] Name of Depository: General Microbiology Center, China Microbial Species Deposit Management Committee Accession Number: CGMCC12856 Date of Deposit: 2016-08-16

Claims

Claim 1 In an L-lysine-producing microorganism belonging to the genus Corynebacterium, the expression of a polynucleotide encoding the amino acid sequence indicated by SEQ ID NO:3 is enhanced, or, the -49th nucleotide of the promoter region indicated by SEQ ID NO:29 is mutated from cytosine (C) to adenine (A), the -51st nucleotide is mutated from guanine (G) to thymine (T), and the -54th to -58th nucleotides are mutated from CTGCA to GGTGT; Herein, the improved expression is enhanced in that the expression of a polynucleotide encoding an amino acid sequence represented by SEQ ID NO:3 is enhanced, or the polynucleotide encoding an amino acid sequence represented by SEQ ID NO:3 has a point mutation, or the expression of a polynucleotide encoding an amino acid sequence represented by SEQ ID NO:3 has a point mutation and is enhanced; the point mutation of the polynucleotide encoding an amino acid sequence represented by SEQ ID NO:3 causes the 176th lysine of the amino acid sequence represented by SEQ ID NO:3 to be substituted with asparagine; and the polynucleotide sequence having the point mutation is formed by a mutation occurring at the 528th base of the polynucleotide sequence represented by SEQ ID NO:1; An L-lysine-producing recombinant microorganism belonging to the genus Corynebacterium, characterized in that the above mutation includes the 528th base of the polynucleotide sequence represented by SEQ ID NO:1, changing from adenine (A) to cytosine (C); and that the microorganism is Corynebacterium glutamicum YP97158. Claim 2 delete Claim 3 An L-lysine-producing recombinant microorganism according to claim 1, characterized in that the polynucleotide encoding the amino acid sequence represented by SEQ ID NO:3 comprises the nucleotide sequence represented by SEQ ID NO:1, or the polynucleotide sequence having the point mutation comprises the polynucleotide sequence represented by SEQ ID NO:

2. Claim 4 delete Claim 5 An L-lysine-producing recombinant microorganism according to claim 1, characterized in that the promoter nucleotide sequence is a nucleotide sequence represented by SEQ ID NO:

30. Claim 6 delete Claim 7 A separated polynucleotide characterized in that the separated polynucleotide comprises a polynucleotide sequence represented by SEQ ID NO:

2. Claim 8 delete Claim 9 A recombinant vector characterized by comprising a polynucleotide according to claim 7. Claim 10 A recombinant strain characterized in that, in a recombinant strain, the host strain is Corynebacterium glutamicum YP97158, and the recombinant strain comprises a polynucleotide sequence according to claim 7. Claim 11 Isolated promoter nucleotides characterized in that, in the isolated promoter nucleotide, the -49th nucleotide of the promoter region indicated by SEQ ID NO:29 is mutated from cytosine (C) to adenine (A), the -51st nucleotide is mutated from guanine (G) to thymine (T), and the -54th to -58th nucleotides are mutated from the nucleotide sequence CTGCA to GGTGT. Claim 12 An isolated promoter nucleotide according to claim 11, characterized by comprising a nucleotide sequence represented by SEQ ID NO:

30. Claim 13 An expression cassette of a promoter, wherein the expression cassette comprises a nucleotide sequence of a promoter according to claim 11 and a coding sequence operably linked to the promoter; wherein the coding sequence is the coding sequence of a dapB gene. Claim 14 A recombinant vector comprising a nucleotide sequence of a promoter according to claim 11; wherein the nucleotide sequence of the promoter is linked to a pK18mobsacB plasmid to construct the recombinant vector. Claim 15 A recombinant strain characterized by comprising a nucleotide sequence of a promoter according to claim 11, an expression cassette according to claim 13, or a recombinant vector according to claim 14; wherein the host strain of the recombinant strain is Corynebacterium glutamicum YP97158. Claim 16 A method for producing L-lysine, characterized by comprising the step of culturing a microorganism according to claim 1 or a recombinant strain according to claim 10, and recovering L-lysine from the culture.

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