Use of tkt gene variants and their L-lysine production

Abstract

This paper provides tkt gene variants and their use in the production of L-lysine. The tkt gene variants are DNA molecules shown in sequence IDs 3, 5, 7, 9, 11, and 13, encoding proteins shown in sequence IDs 4, 6, 8, 10, 12, and 14. The wild-type tkt gene is the DNA molecule shown in sequence ID 1, encoding the protein shown in sequence ID 2. Experiments have shown that the tkt gene and its variants can improve L-lysine production, can be used in L-lysine production, and have good application prospects.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to a Chinese patent application (application number 202310261458.1) filed on March 17, 2023, and all contents of said patent application are incorporated into this application by reference.

[0002] This invention relates to the use of tkt gene variants and their L-lysine in the production of biotechnology. [Background technology]

[0003] L-lysine has physiological effects such as promoting growth, improving immunity, and improving central nervous system function. It is one of the eight essential amino acids that humans and animals cannot synthesize themselves and are indispensable for growth. Currently, L-lysine is the second most abundant amino acid in the world, and its main production method is fermentation. Corynebacterium glutamicum is an important lysine-producing strain. Approximately 90% of industrially produced L-lysine is used as a nutritional fortifier in the feed industry, and 10% is used as a flavor enhancer and sweetener in the food industry, and as a pharmaceutical intermediate in the pharmaceutical industry.

[0004] Improving L-lysine production by fermentation may involve fermentation techniques such as stirring and oxygen supply, the composition of the nutrient medium such as the sugar concentration during fermentation, processing of the fermentation broth into an appropriate product form by methods such as drying and granulation of the fermentation broth or ion exchange chromatography, or the inherent performance characteristics of the microorganisms involved.

[0005] Methods for improving the performance characteristics of these microorganisms include mutagenesis, mutant selection, and screening. The strains thus obtained are either resistant to antimetabolites or nutritionally required for regulatoryally important metabolites and produce L-lysine. [Overview of the project]

[0006] The object of the present invention is to provide a protein that can be used for the production of L-lysine. This protein is named the tkt protein, and the tkt protein comprises the following A1) or A2) or A3): A1) Proteins that include (or are) mutant proteins in which the alanine residue at position 327 of SEQ ID No. 2 is mutated to a threonine residue, serine residue, cysteine ​​residue, proline residue, asparagine residue, glutamine residue, phenylalanine residue, leucine residue, valine residue, isoleucine residue, aspartic acid residue, methionine residue, arginine residue, glutamic acid residue, glycine residue, histidine residue, lysine residue, tryptophan residue, or tyrosine residue. A2) In the amino acid sequence of protein A1), excluding position 327, one or more amino acid residues are substituted and / or deleted and / or added, and the protein has the same function. A3) A fusion protein obtained by ligating a tag to the N-terminus and / or C-terminus of A1) or A2).

[0007] To facilitate the purification of the protein in A1), Poly-Arg, Poly-His, FLAG, Strep-tag II, or c-myc tags can be ligated to its amino or carboxyl terminus.

[0008] The protein in A2) is a protein that has 75% or more identical amino acid sequence to the protein in A1) and has the same function. Having 75% or more identical means having 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical.

[0009] The protein in A2) may be artificially synthesized, or its coding gene may be synthesized and then biologically expressed.

[0010] The protein-coding gene in A2) can be obtained by deleting one or more amino acid codons from the DNA sequence shown in SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, or SEQ ID No. 1, and / or by making one or more base pair missense mutations, and / or by ligating the sequence encoding the tag shown in the table above to its 5' and / or 3' ends. Here, the DNA molecules shown in SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, and SEQ ID No. 1 encode the proteins shown in SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, and SEQ ID No. 2, respectively.

[0011] The present invention further provides biomaterials related to the tkt protein. These biomaterials include any one of the following B1) to B4): B1) Nucleic acid molecule encoding the tkt protein, Expression cassette containing nucleic acid molecules as described in B2)B1), B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2), Recombinant microorganisms containing nucleic acid molecules as described in B4)B1), or recombinant microorganisms containing expression cassettes as described in B2), or recombinant microorganisms containing recombinant vectors as described in B3).

[0012] In the above biomaterial, the nucleic acid molecule described in B1) may contain any one of the following b11) to b19): b11) The DNA molecule shown in SEQ ID No. 3 of the sequence listing, b12) The DNA molecule shown in SEQ ID No. 5 of the sequence listing, b13) DNA molecule shown in SEQ ID No. 7 of the sequence listing, b14) The DNA molecule shown in SEQ ID No. 9 of the sequence listing, b15) The DNA molecule shown in SEQ ID No. 11 of the sequence listing, b16) The DNA molecule shown as SEQ ID No. 13 in the sequence listing, b17) The DNA molecule shown in SEQ ID No. 1 of the sequence listing, A DNA molecule that has 75% or more identity with a nucleotide sequence defined by any one of b18)b11)~b17) and encodes a tkt protein, A genomic DNA molecule that hybridizes under stringent conditions with a nucleotide sequence defined by any one of b19), b11), and b18), and encodes a tkt protein.

[0013] Here, the nucleic acid molecule may be DNA such as cDNA, genomic DNA, or recombinant DNA, or it may be RNA such as mRNA or hnRNA.

[0014] Those skilled in the art can easily mutate the nucleotide sequence encoding the tkt protein of the present invention using known methods such as directed evolution and point mutation. Any artificially modified nucleotide having 75% or more identity with the nucleotide sequence of the tkt protein of the present invention is derived from the nucleotide sequence of the present invention and is equivalent to the sequence of the present invention, as long as it encodes the tkt protein and has the function of the tkt protein.

[0015] As used herein, the term “identity” means sequence similarity to a natural nucleic acid sequence. “Identity” includes nucleotide sequences having 75% or more, 85% or more, 90% or more, or 95% or more identity with the nucleotide sequence encoding the tkt protein of the present invention. Identity can be assessed visually or using computer software. When using computer software, the identity between two or more sequences can be expressed as a percentage (%) and can be used to assess identity between related sequences.

[0016] In the above biomaterial, stringent conditions may be hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, and 1mM EDTA, followed by washing at 50°C in 2×SSC in 0.1% SDS, or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4, and 1mM EDTA, followed by washing at 50°C in 1×SSC. The material may be washed in 1% SDS, or hybridized at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4, and 1mM EDTA, and then washed at 50°C in 0.5×SSC in 0.1% SDS, or hybridized at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4, and 1mM EDTA, and then washed at 50°C in 0.1×SSC in 0.1% SDS. Alternatively, the membrane may be hybridized at 50°C in a mixed solution of 7% SDS, 0.5M NaPO4, and 1mM EDTA, and then washed at 65°C in 0.1×SSC in 0.1% SDS, or hybridized at 65°C in a solution of 6×SSC in 0.5% SDS, and then washed once each with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS. Alternatively, the membrane may be hybridized with 2×SSC in a 0.1% SDS solution at 68°C, washed twice for 5 minutes each, then hybridized with 0.5×SSC in a 0.1% SDS solution at 68°C, washed twice for 15 minutes each, or the membrane may be hybridized with 0.1×SSPE (or 0.1×SSC) in a 0.1% SDS solution at 65°C and washed.

[0017] A 75% or higher degree of identity may also be 80%, 85%, 90%, or 95% or higher.

[0018] In the above biological material, the expression cassette (tkt gene expression cassette) containing the nucleic acid molecule encoding the tkt protein described in B2) refers to DNA that can express the tkt protein in a host cell. This DNA not only contains a promoter that initiates transcription of the tkt gene, but may also contain a terminator that terminates transcription of the tkt gene. Furthermore, the expression cassette may further contain an enhancer sequence.

[0019] In the above biological material, the promoter of the expression cassette described in B2) may be the DNA molecule shown at positions 35 to 437 of SEQ ID No. 15.

[0020] In the above biological material, the vector may be a plasmid, cosmid, phage, or viral vector. Specifically, the plasmid may be pXMJ19 or pK18mobsacB plasmid.

[0021] The recombinant vector described in B3) is pXMJ19-tkt, pXMJ19-tkt A327S 、pXMJ19-tkt A327S 、pXMJ19-tkt A327C 、pXMJ19-tkt A327P 、pXMJ19-tkt A327N 、pXMJ19-tkt A327Q 、or pK18-tkt A327T may be.

[0022] pXMJ19-tkt is a recombinant vector obtained by replacing the DNA fragment between the XbalI recognition sequence and the BamHI recognition sequence of pXMJ19 with the DNA fragment shown in SEQ ID No. 15. pXMJ19-tkt A327T The difference between pXMJ19-tkt A327T and pXMJ19-tkt is that pXMJ19-tkt pXMJ19-tkt A327SThe difference between and pXMJ19-tkt is that pXMJ19-tkt A327S This is a recombinant vector obtained by substituting the gene shown in SEQ ID No. 1 of pXMJ19-tkt with the gene shown in SEQ ID No. 5. pXMJ19-tkt A327C The difference between and pXMJ19-tkt is that pXMJ19-tkt A327C This is a recombinant vector obtained by substituting the gene shown in SEQ ID No. 1 of pXMJ19-tkt with the gene shown in SEQ ID No. 7. pXMJ19-tkt A327P The difference between and pXMJ19-tkt is that pXMJ19-tkt A327P This is a recombinant vector obtained by substituting the gene shown in SEQ ID No. 1 of pXMJ19-tkt with the gene shown in SEQ ID No. 9. pXMJ19-tkt A327N The difference between and pXMJ19-tkt is that pXMJ19-tkt A327N This is a recombinant vector obtained by substituting the gene shown in SEQ ID No. 1 of pXMJ19-tkt with the gene shown in SEQ ID No. 11. pXMJ19-tkt A327Q The difference between and pXMJ19-tkt is that pXMJ19-tkt A327Q This is a recombinant vector obtained by substituting the gene shown in SEQ ID No. 1 of pXMJ19-tkt with the gene shown in SEQ ID No. 13. pK18-tkt A327T This recombinant vector was obtained by replacing the fragment (small fragment) between the XbalI recognition site and the BamHI recognition site of the pK18mobsacB vector with the DNA fragment shown in SEQ ID No. 6 of the sequence listing, without altering any other sequences of the pK18mobsacB vector.

[0023] In the above-mentioned biomaterial, the microorganism may be yeast, bacteria, algae, or fungi. Here, the bacteria may be Escherichia coli, Corynebacterium glutamicum, Brevibacterium lactofermentum, Corynebacterium pekinense, Brevibacterium ammoniagenes, Corynebacterium crenatum, Pantoea, Pantoea ananatis, Bacillus brevis, Brevibacterium brevis lactic acid bacteria, or Brevibacterium flavum. The yeast may be budding yeast or Pichia yeast.

[0024] In one embodiment of the present invention, Corynebacterium glutamicum is Corynebacterium glutamicum YP097158 or Corynebacterium glutamicum ATCC13032.

[0025] The recombinant microorganism described in B4) may be a recombinant microorganism obtained by substituting the tkt gene of a microorganism containing the tkt gene shown in SEQ ID No. 1 with SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, or SEQ ID No. 13, or a recombinant microorganism obtained by introducing and expressing the nucleic acid molecule described in B1) into a microorganism.

[0026] In the embodiment of the present invention, the recombinant microorganism is recombinant strain ATCC13032-pXMJ19-tkt A327T ATCC13032-pXMJ19-tkt A327S ATCC13032-pXMJ19-tkt A327C ATCC13032-pXMJ19-tkt A327P ATCC13032-pXMJ19-tktA327N ATCC13032-pXMJ19-tkt A327Q , YPL-tkt-1, tkt-1, YPL-tkt-2, YPL-tkt-3, tkt-2, tkt-3, YPL-tkt-4, tkt-4, YPL-tkt-5, or tkt-5.

[0027] ATCC13032-pXMJ19-tkt A327T pXMJ19-tkt A327T This is a recombinant strain obtained by introducing it into Corynebacterium glutamicum ATCC13032. ATCC13032-pXMJ19-tkt A327S pXMJ19-tkt A327S This is a recombinant strain obtained by introducing it into Corynebacterium glutamicum ATCC13032. ATCC13032-pXMJ19-tkt A327C pXMJ19-tkt A327C This is a recombinant strain obtained by introducing it into Corynebacterium glutamicum ATCC13032. ATCC13032-pXMJ19-tkt A327P pXMJ19-tkt A327P This is a recombinant strain obtained by introducing it into Corynebacterium glutamicum ATCC13032. ATCC13032-pXMJ19-tkt A327N pXMJ19-tkt A327N This is a recombinant strain obtained by introducing it into Corynebacterium glutamicum ATCC13032. ATCC13032-pXMJ19-tkt A327Q pXMJ19-tkt A327QThis is a recombinant strain obtained by introducing it into Corynebacterium glutamicum ATCC13032. The difference between YPL-tkt-1 and Corynebacterium glutamicum YP097158 is that YPL-tkt-1 is a strain obtained by substituting the gene shown in SEQ ID No. 1 of Corynebacterium glutamicum YP097158 with the gene shown in SEQ ID No. 3, without altering any other sequences. The difference between tkt-1 and Corynebacterium glutamicum ATCC13032 is that tkt-1 is a strain obtained by substituting the gene shown in SEQ ID No. 1 of Corynebacterium glutamicum ATCC13032 with the gene shown in SEQ ID No. 3, without altering any other sequences. YPL-tkt-2 is a recombinant strain obtained by substituting the spacer regions of the upstream homology arm NCgl1741 and the downstream homology arm NCgl1742 in the genome of Corynebacterium glutamicum YP097158 with the DNA fragment shown in SEQ ID No. 16 of the sequence listing, without altering any other nucleotides. YPL-tkt-3 is a recombinant strain obtained by substituting the spacer regions of the upstream homology arm NCgl1741 and the downstream homology arm NCgl1742 in the genome of Corynebacterium glutamicum YP097158 with the DNA fragment shown in SEQ ID No. 17 of the sequence listing, without altering any other nucleotides. tkt-2 is a recombinant strain obtained by substituting the spacer regions of the upstream homology arm NCgl1741 and the downstream homology arm NCgl1742 in the genome of Corynebacterium glutamicum ATCC13032 with the DNA fragment shown in SEQ ID No. 16 of the sequence listing, without altering any other nucleotides. tkt-3 is a recombinant strain obtained by substituting the spacer regions of the upstream homology arm NCgl1741 and the downstream homology arm NCgl1742 in the genome of Corynebacterium glutamicum ATCC13032 with the DNA fragment shown in SEQ ID No. 17 of the sequence listing, without altering any other nucleotides in the genome of Corynebacterium glutamicum ATCC13032. YPL-tkt-4 is a recombinant strain obtained by introducing pXMJ19-tkt into Corynebacterium glutamicum YP097158. YPL-tkt-5 is pXMJ19-tkt for Corynebacterium glutamicum YP097158 A327T This is a recombinant strain obtained by introducing [the specified substance]. tkt-4 is a recombinant strain obtained by introducing pXMJ19-tkt into Corynebacterium glutamicum ATCC13032. tkt-5 is pXMJ19-tkt for Corynebacterium glutamicum ATCC13032. A327T This is a recombinant strain obtained by introducing [the specified substance].

[0028] The present invention further provides a method for producing L-lysine. This method comprises the steps of: obtaining recombinant biological cells by expressing the tkt protein in receptor biological cells, increasing the content or activity of the tkt protein in receptor biological cells, or increasing the content or activity of the protein indicated by SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, or SEQ ID No. 2 in receptor biological cells; and culturing the recombinant biological cells to obtain L-lysine.

[0029] In the above method, the biological cell may be a yeast, bacterium, algae, fungus, plant cell, or animal cell capable of synthesizing L-lysine.

[0030] A biological cell is any biological cell capable of synthesizing a target amino acid.

[0031] In the above method, the bacterium may be Corynebacterium glutamicum.

[0032] The bacteria of the present invention include, but are not limited to, Corynebacterium glutamicum. Any bacterium containing the tkt gene shown in SEQ ID No. 1 of the sequence listing and capable of synthesizing L-lysine can produce L-lysine using the tkt mutant proteins and related biomaterials shown in SEQ ID Nos. 2, 4, 6, 8, 10, 12, or 14 of the present invention. For example, it may be Escherichia coli, Corynebacterium glutamicum, Brevibacterium lactofermentum, Corynebacterium pekinense, Brevibacterium ammoniagenes, Corynebacterium crenatum, Pantoea, Pantoea ananatis, Bacillus brevis, Bacillus brevis lactic acid bacteria, or Brevibacterium flavum. The yeast may be budding yeast or Pichia yeast.

[0033] In one embodiment of the present invention, Corynebacterium glutamicum is Corynebacterium glutamicum YP097158 or Corynebacterium glutamicum ATCC13032.

[0034] The above method is achieved by introducing and expressing the gene encoding the tkt protein into receptor organism cells, or by introducing and expressing the gene encoding the protein shown in SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, or SEQ ID No. 2 into receptor organism cells. Alternatively, the receptor organism contains the DNA molecule shown in SEQ ID No. 1, and this method is achieved by replacing the DNA molecule shown in SEQ ID No. 1 in the receptor organism with the DNA molecule shown in SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, or SEQ ID No. 13.

[0035] In the above method, recombinant biological cells are cultured using a medium capable of growing recombinant biological cells, or and / or culture recombinant biological cells using conditions that enable the proliferation of recombinant biological cells.

[0036] Recombinant biological cells may also be the recombinant microorganisms described above.

[0037] The present invention further provides a product for producing L-lysine. This product includes (or contains as an active ingredient) tkt or biomaterial.

[0038] The use of tkt or biomaterials in the production of L-lysine, or in the manufacture of products for the production of L-lysine, is also included within the scope of protection of this invention.

[0039] The use of tkt or biomaterials or methods for producing L-lysine or products for producing L-lysine in the manufacture of L-lysine-containing foods, feed or pharmaceuticals is also included within the scope of protection of this invention.

[0040] The tkt and its biomaterials of the present invention can be used to produce various products (including, but not limited to, lysine in the examples). The products to be produced may also include glutamic acid, valine, glycine, alanine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, arginine, histidine, shikimic acid, protocatechuic acid, succinic acid, α-ketoglutaric acid, citric acid, ornithine, citrulline, and the like.

[0041] Deposit information for biomaterials Classification name: Corynebacterium glutamicum Strain number: YP097158 Depository name: Center for Ordinary Microorganisms, China Microbial Species Preservation and Storage Administration Abbreviation for depositary institution: CGMCC Depository address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Postal Code: 100101 Deposit date: August 16, 2016 Deposit Center Registration Number: CGMCC No.12856 [Modes for carrying out the invention]

[0042] The present invention will be described in detail below with reference to specific embodiments, but the examples shown are for illustrative purposes only and do not limit the scope of the present invention. The examples provided below can be used by those skilled in the art as a guide for further improvements, but do not limit the present invention in any way.

[0043] The experimental methods in the following examples are all conventional methods unless otherwise specified. The materials, reagents, and equipment used in the following examples are all commercially available unless otherwise specified. In the quantitative experiments in the following examples, three repeated experiments are set up and the average value is used as the result. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5' terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

[0044] The Corynebacterium glutamicum YP097158 used in the following examples was deposited on August 16, 2016, with the Center for Ordinary Microorganisms (CGMCC, address: No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with deposit registration number CGMCC No. 12856. Ningxia Yipin Biotechnology Co., Ltd., the depositor of Corynebacterium glutamicum YP097158, authorized Heilongjiang Yipin Biotechnology Co., Ltd. to use this strain.

[0045] Example 1: Construction of the ATCC13032 strain containing the tkt gene mutant. 1. Construction of a tkt gene mutant plasmid. First, the wild-type tkt gene (SEQ ID No. 1) and its promoter were cloned into the expression vector pXMJ19. Using the genome sequence of Corynebacterium glutamicum ATCC13032, published by NCBI, as a template, PCR amplification was performed using primers tkt-F / tkt-R to obtain the wild-type tkt promoter and a 2689 bp coding region fragment (SEQ ID No. 15). After recovering this fragment, it was enzymatically cleaved with XbalI and BamHI and ligated with the recovered expression vector pXMJ19 (TaKaRa, containing chloramphenicol resistance) using the NEBuilder enzyme (NEB) at 50°C for 30 minutes. The ligated product was transformed into DH5α-competent cells, spread on 2-YT agar medium containing chloramphenicol (34 mg / L), and cultured at 37°C for 12 hours. The amplified single clones were identified by PCR using primers tkt-F / tkt-R. A 2689 bp fragment was amplified by PCR and designated as the positive transformant pXMJ19-tkt, which contained the tkt promoter and coding region sequence.

[0046] To obtain a mutant encoding the tkt gene, a tkt mutant gene plasmid was constructed using a random mutagenesis kit (Agilent Technologies, USA). Using the constructed plasmid pXMJ19-tkt as a template, PCR amplification was performed using primers tkt-F / tkt-R to obtain a tkt gene coding region and promoter region fragment containing random point mutations (the sequence is SEQ ID No. 15, but the tkt coding region has random point mutations).

[0047] The recovered DNA fragments were enzymatically cleaved with XbalI and BamHI and ligated with the expression vector pXMJ19 (TaKaRa, containing chloramphenicol resistance) using NEBuilder enzyme (NEB) at 50°C for 30 minutes. The ligated product was transformed into DH5α and spread on 2-YT agar medium containing chloramphenicol (34 mg / L) and cultured at 37°C for 12 hours. The grown single clones were identified by PCR using primers tkt-F / tkt-R. A 2689 bp fragment (sequence is SEQ ID No. 15, but has a random point mutation in the tkt coding region) was amplified by PCR and designated as a pXMJ19-tkt-MT positive transformant containing a random mutation in the tkt gene.

[0048] Here, positions 35-437 of SEQ ID No. 15 indicate the tkt gene promoter.

[0049] The primer was designed as follows (synthesized by Shanghai Invitrogen): tkt-F:5'- CAGAATAATTAAGCTTGCATGCCTGCAGGTCGAC TTCACAGCGGACGATTTC-3' (The underlined nucleotide sequence is a pXMJ19 homologous sequence, SEQ ID No. 18), tkt-R:5'- CCAAAACAGCCAAGCTGAATTCGAGCTCGGTACC AGGCAAGTAAGGGATGTGC-3' (The underlined nucleotide sequence is homologous to the pXMJ19 sequence, SEQ ID No. 19)

[0050] 2. Construction of a bacterial strain containing a mutant tkt gene plasmid. To confirm the L-lysine productivity of the mutant vector pXMJ19-tkt-MT constructed in Procedure 1, various tkt random mutant plasmids constructed in Procedure 1 were specifically transformed into wild-type Corynebacterium glutamicum ATCC13032 strain by electroporation (see WO2014121669A1 for detailed transformation methods). PCR identification was performed using primers tkt-F / tkt-R, and positive transformants were defined as those with an amplification of 2689 bp by PCR. The positive transformants were cultured for 3 passages in a medium containing chloramphenicol (34 mg / L) (the medium composition and culture conditions are shown in Table 1), then inoculated into a 500 mL Erlenmeyer flask containing 30 mL of nutrient-rich medium, and fermented with shaking at 30°C for 48 hours. After fermentation culture, the concentration of L-amino acids was measured by high-performance liquid chromatography (HPLC), and as shown in Table 2, strains with superior L-amino acid productivity compared to the wild-type Corynebacterium glutamicum ATCC13032 control strain were selected as the ATCC13032-pXMJ19-tkt mutant.

[0051] Nutrient-rich medium: The solvent is water, and the solutes and their concentrations are: glucose 30 g / L, (NH4)2SO4 2 g / L, H3PO4 0.5 g / L, KCl 0.8 g / L, MgSO4·7H2O 0.8 g / L, FeSO4·7H2O 0.05 g / L, MnSO4·H2O 0.05 g / L, FM902 yeast powder 1.5 g / L, corn steep liquor 5 g / L, molasses 17 g / L, betaine 0.5 g / L, citric acid 2 g / L, VH 20 mg / L, VB1 1.5 mg / L, VB3 1.5 mg / L, VB 12 The concentration was 1.5 g / L, and the pH was adjusted to 7.0 with sodium hydroxide.

[0052] TIFF2026510971000001.tif81170

[0053] TIFF2026510971000002.tif172170

[0054] As shown in Table 2, the Corynebacterium glutamicum ATCC13032-pXMJ19-tkt mutant strains possess a certain degree of L-lysine production ability. Of these, ATCC13032-pXMJ19-tkt mutant strain 3 exhibits superior L-lysine production ability, and it has been shown that tkt gene mutant strain 3 has the activity to synthesize L-lysine.

[0055] Plasmids were extracted from ATCC13032-pXMJ19-tkt mutant strain 3, and the tkt gene sequence was determined. The result showed that the guanine (G) at position 979 in the nucleotide sequence of the tkt gene coding region was mutated to adenine (A) (the sequence is shown in SEQ ID No. 3, and the gene containing this mutation was identified as tkt). A327T (referred to as a gene), the alanine (A) at position 327 in the corresponding amino acid sequence is mutated to threonine (T) (the sequence is shown as SEQ ID No. 4, and the protein containing this mutation is tkt A327T It was confirmed that this plasmid is a protein. A327T It states that the mutant strain containing this plasmid is called ATCC13032-pXMJ19-tkt A327T I named it that.

[0056] pXMJ19-tkt A327T The difference between and pXMJ19-tkt is that pXMJ19-tkt A327T This involves the wild-type tkt gene of pXMJ19-tkt being converted to a mutant tkt gene. A327T This is a recombinant vector obtained by substituting the gene sequence, pXMJ19-tkt A327T ga tkt A327T The ability to express mutant proteins. Wild-type TKT gene and mutant TKT A327T The difference from the gene is that the wild-type tkt gene has a GCT at positions 979-981, whereas the mutant tkt A327T The ACT gene is located at positions 979-981. Wild-type tkt protein and tkt A327T The difference from the mutant protein is that the wild-type tkt protein has an alanine residue A at position 327, whereas tkt A327TThe mutant protein has a threonine residue T at position 327.

[0057] 3. Construction of tkt gene mutant strains and recombinant vectors The mutant strain ATCC13032-pXMJ19-tkt was created using the wild-type Corynebacterium glutamicum ATCC13032 through random mutation. A327T To obtain more tkt mutants and improve their L-lysine production capacity, mutants were constructed that had the same tkt mutation site as above but with different amino acids. Specifically, plasmid pXMJ19-tkt sequenced in step two was constructed. A327T Using the template, five mutants were constructed in which the amino acid at position 327 of tkt was replaced with a different amino acid. All of the substituted amino acids were hydrophilic amino acids, and the substituted amino acids in each mutant and the names of the primers used in each mutant are shown in Table 3.

[0058] TIFF2026510971000003.tif86170

[0059] The primer was designed as follows (synthesized by Shanghai Invitrogen): S-PR-1:5'-CCATGCAGCCTTCTTCTGTGCAGAGCGCTCTG-3' (SEQ ID No.20), S-PR-2:5'-CAGAGCGCTCTGCACAGAAGAAGGCTGCATGG-3' (SEQ ID No.21), C-PR-1:5'-CCATGCAGCCTTCTTCTGTGCACAGCGCTCTG-3'(SEQ ID No.22), C-PR-2:5'-CAGAGCGCTGTGCACAGAAGAAGGCTGCATGG-3'(SEQ ID No.23), P-PR-1:5'-CCATGCAGCCTTCTTCTGTGCAGGGCGCTCTG-3' (SEQ ID No.24), P-PR-2: 5'-CAGAGCGCCCTGCACAGAAGAAGGCTGCATGG-3' (SEQ ID No.25), N-PR-1:5'-CCATGCAGCCTTCTTCTGTGCATTGCGCTCTG-3' (SEQ ID No.26), N-PR-2:5'-CAGAGCGCAATGCACAGAAGAAGGCTGCATGG-3'(SEQ ID No.27), Q-PR-1:5'-CCATGCAGCCTTCTTCTGTGCCTGGCGCTCTG-3'(SEQ ID No.28), Q-PR-2:5'-CAGAGCGCCAGGCACAGAAGAAGGCTGCATGG-3' (SEQ ID No. 29).

[0060] Using the wild-type Corynebacterium glutamicum ATCC13032 genome as a template, PCR amplification was performed using the primers tkt-F / S-PR-1 and KAPA HiFi HotStart shown in Table 2 to obtain an Up DNA fragment of 1405 bp containing the tkt mutant base. PCR amplification was then performed using the primers S-PR-2 / tkt-R and KAPA HiFi HotStart to obtain a Down DNA fragment of 1248 bp containing the tkt mutant base. After the PCR reaction, each fragment was recovered by agarose gel electrophoresis using a column-type DNA gel recovery kit. The recovered DNA fragments were ligated with the expression vector pXMJ19, which had been recovered by enzymatic cleavage with XbalI and BamHI, using NEBuilder enzyme (NEB) at 50°C for 30 minutes. The ligated product was transformed into DH5α and spread on 2-YT agar medium containing chloramphenicol (34 mg / L) and cultured at 37°C for 12 hours. The amplified single clone was identified by PCR using primers tkt-F / tkt-R, and a 2689 bp fragment was amplified to form a positive transformant, ATCC13032-pXMJ19-tkt, in which the alanine at position 327 of the protein encoded by the tkt gene was mutated to tryptophan. A327S The other four strains were constructed in the same manner, and five pXMJ19-tkt mutant vectors and strains were created by substituting the alanine at position 327 with the amino acids listed in Table 3. These were named according to the names listed in Table 3, and the mutant strain was named ATCC13032-pXMJ19-tkt. A327CATCC13032-pXMJ19-tkt A327P ATCC13032-pXMJ19-tkt A327N ATCC13032-pXMJ19-tkt A327Q I obtained it.

[0061] ATCC13032-pXMJ19-tkt A327S This is the recombinant vector pXMJ19-tkt A327S Includes pXMJ19-tkt A327S The difference between and pXMJ19-tkt is that pXMJ19-tkt A327S This involves the wild-type tkt gene of pXMJ19-tkt being converted to a mutant tkt gene. A327S This is a recombinant vector obtained by substitution in the gene sequence (shown in SEQ ID No. 5), and is pXMJ19-tkt A327S ga tkt A327S The ability to express the mutant protein (shown in SEQ ID No. 6). Wild-type tkt gene and mutant tkt A327S The difference from the gene is that the wild-type tkt gene has a GCT at positions 979-981, whereas the mutant tkt A327S The TCT is located at positions 979-981 of the gene. Wild-type tkt protein and tkt A327S The difference from the mutant protein is that the wild-type tkt protein has an alanine residue A at position 327, whereas tkt A327S The mutant protein has a serine residue S at position 327.

[0062] ATCC13032-pXMJ19-tkt A327C This is the recombinant vector pXMJ19-tkt A327C Includes pXMJ19-tkt A327C The difference between and pXMJ19-tkt is that pXMJ19-tkt A327C This involves the wild-type tkt gene of pXMJ19-tkt being converted to a mutant tkt gene. A327C This is a recombinant vector obtained by substitution in the gene sequence (shown in SEQ ID No. 7), and is pXMJ19-tkt A327C ga tkt A327CIt is capable of expressing the mutant protein (shown in SEQ ID No. 8). The difference between the wild-type tkt gene and the mutant tkt A327C gene is that positions 979 - 981 of the wild-type tkt gene are GCT, while positions 979 - 981 of the mutant tkt A327C gene are TGT. The difference between the wild-type tkt protein and the tkt A327C mutant protein is that position 327 of the wild-type tkt protein is alanine residue A, while position 327 of the tkt A327C mutant protein is cysteine residue C.

[0063] ATCC13032 - pXMJ19 - tkt A327P contains the recombinant vector pXMJ19 - tkt A327P . The difference between pXMJ19 - tkt A327P and pXMJ19 - tkt is that pXMJ19 - tkt A327P is a recombinant vector obtained by replacing the wild-type tkt gene of pXMJ19 - tkt with the mutant tkt A327P gene sequence (shown in SEQ ID No. 9), and pXMJ19 - tkt A327P is capable of expressing the tkt A327P mutant protein (shown in SEQ ID No. 10). The difference between the wild-type tkt gene and the mutant tkt A327P gene is that positions 979 - 981 of the wild-type tkt gene are GCT, while positions 979 - 981 of the mutant tkt A327P gene are CCT. The difference between the wild-type tkt protein and the tkt A327P mutant protein is that position 327 of the wild-type tkt protein is alanine residue A, while position 327 of the tkt A327P mutant protein is proline residue P.

[0064] ATCC13032 - pXMJ19 - tkt A327N contains the recombinant vector pXMJ19 - tkt A327N . The difference between pXMJ19 - tkt A327N and pXMJ19 - tkt is that pXMJ19 - tkt A327NThis involves the wild-type tkt gene of pXMJ19-tkt being converted to a mutant tkt gene. A327N This is a recombinant vector obtained by substitution in the gene sequence (shown in SEQ ID No. 11), and is pXMJ19-tkt A327N ga tkt A327N The ability to express the mutant protein (shown in SEQ ID No. 12). Wild-type tkt gene and mutant tkt A327N The difference from the gene is that the wild-type tkt gene has a GCT at positions 979-981, whereas the mutant tkt A327N The gene is located at positions 979-981, where AAT is located. Wild-type tkt protein and tkt A327N The difference from the mutant protein is that the wild-type tkt protein has an alanine residue A at position 327, whereas tkt A327N The mutant protein has an asparagine residue N at position 327.

[0065] ATCC13032-pXMJ19-tkt A327Q This is the recombinant vector pXMJ19-tkt A327Q Includes pXMJ19-tkt A327Q The difference between and pXMJ19-tkt is that pXMJ19-tkt A327Q This involves the wild-type tkt gene of pXMJ19-tkt being converted to a mutant tkt gene. A327Q This is a recombinant vector obtained by substitution in the gene sequence (shown as SEQ ID No. 13), and is pXMJ19-tkt A327Q ga tkt A327Q The ability to express the mutant protein (shown in SEQ ID No. 14). Wild-type tkt gene and mutant tkt A327Q The difference from the gene is that the wild-type tkt gene has a GCT at positions 979-981, whereas the mutant tkt A327Q The gene has CAG at positions 979-981. Wild-type tkt protein and tkt A327Q The difference from the mutant protein is that the wild-type tkt protein has an alanine residue A at position 327, whereas tkt A327Q The mutant protein has a glutamine residue Q at position 327.

[0066] IV. Measurement of L-lysine production capacity of tkt mutant strains To confirm the L-lysine productivity of the mutant vector constructed in step three, specifically, the five recombinant strains constructed in step three and the ATCC13032-pXMJ19-tkt from step two were used. A327T Each strain was cultured for three passages in a medium containing chloramphenicol (34 mg / L), then inoculated into a 500 mL Erlenmeyer flask containing 30 mL of nutrient-rich medium, and fermented by shaking at 37°C for 48 hours. After fermentation, the concentration of L-lysine was analyzed by high-performance liquid chromatography (HPLC), with wild-type Corynebacterium glutamicum ATCC13032 used as a control.

[0067] The results are shown in Table 4. L-lysine production in each mutant was significantly higher than that of the wild-type Corynebacterium glutamicum ATCC13032, and the mutant ATCC13032-pXMJ19-tkt A327T The L-lysine production capacity is ATCC13032-pXMJ19-tkt A327S ATCC13032-pXMJ19-tkt A327C ATCC13032-pXMJ19-tkt A327P ATCC13032-pXMJ19-tkt A327N ATCC13032-pXMJ19-tkt A327Q It is superior. This is because the mutation of alanine (A) to threonine (T) at position 327 of the tkt protein has been shown to be advantageous for lysine accumulation.

[0068] TIFF2026510971000004.tif67170

[0069] Example 2: Construction of a recombinant vector of the tkt gene coding region containing point mutations Based on the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, two pairs of primers were designed and synthesized to amplify the tkt gene coding region. A point mutation was introduced by allelic substitution into the tkt gene coding region (SEQ ID No. 1) of Corynebacterium glutamicum YP097158 (Deposit number: CGMCC No. 12856, Deposit date: August 16, 2016, Depositary: Institute of Microbiology, Chinese Academy of Sciences, Room 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807355). This point mutation replaces guanine (G) with adenine (A) at position 979 of the tkt gene nucleotide sequence (SEQ ID No. 1), resulting in the DNA molecule shown in SEQ ID No. 3 (mutated tkt gene, tkt A327T (To be written as "genes") was obtained.

[0070] Here, the DNA molecule shown in SEQ ID No. 1 encodes a protein whose amino acid sequence is SEQ ID No. 2. The DNA molecule shown in SEQ ID No. 3 (tkt A327T The gene is a mutant protein whose amino acid sequence is SEQ ID No. 4 (mutant protein is tkt A327T It codes for a protein. Mutant protein tkt A327T The threonine (T) at position 327 in the amino acid sequence (SEQ ID No. 4) is mutated from alanine (A).

[0071] The recombinant vector was constructed using NEBuilder assembly, and the primers were designed as follows (synthesized by Shanghai Invitrogen), with the bolded bases indicating the mutation sites. P1:5'- CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAG CGTCATTGCTTCTGATGG-3' (The underlined nucleotide sequence is the sequence on pK18, SEQ ID No. 30), P2:5'-CCATGCAGCCTTCTTCTGTGCAGTGCGCTCTG-3'(SEQ ID No.31), P3:5'-CAGAGCGCACTGCACAGAAGAAGGCTGCATGG-3'(SEQ ID No.32), P4:5'- CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCC CGGAGAAGATGAGGAAGGTTC-3' (The underlined nucleotide sequence is the sequence on pK18, SEQ ID No. 33).

[0072] Construction method: Using Corynebacterium glutamicum ATCC13032 as a template, PCR amplification was performed using primers P1 and P2, and P3 and P4, respectively, to obtain two DNA fragments (tktUp and tktDown) of 514 bp and 482 bp containing mutant bases from the tkt gene coding region.

[0073] The two DNA fragments (tktUp and tktDown) were separated and purified by agarose gel electrophoresis. The purified pK18mobsacB plasmid (Addgene) was then ligated using the NEBuilder enzyme (NEB) at 50°C for 30 minutes. The ligated product was transformed, and the resulting single clone was PCR identified using primers P1 / P4. A 964bp fragment was amplified and identified using the positive recombinant vector pK18-tkt containing a kanamycin resistance marker. A327T This was done. The recombinant vector pK18-tkt was correctly enzymatically cleaved. A327T The result was sent to a sequencing company for sequencing, and the recombinant vector pK18-tkt containing the correct point mutation (G979A) was selected. A327T It was stored for use.

[0074] This recombinant vector pK18-tkt A327T The presence of the mutation site (G979A) resulted in a mutation of guanine (G) to adenine (A) at position 979 in the coding region of the tkt gene (SEQ ID No. 1) of the Corynebacterium glutamicum YP097158 strain, ultimately changing alanine (A) at position 327 of the encoded protein to threonine (T).

[0075] Recombinant vector pK18-tktA327T This recombinant vector was obtained by replacing the fragment (small fragment) between the XbalI recognition site and the BamHI recognition site of the pK18mobsacB vector with the DNA fragment shown in SEQ ID No. 6 of the sequence listing, without altering the other sequences of the pK18mobsacB vector. (Recombinant vector pK18-tkt) A327T This is the mutant gene tkt shown in SEQ ID No.3. A327T It includes the mutation site (G979A).

[0076] Example 3: Gene TKT A327T Construction of modified strains including The allele substitution plasmid (pK18-tkt) constructed in Example 2 A327T Corynebacterium glutamicum strain YP097158 (sequencing confirmed that the wild-type tkt gene coding region is retained on the chromosome of this strain) and wild-type Corynebacterium glutamicum strain ATCC13032 were transformed by electroporation and cultured for 40 hours in solid medium containing kanamycin (50 mg / L). Single colonies obtained from the culture were identified using primers P1 / P4 from Example 2, and strains in which a 964 bp band was amplified were designated as positive strains. Positive strains were streaked in medium containing 15% sucrose (medium with the sucrose concentration in the medium in Table 1 increased to 150 g / L), and single colonies obtained from the culture were cultured in medium containing kanamycin and medium without kanamycin. Strains that grew in the medium without kanamycin but not in the medium containing kanamycin were further amplified by PCR using primers P1 / P4. Multiple DNA fragments (964 bp) obtained were sequenced, and by comparing the sequences, strains containing the base sequence mutation (G979A) were identified as positive strains that successfully underwent allelic substitution. The positive strains obtained from Corynebacterium glutamicum YP097158 and the wild-type Corynebacterium glutamicum ATCC13032 strain were named YPL-tkt-1 and tkt-1, respectively.

[0077] Both recombinant strains YPL-tkt-1 and tkt-1 contain the mutant gene tkt shown in SEQ ID No. 3.A327T Includes tkt shown in SEQ ID No. 4 A327T It can express proteins. The difference between recombinant YPL-tkt-1 and Corynebacterium glutamicum YP097158 is that YPL-tkt-1 expresses the tkt gene of Corynebacterium glutamicum YP097158 without altering other sequences. A327T The key difference is that the strain was obtained by gene substitution. The difference between recombinant strain tkt-1 and wild-type Corynebacterium glutamicum ATCC13032 is that tkt-1 replaces the tkt gene of wild-type Corynebacterium glutamicum ATCC13032 without altering any other sequences. A327T This refers to a strain obtained by gene substitution. (mutated tkt) A327T Recombinant strains containing the gene are called tkt A327T It is possible to significantly and stably improve gene expression.

[0078] Example 4: tkt gene or tkt A327T Construction of modified bacterial strains in which genes are overexpressed in the genome. Based on the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, upstream and downstream homology arm fragments and tkt or tkt A327T Three pairs of primers were designed and synthesized to amplify the gene coding region and promoter region of Corynebacterium glutamicum YP97158 and wild-type Corynebacterium glutamicum ATCC13032, and tkt or tkt A327T A gene copy was inserted by homologous recombination.

[0079] The primer was designed as follows (synthesized by Shanghai Invitrogen): P5:5'- CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAG AATGCGTTCTGGACTGAGG-3' (The underlined nucleotide sequence is the sequence on pK18, SEQ ID NO. 34), P6:5'-GAAATCGTCCGCTGTGAAGTGCACCGAGAACAGATG-3'(SEQ ID NO.35), P7:5'-CATCTGTTCTCGGTGCACTTCACAGCGGACGATTTC-3'(SEQ ID NO.36), P8:5'-GATTTAATTGCGCCATCTGAGGCAAGTAAGGGATGTGC-3' (SEQ ID NO.37), P9:5'-GCACATCCCTTACTTGCCTCAGATGGCGCAATTAAATC-3'(SEQ ID NO.38), P10:5'- CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCC GCTATGACACCTTCAACGGATC-3' (The underlined nucleotide sequence is the sequence on pK18, SEQ ID NO. 39).

[0080] Construction Method: Using the genome of Corynebacterium glutamicum ATCC13032 as a template, PCR amplification was performed using primers P5 / P6, P7 / P8, and P9 / P10, respectively, yielding an upstream homology arm fragment of 763 bp (corresponding to the partial coding region of the NCgl1740 gene and the NCgl1741 gene and its promoter region of Corynebacterium glutamicum ATCC13032), a tkt gene and its promoter fragment of 2621 bp (sequence is SEQ ID No. 16), and a downstream homology arm fragment of 596 bp (corresponding to the partial coding region of the NCgl1742 gene of Corynebacterium glutamicum ATCC13032). After the PCR reaction, the three fragments amplified from each template were recovered by electrophoresis using a column-type DNA gel recovery kit. The three recovered fragments were enzymatically cleaved with XbalI and BamHI to purify the pK18mobsacB plasmid (Addgene), and then ligated with the NEBuilder enzyme (NEB) at 50°C for 30 minutes. The ligated product was transformed and a single clone was grown and identified by PCR using primers M13F / M13R to obtain a positive integrated plasmid (recombinant vector). The obtained recombinant vector was pK18-tktOE, and the positive integrated plasmid contained a kanamycin resistance marker, allowing recombinant organisms with the plasmid integrated into the genome to be obtained by kanamycin screening. In SEQ ID No. 16, positions 1-403 are the promoter of the tkt gene, and positions 404-2506 are the tkt gene. M13F:5'-TGTAAAACGACGGCCAGT-3'(SEQ ID No.40), M13R:5'-CAGGAAACAGCTATGACC-3' (SEQ ID No.41).

[0081] Using Corynebacterium glutamicum YPL-tkt-1 as a template, PCR amplification was performed using primers P5 / P6, P7 / P8, and P9 / P10 respectively, resulting in an upstream homology arm fragment of 763 bp (corresponding to the partial coding region of the NCgl1740 gene and the NCgl1741 gene and its promoter region of Corynebacterium glutamicum ATCC13032), tkt A327T The gene and its promoter fragment (2621 bp, sequence SEQ ID No. 17), and a downstream homology arm fragment (596 bp, corresponding to the partial coding region of the NCgl1742 gene of Corynebacterium glutamicum ATCC13032) were obtained. After the PCR reaction, the three amplified fragments from each template were recovered by electrophoresis using a column-type DNA gel recovery kit. The three recovered fragments were enzymatically cleaved with XbalI and BamHI to purify the pK18mobsacB plasmid (Addgene), and ligated with the NEBuilder enzyme (NEB) at 50°C for 30 minutes. The ligated product was transformed and a single clone was grown, which was PCR identified using primers M13F / M13R to obtain a positive embedded plasmid (recombinant vector). The obtained recombinant vector was pK18-tkt A327T It is OE, and the positive integrated plasmid contains a kanamycin resistance marker, allowing recombinants with the plasmid integrated into the genome to be obtained by kanamycin screening. In SEQ ID No. 17, positions 1-403 are tkt A327T It is the promoter of the gene, and positions 404-2506 are tkt A327T It is a gene.

[0082] Correctly sequenced embedded plasmids (pK18-tktOE, pK18-tkt A327TOE) was transformed into Corynebacterium glutamicum YP097158 strain and wild-type Corynebacterium glutamicum ATCC13032 by electroporation and cultured in culture medium for 30 hours. Single colonies obtained from the culture were identified by PCR using primers P11 / P12. Strains in which a 1559 bp fragment was amplified by PCR were designated as positive strains, and those in which the fragment was not amplified were designated as original strains. Positive strains were streaked in solid medium containing 15% sucrose for 30 hours, and single colonies obtained from the culture were further identified by PCR using primers P13 / P14. Strains in which 1559 bp was amplified were designated as tkt or tkt A327T We defined positive strains as those in which the gene and its promoter are integrated into the spacer region between the upstream homology arm NCgl1741 and the downstream homology arm NCgl1742 of the Corynebacterium glutamicum genome. Strains obtained using Corynebacterium glutamicum YP097158 as the starting strain were named YPL-tkt-2 (no mutation) and YPL-tkt-3 (with mutation), respectively, and strains obtained using Corynebacterium glutamicum ATCC13032 as the starting strain were named tkt-2 (no mutation) and tkt-3 (with mutation), respectively.

[0083] Recombinant strain YPL-tkt-2 contains a double copy of the tkt gene shown in SEQ ID No. 1. Specifically, recombinant strain YPL-tkt-2 is a recombinant strain obtained by replacing the spacer region between the upstream homology arm NCgl1741 and the downstream homology arm NCgl1742 of the Corynebacterium glutamicum YP097158 genome with the DNA fragment shown in SEQ ID No. 16 (positions 1-403 of SEQ ID No. 16 are the promoter, and positions 404-2506 are the tkt gene), without altering any other nucleotides in the genome of Corynebacterium glutamicum YP097158. Recombinant strains containing a double copy of the tkt gene can significantly and stably improve tkt gene expression.

[0084] Recombinant strain tkt-2 contains a double copy of the tkt gene shown in SEQ ID No. 1. Specifically, recombinant strain tkt-2 is a recombinant strain obtained by replacing the spacer region between the upstream homology arm NCgl1741 and the downstream homology arm NCgl1742 of the Corynebacterium glutamicum ATCC13032 genome with the DNA fragment shown in SEQ ID No. 16, without altering any other nucleotides in the genome of Corynebacterium glutamicum ATCC13032. Recombinant strains containing a double copy of the tkt gene can significantly and stably improve the expression of the tkt gene.

[0085] Recombinant strain YPL-tkt-3 is a mutant tkt strain as shown in SEQ ID No. 3. A327T It contains genes. Specifically, recombinant strain YPL-tkt-3 modifies the spacer region between the upstream homology arm NCgl1741 and the downstream homology arm NCgl1742 of the Corynebacterium glutamicum YP097158 genome without altering other nucleotides in the genome, as shown in the DNA fragment indicated by SEQ ID No. 17 (positions 1-403 of SEQ ID No. 17 are the promoter, and positions 404-2506 are tkt). A327T This is a recombinant strain obtained by substituting a gene.

[0086] Recombinant strain tkt-3 is a mutant tkt strain as shown in SEQ ID No. 3. A327T It contains genes. Specifically, recombinant strain tkt-3 is a recombinant strain obtained by replacing the spacer region between the upstream homology arm NCgl1741 and the downstream homology arm NCgl1742 of the Corynebacterium glutamicum ATCC13032 genome with the DNA fragment shown in SEQ ID No. 17, without altering any other nucleotides in the genome of Corynebacterium glutamicum ATCC13032.

[0087] The primers for PCR identification are as follows: P11:5'-TCCAAGGAAGATACACGCC-3' (corresponding to the outside of upstream homology arm NCgl1740, SEQ ID No. 42), P12:5'-GCCACAAGAAAGAACGAAG-3' (corresponds to the interior of the tkt gene, SEQ ID No. 43), P13:5'-CATCCTCAACGGCATTTC-3' (corresponds to the interior of the tkt gene, SEQ ID No. 44) P14:5'-TGGTCGTTGGAATCTTGC-3' (corresponding to the outside of downstream homology arm NCgl1742, SEQ ID No. 45).

[0088] Example 5: tkt gene or tkt A327T Construction of modified bacterial strains by overexpressing genes using plasmids. pXMJ19-tkt and pXMJ19-tkt constructed in Example 1 A327T Plasmids were electroporated to transform Corynebacterium glutamicum YP097158 and wild-type Corynebacterium glutamicum ATCC13032, respectively, and cultured in culture medium (see Table 1 for medium components) for 30 hours. Single colonies obtained from the culture were identified by PCR using primers tkt-F / tkt-R, and strains in which a 2689 bp fragment (sequence ID No. 15) was amplified by PCR were designated as positive strains. Strains obtained using Corynebacterium glutamicum YP097158 as the starting strain were designated as YPL-tkt-4 (containing plasmid pXMJ19-tkt) and YPL-tkt-5 (containing plasmid pXMJ19-tkt) respectively. A327T The strains obtained using Corynebacterium glutamicum ATCC13032 as the starting strain were named tkt-4 (containing plasmid pXMJ19-tkt) and tkt-5 (containing plasmid pXMJ19-tkt A327T It was named (including)

[0089] Recombinant strains YPL-tkt-4 and tkt-4 contain the tkt gene plasmid shown in SEQ ID No. 1 and can significantly and stably improve tkt gene expression.

[0090] Recombinant strains YPL-tkt-5 and tkt-5 are mutant tkt strains as shown in SEQ ID No. 3.A327T It contains a plasmid of the gene and can significantly and stably improve the expression of the tkt gene.

[0091] Example 6: Construction of a modified strain in which the tkt gene is deleted in the genome. Based on the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, two pairs of primers were synthesized to amplify the fragments at both ends of the tkt gene coding region as upstream and downstream homology arm fragments. The primers were designed as follows (synthesized by Shanghai Invitrogen): P15:5'- CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAG GGAAATAGATGGGTGTAGACG-3' (The underlined nucleotide sequence is the sequence on pK18, SEQ ID No. 46), P16:5'-GCAAGGAACGGAAACAACGCCTTCAGGTCATCCATCTC-3' (SEQ ID No.47), P17:5'-GAGATGGATGACCTGAAGGCGTTGTTTCCGTTCCTTGC-3' (SEQ ID No.48), P18:5'- CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCC ATCAACCTTTGCCCACAG-3' (The underlined nucleotide sequence is the sequence on pK18, SEQ ID No. 49).

[0092] Construction Method: Using the genome of Corynebacterium glutamicum ATCC13032 as a template, PCR amplification was performed using primers P15 / P16 and P17 / P18, respectively, to obtain an upstream homology arm fragment of 772 bp and a downstream homology arm fragment of 824 bp for tkt knockout. The amplified products were purified by electrophoresis using a column-type DNA gel recovery kit. The recovered DNA fragments were enzymatically cleaved with XbalI / BamHI and purified pK18mobsacB plasmid (Addgene), and ligated with NEBuilder enzyme (NEB) at 50°C for 30 minutes. The ligated product was transformed and a single clone was grown and PCR identified using primers M13F / M13R to obtain the positive knockout vector pK18-Δtkt. This plasmid contains kanamycin resistance as a screening marker. This plasmid was sent for sequencing.

[0093] Correctly sequenced knockout plasmid pK18-Δtkt was used to transform Corynebacterium glutamicum YP097158 and wild-type Corynebacterium glutamicum ATCC13032 by electroporation, and the cells were cultured in culture medium for 30 hours. Single colonies obtained from the culture were identified by PCR using primers P15 / P18. Strains in which both 1596 bp and 3106 bp bands were amplified were designated as positive strains, and strains in which only the 3106 bp band was amplified were designated as original strains. After screening the positive strains in solid medium containing 15% sucrose, they were cultured for 30 hours in medium containing kanamycin and medium without kanamycin. Strains that grew in the medium without kanamycin but not in the medium containing kanamycin were further identified by PCR using primers P15 / P18. Strains in which the 1596 bp band was amplified were designated as positive strains in which the partial coding region of the tkt gene was knocked out. The positive strain tkt fragments were again amplified by PCR using primers P15 / P18 to determine their sequences. Strains that were correctly sequenced were named YPL-tkt-6 (in which the tkt gene on the genome of Corynebacterium glutamicum YP097158 was knocked out) and tkt-6 (in which the tkt gene on the genome of wild-type Corynebacterium glutamicum ATCC13032 was knocked out).

[0094] Example 7: L-Lysine Fermentation Experiment The strains constructed in Examples 2-6, Corynebacterium glutamicum progenitor strain YP097158, and wild-type Corynebacterium glutamicum ATCC13032 were subjected to fermentation experiments in a BLBIO-5GC-4-H fermentation tank (Shanghai Bailun Biotechnology Co., Ltd.) using the culture media shown in Table 5 and the control process shown in Table 6. After fermentation was complete, the amount of L-lysine produced was measured using the ninhydrin colorimetric method. The results of repeating the fermentation process three times for each strain are shown in Table 7.

[0095] TIFF2026510971000005.tif114170

[0096] TIFF2026510971000006.tif107170

[0097] TIFF2026510971000007.tif121170

[0098] As a result, as shown in Table 7, in Corynebacterium glutamicum, a point mutation (G979A) in the tkt gene coding region and tkt or tkt A327T While overexpression of genes generally contributes to increased L-lysine production, weakening or knocking out the tkt gene is detrimental to L-lysine accumulation.

[0099] The present invention has been described in detail above. Those skilled in the art will be able to implement the present invention over a wider range of parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without performing unnecessary experiments. While the present invention has shown specific embodiments, it should be understood that further improvements can be made to the present invention. In short, based on the principles of the present invention, this application is intended to include any modifications, uses, or improvements to the present invention, including those that deviate from the scope disclosed herein and are made using prior art known in the art. Some basic features may be applied within the scope of the appended claims below.

[0100] The arrangement according to the present invention is as follows: SEQ ID No.1: tkt gene wild-type ORF (CDS) sequence (nucleotide sequence 2103bp) SEQ ID NO.2: tkt protein sequence (amino acid sequence 700aa encoded by SEQ ID NO.1) MTTLTLSPELQALTVRNYPSDWSDVDTKAVDTVRVLAADAVENCGSGHPGTAMSLAPLAYTLYQRVMNVDPQDTNWAGRDRFVLSCGHSSLTQYIQLYLGGFGLEMDDLKALRTWDSLTPGHPEYRHTKGVEITTGPLGQGLASAVGMAMAARRERGLFDPTAAEGESPFDHHIY VIASDGDLQEGVTSEASSIAGTQQLGNLIVFWDDNRISIEDNTEIAFNEDVVARYKAYGWQTIEVEAGEDVAAIEAAVEAKKDTKRPTFIRVRTIIGFPAPTMMNTGAVHGAALGAAEVAATKTELGFDPEAHFAIDDEVIAHTRSLAERAAQKKAAWQVKFDEWAAANPENKA LFDRLNSRELPAGYADELPTWDADEKGVATRKASEAALQALGKTLPELWGGSADLAGSNNTVIKGSPSFGPESISTETWSAEPYGRNLHFGIREHAMGSILNGISLHGGTRPYGGTFLIFSDYMRPAVRLAALMETDAYYVWTHDSIGLGEDGPTHQPVETLAALRAIPGLSVLR PADANETAQAWAAALEYKEGPKGLALTRQNVPVLEGTKEKAAEGVRRGGYVLVEGSKETPDVILMGSGSEVQLAVNAAKALEAEGVAARVSVPCMDWFQEQDAEYIESVLPAAVTARVSVEAGIAMPWYRFLGTQGRAVSLEHFGASADYQTLFEKFGITTDAVVAAAKDSING SEQ ID No.3: Genetic variant tkt A327T ORF (CDS) sequence (nucleotide sequence 2103 bp) SEQ ID No. 4: Genetic variant tkt A327T Protein sequence (amino acid sequence 700aa encoded by SEQ ID No.3) MTTLTLSPELQALTVRNYPSDWSDVDTKAVDTVRVLAADAVENCGSGHPGTAMSLAPLAYTLYQRVMNVDPQDTNWAGRDRFVLSCGHSSLTQYIQLYLGGFGLEMDDLKALRTWDSLTPGHPEYRHTKGVEITTGPLGQGLASAVGMAMAARRERGLFDPTAAEGESPFDHHIY VIASDGDLQEGVTSEASSIAGTQQLGNLIVFWDDNRISIEDNTEIAFNEDVVARYKAYGWQTIEVEAGEDVAAIEAAVEAKKDTKRPTFIRVRTIIGFPAPTMMNTGAVHGAALGAAEVAATKTELGFDPEAHFAIDDEVIAHTRSLAERTAQKKAAWQVKFDEWAAANPENKA LFDRLNSRELPAGYADELPTWDADEKGVATRKASEAALQALGKTLPELWGGSADLAGSNNTVIKGSPSFGPESISTETWSAEPYGRNLHFGIREHAMGSILNGISLHGGTRPYGGTFLIFSDYMRPAVRLAALMETDAYYVWTHDSIGLGEDGPTHQPVETLAALRAIPGLSVLR PADANETAQAWAAALEYKEGPKGLALTRQNVPVLEGTKEKAAEGVRRGGYVLVEGSKETPDVILMGSGSEVQLAVNAAKALEAEGVAARVSVPCMDWFQEQDAEYIESVLPAAVTARVSVEAGIAMPWYRFLGTQGRAVSLEHFGASADYQTLFEKFGITTDAVVAAAKDSING SEQ ID No. 5: Genetic variant tkt A327S ORF (CDS) sequence (nucleotide sequence 2103 bp) SEQ ID No. 6: Genetic variant tkt A327S Protein sequence (amino acid sequence 700aa encoded by SEQ ID No. 5) MTTLTLSPELQALTVRNYPSDWSDVDTKAVDTVRVLAADAVENCGSGHPGTAMSLAPLAYTLYQRVMNVDPQDTNWAGRDRFVLSCGHSSLTQYIQLYLGGFGLEMDDLKALRTWDSLTPGHPEYRHTKGVEITTGPLGQGLASAVGMAMAARRERGLFDPTAAEGESPFDHHIY VIASDGDLQEGVTSEASSIAGTQQLGNLIVFWDDNRISIEDNTEIAFNEDVVARYKAYGWQTIEVEAGEDVAAIEAAVEAKKDTKRPTFIRVRTIIGFPAPTMMNTGAVHGAALGAAEVAATKTELGFDPEAHFAIDDEVIAHTRSLAERSAQKKAAWQVKFDEWAAANPENKA LFDRLNSRELPAGYADELPTWDADEKGVATRKASEAALQALGKTLPELWGGSADLAGSNNTVIKGSPSFGPESISTETWSAEPYGRNLHFGIREHAMGSILNGISLHGGTRPYGGTFLIFSDYMRPAVRLAALMETDAYYVWTHDSIGLGEDGPTHQPVETLAALRAIPGLSVLR PADANETAQAWAAALEYKEGPKGLALTRQNVPVLEGTKEKAAEGVRRGGYVLVEGSKETPDVILMGSGSEVQLAVNAAKALEAEGVAARVSVPCMDWFQEQDAEYIESVLPAAVTARVSVEAGIAMPWYRFLGTQGRAVSLEHFGASADYQTLFEKFGITTDAVVAAAKDSING SEQ ID No. 7: Genetic variant tkt A327C ORF (CDS) sequence (nucleotide sequence 2103 bp) SEQ ID No. 8: Genetic variant tkt A327C Protein sequence (amino acid sequence 700aa encoded by SEQ ID No. 7) MTTLTLSPELQALTVRNYPSDWSDVDTKAVDTVRVLAADAVENCGSGHPGTAMSLAPLAYTLYQRVMNVDPQDTNWAGRDRFVLSCGHSSLTQYIQLYLGGFGLEMDDLKALRTWDSLTPGHPEYRHTKGVEITTGPLGQGLASAVGMAMAARRERGLFDPTAAEGESPFDHHIY VIASDGDLQEGVTSEASSIAGTQQLGNLIVFWDDNRISIEDNTEIAFNEDVVARYKAYGWQTIEVEAGEDVAAIEAAVAEAKKDTKRPTFIRVRTIIGFPAPTMMNTGAVHGAALGAAEVAATKTELGFDPEAHFAIDDEVIAHTRSLAERCAQKKAAWQVKFDEWAAANPENKA LFDRLNSRELPAGYADELPTWDADEKGVATRKASEAALQALGKTLPELWGGSADLAGSNNTVIKGSPSFGPESISTETWSAEPYGRNLHFGIREHAMGSILNGISLHGGTRPYGGTFLIFSDYMRPAVRLAALMETDAYYVWTHDSIGLGEDGPTHQPVETLAALRAIPGLSVLR PADANETAQAWAAALEYKEGPKGLALTRQNVPVLEGTKEKAAEGVRRGGYVLVEGSKETPDVILMGSGSEVQLAVNAAKALEAEGVAARVSVPCMDWFQEQDAEYIESVLPAAVTARVSVEAGIAMPWYRFLGTQGRAVSLEHFGASADYQTLFEKFGITTDAVVAAAKDSING SEQ ID No. 9: Genetic variant tkt A327P ORF (CDS) sequence (nucleotide sequence 2103 bp) SEQ ID No. 10: Genetic variant tkt A327P Protein sequence (amino acid sequence 700aa encoded by SEQ ID No. 9) MTTLTLSPELQALTVRNYPSDWSDVDTKAVDTVRVLAADAVENCGSGHPGTAMSLAPLAYTLYQRVMNVDPQDTNWAGRDRFVLSCGHSSLTQYIQLYLGGFGLEMDDLKALRTWDSLTPGHPEYRHTKGVEITTGPLGQGLASAVGMAMAARRERGLFDPTAAEGESPFDHHIY VIASDGDLQEGVTSEASSIAGTQQLGNLIVFWDDNRISIEDNTEIAFNEDVVARYKAYGWQTIEVEAGEDVAAIEAAVEAKKDTKRPTFIRVRTIIGFPAPTMMNTGAVHGAALGAAEVAATKTELGFDPEAHFAIDDEVIAHTRSLAERPAQKKAAWQVKFDEWAAANPENKA LFDRLNSRELPAGYADELPTWDADEKGVATRKASEAALQALGKTLPELWGGSADLAGSNNTVIKGSPSFGPESISTETWSAEPYGRNLHFGIREHAMGSILNGISLHGGTRPYGGTFLIFSDYMRPAVRLAALMETDAYYVWTHDSIGLGEDGPTHQPVETLAALRAIPGLSVLR PADANETAQAWAAALEYKEGPKGLALTRQNVPVLEGTKEKAAEGVRRGGYVLVEGSKETPDVILMGSGSEVQLAVNAAKALEAEGVAARVSVPCMDWFQEQDAEYIESVLPAAVTARVSVEAGIAMPWYRFLGTQGRAVSLEHFGASADYQTLFEKFGITTDAVVAAAKDSING SEQ ID No. 11: Genetic variant tkt A327N ORF (CDS) sequence (nucleotide sequence 2103 bp) SEQ ID No. 12: Genetic variant tkt A327N Protein sequence (amino acid sequence 700aa encoded by SEQ ID No. 11) MTTLTLSPELQALTVRNYPSDWSDVDTKAVDTVRVLAADAVENCGSGHPGTAMSLAPLAYTLYQRVMNVDPQDTNWAGRDRFVLSCGHSSLTQYIQLYLGGFGLEMDDLKALRTWDSLTPGHPEYRHTKGVEITTGPLGQGLASAVGMAMAARRERGLFDPTAAEGESPFDHHIY VIASDGDLQEGVTSEASSIAGTQQLGNLIVFWDDNRISIEDNTEIAFNEDVVARYKAYGWQTIEVEAGEDVAAIEAAVEAKKDTKRPTFIRVRTIIGFPAPTMMNTGAVHGAALGAAEVAATKTELGFDPEAHFAIDDEVIAHTRSLAERNAQKKAAWQVKFDEWAAANPENKA LFDRLNSRELPAGYADELPTWDADEKGVATRKASEAALQALGKTLPELWGGSADLAGSNNTVIKGSPSFGPESISTETWSAEPYGRNLHFGIREHAMGSILNGISLHGGTRPYGGTFLIFSDYMRPAVRLAALMETDAYYVWTHDSIGLGEDGPTHQPVETLAALRAIPGLSVLR PADANETAQAWAAALEYKEGPKGLALTRQNVPVLEGTKEKAAEGVRRGGYVLVEGSKETPDVILMGSGSEVQLAVNAAKALEAEGVAARVSVPCMDWFQEQDAEYIESVLPAAVTARVSVEAGIAMPWYRFLGTQGRAVSLEHFGASADYQTLFEKFGITTDAVVAAAKDSING SEQ ID No. 13: Genetic variant tkt A327Q ORF (CDS) sequence (nucleotide sequence 2103 bp) SEQ ID No. 14: Genetic variant tkt A327Q Protein sequence (amino acid sequence 700aa encoded by SEQ ID No. 13) MTTLTLSPELQALTVRNYPSDWSDVDTKAVDTVRVLAADAVENCGSGHPGTAMSLAPLAYTLYQRVMNVDPQDTNWAGRDRFVLSCGHSSLTQYIQLYLGGFGLEMDDLKALRTWDSLTPGHPEYRHTKGVEITTGPLGQGLASAVGMAMAARRERGLFDPTAAEGESPFDHHIY VIASDGDLQEGVTSEASSIAGTQQLGNLIVFWDDNRISIEDNTEIAFNEDVVARYKAYGWQTIEVEAGEDVAAIEAAVEAKKDTKRPTFIRVRTIIGFPAPTMMNTGAVHGAALGAAEVAATKTELGFDPEAHFAIDDEVIAHTRSLAERQAQKKAAWQVKFDEWAAANPENKA LFDRLNSRELPAGYADELPTWDADEKGVATRKASEAALQALGKTLPELWGGSADLAGSNNTVIKGSPSFGPESISTETWSAEPYGRNLHFGIREHAMGSILNGISLHGGTRPYGGTFLIFSDYMRPAVRLAALMETDAYYVWTHDSIGLGEDGPTHQPVETLAALRAIPGLSVLR PADANETAQAWAAALEYKEGPKGLALTRQNVPVLEGTKEKAAEGVRRGGYVLVEGSKETPDVILMGSGSEVQLAVNAAKALEAEGVAARVSVPCMDWFQEQDAEYIESVLPAAVTARVSVEAGIAMPWYRFLGTQGRAVSLEHFGASADYQTLFEKFGITTDAVVAAAKDSING SEQ ID No. 15: Primer tkt-F / tkt-R amplification sequence (2689 bp) SEQ ID No. 16: P7 / P8tkt and its promoter sequence (2621 bp) integrated into the genome. SEQ ID No. 17: P7 / P8tkt integrated into the genome A327T and its promoter sequence (2621 bp) [Industrial applicability]

[0101] The protein and related biomaterials of the present invention can improve L-lysine production, can be used for L-lysine production, and have good application prospects.

Claims

1. Proteins containing A1) or A2) or A3) below: A1) Proteins including mutant proteins in which the alanine residue at position 327 of SEQ ID No. 2 is mutated to a threonine residue, serine residue, cysteine ​​residue, proline residue, asparagine residue, glutamine residue, phenylalanine residue, leucine residue, valine residue, isoleucine residue, aspartic acid residue, methionine residue, arginine residue, glutamic acid residue, glycine residue, histidine residue, lysine residue, tryptophan residue, or tyrosine residue. A2) A protein in which one or more amino acid residues are substituted and / or deleted and / or added in the amino acid sequence excluding position 327 of protein A1), and which has the same function, A3) A fusion protein obtained by ligating a tag to the N-terminus and / or C-terminus of A1) or A2).

2. A biomaterial relating to a protein according to claim 1, comprising any one of the following B1) to B4): B1) A nucleic acid molecule encoding the protein described in claim 1, B2) Expression cassette containing the nucleic acid molecule described in B1), B3) Recombinant vectors containing nucleic acid molecules as described in B1), or recombinant vectors containing expression cassettes as described in B2), B4) Recombinant microorganisms containing the nucleic acid molecule described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

3. The biomaterial according to claim 2, characterized in that the nucleic acid molecule described in B1) contains any one of the following b11) to b19): b11) DNA molecule shown in SEQ ID No. 3 of the sequence listing, b12) DNA molecule shown in SEQ ID No. 5 of the sequence listing, b13) DNA molecule shown in SEQ ID No. 7 of the sequence listing, b14) DNA molecule shown in SEQ ID No. 9 of the sequence listing, b15) DNA molecule shown in SEQ ID No. 11 of the sequence listing, b16) DNA molecule shown in SEQ ID No. 13 of the sequence listing, b17) DNA molecule shown in SEQ ID No. 1 of the sequence listing, A DNA molecule that has 75% or more identity with a nucleotide sequence defined by any one of b18) b11) to b17), and that encodes the protein described in claim 1, A genomic DNA molecule that hybridizes under stringent conditions with a nucleotide sequence defined by any one of b19) b11) to b18), and encodes the protein described in claim 1.

4. The promoter of the expression cassette described in B2) is the DNA molecule shown at positions 35-437 of SEQ ID No.

15. The biomaterial according to claim 2 or 3, characterized in that the recombinant microorganism described in B4) is a recombinant microorganism obtained by substituting the tkt gene of a microorganism containing the tkt gene shown in SEQ ID No. 1 with SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, or SEQ ID No. 13, or a recombinant microorganism obtained by introducing and expressing the nucleic acid molecule described in B1) into a microorganism.

5. A method for producing L-lysine, A method for producing L-lysine, comprising the steps of: expressing the protein described in claim 1 in receptor biological cells, increasing the content or activity of the protein described in claim 1 in receptor biological cells, or increasing the content or activity of the protein shown in SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, or SEQ ID No. 2 in receptor biological cells to obtain recombinant biological cells; and culturing the recombinant biological cells to obtain L-lysine.

6. The method according to claim 5, characterized in that the biological cell is a yeast, bacterium, algae, fungus, plant cell, or animal cell capable of synthesizing L-lysine.

7. The method according to claim 6, characterized in that the bacterium is Corynebacterium glutamicum.

8. A product for producing L-lysine, comprising the protein described in claim 1 or the biomaterial described in any one of claims 2 to 4.

9. Use of the protein according to claim 1 in the production of L-lysine, or in the manufacture of a product for the production of L-lysine.

10. Use of the biomaterial according to any one of claims 2 to 4 in the production of L-lysine, or in the manufacture of a product for the production of L-lysine.

11. Use of the protein described in claim 1 in the manufacture of a food, feed, or pharmaceutical product containing L-lysine.

12. Use of the biomaterial according to any one of claims 2 to 4 in the manufacture of food, feed, or pharmaceuticals containing L-lysine.

13. Use of the method according to any one of claims 5 to 7 in the manufacture of a food, feed or pharmaceutical product containing L-lysine.

14. Use of the product according to claim 8 in the manufacture of food, feed, or pharmaceuticals containing L-lysine.

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