Galactose-1-phosphate uridyltransferase mutants and their use in the production of L-lysine

Abstract

The present invention provides a galactose-1-phosphate uridyl transferase mutant and its use in producing L-lysine. This galactose-1-phosphate uridyl transferase comprises the following A1) or A2): A1) a protein having the amino acid sequence set forth in SEQ ID NO: 2, and A2) a fusion protein in which a tag is linked to the N-terminus and / or C-terminus of A1). The present invention has discovered that the amount of L-lysine produced in cells can be increased by terminating or knocking out the gene encoding galactose-1-phosphate uridyl transferase in advance. L-lysine can be produced by mutating or knocking out the gene encoding galactose-1-phosphate uridyl acyl transferase in cells. The present invention has excellent applicability.

Description

[Technical Field]

[0001] The present invention relates to a galactose-1-phosphate uridyltransferase mutant and its use in the production of L-lysine in the field of biotechnology. [Background technology]

[0002] L-lysine is one of the eight essential amino acids that humans and animals cannot synthesize on their own and is essential for growth, possessing physiological effects such as promoting growth, improving immunity, and improving central nervous system function. Currently, L-lysine is the second most abundant amino acid in the world, and its main production method is fermentation, with Corynebacterium glutamicum being an important lysine-producing strain. Approximately 90% of the industrially produced L-lysine is used as a nutritional enhancer in the feed industry, and 10% is used as an umami seasoning and sweetener in the food industry and as a pharmaceutical intermediate in the pharmaceutical industry.

[0003] Currently, L-lysine is mainly produced by direct fermentation, which uses a strain with a complete L-lysine biosynthetic pathway to produce it through aerobic fermentation using molasses, starch hydrolysate, etc. as substrates. Currently, L-lysine fermentation strains both domestically and internationally are mainly mutant strains of Corynebacterium glutamicum, and since the main factor affecting L-lysine production volume is the producing bacteria, the current focus of research is on improving the productivity of L-lysine-producing strains. Summary of the Invention

[0004] The technical problem that the present invention aims to solve is how to produce L-lysine.

[0005] In order to solve the above technical problems, the present invention first provides the use of a substance that knocks out a gene encoding a protein or inhibits the content or activity of a protein in the production of L-lysine.

[0006] The protein comprises A1) or A2) below: A1) a protein whose amino acid sequence is SEQ ID NO: 2; A2) A fusion protein in which a tag is linked to the N-terminus and / or C-terminus of A1).

[0007] In the above use, the encoding gene may include the following b1), b2), or b3): b1) a DNA molecule shown in SEQ ID NO: 1 of the Sequence Listing; b2) a DNA molecule having 75% or more identity with the nucleotide sequence defined in b1) and encoding a protein; b3) A DNA molecule that hybridizes under stringent conditions with a nucleotide sequence defined in b1) or b2) and encodes a protein.

[0008] The term "identity" as used herein refers to sequence similarity with a natural nucleic acid sequence. "Identity" includes nucleotide sequences that exhibit 75% or more, 85% or more, 90% or more, or 95% or more identity with the nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 of the present invention. Identity can be evaluated visually or using computer software. When using computer software, identity between two or more sequences can be expressed as a percentage (%) and can be used to evaluate identity between related sequences.

[0009] Stringent conditions are hybridization at 50°C in a mixture of 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, and 1 mM EDTA, followed by rinsing in 2x SSC with 0.1% SDS at 50°C, or hybridization at 50°C in a mixture of 7% SDS, 0.5 M NaPO4, and 1 mM EDTA, followed by rinsing in 1x SSC with 0.1% SDS at 50°C. or hybridizing in a mixture of 7% SDS, 0.5 M NaPO4, and 1 mM EDTA at 50°C and rinsing in 0.5x SSC and 0.1% SDS at 50°C; or hybridizing in a mixture of 7% SDS, 0.5 M NaPO4, and 1 mM EDTA at 50°C and rinsing in 0.1x SSC and 0.1% SDS at 50°C. Alternatively, hybridize in a mixed solution of 7% SDS, 0.5 M NaPO4, and 1 mM EDTA at 50°C and rinse in 0.1×SSC, 0.1% SDS at 65°C, or hybridize in a solution of 6×SSC, 0.5% SDS at 65°C, and then wash the membrane once each with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS, or Alternatively, hybridization can be performed in a solution of 0.1x SSPE (or 0.1x SSC) and 0.1% SDS at 65°C and the membrane can be washed twice for 5 minutes each, followed by hybridization in a solution of 0.5x SSC and 0.1% SDS at 68°C and the membrane can be washed twice for 15 minutes each, or hybridization can be performed in a solution of 0.1x SSPE (or 0.1x SSC) and 0.1% SDS at 65°C and the membrane can be washed.

[0010] 75% or greater identity may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0011] In the above uses, the substance may comprise B1) or B2): B1) a nucleic acid molecule that reduces the expression level of a protein; B2) An expression cassette, a recombinant vector, a recombinant microorganism or a genetically modified cell line comprising a nucleic acid molecule according to B1).

[0012] The nucleic acid molecule described in B1) may be DNA such as cDNA, genomic DNA, or recombinant DNA, or may be RNA such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0013] The expression cassette described in B2) is DNA capable of expressing a gene in a host cell. The DNA may contain not only a promoter for initiating transcription of the gene but also a terminator for terminating transcription of the gene. Furthermore, the expression cassette may contain an enhancer sequence.

[0014] Plant expression vectors can be used to construct recombinant vectors containing gene expression cassettes.

[0015] In the above use, the substance may be a substance in which the codon for the glutamic acid residue at position 414 of SEQ ID NO: 2 has been mutated to a terminator; Alternatively, the substance may be a substance in which the guanine nucleotide at position 1240 of SEQ ID NO: 1 has been mutated to a thymine nucleotide.

[0016] The present invention further provides a method for producing L-lysine, which includes the steps of reducing the content or activity of a protein in a recipient organism cell or knocking out a gene encoding the protein in the recipient organism cell to obtain a recombinant organism cell, and culturing the recombinant organism cell to obtain L-lysine.

[0017] The biological cells contain a gene encoding the protein.

[0018] In the above, knocking out a gene encoding a protein in a recipient organism cell can be achieved by methods such as gene knockout or gene silencing.

[0019] In the above, gene knockout means the following: Knockout is a foreign DNA introduction technique in which a DNA fragment containing a specific known sequence is used to cause homologous recombination with a gene with the same or similar sequence in the recipient cell genome, resulting in its integration into the recipient cell genome and its expression. By modifying the genes of an organism and disabling specific gene functions, it is possible to inhibit some functions.

[0020] In the above, gene silencing means the following: Gene silencing, also known as gene suppression, is a special physiological phenomenon in the control of gene expression in eukaryotic cells, in which cellular genes are affected by a complex of various factors during their expression process, causing some genes to be "silenced," resulting in a loss of transcriptional activity and the cessation or reduction of expression.

[0021] In the above method, the biological cells may be yeast, bacteria, algae, fungi, plant cells, or animal cells capable of synthesizing L-lysine.

[0022] In the above method, the bacterium may be Corynebacterium glutamicum, for example, Corynebacterium glutamicum YP097158.

[0023] The bacterium of the present invention includes, but is not limited to, Corynebacterium glutamicum. L-lysine can be produced by mutating or knocking out any gene set forth in SEQ ID NO: 1 in the Sequence Listing. For example, the bacterium may be Corynebacterium glutamicum, Escherichia coli, Pantoea ananatis, Bacillus brevis, or lactobacillus brevis.

[0024] The above method can be achieved by mutating the codon for the glutamic acid residue at position 414 of SEQ ID NO: 2 to a terminator in a recipient organism cell, or by mutating the guanine nucleotide at position 1240 of SEQ ID NO: 1 to a thymine nucleotide, or by knocking out the gene shown in SEQ ID NO: 1.

[0025] In the above method, the recombinant biological cells are cultured using a medium in which the recombinant biological cells can grow, And / or, the recombinant biological cells are cultured under conditions that allow the recombinant biological cells to grow.

[0026] Recombinant biological cells can be used to produce a variety of products, including, but not limited to, lysine in the examples, and may further 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.

[0027] The present invention further provides a biomaterial comprising the following b1), b2), b3), b4), or b5): b1) a DNA molecule encoding a protein having the amino acid sequence set forth in SEQ ID NO: 6; b2) a DNA molecule having 75% or more identity with the sequence of the DNA molecule defined in b1) and encoding the protein set forth in SEQ ID NO: 6; b3) a DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in b1) or b2) and encodes the protein set forth in SEQ ID NO: 6; b4) an expression cassette, a recombinant vector, a recombinant microorganism or a genetically engineered cell line comprising a DNA molecule according to b1), b2) or b3); b5) Recombinant biological cells.

[0028] The cell line may or may not include propagation material.

[0029] The present invention further provides a product for producing L-lysine, which contains (or has as an active ingredient) the above-mentioned substance or biomaterial.

[0030] Deposit Information of Biological Materials Classification name: Corynebacterium glutamicum Strain number: YP097158 Depository name: Center for Ordinary Microorganisms, China Committee for the Preservation and Management of Microorganisms Depository institution abbreviation: CGMCC Address of depository institution: No. 3, Courtyard, No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101 Deposit date: August 16, 2016 Depository Center Registration Number: CGMCC No. 12856 DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention will be described in detail below with reference to specific embodiments, but the examples shown are merely for the purpose of illustrating the present invention and do not limit the scope of the present invention. The examples provided below can be used as a guide for those skilled in the art to make further improvements, but do not limit the present invention in any way.

[0032] Unless otherwise specified, the experimental methods in the following examples are all conventional methods and are performed according to the techniques or conditions described in literature in the field or in accordance with the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc. used in the following examples are all commercially available. The quantitative experiments in the following examples were all performed three times, and the average value was 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 / RNA, and the last position is the 3'-terminal nucleotide of the corresponding DNA / RNA.

[0033] In the examples below, data was processed using SPSS1 1.5 statistical software and tested using One-way ANOVA.

[0034] Example 1: Construction and screening of mutants of the galactose-1-phosphate uridyltransferase NCgl2002 gene favorable for L-lysine synthesis 1. Construction of a mutant plasmid for galactose-1-phosphate uridyltransferase NCgl2002 First, the wild-type NCgl2002 gene (SEQ ID NO: 1) and its promoter sequence 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 pXMJ19-PF and pXMJ19-PR to obtain the wild-type NCgl2002 gene and its promoter sequence (SEQ ID NO: 3). The isolated vector was ligated with the expression vector pXMJ19 (TaKaRa, containing chloramphenicol resistance) digested with BamHI / EcoRI and recovered using NEBuilder enzyme (NEB) at 50°C for 30 minutes. The ligation product was transformed into DH5α and plated on 2-YT agar medium containing chloramphenicol (34 mg / L) and cultured at 37°C to obtain pXMJ19-NCgl2002 (SEQ ID NO: 3), a pXMJ19 transformant containing the NCgl2002 gene and its promoter sequence. A single clone was identified by PCR using primers M13R(-48) / P1 and rTaq. A PCR-amplified 1465 bp fragment (SEQ ID NO: 4) was identified as the positive pXMJ19 transformant containing the NCgl2002 gene and its promoter sequence, pXMJ19-NCgl2002.

[0035] In SEQ ID NO: 3, positions 42 to 113 are the promoter sequence.

[0036] To obtain mutants of the NCgl2002 gene encoding galactose-1-phosphate uridyltransferase, a random mutagenesis kit (Agilent Technologies, USA) was used to generate NCgl2002 mutant gene plasmids. PCR amplification was performed using the pXMJ19-NCgl2002 plasmid as a template and primers pXMJ19-PF and pXMJ19-PR to obtain a 1441-bp NCgl2002 gene fragment containing random point mutations. A positive transformant of pXMJ19 containing the NCgl2002 gene, pXMJ19-NCgl2002-MT (the sequence of which is SEQ ID NO: 3, but which contains random point mutations in the NCgl2002 coding region), was also obtained.

[0037] The recovered DNA fragment was ligated with the expression vector pXMJ19 (TaKaRa, containing chloramphenicol resistance) that had been digested with BamHI / EcoRI and recovered using NEBuilder enzyme (NEB) at 50°C for 30 minutes. The ligation product was transformed into DH5α, plated on 2-YT agar medium containing chloramphenicol (34 mg / L), and cultured at 37°C. The grown single clones were identified by PCR using primers M13R(-48) / P1 and rTaq. Those containing the PCR-amplified 1465 bp fragment (sequence of SEQ ID NO: 4, but with random point mutations in the NCgl2002 coding region) were identified as positive transformants of pXMJ19 containing the NCgl2002 random mutations.

[0038] The primers were designed as follows (synthesized by Shanghai Invitrogen): pXMJ19-PF:5'- AATTAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCC aacaccacagtagacaatagccttg-3' (the underlined nucleotide sequence is the sequence of the homology arm of pXMJ19) (SEQ ID NO: 9), pXMJ19-PR:5'- GAAAATCTTCTCTCATCCGCCAAAACAGCCAAGCTGAATTC ttataggaggggattgtatttaagg-3' (the underlined nucleotide sequence is the sequence of the homology arm of pXMJ19) (SEQ ID NO: 10), M13R(-48): 5'-AGCGGATAACAATTTCACACAGGA-3' (SEQ ID NO: 11), P1: 5'-CTCTCATCCGCCAAAACAG-3' (SEQ ID NO: 12).

[0039] 2. Screening for mutants of the galactose-1-phosphate uridyltransferase NCgl2002 gene that favor L-lysine synthesis To confirm the L-lysine productivity of the mutant vector constructed in step 1, the NCgl2002 randomly mutated plasmid constructed in step 1 was transferred to Corynebacterium glutamicum YP097158 (accession number: CGMCC). No. 12856, deposit date: August 16, 2016, depository institution: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807355), was electroporated and cultured in medium containing chloramphenicol (34 mg / L) (medium composition and culture conditions are shown in Table 1). The grown single clones were identified by PCR using primers M13R(-48) / P1 and rTaq. The PCR-amplified clone containing a 1465 bp fragment (sequence is SEQ ID NO: 4, but with random point mutations in the NCgl2002 coding region) was determined to be a positive transformant of pXMJ19 containing the NCgl2002 random mutation.

[0040] The positive transformant was cultured in a medium containing chloramphenicol (34 mg / L) (the medium composition and culture conditions are shown in Table 1). After three subcultures, it was inoculated into a 500 mL Erlenmeyer flask containing 30 mL of rich medium and subjected to shaking fermentation at 37°C for 24 hours. The fermented bacteria reached an OD 600 When the RI=0.1 was reached, IPTG was added to a final concentration of 0.1 mM to induce overexpression of the NCgl2002 protein.

[0041] After fermentation, the concentrations of L-amino acids were analyzed by high-performance liquid chromatography (HPLC), and the results are shown in Table 2. A strain with superior L-amino acid productivity to the control Corynebacterium glutamicum YP097158 was selected as the YP097158-pXMJ19-NCgl2002 mutant.

[0042] Rich medium: The solvent is water, and the solutes and their concentrations are glucose 30g / L, (NH4)2SO4 2g / L, H3PO4 0.5g / L, KCl 0.8g / L, MgSO4·7H2O 0.8g / L, FeSO4·7H2O 0.05g / L, MnSO4·H2O 0.05g / L, FM902 yeast powder 1.5g / L, corn steep liquor 5g / L, molasses 17g / L, betaine 0.5g / L, citric acid 2g / L, vitamin H 20mg / L, vitamin B 11.5mg / L, vitamin B 31.5mg / L, vitamin B 12 The concentration was 1.5 g / L and the pH was adjusted to 7.0 with sodium hydroxide.

[0043] TIFF2026501022000001.tif73170

[0044] TIFF2026501022000002.tif172170

[0045] As shown in Table 2, among the Corynebacterium glutamicum YP097158-NCgl2002 mutant strains, YP097158-NCgl2002 mutant strain 3 maintains L-lysine productivity, while the others have reduced L-lysine productivity. This gene inhibits L-lysine production, and NCgl2002 mutant strain 3 maintains its L-lysine synthetic ability by inactivating this gene.

[0046] A plasmid was extracted from Corynebacterium glutamicum YP097158 mutant strain 3, and the sequence of the NCgl2002 gene was analyzed. As a result, the mutation site of NCgl2002 was found to be a mutation of guanine (G) to thymine (T) at position 1240 of the gene coding region (the gene containing this mutation was called NCgl2002). G1240T The glutamic acid (E) at position 414 in the amino acid sequence of the mutant protein NCgl2002 is mutated to a terminator (*) (the protein containing this mutation is called NCgl2002 G1240T The DNA sequence shown in SEQ ID NO: 1 is the wild-type NCgl2002 gene, the amino acid sequence of the encoded protein is SEQ ID NO: 2 (the name of this protein is wild-type NCgl2002 protein), and the DNA sequence shown in SEQ ID NO: 5 is the mutant NCgl2002 protein. G1240T gene, and mutant NCgl2002 G1240T The thymine (T) at position 1240 in the gene sequence (SEQ ID NO: 5) was mutated from a guanine (G), and the amino acid sequence of the encoded protein was SEQ ID NO: 6 (the name of this mutant protein is mutant NCgl2002 E414* protein), and the mutant protein NCgl2002 E414* The terminator (*) at position 414 in the amino acid sequence of (SEQ ID NO: 6) is mutated from glutamic acid (E).

[0047] Example 2: Construction of a modified strain containing the NCgl2002 gene mutation in its genome Based on the genome sequence of Corynebacterium glutamicum YP097158 or wild-type Corynebacterium glutamicum strain ATCC13032, the NCgl2002 gene and mutant NCgl2002 were isolated in high-yielding strains by allelic replacement. E414* The effect of the gene on L-lysine production was further investigated.

[0048] A point mutation was introduced into the coding region of the NCgl2002 gene (SEQ ID NO: 1), where the guanine (G) at position 1240 in the nucleotide sequence of the NCgl2002 gene (SEQ ID NO: 1) was mutated to thymine (T), resulting in the DNA molecule shown in SEQ ID NO: 5 (mutant NCgl2002 gene, named mutant NCgl2002 E414* The goal is to obtain the gene.

[0049] Here, the amino acid sequence of the protein encoded by the DNA molecule shown in SEQ ID NO: 1 is SEQ ID NO: 2 (the name of this protein is wild-type NCgl2002 protein). The amino acid sequence of the protein encoded by the DNA molecule shown in SEQ ID NO: 5 is SEQ ID NO: 6 (the name of this protein is mutant NCgl2002 protein). E414* Mutant protein NCgl2002 E414* The glutamic acid (E) at position 414 in the amino acid sequence (SEQ ID NO: 6) is mutated to a terminator (*).

[0050] 1. Mutant NCgl2002 gene E414* Construction of recombinant vectors for gene coding regions Using the genomic DNA of Corynebacterium glutamicum YP097158 or the wild-type Corynebacterium glutamicum strain ATCC13032 as a template, PCR amplification was performed using primers P2 / P3, P4 / P5, and KAPA HiFi HotStart, respectively, to obtain two NCgl2002 clones with 525-bp and 533-bp mutant bases, respectively. E414* DNA fragment (NCgl2002 E414* Up and NCgl2002 E414* After the PCR reaction was completed, NCgl2002 was isolated by agarose gel electrophoresis using a column-type DNA gel recovery kit. E414* Up and NCgl2002 E414* The Down fragment was recovered. The recovered DNA was subjected to overlap PCR using primers P2 / P5 to generate the point mutation integrated homology arm DNA fragment Up-NCgl2002E414* -Down (SEQ ID NO: 7) 1022 bp was obtained.

[0051] The primers were designed as follows (synthesized by Shanghai Invitrogen): P2:5'- CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAG GAACTGCATCATCTACGTGG-3' (the underlined nucleotide sequence is the sequence on pK18) (SEQ ID NO: 13), P3: 5'-GCGTTCAACGGAGCATTACATGGCGATGCG-3' (SEQ ID NO: 14), P4: 5'-GATCTG CGCATCGCCATGTAATGCTCCGTTG -3' (SEQ ID NO: 15), P5:5'- CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCC GCACCAAGCAGCGCGGTGAC-3', (the underlined nucleotide sequence is the sequence on pK18) (SEQ ID NO: 16).

[0052] The point mutation integrated homology arm DNA fragment obtained by overlap PCR (Up-NCgl2002 E414* The resulting plasmid (NCgl2002) was purified by agarose gel electrophoresis and then ligated with pK18mobsacB plasmid (Addgene) that had been digested with XbaI and BamHI and purified using NEBuilder enzyme (NEB) at 50°C for 30 minutes. The ligated product was transformed into E. coli DH5a and the resulting single clones were identified by PCR using primers M13F / M13R (M13F: 5'-TGTAAAACGACGGCCAGT-3' (SEQ ID NO: 17), M13R: 5'-CAGGAAACAGCTATGACC-3' (SEQ ID NO: 18)). The resulting plasmid was extracted and a positive recombinant vector with the correct sequence was designated pK18-NCgl2002. G1240T This recombinant vector contains a kanamycin resistance marker.

[0053] This recombinant vector, pK18-NCgl2002 G1240T in NCgl2002 G1240TThe Up-Down DNA is 1022 bp (SEQ ID NO: 7) and contains a mutation site (GT) that mutates guanine (G) at position 1240 in the coding region of the NCgl2002 gene of Corynebacterium glutamicum YP097158 and the wild-type Corynebacterium glutamicum strain ATCC13032 to thymine (T), ultimately mutating glutamic acid (E) at position 414 in the encoded protein to a terminator (*).

[0054] Recombinant vector pK18-NCgl2002 G1240T is a recombinant vector obtained by replacing the fragment (small fragment) between the XbaI recognition site and the BamHI recognition site of the pK18mobsacB vector with the DNA fragment shown in SEQ ID NO: 7 in the Sequence Listing, without changing the other sequences of the pK18mobsacB vector. G1240T is the mutant gene NCgl2747 shown in SEQ ID NO: 5 G1240T Contains the mutation site (GT).

[0055] 2. NCgl2002 in the genome G1240T Construction of engineered strains containing The allele replacement plasmid (pK18-NCgl2002 G1240TThe L-lysine-producing bacterium Corynebacterium glutamicum YP097158 and wild-type Corynebacterium glutamicum strain ATCC13032 were transformed with the same transformation method as above by electroporation and cultured on solid culture plates containing kanamycin (see Table 1 for medium composition and culture conditions). Single colonies arising from the culture were identified using the above primer P2 and universal primer M13R, respectively, and strains that amplified a 1070 bp band were designated as positive strains. Positive strains were cultured in a medium containing 15% sucrose (a medium in which the sucrose concentration in Table 1 was increased to 15 g / L), and the resulting single colonies were cultured in a medium containing and without kanamycin, respectively. Strains that grew in the kanamycin-free medium but not in the kanamycin-containing medium were selected for further PCR amplification using the following primers (synthesized by Shanghai Invitrogen). P6: 5'-CTACCCTGGCAGGTTTTGAAG-3' (SEQ ID NO: 19), P7: 5'-GAAGTTCTGAATGCGGCTC-3' (SEQ ID NO: 20).

[0056] The resulting DNA fragment (256 bp) was denatured at 95°C for 10 minutes, followed by ice bath for 5 minutes, and then subjected to SSCP (Single-Strand Conformation Polymorphism) electrophoresis (plasmid pK18-NCgl2002 G1240T The amplified fragment was used as the positive control, the Corynebacterium glutamicum ATCC13032 amplified fragment as the negative control, and water as the blank control. The preparation of PAGE for SSCP electrophoresis and the electrophoresis conditions are shown in Table 3. Because the fragment structures differ and their electrophoretic positions also differ, strains whose electrophoretic positions did not match those of the negative control fragments but matched those of the positive control fragments were strains in which allelic replacement was successful. Positive strain NCgl2002 G1240TThe gene fragment was amplified again by PCR using primers P6 / P7, ligated into the PMD19-T vector, and sequenced. Based on sequence comparison, strains with a nucleotide sequence mutation (GT) were identified as positive for successful allelic replacement. The positive strains obtained from Corynebacterium glutamicum YP097158 and wild-type Corynebacterium glutamicum ATCC13032 were designated YPL-NCgl2002-1 and L2002-1, respectively.

[0057] Both recombinant strains YPL-NCgl2002-1 and YPL-NCgl2002-1 contain the mutant gene NCgl2002 shown in SEQ ID NO: 5. G1240T and capable of expressing the protein shown in SEQ ID NO: 6. The recombinant strain YPL-NCgl2002-1 differs from Corynebacterium glutamicum YP097158 in that YPL-NCgl2002-1 has the NCgl2002 gene of Corynebacterium glutamicum YP097158 G1240T The recombinant strain L2002-1 differs from ATCC13032 in that the NCgl2002 gene of ATCC13032 was replaced with NCgl2002 G1240T The strain was obtained by replacing the gene without changing any other sequences.

[0058] TIFF2026501022000003.tif56170

[0059] Example 3: Construction of a modified strain lacking the NCgl2002 gene in its genome Based on the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, two pairs of primers were synthesized to amplify the upstream and downstream homology arm fragments at both ends of the NCgl2002 gene coding region. The primers were designed as follows (synthesized by Shanghai Invitrogen): P8:5'- CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAG CGTGATGCAGGCCGAAGGATC-3' (the underlined nucleotide sequence is the sequence on pK18) (SEQ ID NO: 21), P9: 5'-GCGACACTAAAACTCTTGGCGGTGCGAATGGGGGTGACAG-3' (SEQ ID NO: 22), P10: 5'-CTGTCACCCCCATTCGCACCGCCAAGAGTTTTAGTGTCGC-3' (SEQ ID NO: 23), P11:5'- CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCC GGAGTTTTCCTCCGATGGCTG-3' (the underlined nucleotide sequence is the sequence on pK18) (SEQ ID NO: 24).

[0060] Construction method: Using Corynebacterium glutamicum ATCC13032 as the template, PCR amplification was performed using primers P8 / P9 and P10 / P11, respectively, to obtain a 571-bp upstream homology arm fragment and a 566-bp downstream homology arm fragment for the NCgl2002 knockout. The amplified products were electrophoresed and purified using a column-based DNA gel collection kit. The recovered DNA fragments were ligated with pK18mobsacB plasmid (Addgene) that had been digested with XbaI and BamHI and purified using NEBuilder enzyme (NEB) at 50°C for 30 minutes. After transformation of the ligated product, a single clone was grown and identified by PCR using M13 primers to obtain the positive knockout vector pK18-ΔNCgl2002. This plasmid contains the complete 1097-bp NCgl2002 knockout homology arm fragment (sequence shown in SEQ ID NO: 8) and a kanamycin resistance marker. This plasmid was sent for sequencing.

[0061] The correctly sequenced knockout plasmid pK18-ΔNCgl2002 was transformed into Corynebacterium glutamicum YP097158 and wild-type Corynebacterium glutamicum ATCC13032 by electroporation and cultured in medium (see Table 1 for medium composition and culture conditions). Single colonies resulting from the culture were identified by PCR using primers P8 / P11. Strains that simultaneously amplified the 1097 bp and 2384 bp bands were designated as positive strains, while strains that amplified only the 2384 bp band were designated as original strains. Positive strains were screened on 15% sucrose solid medium and then cultured in medium with and without kanamycin, respectively. Strains that grew in the kanamycin-free medium but not in the kanamycin-containing medium were further identified by PCR using primers P8 / P11. Strains that amplified a 1097-bp band were designated as positive strains with the NCgl2002 gene knockout. The NCgl2002 fragment from the positive strains was amplified again by PCR using primers P8 / P11, ligated into the pMD19-T vector, and sequenced. Correctly sequenced strains were designated YPL-NCgl2002-2 (the NCgl2002 gene in the genome of Corynebacterium glutamicum YP097158 was knocked out) and L2002-2 (the NCgl2002 gene in the genome of wild-type Corynebacterium glutamicum ATCC13032 was knocked out).

[0062] Example 4: L-lysine fermentation experiment Fermentation experiments were carried out using the strains constructed in Examples 2 and 3, the original Corynebacterium glutamicum strains YP097158 and ATCC13032, in a BLBIO-5GC-4-H fermentation tank (Shanghai Bailun Biotechnology Co., Ltd.) using the media shown in Table 4 and the controlled process shown in Table 5. After fermentation, the amount of L-lysine produced was measured using the ninhydrin colorimetric method. The results of three replicates for each strain are shown in Table 6.

[0063] TIFF2026501022000004.tif96170

[0064] TIFF2026501022000005.tif83170

[0065] TIFF2026501022000006.tif46170

[0066] As a result, as shown in Table 6, the point mutation NCgl2002 in the NCgl2002 gene coding region was detected in Corynebacterium glutamicum. G1240T and knockout contributes to improving L-lysine production and growth rate.

[0067] The present invention has been described in detail above. Those skilled in the art can practice the present invention to a broader extent under equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without undue experimentation. Although the present invention has been described with specific examples, it is understood that the present invention can be further improved. In short, based on the principles of the present invention, the present application is intended to cover any changes, uses, or improvements to the present invention that depart from the scope disclosed herein, including improvements made using conventional techniques known in the art. Some basic features may be applied within the scope of the following appended claims.

[0068] Sequences 1 to 8 according to the above examples are as follows: SEQ ID NO: 1: Wild-type ORF (CDS) sequence of the NCgl2002 gene (nucleotide sequence 1287 bp) SEQ ID NO: 2: NCgl2002 protein sequence (428 aa amino acid sequence encoded by SEQ ID NO: 1) MADGTIKQIHPFTGTEVWTVPGRGNRPLSHPASTIVELSAHDHTSYCAFCSDNMLSTPPEKSRIIIDSSGDFDILPGALPGELSETTPEFRRVPNLFEIVSFDYWHQNFGFDMDSETAMRMAQYLAIPEGREHVLAIVRTRLSAAGEDPAHMTDGELLEKAPSYFAGGHDVIIGRRHFVDDATTSDQLASSGTLTVKEHEAFIRLTVDGIRDLY HRNRYAPYVVAFQNWLKPAGASFDHLHKQLVAIDERGRLIADELHHLRGNPNMYNELAVDYAGYHNLIIAENDHAVAFAGFGHRYPTIEIYSKSAIPEPWLQSDEEI QAMSNLIHACHAATGADVPCNEEWVHKPIDVDMPMPWHVMIKWRVSTLAGFEGGTKVYLNTLSPHKVRDRVVKEMYRLRDEELIASDLRIAMECSVERNSLKYNPLL SEQ ID NO: 3: pXMJ19-NCgl2002 sequence of the pXMJ19-integrated NCgl2002 gene and its promoter (nucleotide sequence 1441 bp) AATTAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCGAATTCAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTC SEQ ID NO: 4: Amplified fragment of identification primer M13R(-48) / P1 (nucleotide sequence 1465 bp) SEQ ID NO: 5: Gene mutant type NCgl2002 G1240T ORF (CDS) sequence (nucleotide sequence 1287 bp) SEQ ID NO: 6: Gene mutant type NCgl2002 E414* Protein sequence (428 aa amino acid sequence encoded by sequence 5) MADGTIKQIHPFTGTEVWTVPGRGNRPLSHPASTIVELSAHDHTSYCAFCSDNMLSTPPEKSRIIIDSSGDFDILPGALPGELSETTPEFRRVPNLFEIVSFDYWHQNFGFDMDSETAMRMAQYLAIPEGREHVLAIVRTRLSAAGEDPAHMTDGELLEKAPSYFAGGHDVIIGRRHFVDDATTSDQLASSGTLTVKEHEAFIRLTVDGIRDLY HRNRYAPYVVAFQNWLKPAGASFDHLHKQLVAIDERGRLIADELHHLRGNPNMYNELAVDYAGYHNLIIAENDHAVAFAGFGHRYPTIEIYSKSAIPEPWLQSDEEI QAMSNLIHACHAATGADVPCNEEWVHKPIDVDMPMPWHVMIKWRVSTLAGFEGGTKVYLNTLSPHKVRDRVVKEMYRLRDEELIASDLRIAM*CSVERNSLKYNPLL SEQ ID NO: 7: Point mutation integrated homology arm Up-NCgl2002 obtained by overlap PCR using P2 / P5 E414* -Down sequence (nucleotide sequence 1022 bp) SEQ ID NO: 8: Homology arm DNA sequence of NCgl2002 knockout obtained in P8 / P11 (nucleotide sequence 1097 bp) [Industrial Applicability]

[0069] Experiments have shown that intracellular L-lysine production can be improved by mutating or knocking out the gene encoding the protein shown in SEQ ID NO: 2. Since L-lysine can be produced by mutating or knocking out the intracellular gene encoding the protein shown in SEQ ID NO: 2, the present invention has good prospects for application.

Claims

1. The use of a substance in the production of L-lysine that knocks out a gene encoding a protein, or inhibits the content or activity of said protein, The aforementioned protein is used in the following ways: A1) or A2): A1) A protein whose amino acid sequence is sequence number 2, A2) A fusion protein in which a tag is attached to the N-terminus and / or C-terminus of A1).

2. The use according to claim 1, characterized in that the gene encoding comprises the following b1) or b2) or b3): b1) The DNA molecule shown in sequence number 1 of the sequence listing, b2) A DNA molecule having 75% or more identity with the nucleotide sequence defined in b1), and encoding the protein, b3) A DNA molecule that hybridizes under stringent conditions with a nucleotide sequence defined in b1) or b2) and encodes the protein.

3. The use according to claim 1, characterized in that the substance comprises B1) or B2): B1) A nucleic acid molecule that reduces the expression level of the protein described in claim 1, B2) Expression cassettes, recombinant vectors, recombinant microorganisms, or genetically modified cell lines containing the nucleic acid molecules described in B1).

4. The substance includes a substance in which the codon of the glutamic acid residue at position 414 of Sequence ID No. 2 has been mutated into a terminator, Alternatively, the use according to any one of claims 1 to 3, characterized in that the substance includes a substance in which the guanine nucleotide at position 1240 of Sequence ID No. 1 is mutated to a thymine nucleotide.

5. A method for producing L-lysine, A method for producing L-lysine, comprising the steps of: obtaining recombinant biological cells by reducing the content or activity of the protein described in claim 1 in receptor biological cells, or by knocking out the gene encoding the protein described in claim 1 in receptor biological cells; and culturing the recombinant biological cells to obtain L-lysine.

6. The method according to claim 5, characterized in that the biological cells include yeast, bacteria, algae, fungi, plant cells, or animal cells capable of synthesizing L-lysine.

7. The method according to claim 6, characterized in that the bacteria include Corynebacterium glutamicum.

8. The recombinant biological cells are cultured using a culture medium capable of growing the recombinant biological cells, The method according to any one of claims 5 to 7, characterized in that the recombinant biological cells are cultured using conditions that enable the proliferation of the recombinant biological cells.

9. The method according to any one of claims 5 to 7, characterized in that the method is achieved by mutating the codon of the glutamic acid residue at position 414 of SEQ ID NO: 2 in a receptor biological cell to a terminator, or by mutating the guanine nucleotide at position 1240 of SEQ ID NO: 1 to a thymine nucleotide, or by knocking out the gene shown in SEQ ID NO:

1.

10. The recombinant biological cells are cultured using a culture medium capable of growing the recombinant biological cells, The method according to claim 9, characterized in that the recombinant biological cells are cultured using conditions that enable the proliferation of the recombinant biological cells.

11. Biomaterials containing the following b1) or b2) or b3) or b4) or b5): b1) A DNA molecule whose amino acid sequence codes for the protein shown in SEQ ID NO: 6, b2) A DNA molecule having 75% or more identity with the sequence of the DNA molecule defined in b1), and encoding the protein shown in Sequence ID No. 6, b3) A DNA molecule that hybridizes under stringent conditions with a nucleotide sequence defined in b1) or b2) and encodes the protein shown in Sequence ID No. 6, b4) Expression cassettes, recombinant vectors, recombinant microorganisms or genetically modified cell lines containing the DNA molecules described in b1) or b2) or b3), b5) Recombinant biological cells according to any one of claims 5 to 7.