WO2022163904A1 - Nouveau variant de protéine et procédé de production de l-lysine l'utilisant - Google Patents

Nouveau variant de protéine et procédé de production de l-lysine l'utilisant Download PDF

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WO2022163904A1
WO2022163904A1 PCT/KR2021/003261 KR2021003261W WO2022163904A1 WO 2022163904 A1 WO2022163904 A1 WO 2022163904A1 KR 2021003261 W KR2021003261 W KR 2021003261W WO 2022163904 A1 WO2022163904 A1 WO 2022163904A1
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seq
amino acid
substituted
acid sequence
set forth
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PCT/KR2021/003261
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권나라
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씨제이제일제당 (주)
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Priority claimed from KR1020210013691A external-priority patent/KR20220110412A/ko
Priority claimed from KR1020210013690A external-priority patent/KR20220110411A/ko
Priority claimed from KR1020210013694A external-priority patent/KR20220110413A/ko
Application filed by 씨제이제일제당 (주) filed Critical 씨제이제일제당 (주)
Priority to PCT/KR2021/007122 priority Critical patent/WO2022163951A1/fr
Publication of WO2022163904A1 publication Critical patent/WO2022163904A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine

Definitions

  • the present application relates to a novel protein variant, a Corynebacterium glutamicum strain comprising the variant, and a method for producing L- lysine using the strain.
  • One object of the present application is to provide a protein variant selected from the group consisting of the following (1) to (15):
  • an endogenous membrane transport protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which phenylalanine, an amino acid corresponding to position 220 of SEQ ID NO: 3, is substituted with cysteine;
  • DNA polymerase III gamma and tau subunit variants consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which proline, an amino acid corresponding to position 43 of SEQ ID NO: 7, is substituted with leucine;
  • a transcriptional anti-termination protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 13 in which asparagine, an amino acid corresponding to position 210 of SEQ ID NO: 15, is substituted with aspartic acid;
  • (6) a malate dehydrogenase variant consisting of the amino acid sequence set forth in SEQ ID NO: 21 in which threonine, an amino acid corresponding to position 272 of SEQ ID NO: 23, is substituted with isoleucine;
  • a primosome assembly protein variant consisting of the amino acid sequence shown in SEQ ID NO: 25, in which arginine, an amino acid corresponding to position 304 of SEQ ID NO: 27, is substituted with glutamine;
  • a Co/Zn/Cd efflux system component variant consisting of the amino acid sequence set forth in SEQ ID NO: 41 in which glycine, an amino acid corresponding to position 130 of SEQ ID NO: 43, is substituted with aspartic acid;
  • DAHP synthase variant consisting of the amino acid sequence set forth in SEQ ID NO: 45 in which glycine, an amino acid corresponding to position 382 of SEQ ID NO: 47, is substituted with cysteine;
  • a helicase variant consisting of the amino acid sequence set forth in SEQ ID NO: 53 in which tyrosine, an amino acid corresponding to position 592 of SEQ ID NO: 55, is substituted with phenylalanine;
  • a protein variant consisting of the amino acid sequence shown in SEQ ID NO: 57, wherein glutamic acid, which is an amino acid corresponding to position 170 of SEQ ID NO: 59, is substituted with lysine.
  • Another object of the present application is to provide a polynucleotide encoding the variant of the present application.
  • Another object of the present application is to include two or more protein variants selected from the group consisting of the following (1) to (15) or a polynucleotide encoding the variant, and having L-lysine-producing ability, Corynebacter Leum glutamicum ( Corynebacterium glutamicum ) To provide a strain:
  • an endogenous membrane transport protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which phenylalanine, an amino acid corresponding to position 220 of SEQ ID NO: 3, is substituted with cysteine;
  • DNA polymerase III gamma and tau subunit variants consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which proline, an amino acid corresponding to position 43 of SEQ ID NO: 7, is substituted with leucine;
  • a transcriptional anti-termination protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 13 in which asparagine, an amino acid corresponding to position 210 of SEQ ID NO: 15, is substituted with aspartic acid;
  • (6) a malate dehydrogenase variant consisting of the amino acid sequence set forth in SEQ ID NO: 21 in which threonine, an amino acid corresponding to position 272 of SEQ ID NO: 23, is substituted with isoleucine;
  • a primosome assembly protein variant consisting of the amino acid sequence shown in SEQ ID NO: 25, in which arginine, an amino acid corresponding to position 304 of SEQ ID NO: 27, is substituted with glutamine;
  • a Co/Zn/Cd efflux system component variant consisting of the amino acid sequence set forth in SEQ ID NO: 41 in which glycine, an amino acid corresponding to position 130 of SEQ ID NO: 43, is substituted with aspartic acid;
  • DAHP synthase variant consisting of the amino acid sequence set forth in SEQ ID NO: 45 in which glycine, an amino acid corresponding to position 382 of SEQ ID NO: 47, is substituted with cysteine;
  • a helicase variant consisting of the amino acid sequence set forth in SEQ ID NO: 53 in which tyrosine, an amino acid corresponding to position 592 of SEQ ID NO: 55, is substituted with phenylalanine;
  • a protein variant consisting of the amino acid sequence shown in SEQ ID NO: 57, wherein glutamic acid, which is an amino acid corresponding to position 170 of SEQ ID NO: 59, is substituted with lysine.
  • Another object of the present application is to provide a method for producing L-lysine, comprising culturing the Corynebacterium glutamicum strain of the present application in a medium.
  • One aspect of the present application is to provide a protein variant selected from the group consisting of the following (1) to (15):
  • an endogenous membrane transport protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which phenylalanine, an amino acid corresponding to position 220 of SEQ ID NO: 3, is substituted with cysteine;
  • DNA polymerase III gamma and tau subunit variants consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which proline, an amino acid corresponding to position 43 of SEQ ID NO: 7, is substituted with leucine;
  • a transcriptional anti-termination protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 13 in which asparagine, an amino acid corresponding to position 210 of SEQ ID NO: 15, is substituted with aspartic acid;
  • (6) a malate dehydrogenase variant consisting of the amino acid sequence set forth in SEQ ID NO: 21 in which threonine, an amino acid corresponding to position 272 of SEQ ID NO: 23, is substituted with isoleucine;
  • a primosome assembly protein variant consisting of the amino acid sequence shown in SEQ ID NO: 25, in which arginine, an amino acid corresponding to position 304 of SEQ ID NO: 27, is substituted with glutamine;
  • a Co/Zn/Cd efflux system component variant consisting of the amino acid sequence set forth in SEQ ID NO: 41 in which glycine, an amino acid corresponding to position 130 of SEQ ID NO: 43, is substituted with aspartic acid;
  • DAHP synthase variant consisting of the amino acid sequence set forth in SEQ ID NO: 45 in which glycine, an amino acid corresponding to position 382 of SEQ ID NO: 47, is substituted with cysteine;
  • a helicase variant consisting of the amino acid sequence set forth in SEQ ID NO: 53 in which tyrosine, an amino acid corresponding to position 592 of SEQ ID NO: 55, is substituted with phenylalanine;
  • a protein variant consisting of the amino acid sequence shown in SEQ ID NO: 57, wherein glutamic acid, which is an amino acid corresponding to position 170 of SEQ ID NO: 59, is substituted with lysine.
  • the variant of the present application has an amino acid sequence set forth in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, and 57 It may include, or may consist essentially of, the amino acid sequence.
  • the variant of the present application is SEQ ID NO: 3 in the amino acid sequence set forth in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, or 57, respectively, Based on the amino acid sequence of 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, or 59, 220, 43, 33, 210, 199, respectively,
  • the amino acids corresponding to positions 272, 304, 169, 294, 358, 130, 382, 209, 592, or 170 are cysteine, leucine, serine, aspartic acid, methionine, isoleucine, glutamine.
  • valine valine, lysine, serine, aspartic acid, cysteine, leucine, phenylalanine, or lysine, wherein SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53 , and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7 with the amino acid sequence set forth in SEQ ID NO: % or 99.9% or more homology or identity.
  • Table 1 Each amino acid sequence of the variants of the present application, positions, and correspondences to amino acid types corresponding to positions are shown in Table 1 below.
  • variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident
  • amino acid sequence The amino acid sequence as a reference for counting positions location type of amino acid
  • SEQ ID NO: 1 SEQ ID NO: 3 220 cysteine 2 SEQ ID NO: 5 SEQ ID NO: 7 43 leucine 3 SEQ ID NO: 9 SEQ ID NO: 11 33 serine 4 SEQ ID NO: 13 SEQ ID NO: 15 210 aspartic acid 5 SEQ ID NO: 17 SEQ ID NO: 19 199 methionine 6 SEQ ID NO: 21 SEQ ID NO: 23 272 isoleucine 7 SEQ ID NO: 25 SEQ ID NO: 27 304 glutamine 8 SEQ ID NO: 29 SEQ ID NO: 31 169 valine 9 SEQ ID NO: 33 SEQ ID NO: 35 294 lysine 10 SEQ ID NO: 37 SEQ ID NO: 39 358 serine 11 SEQ ID NO: 41 SEQ ID NO: 43 130 aspartic acid 12 SEQ ID NO: 45 SEQ ID NO: 47 382 cysteine 13 SEQ ID NO: 49 SEQ ID NO: 51 209 leucine 14
  • sequence additions or deletions naturally occurring mutations, silent mutations or conservation within the N-terminus, C-terminus and/or within the amino acid sequence that do not alter the function of the variants of the present application It is a case of having an enemy substitution.
  • conservative substitution means substituting an amino acid for another amino acid having similar structural and/or chemical properties. Such amino acid substitutions may generally occur based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of the protein or polypeptide.
  • variant means that one or more amino acids are conservatively substituted and/or modified so that they differ from the amino acid sequence before the mutation of the variant, but have functions or properties. refers to a polypeptide that is maintained. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the polypeptide before the mutation. In addition, some variants may include variants in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, have been removed.
  • variants may include variants in which a portion is removed from the N- and/or C-terminus of the mature protein.
  • variant may be used interchangeably with terms such as mutant, modified, mutant polypeptide, mutated protein, mutant and mutant (in English, modified, modified polypeptide, modified protein, mutant, mutein, divergent, etc.) and, as long as it is a term used in a mutated sense, it is not limited thereto.
  • the variant is at position 220, 43 of the amino acid sequence of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, or 59; phenylalanine, proline, amino acids corresponding to positions 33, 210, 199, 272, 304, 169, 294, 358, 130, 382, 209, 592, or 170, respectively; Cysteine, asparagine, valine, threonine, arginine, alanine, glutamic acid, proline, glycine, glycine, proline, tyrosine, or glutamic acid to cysteine, leucine, serine, aspartic acid, methionine, isoleucine, glutamine, valine, lysine, serine, aspartic acid , cysteine, leucine, phenylalanine, or the amino acid sequence set forth in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41,
  • amino acid sequence The amino acid sequence as a reference for counting positions mutation position Type of amino acid before substitution
  • amino acid sequence One SEQ ID NO: 1 SEQ ID NO: 3 220 phenylalanine cysteine 2 SEQ ID NO: 5 SEQ ID NO: 7 43 proline leucine 3 SEQ ID NO: 9 SEQ ID NO: 11 33 cysteine serine 4 SEQ ID NO: 13 SEQ ID NO: 15 210 asparagine aspartic acid 5 SEQ ID NO: 17 SEQ ID NO: 19 199 valine methionine 6 SEQ ID NO: 21 SEQ ID NO: 23 272 threonine isoleucine 7 SEQ ID NO: 25 SEQ ID NO: 27 304 arginine glutamine 8 SEQ ID NO: 29 SEQ ID NO: 31 169 alanine valine 9 SEQ ID NO: 33 SEQ ID NO: 35 294 glutamic acid lysine 10 SEQ ID NO: 37 SEQ ID NO: 39 358 proline serine 11 SEQ ID NO: 41
  • variants may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide.
  • a signal (or leader) sequence involved in protein translocation may be conjugated to the N-terminus of the mutant, either co-translationally or post-translationally.
  • the variants may also be conjugated with other sequences or linkers for identification, purification, or synthesis.
  • the term 'homology' or 'identity' refers to the degree of similarity between two given amino acid sequences or base sequences and may be expressed as a percentage.
  • the terms homology and identity can often be used interchangeably.
  • Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, with default gap penalties established by the program used may be used. Substantially homologous or identical sequences are generally capable of hybridizing with all or part of a sequence under moderate or high stringent conditions. It is apparent that hybridization also includes hybridization with polynucleotides containing common codons or codons taking codon degeneracy into account in the polynucleotide.
  • a GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly aligned symbols (ie, nucleotides or amino acids).
  • Default parameters for the GAP program are: (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.
  • the variant of the present application is an integral membrane transport protein; DNA polymerase III subunits gamma and tau; a polypeptide comprising the amino acid sequence of SEQ ID NO: 11 and/or a protein encoded by the NCgl3069 gene; transcription antitermination protein; ABC transporter ATP-binding protein; malate dehydrogenase; primosome assembly protein; type II citrate synthase; membrane protein (TerC); mycothione reductase; Co/Zn/Cd efflux system component; DAHP synthase (3-deoxy-D-arabinoheptulosonate-7-phosphate synthase; DAHP synthase); N-succinyldiaminopimelate aminotransferase; Helicase; and/or a polypeptide comprising the amino acid sequence of SEQ ID NO: 59 and/or a protein encoded by the NCgl1098 gene.
  • the term "integral membrane transport protein” refers to the formation of macromolecules, small molecules, and ions, such as other proteins across biological membranes. It is a membrane protein that is involved in migration and is attached to a biological membrane.
  • the integral membrane transport protein of the present application can be used interchangeably as an integral membrane transport protein.
  • the internal membrane transport protein is a known data
  • GenBank of NCBI which is the base, for example, it can be GenBank Accession No. YP_227155.1, WP_011015489.1.
  • it can be a polypeptide having an intrinsic membrane transport protein activity encoded by the NCgl2816 gene. However, it is not limited thereto.
  • DNA polymerase III subunits gamma and tau is a major enzyme responsible for DNA replication
  • the DNA polymerase III tau subunit is a core (alpha ( alpha), epsilon, and theta (consisting of chains) bind to dimerization of the core
  • DNA polymerase III gamma subunit is a clamp loader that obtains beta subunit to lagging strand.
  • the DNA polymerase III gamma and tau subunit of the present application may be used interchangeably as DNA polymerase III gamma and tau subunits, or DNA polymerase III subunit gamma/tau.
  • Polymerase III gamma and tau subunits can be sequenced from GenBank of NCBI, which is a known database, for example, GenBank Accession No. YP_224542.1, WP_011013499.1. Specifically, the dnaZX gene (or NCgl0239 gene) encoded by DNA polymerase III gamma and tau subunit activity, but is not limited thereto.
  • transcription antitermination protein is a transcription complex, capable of interacting with the termination factor Rho and RNA polymerase, transcription termination and/or anti-termination It is a polypeptide that is a transcriptional elongation factor involved in Specifically, the transcriptional anti-termination protein of the present application may be used interchangeably as a transcription termination/antitermination protein, NusG protein, or NusG.
  • the transcriptional anti-termination protein can be sequenced from GenBank of NCBI, a known database, for example, GenBank Accession No. It may be WP_011013678.1, YP_224775.1. Specifically, it may be a polypeptide having transcriptional anti-termination protein activity encoded by the nusG gene (or NCgl0458 gene), but is not limited thereto.
  • the term "ABC transporter ATP-binding protein” is a transmembrane protein that uses the energy obtained by hydrolyzing ATP to move and/or transport various substrates across the membrane, and Polypeptides with biological functions such as repair of unrelated DNA and RNA.
  • the ABC transporter ATP-binding protein of the present application may be used in combination as the ABC transporter ATP-binding protein.
  • the ABC transporter ATP-binding protein may have its sequence obtained from GenBank of NCBI, a known database, for example, GenBank Accession No. It may be WP_011013802.1, YP_224957.1. Specifically, it may be a polypeptide having an ABC transporter ATP-binding protein activity encoded by the NCgl0636 gene, but is not limited thereto.
  • N-succinyldiaminopimelate aminotransferase refers to L-glutamate and N-succinyl-2-amino-6-ketopimelate as substrates, 2-oxoglutarate and N-succinyl-L,L It is an enzyme involved in the lysine biosynthetic pathway to produce -2,6-diaminopimelate.
  • the N-succinyldiaminopimelate aminotransferase of the present application can be used in combination with N-succinyldiaminopimelate aminotransferase.
  • N-succinyldiaminopimelate aminotransferase sequence can be obtained from GenBank of NCBI, a known database, for example, GenBank Accession No. WP_011014123.1, YP_225395.1.
  • NCgl1058 It may be a polypeptide having N-succinyldiaminopimelate aminotransferase activity encoded by the gene (or dapC gene), but is not limited thereto.
  • malate dehydrogenase is an enzyme that reversibly catalyzes the oxidation of malate (malic acid) to oxaloacetic acid and the reduction of NAD+ to NADH in this process.
  • the malate dehydrogenase of the application can be used interchangeably with malate dehydrogenase, MDH, or malate dehydrogenase
  • the malate dehydrogenase sequence can be obtained from NCBI's GenBank, a known database. and may be, for example, GenBank Accession No. WP_011015079.1, YP_226625.1. Specifically, it may be a polypeptide having malate dehydrogenase activity encoded by NCgl2297 gene (or mdh gene), but is not limited thereto. does not
  • primosome assembly protein refers to a protein having a helicase activity and involved in the DNA replication process.
  • the primosome assembly protein of the present application may be used interchangeably as primosome assembly protein, PriA protein, PriA, or primosomal protein N'.
  • sequence of the primosome assembly protein can be obtained from GenBank of NCBI, a known database, for example, GenBank Accession No. It may be WP_011014474.1, YP_225886.1. Specifically, it may be a polypeptide having a primosome assembly protein activity encoded by the NCgl1540 gene (or priA gene), but is not limited thereto.
  • helicase is a polypeptide capable of separating a DNA double helix or a strand of a self-associated RNA molecule.
  • the helicase of the present application may be used in combination with a helicase.
  • the helicase sequence can be obtained from GenBank of NCBI, a known database, for example, GenBank Accession No. It can be WP_011014500.1, YP_225922.1. Specifically, it may be a polypeptide having a helicase activity encoded by the NCgl1575 gene, but is not limited thereto.
  • Type II citrate synthase of the present application may be used in combination with integral type II citrate synthase, citrate synthase, or citrate synthase.
  • the type II citrate synthase may have its sequence obtained from GenBank of NCBI, which is a known database, for example, GenBank Accession No. It may be YP_225121.1 or WP_011013914.1. Specifically, it may be a polypeptide having a type II citrate synthase activity encoded by the gltA gene (or NCgl0795 gene), but is not limited thereto.
  • the membrane protein of the present application may be used interchangeably as a membrane protein, a membrane protein, TerC, or a TerC protein.
  • the sequence of the membrane protein can be obtained from GenBank of NCBI, which is a known database, for example, GenBank Accession No. It may be YP_226209.1 or WP_011014789.1. Specifically, it may be a polypeptide having a membrane protein activity encoded by the terC gene (or NCgl1892 gene), but is not limited thereto.
  • Mycothione reductase of the present application may be used in combination with mycothione reductase, or mycothione reductase.
  • the mycothione reductase sequence can be obtained from GenBank of NCBI, a known database, for example, GenBank Accession No. It may be YP_226245.1 or WP_011014816.1. Specifically, it may be a polypeptide having mycothione reductase activity encoded by the mtr gene (or NCgl1928 gene), but is not limited thereto.
  • the Co/Zn/Cd efflux system component of the present application may be used in combination as a Co/Zn/Cd efflux system component.
  • the Co/Zn/Cd efflux system component can obtain its sequence from NCBI's GenBank, a known database, for example, GenBank Accession No. It may be YP_226309.1, WP_011014862.1. Specifically, it may be a polypeptide having a Co/Zn/Cd efflux system component activity encoded by the NCgl1992 gene, but is not limited thereto.
  • DAHP synthase of the present application is 3-deoxy-D-arabinoheptulosonic acid 7-phosphate synthase, 3-deoxy-D-arabinoheptulosonic acid 7-phosphate synthase, 3-deoxy-D-arabinoheptulosonate -7-phosphate synthase, or DAHP synthase may be used in combination.
  • the sequence of the DAHP synthase can be obtained from GenBank of NCBI, a known database, for example, GenBank Accession No. It may be YP_226420.1, WP_011014936.1. Specifically, it may be a polypeptide having DAHP synthase activity encoded by the aroG gene (or NCgl2098 gene), but is not limited thereto.
  • the mutant of the present application may have an activity to increase L-lysine production capacity compared to the wild-type polypeptide.
  • SEQ ID NO: 8 Protein encoded by the NCgl3069 gene SEQ ID NO: 11 SEQ ID NO: 12 transcriptional anti-termination protein SEQ ID NO: 15 SEQ ID NO: 16 ABC transporter ATP-binding protein SEQ ID NO: 19 SEQ ID NO: 20 malate dehydrogenase SEQ ID NO: 23 SEQ ID NO: 24 primosome assembly protein SEQ ID NO: 27 SEQ ID NO: 28 Type II citrate synthase SEQ ID NO: 31 SEQ ID NO: 32 membrane protein SEQ ID NO: 35 SEQ ID NO: 36 mycothion reductase SEQ ID NO: 39 SEQ ID NO: 40 Co/Zn/Cd effluent system components SEQ ID NO: 43 SEQ ID NO: 44 DAHP synthase SEQ ID NO: 47 SEQ
  • corresponding to refers to an amino acid residue at a position listed in a polypeptide, or an amino acid residue similar to, identical to, or homologous to a residue listed in a polypeptide. Identifying an amino acid at a corresponding position may be determining a specific amino acid in a sequence that refers to a specific sequence.
  • corresponding region generally refers to a similar or corresponding position in a related protein or reference protein.
  • any amino acid sequence is aligned with SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, or 59, and based on Each amino acid residue in the amino acid sequence is a number of amino acid residues corresponding to those of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, or 59 You can number by referring to the location.
  • a sequence alignment algorithm such as that described in this application can identify the position of an amino acid, or a position at which modifications, such as substitutions, insertions, or deletions, occur compared to a query sequence (also referred to as a "reference sequence").
  • Such alignments include, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al. , 2000), Trends Genet. 16: 276-277), etc., but is not limited thereto, and a sequence alignment program, pairwise sequence comparison algorithm, etc. known in the art may be appropriately used.
  • Another aspect of the present application is to provide a polynucleotide encoding the variant of the present application.
  • polynucleotide refers to a DNA or RNA strand of a certain length or longer as a polymer of nucleotides in which nucleotide monomers are linked in a long chain by covalent bonds, and more specifically, encoding the variant. polynucleotide fragments.
  • the polynucleotide encoding the variant of the present application is a base encoding the amino acid sequence set forth in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, or 57 sequence may be included.
  • the polynucleotide of the present application has or comprises the sequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, or 58 can do.
  • the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, or 58.
  • the polynucleotide of the present application is each in the nucleic acid sequence set forth in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, or 58 659, 128, 97, 628, based on the nucleic acid sequence of SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, or 60;
  • the bases corresponding to positions 595, 815, 911, 506, 880, 1072, 389, 1144, 626, 1775, or 508, respectively, are G, T, A, G, A, T, A, T, A, T, T, T, T, or A, wherein SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50 , 54, or 58 and at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more nu
  • a polynucleotide having a nucleic acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added is also It is obvious that they are included within the scope of the present application.
  • Table 4 below shows the correspondence of each nucleic acid sequence, position, and base type corresponding to the position of the polynucleotide of the present application.
  • nucleic acid sequence Nucleic acid sequence as a reference for counting positions location base type One SEQ ID NO: 2 SEQ ID NO: 4 659 G 2 SEQ ID NO: 6 SEQ ID NO: 8 128 T 3 SEQ ID NO: 10 SEQ ID NO: 12 97 A 4 SEQ ID NO: 14 SEQ ID NO: 16 628 G 5 SEQ ID NO: 18 SEQ ID NO: 20 595 A 6 SEQ ID NO: 22 SEQ ID NO: 24 815 T 7 SEQ ID NO: 26 SEQ ID NO: 28 911 A 8 SEQ ID NO: 30 SEQ ID NO: 32 506 T 9 SEQ ID NO: 34 SEQ ID NO: 36 880 A 10 SEQ ID NO: 38 SEQ ID NO: 40 1072 T 11 SEQ ID NO: 42 SEQ ID NO: 44 389 A 12 SEQ ID NO: 46 SEQ ID NO: 48 1144 T 13 SEQ ID NO: 50 SEQ ID NO: 52 626 T 14 SEQ ID NO: 54 SEQ ID NO: 56 1775 T 15 SEQ ID NO: 58 SEQ ID
  • the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made.
  • the polynucleotide of the present application has 70 homology or identity to the sequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, or 58 % or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% of the base sequence, or SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, or 58 at least 70%, at least 75%, at least 80% homology or identity to the sequence of 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% of the base sequence may consist of or consist essentially of, but is not limited thereto.
  • 220 times, 43 times of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, or 57 codons encoding amino acids corresponding to positions 33, 210, 199, 272, 304, 169, 294, 358, 130, 382, 209, 592, and 170, respectively may be one of codons encoding cysteine, leucine, serine, aspartic acid, methionine, isoleucine, glutamine, valine, lysine, serine, aspartic acid, cysteine, leucine, phenylalanine, and lysine, respectively.
  • the polynucleotide may be included without limitation as long as it can hybridize under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to all or part of the polynucleotide sequence of the present application.
  • the “stringent condition” refers to a condition that enables specific hybridization between polynucleotides. These conditions are described in J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8).
  • polynucleotides with high homology or identity 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or a condition in which polynucleotides having 99% or more homology or identity hybridize with each other, and polynucleotides having lower homology or identity do not hybridize, or 60 ° C., which is a washing condition of conventional Southern hybridization, 1 ⁇ SSC, 0.1% SDS, specifically 60°C, 0.1 ⁇ SSC, 0.1% SDS, more specifically 68°C, 0.1 ⁇ SSC, 0.1% SDS at a salt concentration and temperature equivalent to once, specifically twice The conditions for washing to 3 times can be enumerated.
  • Hybridization requires that two nucleic acids have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization.
  • the term “complementary” is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the polynucleotides of the present application may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments complementary to the overall sequence.
  • a polynucleotide having homology or identity with the polynucleotide of the present application can be detected using the hybridization conditions including a hybridization step at a Tm value of 55°C and using the above-described conditions.
  • the Tm value may be 60 °C, 63 °C, or 65 °C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.
  • Appropriate stringency for hybridizing the polynucleotide depends on the length of the polynucleotide and the degree of complementarity, and the variables are well known in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; see F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8).
  • Another aspect of the present application is to provide a vector comprising the polynucleotide of the present application.
  • the vector may be an expression vector for expressing the polynucleotide in a host cell, but is not limited thereto.
  • vector refers to a DNA preparation comprising the base sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable expression control region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host.
  • suitable expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation.
  • the vector After transformation into an appropriate host cell, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself.
  • the vector used in the present application is not particularly limited, and any vector known in the art may be used.
  • Examples of commonly used vectors include plasmids, cosmids, viruses and bacteriophages in a natural or recombinant state.
  • pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, and pDZ-based, pBR-based, and pUC-based plasmid vectors may be used.
  • pBluescript II-based pGEM-based, pTZ-based, pCL-based, pET-based and the like
  • pDZ pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like
  • pC1BAC vectors and the like can be used.
  • a polynucleotide encoding a target polypeptide may be inserted into a chromosome through a vector for intracellular chromosome insertion.
  • the insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto.
  • It may further include a selection marker (selection marker) for confirming whether the chromosome is inserted.
  • the selection marker is used to select cells transformed with the vector, that is, to determine whether a target nucleic acid molecule is inserted, and selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or surface polypeptide expression. Markers to be given can be used. In an environment treated with a selective agent, only the cells expressing the selectable marker survive or exhibit other expression traits, so that the transformed cells can be selected.
  • the term "transformation” refers to introducing a vector including a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell.
  • the transformed polynucleotide may include all of them regardless of whether they are inserted into the chromosome of the host cell or located outside the chromosome, as long as they can be expressed in the host cell.
  • the polynucleotide includes DNA and/or RNA encoding a target polypeptide.
  • the polynucleotide may be introduced in any form as long as it can be introduced and expressed into a host cell.
  • the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements necessary for self-expression.
  • the expression cassette may include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal.
  • the expression cassette may be in the form of an expression vector capable of self-replication.
  • the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.
  • operably linked means that a promoter sequence that initiates and mediates transcription of a polynucleotide encoding the target variant of the present application and the polynucleotide sequence are functionally linked.
  • Corynebacterium glutamicum ( Corynebacterium glutamicum ) comprising the mutant of the present application or the polynucleotide of the present application It is to provide a strain.
  • One example is one or more (eg, one or more or two or more) protein variants selected from the group consisting of (1) to (15) or a polynucleotide encoding the variant, Corynebacterium Glutamicum strain ( Corynebacterium glutamicum ) can be provided:
  • an endogenous membrane transport protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which phenylalanine, an amino acid corresponding to position 220 of SEQ ID NO: 3, is substituted with cysteine;
  • DNA polymerase III gamma and tau subunit variants consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which proline, an amino acid corresponding to position 43 of SEQ ID NO: 7, is substituted with leucine;
  • (6) a malate dehydrogenase variant consisting of the amino acid sequence set forth in SEQ ID NO: 21 in which threonine, an amino acid corresponding to position 272 of SEQ ID NO: 23, is substituted with isoleucine;
  • a primosome assembly protein variant consisting of the amino acid sequence shown in SEQ ID NO: 25, in which arginine, an amino acid corresponding to position 304 of SEQ ID NO: 27, is substituted with glutamine;
  • a Co/Zn/Cd efflux system component variant consisting of the amino acid sequence set forth in SEQ ID NO: 41 in which glycine, an amino acid corresponding to position 130 of SEQ ID NO: 43, is substituted with aspartic acid;
  • DAHP synthase variant consisting of the amino acid sequence set forth in SEQ ID NO: 45 in which glycine, an amino acid corresponding to position 382 of SEQ ID NO: 47, is substituted with cysteine;
  • N-succinyldiaminopimelate aminotransferase variant consisting of the amino acid sequence set forth in SEQ ID NO: 49, wherein proline, which is the amino acid corresponding to position 209 of SEQ ID NO: 51, is substituted with leucine;
  • a helicase variant consisting of the amino acid sequence set forth in SEQ ID NO: 53 in which tyrosine, an amino acid corresponding to position 592 of SEQ ID NO: 55, is substituted with phenylalanine;
  • a protein variant consisting of the amino acid sequence shown in SEQ ID NO: 57, wherein glutamic acid, which is an amino acid corresponding to position 170 of SEQ ID NO: 59, is substituted with lysine.
  • An example is a protein variant of the following (a) and (b) or a polynucleotide encoding the variant (eg, (a) a polypeptide encoding the variant, (b) a polypeptide encoding the variant, and / or (a) ) and (b) can provide a Corynebacterium glutamicum strain comprising a polypeptide encoding:
  • an endogenous membrane transport protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which phenylalanine, an amino acid corresponding to position 220 of SEQ ID NO: 3, is substituted with cysteine;
  • DNA polymerase III gamma and tau subunit variants consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which proline, an amino acid corresponding to position 43 of SEQ ID NO: 7, is substituted with leucine;
  • (6) a malate dehydrogenase variant consisting of the amino acid sequence set forth in SEQ ID NO: 21 in which threonine, an amino acid corresponding to position 272 of SEQ ID NO: 23, is substituted with isoleucine;
  • a primosome assembly protein variant consisting of the amino acid sequence shown in SEQ ID NO: 25, in which arginine, an amino acid corresponding to position 304 of SEQ ID NO: 27, is substituted with glutamine;
  • a Co/Zn/Cd efflux system component variant consisting of the amino acid sequence set forth in SEQ ID NO: 41 in which glycine, an amino acid corresponding to position 130 of SEQ ID NO: 43, is substituted with aspartic acid;
  • DAHP synthase variant consisting of the amino acid sequence set forth in SEQ ID NO: 45 in which glycine, an amino acid corresponding to position 382 of SEQ ID NO: 47, is substituted with cysteine;
  • a helicase variant consisting of the amino acid sequence shown in SEQ ID NO: 53, wherein tyrosine, which is an amino acid corresponding to position 592 of SEQ ID NO: 55, is substituted with phenylalanine.
  • One example is a protein variant of the following (A) and (B) or a polynucleotide encoding the variant (eg, (A) a polypeptide encoding the variant, (B) a polypeptide encoding the variant, and / or (A) ) and (B) a polypeptide encoding), it is possible to provide a Corynebacterium glutamicum strain comprising:
  • (A) a helicase variant consisting of the amino acid sequence set forth in SEQ ID NO: 53 in which tyrosine, an amino acid corresponding to position 592 of SEQ ID NO: 55, is substituted with phenylalanine, and
  • an endogenous membrane transport protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which phenylalanine, an amino acid corresponding to position 220 of SEQ ID NO: 3, is substituted with cysteine;
  • DNA polymerase III gamma and tau subunit variants consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which proline, an amino acid corresponding to position 43 of SEQ ID NO: 7, is substituted with leucine;
  • (6) a malate dehydrogenase variant consisting of the amino acid sequence set forth in SEQ ID NO: 21 in which threonine, an amino acid corresponding to position 272 of SEQ ID NO: 23, is substituted with isoleucine;
  • a primosome assembly protein variant consisting of the amino acid sequence shown in SEQ ID NO: 25, in which arginine, an amino acid corresponding to position 304 of SEQ ID NO: 27, is substituted with glutamine;
  • a Co/Zn/Cd efflux system component variant consisting of the amino acid sequence set forth in SEQ ID NO: 41 in which glycine, an amino acid corresponding to position 130 of SEQ ID NO: 43, is substituted with aspartic acid;
  • a DAHP synthase variant consisting of the amino acid sequence set forth in SEQ ID NO: 45 in which glycine, an amino acid corresponding to position 382 of SEQ ID NO: 47, is substituted with cysteine;
  • a protein variant of the following (I) and (II) or a polynucleotide encoding the variant can provide a Corynebacterium glutamicum strain:
  • an endogenous membrane transport protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which phenylalanine, an amino acid corresponding to position 220 of SEQ ID NO: 3, is substituted with cysteine;
  • DNA polymerase III gamma and tau subunit variants consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which proline, an amino acid corresponding to position 43 of SEQ ID NO: 7, is substituted with leucine;
  • (6) a malate dehydrogenase variant consisting of the amino acid sequence set forth in SEQ ID NO: 21 in which threonine, an amino acid corresponding to position 272 of SEQ ID NO: 23, is substituted with isoleucine;
  • a primosome assembly protein variant consisting of the amino acid sequence shown in SEQ ID NO: 25, in which arginine, an amino acid corresponding to position 304 of SEQ ID NO: 27, is substituted with glutamine;
  • a Co/Zn/Cd efflux system component variant consisting of the amino acid sequence set forth in SEQ ID NO: 41 in which glycine, an amino acid corresponding to position 130 of SEQ ID NO: 43, is substituted with aspartic acid;
  • a DAHP synthase variant consisting of the amino acid sequence shown in SEQ ID NO: 45, wherein glycine, which is an amino acid corresponding to position 382 of SEQ ID NO: 47, is substituted with cysteine.
  • the strain of the present application may include a vector comprising the mutant polypeptide of the present application, a polynucleotide encoding the polypeptide, or the polynucleotide of the present application.
  • strain or microorganism
  • strain includes both wild-type microorganisms and microorganisms in which genetic modification has occurred naturally or artificially.
  • a specific mechanism is weakened or enhanced as a microorganism, and may be a microorganism including genetic modification for the production of a desired polypeptide, protein or product.
  • the strain of the present application includes any one or more (eg, one or more or two or more) of the variant of the present application, the polynucleotide of the present application, and a vector including the polynucleotide of the present application; a strain modified to express a variant of the present application or a polynucleotide of the present application; a strain expressing the variant of the present application or the polynucleotide of the present application (eg, a recombinant strain); Or it may be a strain having the mutant activity of the present application (eg, a recombinant strain), but is not limited thereto.
  • the strain of the present application may be a strain having L-lysine-producing ability.
  • the strain of the present application naturally comprises (i) an integral membrane transport protein; (ii) DNA polymerase III subunits gamma and tau; (iii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 11 and/or a protein encoded by the NCgl3069 gene; (iv) transcription antitermination protein; (v) ABC transporter ATP-binding protein; (vi) malate dehydrogenase; (vii) primosome assembly protein; (viii) type II citrate synthase; (ix) membrane protein (TerC); (x) mycothione reductase; (xi) Co/Zn/Cd efflux system component; (xii) DAHP synthase (3-deoxy-D-arabinoheptulosonate-7-phosphate synthase; DAHP synthase); (xiii) N-succinyldiaminopimelate aminotransferase; (xiv) helicase; and
  • the strain of the present application is transformed with a vector containing the polynucleotide of the present application or a polynucleotide encoding the variant of the present application, and expresses the variant of the present application as a cell or microorganism
  • the strains of the application may include all microorganisms capable of producing L-lysine, including the variants of the present application.
  • the strain of the present application is a recombinant strain in which the protein variant is expressed by introducing a polynucleotide encoding the variant of the present application into a natural wild-type microorganism or a microorganism producing L-lysine, and L-lysine-producing ability is increased.
  • the strain of the present application has an increased L-lysine-producing ability compared to a strain (eg, Corynebacterium glutamicum) that does not include a protein variant or a polynucleotide encoding the variant of the present application (eg, Corynebacterium glutamicum). It may be a recombinant strain.
  • the recombinant strain having an increased ability to produce L-amino acids is a natural wild-type microorganism or a microorganism in which one or more proteins selected from the group consisting of (i) to (xv) are unmodified (that is, (i) to (xv) above.
  • a microorganism expressing one or more wild-type proteins selected from the group consisting of; for example, SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, Or 59 polypeptide and / or microorganism comprising a polynucleotide encoding the same) compared to the L-lysine-producing ability may be increased compared to the microorganism, but is not limited thereto.
  • the non-modified microorganism which is the target strain to compare whether the increase in the L-lysine production ability, is ATCC13032 strain and / or Corynebacterium glutamicum CJ3P (US 9556463 B2; the entire document is incorporated herein by reference) ), but is not limited thereto.
  • the recombinant strain with increased production capacity has an L-lysine production capacity of about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, about 7% compared to the parent strain or unmodified microorganism before mutation.
  • the recombinant strain with increased production capacity has an L-lysine production capacity of about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, 1.15 times or more, 1.16 times or more, compared to the parent strain or unmodified microorganism before mutation. , 1.17 times or more, 1.18 times or more, 1.19 times or more, about 1.2 times or more, 1.25 times or more, or about 1.3 times or more (the upper limit is not particularly limited, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less) may be increased.
  • the term “about” is a range including all of ⁇ 0.5, ⁇ 0.4, ⁇ 0.3, ⁇ 0.2, ⁇ 0.1, etc. not limited
  • the term "unmodified microorganism” does not exclude a strain containing a mutation that can occur naturally in a microorganism, it is a wild-type strain or a natural-type strain itself, or a genetic variation caused by natural or artificial factors. It may mean the strain before being changed.
  • the unmodified microorganism may refer to a strain in which the transcriptional anti-termination protein variant described herein is not introduced or before it is introduced.
  • the "unmodified microorganism” may be used interchangeably with "strain before modification", “microbe before modification”, “unmodified strain”, “unmodified strain”, "unmodified microorganism” or "reference microorganism”.
  • the microorganism of the present application is Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium crudilactis ( Corynebacterium crudilactis ), Corynebacterium deserti ( Corynebacterium deserti ), Cory Nebacterium efficiens ( Corynebacterium efficiens ), Corynebacterium callunae ), Corynebacterium stationis , Corynebacterium stationis ), Corynebacterium singulare ( Corynebacterium singulare ), Corynebacterium halo Tolerans ( Corynebacterium halotolerans ), Corynebacterium striatum ( Corynebacterium striatum ), Corynebacterium ammoniagenes ( Corynebacterium ammoniagenes ), Corynebacterium pollutisoli ( Corynebacterium pollutisoli ), Corynebacterium pol
  • the term “weakened” of a polypeptide is a concept that includes both reduced or no activity compared to intrinsic activity.
  • the attenuation may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, attenuation, and the like.
  • the attenuation is when the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide possessed by the original microorganism due to mutation of the polynucleotide encoding the polypeptide, etc.
  • the overall polypeptide activity level and/or concentration (expression amount) in the cell is lower than that of the native strain due to (translation) inhibition, etc., when the expression of the polynucleotide is not made at all, and/or when the expression of the polynucleotide is Even if there is no activity of the polypeptide, it may also be included.
  • the “intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain, wild-type or unmodified microorganism before transformation when the trait is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activity before modification”. “Inactivation, deficiency, reduction, downregulation, reduction, attenuation” of the activity of a polypeptide compared to the intrinsic activity means that the activity of the specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation is lowered.
  • Attenuation of the activity of such a polypeptide may be performed by any method known in the art, but is not limited thereto, and may be achieved by application of various methods well known in the art (eg, Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).
  • the attenuation of the polypeptide of the present application is
  • an antisense oligonucleotide eg, antisense RNA
  • an antisense oligonucleotide that complementarily binds to the transcript of said gene encoding the polypeptide
  • deletion of a part or all of the gene encoding the polypeptide may be the removal of the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, replacement with a polynucleotide in which some nucleotides are deleted, or replacement with a marker gene.
  • the expression control region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.
  • the base sequence modification encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a base encoding another start codon having a lower polypeptide expression rate than the intrinsic start codon It may be substituted with a sequence, but is not limited thereto.
  • the modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above is a deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to weaken the activity of the polypeptide.
  • a combination thereof may result in sequence mutation, or replacement with an amino acid sequence or polynucleotide sequence improved to have weaker activity or an amino acid sequence or polynucleotide sequence improved to have no activity, but is not limited thereto.
  • the expression of a gene may be inhibited or attenuated, but is not limited thereto.
  • antisense oligonucleotide eg, antisense RNA
  • antisense RNA an antisense oligonucleotide that complementarily binds to the transcript of the gene encoding the polypeptide
  • Weintraub, H. et al. Antisense-RNA as a molecular tool. for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].
  • RTE reverse transcription engineering
  • the term “enhancement” of a polypeptide activity means that the activity of the polypeptide is increased compared to the intrinsic activity.
  • the reinforcement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase.
  • activation, enhancement, up-regulation, overexpression, and increase may include all of those exhibiting an activity that was not originally possessed, or exhibiting an improved activity compared to an intrinsic activity or an activity prior to modification.
  • intrinsic activity refers to the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before the transformation when the trait is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activity before modification”.
  • Enhancement”, “up-regulation”, “overexpression” or “increase” in the activity of a polypeptide compared to its intrinsic activity means that the activity and/or concentration (expression) of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation. amount), which means improved.
  • the enrichment can be achieved by introducing an exogenous polypeptide, or by enhancing the activity and/or concentration (expression amount) of the endogenous polypeptide. Whether or not the activity of the polypeptide is enhanced can be confirmed from the increase in the level of activity, expression level, or the amount of product excreted from the polypeptide.
  • the enhancement of the activity of the polypeptide can be applied by various methods well known in the art, and is not limited as long as it can enhance the activity of the target polypeptide compared to the microorganism before modification. Specifically, it may be one using genetic engineering and/or protein engineering well known to those skilled in the art, which is a routine method of molecular biology, but is not limited thereto (eg, Sitnicka et al. Functional Analysis of Genes. Advances in Cell). Biology 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).
  • modification of the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide eg, modification of the polynucleotide sequence of the polypeptide gene to encode a polypeptide that has been modified to enhance the activity of the polypeptide;
  • the increase in the intracellular copy number of the polynucleotide encoding the polypeptide is achieved by introduction into the host cell of a vector to which the polynucleotide encoding the polypeptide is operably linked, which can replicate and function independently of the host it may be Alternatively, the polynucleotide encoding the polypeptide may be achieved by introducing one copy or two or more copies into a chromosome in a host cell.
  • the introduction into the chromosome may be performed by introducing a vector capable of inserting the polynucleotide into the chromosome in the host cell into the host cell, but is not limited thereto.
  • the vector is the same as described above.
  • Replacing the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide with a sequence with strong activity is, for example, deletion, insertion, non-conservative or Conservative substitution or a combination thereof may result in a mutation in the sequence, or replacement with a sequence having a stronger activity.
  • the expression control region is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence controlling the termination of transcription and translation.
  • the original promoter may be replaced with a strong promoter, but is not limited thereto.
  • Examples of known strong promoters include CJ1 to CJ7 promoters (US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Patent US 10584338 B2), O2 promoter (US Patent US 10273491 B2), tkt promoter, yccA promoter, etc., but is not limited thereto.
  • Modification of the nucleotide sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a nucleotide sequence encoding another start codon having a higher expression rate of the polypeptide compared to the intrinsic start codon. It may be a substitution, but is not limited thereto.
  • the modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above may include deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide;
  • a combination thereof may result in sequence mutation, or replacement with an amino acid sequence or polynucleotide sequence improved to have stronger activity or an amino acid sequence or polynucleotide sequence improved to increase activity, but is not limited thereto.
  • the replacement may be specifically performed by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited thereto.
  • the vector used may further include a selection marker for confirming whether or not the chromosome is inserted.
  • the selection marker is the same as described above.
  • the introduction of the foreign polynucleotide exhibiting the activity of the polypeptide may be the introduction of the foreign polynucleotide encoding the polypeptide exhibiting the same/similar activity as the polypeptide into a host cell.
  • the foreign polynucleotide is not limited in origin or sequence as long as it exhibits the same/similar activity as the polypeptide.
  • the method used for the introduction may be performed by appropriately selecting a known transformation method by those skilled in the art, and the introduced polynucleotide is expressed in a host cell to generate a polypeptide and increase its activity.
  • Codon optimization of the polynucleotide encoding the polypeptide is codon-optimized so that the transcription or translation of the endogenous polynucleotide is increased in the host cell, or the transcription and translation of the foreign polynucleotide is optimized in the host cell. It may be that its codons are optimized so that the
  • Selecting an exposed site by analyzing the tertiary structure of the polypeptide and modifying or chemically modifying it for example, compares the sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored to determine the degree of sequence similarity. Accordingly, it may be to determine a template protein candidate, check the structure based on this, and select an exposed site to be modified or chemically modified and modified or modified.
  • Such enhancement of polypeptide activity is to increase the activity or concentration of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild-type or unmodified microbial strain, or increase the amount of product produced from the polypeptide.
  • the present invention is not limited thereto.
  • Modification of some or all of the polynucleotide in the microorganism of the present application is (a) homologous recombination using a vector for chromosomal insertion in the microorganism or engineered nuclease (e.g., CRISPR) -Cas9) and/or (b) induced by light and/or chemical treatment such as ultraviolet and radiation, but not limited thereto.
  • the method for modifying part or all of the gene may include a method by DNA recombination technology.
  • a part or all of the gene may be deleted.
  • the injected nucleotide sequence or vector may include a dominant selection marker, but is not limited thereto.
  • Corynebacterium glutamicum strain comprising a mutant of the present application or a polynucleotide of the present application (eg, one selected from the group consisting of (1) to (15) above) It provides a method for producing L-amino acids, comprising the step of culturing in a medium (for example, one or more or two or more) protein variants or a strain comprising a polynucleotide encoding the variant.
  • it may provide a method for producing L- amino acids, comprising the step of culturing the Corynebacterium glutamicum strain of the present application in a medium.
  • the L-amino acid production method of the present application is a medium of the Corynebacterium glutamicum (or Corynebacterium glutamicum comprising the mutant of the present application or the polynucleotide of the present application or the vector of the present application) of the present application. It may include the step of culturing in
  • L-amino acid of the present application may be L-lysine.
  • the term "culture” means growing the Corynebacterium glutamicum strain of the present application in an appropriately controlled environmental condition.
  • the culture process of the present application may be performed according to a suitable medium and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art according to the selected strain.
  • the culture may be a batch, continuous and/or fed-batch, but is not limited thereto.
  • the term "medium” refers to a material in which nutrients required for culturing the Corynebacterium glutamicum strain of the present application are mixed as a main component, and nutrients including water essential for survival and growth and growth factors.
  • any medium and other culture conditions used for culturing the Corynebacterium glutamicum strain of the present application may be used without particular limitation as long as it is a medium used for culturing conventional microorganisms, but the Corynebacterium glutamicum of the present application Lium glutamicum strain can be cultured while controlling the temperature, pH, etc. under aerobic conditions in a conventional medium containing an appropriate carbon source, nitrogen source, phosphorus, inorganic compound, amino acid and / or vitamin and the like.
  • the culture medium for the Corynebacterium sp. strain can be found in the literature ["Manual of Methods for General Bacteriology” by the American Society for Bacteriology (Washington D.C., USA, 1981)].
  • the carbon source includes carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, and the like; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, citric acid and the like; Amino acids such as glutamic acid, methionine, lysine, and the like may be included.
  • natural organic nutrient sources such as starch hydrolyzate, molasses, blackstrap molasses, rice winter, cassava, sugar cane offal and corn steep liquor can be used, specifically glucose and sterilized pre-treated molasses (i.e., converted to reducing sugar). molasses) may be used, and other appropriate amounts of carbon sources may be variously used without limitation. These carbon sources may be used alone or in combination of two or more, but is not limited thereto.
  • nitrogen source examples include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or degradation products thereof, defatted soybean cake or degradation products thereof, etc. can be used These nitrogen sources may be used alone or in combination of two or more, but is not limited thereto.
  • inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate
  • Amino acids such as glutamic acid, methionine, glutamine
  • organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract
  • the phosphorus may include potassium monobasic phosphate, dipotassium phosphate, or a sodium-containing salt corresponding thereto.
  • potassium monobasic phosphate dipotassium phosphate
  • sodium-containing salt corresponding thereto.
  • sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. may be used, and in addition, amino acids, vitamins and/or suitable precursors may be included. These components or precursors may be added to the medium either batchwise or continuously. However, the present invention is not limited thereto.
  • compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. may be added to the medium in an appropriate manner to adjust the pH of the medium.
  • an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation.
  • oxygen or oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen or carbon dioxide gas may be injected without injection of gas or without injection of gas to maintain anaerobic and microaerobic conditions, which are limited thereto it is not
  • the culture temperature may be maintained at 20 to 45° C., specifically, 25 to 40° C., and may be cultured for about 10 to 160 hours, but is not limited thereto.
  • the L-amino acid produced by the culture of the present application may be secreted into the medium or may remain in the cell.
  • the L-amino acid production method of the present application includes the steps of preparing the Corynebacterium glutamicum strain of the present application, preparing a medium for culturing the strain, or a combination thereof (regardless of the order, in any order) ), for example, prior to the culturing step, may further include.
  • the method for producing L-amino acids of the present application may further include recovering L-amino acids from the culture medium (the culture medium) or the Corynebacterium glutamicum strain.
  • the recovering step may be further included after the culturing step.
  • the recovery may be to collect the desired L-amino acid using a suitable method known in the art according to the culture method of the microorganism of the present application, for example, a batch, continuous or fed-batch culture method, etc. .
  • a suitable method known in the art according to the culture method of the microorganism of the present application, for example, a batch, continuous or fed-batch culture method, etc. .
  • chromatography such as island chromatography, HPLC, or a combination thereof may be used, and a desired L-amino acid may be recovered from a medium or a microorganism using a suitable method known in the art.
  • the L-amino acid production method of the present application may include an additional purification step.
  • the purification may be performed using a suitable method known in the art.
  • the recovery step and the purification step are performed continuously or discontinuously, regardless of the order, or integrated into one step. may be performed, but is not limited thereto.
  • variants, polynucleotides, vectors, strains, and the like are as described in the other aspects above.
  • Corynebacterium glutamicum strain comprising a variant of the present application, a polynucleotide encoding the variant, a vector including the polynucleotide or the polynucleotide of the present application; the culture medium; Or to provide a composition for producing L- amino acids comprising a combination of two or more of them.
  • composition of the present application may further include any suitable excipients commonly used in compositions for the production of amino acids, and such excipients may be, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents or isotonic agents, etc.
  • excipients may be, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents or isotonic agents, etc.
  • the present invention is not limited thereto.
  • composition of the present application variants, polynucleotides, vectors, strains, media and L-amino acids are the same as those described in the other aspects above.

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Abstract

La présente invention concerne un nouveau variant de protéine, une souche de Corynebacterium glutamicum comprenant le variant, et un procédé de production de L-lysine à l'aide de la souche.
PCT/KR2021/003261 2021-01-29 2021-03-16 Nouveau variant de protéine et procédé de production de l-lysine l'utilisant WO2022163904A1 (fr)

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PCT/KR2021/007122 WO2022163951A1 (fr) 2021-01-29 2021-06-08 Nouveau variant de protéine et procédé de production de l-lysine l'utilisant

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
KR10-2021-0013694 2021-01-29
KR1020210013691A KR20220110412A (ko) 2021-01-29 2021-01-29 신규한 단백질 변이체 및 이를 이용한 l-라이신 생산 방법
KR10-2021-0013690 2021-01-29
KR1020210013690A KR20220110411A (ko) 2021-01-29 2021-01-29 신규한 단백질 변이체 및 이를 이용한 l-라이신 생산 방법
KR1020210013694A KR20220110413A (ko) 2021-01-29 2021-01-29 신규한 단백질 변이체 및 이를 이용한 l-라이신 생산 방법
KR10-2021-0013691 2021-01-29
KR10-2021-0013693 2021-01-29
KR20210013693 2021-01-29

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Cited By (1)

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EP4056694A4 (fr) * 2021-01-29 2024-01-24 Cj Cheiljedang Corp Nouveau variant de citrate synthase de type ii, et procédé de production de l-lysine l'utilisant

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EP4056694A4 (fr) * 2021-01-29 2024-01-24 Cj Cheiljedang Corp Nouveau variant de citrate synthase de type ii, et procédé de production de l-lysine l'utilisant

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