WO2022163904A1

WO2022163904A1 - Novel protein variant and method for producing l-lysine using same

Info

Publication number: WO2022163904A1
Application number: PCT/KR2021/003261
Authority: WO
Inventors: 권나라
Original assignee: 씨제이제일제당 (주)
Priority date: 2021-01-29
Filing date: 2021-03-16
Publication date: 2022-08-04

Abstract

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.

Description

Novel protein variant and L-lysine production method using the same

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.

In order to produce L-amino acids and other useful substances, various studies are being conducted for the development of high-efficiency production microorganisms and fermentation process technology. For example, a target substance-specific approach such as increasing the expression of a gene encoding an enzyme involved in L-lysine biosynthesis or removing a gene unnecessary for biosynthesis is mainly used (WO2008-082181 A1).

However, as the demand for L-lysine increases, there is still a need for research to increase the effective production capacity of L-lysine.

One object of the present application is to provide a protein variant selected from the group consisting of the following (1) to (15):

(1) 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;

(2) 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;

(3) a protein variant consisting of the amino acid sequence shown in SEQ ID NO: 9, in which cysteine, an amino acid corresponding to position 33 of SEQ ID NO: 11, is substituted with serine;

(4) 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;

(5) an ABC transporter ATP-binding protein variant consisting of the amino acid sequence shown in SEQ ID NO: 17 in which valine, an amino acid corresponding to position 199 of SEQ ID NO: 19, is substituted with methionine;

(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;

(7) 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;

(8) a type II citrate synthase variant consisting of the amino acid sequence shown in SEQ ID NO: 29, in which alanine, an amino acid corresponding to position 169 of SEQ ID NO: 31, is substituted with valine;

(9) a membrane protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 33 in which glutamic acid, an amino acid corresponding to position 294 of SEQ ID NO: 35, is substituted with lysine;

(10) a mycothione reductase variant consisting of the amino acid sequence set forth in SEQ ID NO: 37 in which proline, which is the amino acid corresponding to position 358 of SEQ ID NO: 39, is substituted with serine;

(11) 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;

(12) 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;

(13) an N-succinyldiaminopimelate aminotransferase variant consisting of the amino acid sequence set forth in SEQ ID NO: 49, in which proline, an amino acid corresponding to position 209 of SEQ ID NO: 51, is substituted with leucine;

(14) 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

(15) 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:

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.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, it cannot be seen that the scope of the present application is limited by the specific descriptions described below. In addition, a number of papers and patent documents are referenced throughout this specification and their citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention pertains and the content of the present invention.

One aspect of the present application is to provide a protein variant selected from the group consisting of the following (1) to (15):

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.

In addition, 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, 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. 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. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, 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

	아미노산 서열amino acid sequence	위치를 카운팅하기 위해 기준이 되는 아미노산 서열The amino acid sequence as a reference for counting positions	위치location	아미노산 종류type of amino acid
1One	서열번호 1SEQ ID NO: 1	서열번호 3SEQ ID NO: 3	220220	시스테인cysteine
22	서열번호 5SEQ ID NO: 5	서열번호 7SEQ ID NO: 7	4343	류신leucine
33	서열번호 9SEQ ID NO: 9	서열번호 11SEQ ID NO: 11	3333	세린serine
44	서열번호 13SEQ ID NO: 13	서열번호 15SEQ ID NO: 15	210210	아스파르트산aspartic acid
55	서열번호 17SEQ ID NO: 17	서열번호 19SEQ ID NO: 19	199199	메티오닌methionine
66	서열번호 21SEQ ID NO: 21	서열번호 23SEQ ID NO: 23	272272	이소류신isoleucine
77	서열번호 25SEQ ID NO: 25	서열번호 27SEQ ID NO: 27	304304	글루타민glutamine
88	서열번호 29SEQ ID NO: 29	서열번호 31SEQ ID NO: 31	169169	발린valine
99	서열번호 33SEQ ID NO: 33	서열번호 35SEQ ID NO: 35	294294	라이신lysine
1010	서열번호 37SEQ ID NO: 37	서열번호 39SEQ ID NO: 39	358358	세린serine
1111	서열번호 41SEQ ID NO: 41	서열번호 43SEQ ID NO: 43	130130	아스파르트산aspartic acid
1212	서열번호 45SEQ ID NO: 45	서열번호 47SEQ ID NO: 47	382382	시스테인cysteine
1313	서열번호 49SEQ ID NO: 49	서열번호 51SEQ ID NO: 51	209209	류신leucine
1414	서열번호 53SEQ ID NO: 53	서열번호 55SEQ ID NO: 55	592592	페닐알라닌phenylalanine
1515	서열번호 57SEQ ID NO: 57	서열번호 59SEQ ID NO: 59	170170	라이신lysine

For example, 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. The term “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.

As used herein, the term "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. Other variants may include variants in which a portion is removed from the N- and/or C-terminus of the mature protein. The term “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. For the purposes of the present application, 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, 45, 49, 53, or 57, respectively, substituted with It may be a polypeptide comprising Table 2 below shows the correspondence between amino acid sequences, mutation positions, amino acids before substitution corresponding to the positions, and types of amino acids after substitution of the variants of the present application.

	아미노산 서열amino acid sequence	위치를 카운팅하기 위해 기준이 되는 아미노산 서열The amino acid sequence as a reference for counting positions	변이 위치mutation position	치환 전 아미노산 종류Type of amino acid before substitution	치환된 아미노산 종류Substituted Amino Acid Types
1One	서열번호 1SEQ ID NO: 1	서열번호 3SEQ ID NO: 3	220220	페닐알라닌phenylalanine	시스테인cysteine
22	서열번호 5SEQ ID NO: 5	서열번호 7SEQ ID NO: 7	4343	프롤린proline	류신leucine
33	서열번호 9SEQ ID NO: 9	서열번호 11SEQ ID NO: 11	3333	시스테인cysteine	세린serine
44	서열번호 13SEQ ID NO: 13	서열번호 15SEQ ID NO: 15	210210	아스파라긴asparagine	아스파르트산aspartic acid
55	서열번호 17SEQ ID NO: 17	서열번호 19SEQ ID NO: 19	199199	발린valine	메티오닌methionine
66	서열번호 21SEQ ID NO: 21	서열번호 23SEQ ID NO: 23	272272	트레오닌threonine	이소류신isoleucine
77	서열번호 25SEQ ID NO: 25	서열번호 27SEQ ID NO: 27	304304	아르기닌arginine	글루타민glutamine
88	서열번호 29SEQ ID NO: 29	서열번호 31SEQ ID NO: 31	169169	알라닌alanine	발린valine
99	서열번호 33SEQ ID NO: 33	서열번호 35SEQ ID NO: 35	294294	글루탐산glutamic acid	라이신lysine
1010	서열번호 37SEQ ID NO: 37	서열번호 39SEQ ID NO: 39	358358	프롤린proline	세린serine
1111	서열번호 41SEQ ID NO: 41	서열번호 43SEQ ID NO: 43	130130	글리신glycine	아스파르트산aspartic acid
1212	서열번호 45SEQ ID NO: 45	서열번호 47SEQ ID NO: 47	382382	글리신glycine	시스테인cysteine
1313	서열번호 49SEQ ID NO: 49	서열번호 51SEQ ID NO: 51	209209	프롤린proline	류신leucine
1414	서열번호 53SEQ ID NO: 53	서열번호 55SEQ ID NO: 55	592592	티로신tyrosine	페닐알라닌phenylalanine
1515	서열번호 57SEQ ID NO: 57	서열번호 59SEQ ID NO: 59	170170	글루탐산glutamic acid	라이신lysine

In addition, variants may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, 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.

In the case of culturing a Corynebacterium glutamicum strain comprising a variant of the protein of the present application, it is possible to produce a high yield of L-lysine compared to a microorganism having an existing unmodified polypeptide.

As used herein, 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.

Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444, using a known computer algorithm such as the "FASTA" program. or, as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later), Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine (GCG program package (Devereux, J., et al, Nucleic Acids) Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop , [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA/.] (1988) SIAM J Applied Math 48: 1073).For example, BLAST of the National Center for Biotechnology Information Database, or ClustalW can be used to determine homology, similarity or identity.

Homology, similarity or identity of polynucleotides or polypeptides is described, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482, see, for example, Needleman et al. (1970), J Mol Biol. can be determined by comparing sequence information using a GAP computer program such as 48:443. In summary, 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.

As an example of the present application, 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.

As used herein, 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.Specifically, the integral membrane transport protein of the present application can be used interchangeably as an integral membrane transport protein.In the present application, the internal membrane transport protein is a known data The sequence can be obtained from GenBank of NCBI, which is the base, for example, it can be GenBank Accession No. YP_227155.1, WP_011015489.1. Specifically, it can be a polypeptide having an intrinsic membrane transport protein activity encoded by the NCgl2816 gene. However, it is not limited thereto.

As used herein, the term "DNA polymerase Ⅲ subunits gamma and tau" is a major enzyme responsible for DNA replication, and the DNA polymerase Ⅲ tau subunit is a core (alpha ( alpha), epsilon, and theta (consisting of chains) bind to dimerization of the core, and DNA polymerase III gamma subunit is a clamp loader that obtains beta subunit to lagging strand. Specifically, 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.

As used herein, the term "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. In the present application, 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.

As used herein, 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. Specifically, the ABC transporter ATP-binding protein of the present application may be used in combination as the ABC transporter ATP-binding protein. In the present application, 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.

As used herein, the term "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. Specifically, the N-succinyldiaminopimelate aminotransferase of the present application can be used in combination with N-succinyldiaminopimelate aminotransferase. The 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. Specifically, NCgl1058 It may be a polypeptide having N-succinyldiaminopimelate aminotransferase activity encoded by the gene (or dapC gene), but is not limited thereto.

As used herein, the term “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 In the present application, 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

As used herein, the term "primosome assembly protein" refers to a protein having a helicase activity and involved in the DNA replication process. Specifically, the primosome assembly protein of the present application may be used interchangeably as primosome assembly protein, PriA protein, PriA, or primosomal protein N'. In the present application, the 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.

As used herein, the term "helicase" is a polypeptide capable of separating a DNA double helix or a strand of a self-associated RNA molecule. Specifically, the helicase of the present application may be used in combination with a helicase. In the present application, 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. In the present application, 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. In the present application, 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. In the present application, 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. In the present application, 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. In the present application, 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.

As an example of the present application, the mutant of the present application may have an activity to increase L-lysine production capacity compared to the wild-type polypeptide.

said endogenous membrane transport protein; DNA polymerase III gamma and tau subunits; a protein encoded by the NCgl3069 gene; transcriptional anti-termination protein; ABC transporter ATP-binding protein; malate dehydrogenase; primosome assembly protein; type II citrate synthase; membrane protein; mycothion reductase; Co/Zn/Cd effluent system components; DAHP synthase; N-succinyldiaminopimelate aminotransferase; helicase; and/or an amino acid sequence of a wild-type polypeptide of a polypeptide comprising the amino acid sequence of SEQ ID NO: 59 and/or a protein encoded by the NCgl1098 gene and an example of a nucleic acid sequence encoding the same are shown in Table 3 below.

단백질protein	아미노산 서열amino acid sequence	단백질을 코딩하는 핵산 서열a nucleic acid sequence encoding a protein
내재성 막 수송 단백질endogenous membrane transport protein	서열번호 3SEQ ID NO: 3	서열번호 4SEQ ID NO: 4
DNA 중합효소 Ⅲ 감마 및 타우 서브유닛DNA Polymerase III Gamma and Tau Subunits	서열번호 7SEQ ID NO: 7	서열번호 8SEQ ID NO: 8
NCgl3069 유전자에 의해 코딩되는 단백질Protein encoded by the NCgl3069 gene	서열번호 11SEQ ID NO: 11	서열번호 12SEQ ID NO: 12
전사 안티터미네이션 단백질transcriptional anti-termination protein	서열번호 15SEQ ID NO: 15	서열번호 16SEQ ID NO: 16
ABC 트랜스포터 ATP-결합 단백질ABC transporter ATP-binding protein	서열번호 19SEQ ID NO: 19	서열번호 20SEQ ID NO: 20
말레이트 디하이드로게나제malate dehydrogenase	서열번호 23SEQ ID NO: 23	서열번호 24SEQ ID NO: 24
프리모솜 조립 단백질primosome assembly protein	서열번호 27SEQ ID NO: 27	서열번호 28SEQ ID NO: 28
타입 II 시트레이트 신타아제Type II citrate synthase	서열번호 31SEQ ID NO: 31	서열번호 32SEQ ID NO: 32
막 단백질membrane protein	서열번호 35SEQ ID NO: 35	서열번호 36SEQ ID NO: 36
미코티온 리덕타제mycothion reductase	서열번호 39SEQ ID NO: 39	서열번호 40SEQ ID NO: 40
Co/Zn/Cd 유출 시스템 컴포넌트Co/Zn/Cd effluent system components	서열번호 43SEQ ID NO: 43	서열번호 44SEQ ID NO: 44
DAHP 신타아제DAHP synthase	서열번호 47SEQ ID NO: 47	서열번호 48SEQ ID NO: 48
N-숙시닐디아미노피멜레이트 아미노트랜스퍼라제N-succinyldiaminopimelate aminotransferase	서열번호 51SEQ ID NO: 51	서열번호 52SEQ ID NO: 52
헬리카제helicase	서열번호 55SEQ ID NO: 55	서열번호 56SEQ ID NO: 56
NCgl1098 유전자에 의해 코딩되는 단백질Protein encoded by the NCgl1098 gene	서열번호 59SEQ ID NO: 59	서열번호 60SEQ ID NO: 60

As used herein, the term “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. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related protein or reference protein.

For example, 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. For example, 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.

As used herein, the term "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. In one example of the present application, 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. In addition, 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. can In another example, 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, A, 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 nucleic acid sequences having homology or identity. In addition, if the sequence encoding a polypeptide or protein having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, 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.

	핵산 서열nucleic acid sequence	위치를 카운팅하기 위해 기준이 되는 핵산 서열Nucleic acid sequence as a reference for counting positions	위치location	염기 종류base type
1One	서열번호 2SEQ ID NO: 2	서열번호 4SEQ ID NO: 4	659659	GG
22	서열번호 6SEQ ID NO: 6	서열번호 8SEQ ID NO: 8	128128	TT
33	서열번호 10SEQ ID NO: 10	서열번호 12SEQ ID NO: 12	9797	AA
44	서열번호 14SEQ ID NO: 14	서열번호 16SEQ ID NO: 16	628628	GG
55	서열번호 18SEQ ID NO: 18	서열번호 20SEQ ID NO: 20	595595	AA
66	서열번호 22SEQ ID NO: 22	서열번호 24SEQ ID NO: 24	815815	TT
77	서열번호 26SEQ ID NO: 26	서열번호 28SEQ ID NO: 28	911911	AA
88	서열번호 30SEQ ID NO: 30	서열번호 32SEQ ID NO: 32	506506	TT
99	서열번호 34SEQ ID NO: 34	서열번호 36SEQ ID NO: 36	880880	AA
1010	서열번호 38SEQ ID NO: 38	서열번호 40SEQ ID NO: 40	10721072	TT
1111	서열번호 42SEQ ID NO: 42	서열번호 44SEQ ID NO: 44	389389	AA
1212	서열번호 46SEQ ID NO: 46	서열번호 48SEQ ID NO: 48	11441144	TT
1313	서열번호 50SEQ ID NO: 50	서열번호 52SEQ ID NO: 52	626626	TT
1414	서열번호 54SEQ ID NO: 54	서열번호 56SEQ ID NO: 56	17751775	TT
1515	서열번호 58SEQ ID NO: 58	서열번호 60SEQ ID NO: 60	508508	AA

In consideration of codon degeneracy or preferred codons in organisms that want to express the variants of the present application, 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. Specifically, 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. In this case, in the sequence having the homology or identity, 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). For example, 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.

Specifically, 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. In addition, 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.

In the present application, "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. may include The 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. 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. For example, 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 can be used. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like can be used.

For example, 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.

As used herein, 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. In addition, 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. For example, 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. In addition, 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.

In addition, the term “operably linked” as used herein 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.

Another aspect of the present application, 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:

(4) a transcriptional anti-termination protein variant consisting of the amino acid sequence shown 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;

(13) an 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;

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:

(a) a protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 57, in which glutamic acid, an amino acid corresponding to position 170 of SEQ ID NO: 59, is substituted with lysine; and

(b) at least one protein variant selected from the group consisting of (1) to (14):

(13) an 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; and

(14) 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

(B) at least one protein variant selected from the group consisting of (1) to (13):

(12) 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; and

(13) An N-succinyldiaminopimelate aminotransferase mutant consisting of the amino acid sequence shown 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.

One example is a protein variant of the following (I) and (II) or a polynucleotide encoding the variant (e.g., (I) a polypeptide encoding a variant, (II) a polypeptide encoding a variant, and/or (I) ) and a polypeptide encoding (II)), can provide a Corynebacterium glutamicum strain:

(I) an N-succinyldiaminopimelate aminotransferase variant consisting of the amino acid sequence set forth in SEQ ID NO: 49 in which proline, which is the amino acid corresponding to position 209 of SEQ ID NO: 51, is substituted with leucine, and

(II) at least one protein variant selected from the group consisting of the following (1) to (12):

(11) 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; and

(12) 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.

As used herein, the term "strain (or microorganism)" includes both wild-type microorganisms and microorganisms in which genetic modification has occurred naturally or artificially. As a result, 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/or (xv) a polypeptide comprising the amino acid sequence of SEQ ID NO: 59 and/or a microorganism having an activity and/or L-lysine-producing ability of a protein encoded by the NCgl1098 gene, or the above (i) to (xv) A mutant of the present application or a polynucleotide encoding the same in the parent strain without the activity and/or L-lysine-producing ability of one or more (for example, one or more or two or more) proteins selected from the group consisting of (or The vector including the polynucleotide) may be introduced and/or a microorganism to which L-lysine-producing ability is imparted, but is not limited thereto.

As an example, 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. For example, 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. can In one example, 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. For example, 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.

For example, 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. or more, about 8% or more, about 9% or more, about 10% or more, about 10.5% or more, about 11% or more, about 11.5% or more, about 12% or more, about 12.5% or more, about 13% or more, about 13.5% or more, about 14% or more, about 14.5% or more, about 15% or more, about 15.5% or more, about 16% or more, about 16.5% or more, about 17% or more, about 17.5% or more, about 18% or more, about 18.5% or more, about 19% or more, about 19.5% or more, about 20% or more, about 20.5% or more, about 21% or more, about 21.5% or more, about 22% or more, about 22.5% or more, about 23% or more, about 23.5% or more, about 24% or more, about 24.5% or more, about 25% or more, about 25.5% or more, about 26% or more, about 26.5% or more, about 27% or more, about 27.5% or more, about 28% or more, about 28.5% or more, about 29% or more, about 29.5% or more, about 30% or more, about 31% or more, about 32% or more, about 33% or more, about 34% or more, or about 35% or more (the upper limit is not particularly limited, For example, it may be about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 45% or less, about 40% or less, or about 35% or less). In another example, 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

As used herein, 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. For example, 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".

As another example of the present application, 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 imitans ( Corynebacterium imitans ) imitans ), Corynebacterium testudinoris ) or Corynebacterium flavescens ).

As used herein, 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. When 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.).

Specifically, the attenuation of the polypeptide of the present application is

1) deletion of all or part of a gene encoding a polypeptide;

2) modification of the expression control region (or expression control sequence) to reduce the expression of the gene encoding the polypeptide;

3) modification of the amino acid sequence constituting the polypeptide such that the activity of the polypeptide is eliminated or attenuated (eg, deletion/substitution/addition of one or more amino acids on the amino acid sequence);

4) modification of the gene sequence encoding the polypeptide such that the activity of the polypeptide is eliminated or attenuated (eg, one or more nucleobases on the nucleotide sequence of the polypeptide gene to encode a polypeptide modified such that the activity of the polypeptide is eliminated or attenuated) deletion/replacement/addition of);

5) modification of the nucleotide sequence encoding the initiation codon or 5'-UTR region of the gene transcript encoding the polypeptide;

6) introduction of an antisense oligonucleotide (eg, antisense RNA) that complementarily binds to the transcript of said gene encoding the polypeptide;

7) adding a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide to form a secondary structure that cannot be attached to the ribosome;

8) addition of a promoter transcribed in the opposite direction to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE); or

9) It may be a combination of two or more selected from 1) to 8) above, but is not particularly limited thereto.

for example,

1) The 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.

In addition, the above 2) modification of the expression control region (or expression control sequence), deletion, insertion, non-conservative or conservative substitution, or a combination thereof, mutation in the expression control region (or expression control sequence) occurs, or weaker replacement with an active sequence. 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.

In addition, 3) 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.

In addition, 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. Or 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. For example, by introducing a mutation in the polynucleotide sequence to form a stop codon, the expression of a gene may be inhibited or attenuated, but is not limited thereto.

6) The introduction of an antisense oligonucleotide (eg, antisense RNA) that complementarily binds to the transcript of the gene encoding the polypeptide is described, for example, in Weintraub, H. et al., Antisense-RNA as a molecular tool. for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

7) Addition of a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide to form a secondary structure that cannot be attached to the ribosome is mRNA translation It may make it impossible or slow it down.

8) the addition of a promoter transcribed in the opposite direction to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE) is an antisense complementary to the transcript of the gene encoding the polypeptide It may be to attenuate activity by making nucleotides.

As used herein, 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. Herein, 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. The “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.).

Specifically, the enrichment of the polypeptide of the present application is

1) increasing the intracellular copy number of a polynucleotide encoding the polypeptide;

2) replacing the gene expression control region on the chromosome encoding the polypeptide with a sequence with strong activity;

3) modification of the nucleotide sequence encoding the initiation codon or 5'-UTR region of the gene transcript encoding the polypeptide;

4) modification of the amino acid sequence of said polypeptide to enhance polypeptide activity;

5) 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);

6) introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same;

7) codon optimization of the polynucleotide encoding the polypeptide;

8) by analyzing the tertiary structure of the polypeptide to select an exposed site for modification or chemical modification; or

More specifically,

1) 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.

2) 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. As an example, 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.

3) 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. In this case, 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.

6) 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.

7) 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

8) 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. However, the present invention is not limited thereto.

Modification of some or all of the polynucleotide in the microorganism of the present application (eg, modification for encoding the protein variant described above) 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. For example, by injecting a nucleotide sequence or a vector containing a nucleotide sequence homologous to a target gene into the microorganism to cause homologous recombination, 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.

In the microorganism of the present application, variants, polynucleotides, L-lysine, and the like are as described in the other aspects above.

Another aspect of the present application is a 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.

In one example, 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

In addition, the L-amino acid of the present application may be L-lysine.

In the present application, 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. Specifically, the culture may be a batch, continuous and/or fed-batch, but is not limited thereto.

As used herein, 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. Specifically, 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.

Specifically, 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)].

In the present application, 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. In addition, 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.

Examples of the nitrogen source 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.

The phosphorus may include potassium monobasic phosphate, dipotassium phosphate, or a sodium-containing salt corresponding thereto. As the inorganic compound, 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.

In addition, during the culture of the Corynebacterium glutamicum strain of the present application, 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. In addition, during culturing, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation. In addition, in order to maintain the aerobic state of the medium, 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

In the culture of the present application, 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. . For example, centrifugation, filtration, treatment with a crystallized protein precipitating agent (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity Various 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.

In addition, 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. In one example, when the L-amino acid production method of the present application includes both the recovery step and the purification step, 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.

In the method of the present application, variants, polynucleotides, vectors, strains, and the like are as described in the other aspects above.

Another aspect of the present application is a 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.

The 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. However, the present invention is not limited thereto.

In the 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.

Claims

Protein variants selected from the group consisting of the following (1) to (15):

(1) 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;

(2) 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;

(3) a protein variant consisting of the amino acid sequence shown in SEQ ID NO: 9, in which cysteine, an amino acid corresponding to position 33 of SEQ ID NO: 11, is substituted with serine;

(4) a transcriptional anti-termination protein variant consisting of the amino acid sequence shown 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;

(5) an ABC transporter ATP-binding protein variant consisting of the amino acid sequence shown in SEQ ID NO: 17 in which valine, an amino acid corresponding to position 199 of SEQ ID NO: 19, is substituted with methionine;

(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;

(7) 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;

(8) a type II citrate synthase variant consisting of the amino acid sequence shown in SEQ ID NO: 29, in which alanine, an amino acid corresponding to position 169 of SEQ ID NO: 31, is substituted with valine;

(9) a membrane protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 33 in which glutamic acid, an amino acid corresponding to position 294 of SEQ ID NO: 35, is substituted with lysine;

(10) a mycothione reductase variant consisting of the amino acid sequence set forth in SEQ ID NO: 37 in which proline, which is the amino acid corresponding to position 358 of SEQ ID NO: 39, is substituted with serine;

(11) 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;

(12) 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;

(13) an N-succinyldiaminopimelate aminotransferase variant consisting of the amino acid sequence set forth in SEQ ID NO: 49, in which proline, an amino acid corresponding to position 209 of SEQ ID NO: 51, is substituted with leucine;

(14) 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

(15) 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.
A polynucleotide encoding the variant of claim 1 .
A Corynebacterium glutamicum strain comprising two or more protein variants selected from the group consisting of the following (1) to (15) or a polynucleotide encoding the variant:

(1) 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;

(2) 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;

(3) a protein variant consisting of the amino acid sequence shown in SEQ ID NO: 9, in which cysteine, an amino acid corresponding to position 33 of SEQ ID NO: 11, is substituted with serine;

(4) a transcriptional anti-termination protein variant consisting of the amino acid sequence shown 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;

(5) an ABC transporter ATP-binding protein variant consisting of the amino acid sequence shown in SEQ ID NO: 17 in which valine, an amino acid corresponding to position 199 of SEQ ID NO: 19, is substituted with methionine;

(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;

(7) 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;

(8) a type II citrate synthase variant consisting of the amino acid sequence shown in SEQ ID NO: 29, in which alanine, an amino acid corresponding to position 169 of SEQ ID NO: 31, is substituted with valine;

(9) a membrane protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 33 in which glutamic acid, an amino acid corresponding to position 294 of SEQ ID NO: 35, is substituted with lysine;

(10) a mycothione reductase variant consisting of the amino acid sequence set forth in SEQ ID NO: 37 in which proline, which is the amino acid corresponding to position 358 of SEQ ID NO: 39, is substituted with serine;

(11) 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;

(12) 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;

(13) an N-succinyldiaminopimelate aminotransferase variant consisting of the amino acid sequence set forth in SEQ ID NO: 49, in which proline, an amino acid corresponding to position 209 of SEQ ID NO: 51, is substituted with leucine;

(14) 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

(15) 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 strain of claim 3, wherein the L-lysine-producing ability is increased compared to Corynebacterium glutamicum that does not include the protein variant or the polynucleotide encoding the variant.
A method for producing L-lysine, comprising the step of culturing the Corynebacterium glutamicum strain of claim 3 or 4 in a medium.