Protein variants and method for producing l-lysine using same

Abstract

The present invention provides a protein variant, a Corynebacterium genus microorganism comprising the protein variant, and a method for producing L-lysine using the microorganism of the present disclosure.

Description

Technical Field

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present disclosure claims the benefit of priority based on Korean Patent Application No. 10-2023-0096086, filed on Jul. 24, 2023, and all contents disclosed in the corresponding Korean patent application document are incorporated as a part of the present disclosure.

[0003] Throughout the present disclosure, many papers and patent documents are cited, and their citations are indicated. The disclosures of the cited papers and patent documents are incorporated into the present disclosure by reference in their entirety to more clearly describe the level of the technical field to which the present invention belongs and the content of the present invention.

[0004] The present invention relates to a protein variant, a Corynebacterium microorganism comprising the protein variant, and a method for producing L-lysine using the microorganism. Background Art

[0005] In order to produce L-amino acid and other useful substances, people carry out various researches to develop efficient production microorganisms and fermentation process technology.For example, mainly use the target substance specific approach method, for example increase the expression of the gene of the enzyme related to the biosynthesis of L-lysine coding or remove the unwanted gene of biosynthesis (WO2008-082181A1).

[0006] However, according to the increase in demand for L-lysine, research is still needed to effectively improve the production capacity of L-lysine. Summary of the invention

[0007] Technical issues

[0008] One object of the present disclosure is to provide a protein variant.

[0009] Another object of the present disclosure is to provide a polypeptide encoding a protein variant.

[0010] Still another object of the present disclosure is to provide a microorganism of the genus Corynebacterium, comprising at least one selected from the group consisting of a protein variant and a polynucleotide encoding the protein variant.

[0011] Still another object of the present disclosure is to provide a composition for producing L-lysine, comprising a microorganism of the genus Corynebacterium.

[0012] Still another object of the present disclosure is to provide a method for producing L-lysine, comprising culturing a microorganism of the genus Corynebacterium in a culture medium.

[0013] Another object of the present disclosure is to provide a use of a microorganism in producing L-lysine.

[0014] Technical Solution

[0015] The technical solution is described in detail as follows. On the other hand, each description, aspect and embodiment disclosed in the present disclosure may also be applied to each other description, aspect and embodiment. In other words, all combinations of the various elements disclosed in the present disclosure belong to the scope of the present disclosure. In addition, the scope of the present disclosure should not be considered to be limited by the specific description described below. In addition, those skilled in the art can recognize or confirm many equivalents of the specific aspects of the present application disclosed in the present application using only common experiments. In addition, these equivalents are intended to be included in the present application.

[0016] One aspect of the present disclosure provides a protein variant comprising the following amino acid sequence, in which the amino acid at position 75 from the N-terminus in the amino acid sequence corresponding to SEQ ID NO: 105 is substituted by an amino acid different from the original amino acid; or comprising the following amino acid sequence, in which the amino acid at position 35 from the N-terminus in the amino acid sequence corresponding to SEQ ID NO: 106 is substituted by an amino acid different from the original amino acid.

[0017] In the present disclosure, the sequence of SEQ ID NO: 105 is as follows:

[0018] MVSQTERQHAIASLLAPTGAVSVGDLAEHFHVTTETVRRDLRIMESLGLLQRVHGGAISPEPMGTSPPRLKPALGKGMPPEPRVLELAETAVSLITPLARSIFLDSGLACTAIATVLGDPPEDARWTVVTSS PGAVIALSATDATSTVVLHGQVHGNCSSIIGSTAVDMISQLRADIAFVEVDAIQSDTSLCTFFPETIPIKQAMIKNAAFTVAVLSPRSPQDQELQLLKHPFSTLADFDALVTDDHTLDFPVLPDHNFQVVTP.

[0019] In the present disclosure, the sequence of SEQ ID NO: 106 is as follows:

[0020] MTTNAPDGATNNINNAHSGAVGKPKVQLSDAEIQEYTAAFAGTTTTKPWELEVTTKFLKKIAWVAVVVIMAVHIFMGAVVDVDFTGAAVTFVDTLAFPALGIIFSVLVFLGLTRPRVRANEDGVEVRNFIGTRFYPWVVIYGMSFPKGSSVARLELPDFEFVPMWAFQSRDGEDVVRAVATFRDLENKYMPED.

[0021] In one embodiment, the aforementioned protein variants of the present disclosure may consist essentially of the following amino acid sequence: wherein the amino acid at position 75 from the N-terminus in the amino acid sequence corresponding to SEQ ID NO: 105 is substituted with an amino acid different from the original amino acid; or consist essentially of the following amino acid sequence: wherein the amino acid at position 35 from the N-terminus in the amino acid sequence corresponding to SEQ ID NO: 106 is substituted with an amino acid different from the original amino acid. In another embodiment, the aforementioned protein variants of the present disclosure may consist of the following amino acid sequence: wherein the amino acid at position 75 from the N-terminus in the amino acid sequence corresponding to SEQ ID NO: 105 is substituted with an amino acid different from the original amino acid; or consist of the following amino acid sequence: wherein the amino acid at position 35 from the N-terminus in the amino acid sequence corresponding to SEQ ID NO: 106 is substituted with an amino acid different from the original amino acid.

[0022] In the present disclosure, the amino acid sequence of SEQ ID NO: 105 may be the amino acid sequence of FruR (transcriptional regulator of carbohydrate metabolism) protein. In one embodiment, the protein may be a protein encoded by the NCgl1859 gene.

[0023] In the present disclosure, the amino acid sequence of SEQ ID NO: 106 may be the amino acid sequence of a protein encoded by the NCg11531 gene.

[0024] In one embodiment, the 75th amino acid from the N-terminus in the amino acid sequence corresponding to SEQ ID NO: 105 may be glycine (Gly, G), but is not limited thereto.

[0025] In one embodiment, the 35th amino acid from the N-terminus in the amino acid sequence corresponding to SEQ ID NO: 106 may be glutamic acid (Glu, E), but is not limited thereto.

[0026] In one embodiment, the amino acid at position 75 from the N-terminus in the amino acid sequence corresponding to SEQ ID NO: 105 may be substituted with valine (Val, V), but is not limited thereto.

[0027] In one embodiment, the 35th amino acid from the N-terminus in the amino acid sequence corresponding to SEQ ID NO: 106 may be substituted with lysine (Lys, K), but is not limited thereto.

[0028] The protein variants of the present disclosure may have an activity of increasing L-lysine-producing capacity compared to the wild-type protein (polypeptide).

[0029] The protein variant of the present disclosure may comprise an amino acid sequence in which the amino acid corresponding to the 75th amino acid from the N-terminus in the amino acid sequence from SEQ ID NO: 105 is substituted with an amino acid different from the original amino acid (e.g., valine); or an amino acid sequence in which the amino acid corresponding to the 35th amino acid from the N-terminus in the amino acid sequence of SEQ ID NO: 106 is substituted with an amino acid different from the original amino acid (e.g., lysine); or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.7% or more homology or identity with the amino acid sequence. In addition, it is obvious that even variants of amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included in the scope of the present disclosure, as long as the amino acid sequence is an amino acid sequence having such homology or identity and exhibits the efficacy corresponding to the variant of the present disclosure (efficacy of increasing L-lysine production).

[0030] For example, the amino acid sequence has sequence additions or deletions, naturally occurring mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or within the amino acid sequence that do not alter the function of the variant of the present disclosure.

[0031] A "conservative substitution" refers to the replacement of an amino acid with an amino acid having similar structure and / or chemical properties that is different from the original amino acid. Such amino acid substitutions can generally occur based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues. Typically, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0032] In the present disclosure, the term "variant" refers to a polypeptide that is different from the amino acid sequence before the variant mutation due to conservative substitution and / or modification of at least one amino acid, but whose function or property remains unchanged. Such a variant can generally be identified by modifying at least one amino acid in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. In other words, the ability of the variant may be increased or unchanged or decreased compared to the polypeptide before the mutation. In addition, some variants may include variants in which at least one part such as an N-terminal leader sequence or a transmembrane domain is removed. Other variants may include variants in which some parts are removed from the N- and / or C-terminus of the mature protein. The term "variant" may be used interchangeably with terms such as mutant, modification, modified polypeptide, mutant protein, mutation and variant (modification, modified polypeptide, modified protein, mutant, mutein, divergent as English expressions), and even if it is a term used as a mutation meaning, it is not limited thereto.

[0033] 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 associated with co-translational or post-translational translocation of the protein may be conjugated to the N-terminus. In addition, variants may be conjugated to other sequences or linkers for identification, purification or synthesis.

[0034] In one embodiment, the aforementioned protein variants disclosed herein may have 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more, but less than 100% homology or identity with the amino acid sequence of SEQ ID NO: 105, or may have 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more, but less than 100% homology or identity with the amino acid sequence of SEQ ID NO: 106, but are not limited thereto. In other words, the aforementioned protein variants disclosed herein may have 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more, but less than 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 105 or SEQ ID NO: 106, respectively, but are not limited thereto.

[0035] Another aspect of the present disclosure provides a protein variant comprising at least one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 3 to SEQ ID NO: 5, SEQ ID NO: 7 to SEQ ID NO: 16, and SEQ ID NO: 18. In one embodiment, the protein variant may be a protein variant comprising the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15. In another embodiment, the protein variant may be a protein variant comprising the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15. In another embodiment, the protein variant may consist essentially of the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15. In another embodiment, the protein variant may consist of the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15.

[0036] The protein variant of the present disclosure may comprise any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 to SEQ ID NO: 5, SEQ ID NO: 7 to SEQ ID NO: 16 and SEQ ID NO: 18, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity with the amino acid sequence. In addition, it is obvious that even variants having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included in the scope of the present disclosure, as long as the amino acid sequence is an amino acid sequence having such homology or identity and exhibits the efficacy corresponding to the variant of the present disclosure (efficacy of increasing L-lysine production).

[0037] In the present disclosure, the amino acid sequence of SEQ ID NO: 1 may be a sequence of a protein variant in which the 184th amino acid of the amino acid sequence of the ABC transporter permease is substituted from asparagine (Asn, N) to isoleucine (Ile, I). The ABC transporter permease may be a protein encoded by the NCg10030 gene.

[0038] In the present disclosure, the amino acid sequence of SEQ ID NO: 3 may be a sequence of a protein variant in which the 86th amino acid of the nth (Endo III-related nuclease) protein is substituted from alanine (Ala, A) to valine (Val, V). The nth protein may be a protein encoded by the NCgl0288 gene.

[0039] In the present disclosure, the amino acid sequence of SEQ ID NO: 4 may be a sequence of a protein variant in which the 363rd amino acid of the wzz (chromosome segregating ATPase) protein is substituted from glutamic acid (Glu, E) to aspartic acid (Asp, D). The wzz protein may be a protein encoded by the NCgl0337 gene.

[0040] In the present disclosure, the amino acid sequence of SEQ ID NO: 5 may be a sequence of a protein variant in which the 210th amino acid of the NusG (transcription anti-termination protein NusG) protein is substituted from asparagine (Asn, N) to aspartic acid (Asp, D). The NusG protein may be a protein encoded by the NCgl0458 gene.

[0041] In the present disclosure, the amino acid sequence of SEQ ID NO: 7 may be a sequence of a protein variant in which the 23rd amino acid of the protein (hypothetical protein) encoded by the NCgl0686 gene is substituted from valine (Val, V) to alanine (Ala, A).

[0042] In the present disclosure, the amino acid sequence of SEQ ID NO: 8 may be a sequence of a protein variant, in which the 1112th amino acid of the type II restriction enzyme, methylase subunit protein is substituted from leucine (Leu, L) to phenylalanine (Phe, F). The type II restriction enzyme, methylase subunit protein may be a protein encoded by the NCgl0706 gene.

[0043] In the present disclosure, the amino acid sequence of SEQ ID NO: 9 may be a sequence of a protein variant in which the 101st amino acid of the mctC (Na+ / proline, Na+ / pantothenate symporter or related permease) protein is substituted from tryptophan (Trp, W) to arginine (Arg, R). The mctC protein may be a protein encoded by the NCg10799 gene.

[0044] In the present disclosure, the amino acid sequence of SEQ ID NO: 10 may be a sequence of a protein variant, in which the 461st amino acid of the atpD (ATP synthase F0F1 subunit β) protein is substituted from aspartic acid (Asp, D) to glutamic acid (Glu, E). The atpD protein may be a protein encoded by the NCg11165 gene.

[0045] In the present disclosure, the amino acid sequence of SEQ ID NO: 11 may be a sequence of a protein variant in which the amino acid 456 of the leuC (isopropylmalate isomerase large subunit) protein is substituted from glycine (Gly, G) to aspartic acid (Asp, D). The leuC protein may be a protein encoded by the NCgl1262 gene.

[0046] In the present disclosure, the amino acid sequence of SEQ ID NO: 12 may be a sequence of a protein variant in which the 697th amino acid of the helicase protein is substituted from histidine (His, H) to tyrosine (Tyr, Y). The helicase protein may be a protein encoded by the NCgl1432 gene.

[0047] In the present disclosure, the amino acid sequence of SEQ ID NO: 13 may be the sequence of a protein variant in which the 35th amino acid of the protein (hypothetical protein) encoded by the NCg11531 gene is substituted from glutamic acid (Glu, E) to lysine (Lys, K).

[0048] In the present disclosure, the amino acid sequence of SEQ ID NO: 14 may be a sequence of a protein variant in which the 81st amino acid of the protein encoded by the NCgl1588 gene (hypothetical protein) is substituted from valine (Val, V) to isoleucine (Ile, I).

[0049] In the present disclosure, the amino acid sequence of SEQ ID NO: 15 may be a sequence of a protein variant in which the 75th amino acid of the fruR (transcriptional regulator of sugar metabolism) protein is substituted from glycine (Gly, G) to valine (Val, V). The fruR may be a protein encoded by the NCgl1859 gene.

[0050] In the present disclosure, the amino acid sequence of SEQ ID NO: 16 may be a sequence of a protein variant in which the 164th amino acid of the miaB ((dimethylallyl)adenosine tRNA methylsulfide transferase) protein is substituted from serine (Ser, S) to proline (Pro, P). The miaB protein may be a protein encoded by the NCgl1874 gene.

[0051] In the present disclosure, the amino acid sequence of SEQ ID NO:18 can be the sequence of a protein variant of the protein (hypothetical protein) encoded by NCgl2356, in which sequence, the 456th amino acid of the protein is substituted from valine (Val, V) to alanine (Ala, A), the 464th amino acid of the protein is substituted from serine (Ser, S) to proline (Pro, P), the 465th amino acid of the protein is substituted from alanine (Ala, A) to valine (Val, V), the 466th amino acid of the protein is substituted from serine (Ser, S) to glycine (Gly, G), the 496th amino acid of the protein is substituted from glutamic acid (Glu, E) to threonine (Thr, T), and the 499th amino acid of the protein is substituted from serine (Ser, S) to aspartic acid (Asp, D).

[0052] The protein variant of the present disclosure may comprise any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 13 or SEQ ID NO: 15 (e.g., the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15): the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3 to SEQ ID NO: 5, SEQ ID NO: 7 to SEQ ID NO: 16 and SEQ ID NO: 18, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity with the amino acid sequence. In addition, it is obvious that even if some sequences are deleted, modified, substituted, conservatively substituted or added in the amino acid sequence possessed by the variant, the variant is still included in the scope of the present disclosure, as long as the amino acid sequence is an amino acid sequence having such homology or identity and exhibits the efficacy (efficacy of increasing L-lysine production) corresponding to the variant of the present disclosure.

[0053] For example, the amino acid sequence may have sequence additions or deletions, naturally occurring mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or within the amino acid sequence that do not alter the function of the variant of the present disclosure.

[0054] In protein variants, conservative substitutions, variations, etc. are as described above.

[0055] In the present disclosure, the term "homology" or "identity" refers to the degree of similarity between two given amino acid sequences or nucleotide sequences, and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0056] The sequence homology or identity of conservative polynucleotides or polypeptides can be determined by standard comparison algorithms, and can be used together with the default gap penalty set up by the program used. Basically, sequences of homology or identity can hybridize with the entire or part of the sequence under medium or high stringency conditions usually. Obviously, hybridization also includes polynucleotides and polynucleotide hybridization containing common codons or codons that take into account codon degeneracy.

[0057] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using a known computer algorithm, such as the "FASTA" program using default parameters, such as the "FASTA" program using default parameters in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Otherwise, the Needlean-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented 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) (GCG program package (including 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 et al.] (1988) SIAM J Applied Math 48:1073). For example, BLAST or ClustalW of the National Database Center of Biotechnology Information can be used to determine homology, similarity or identity.

[0058] Homology, similarity or identity of polynucleotides or polypeptides can be determined, for example, as known in Smith and Waterman, Adv. Appl. Math (1981) 2: 482, by comparing sequence information using, for example, a GAP computer program such as Needleman et al. (1970), J Mol Biol. 48: 443. In summary, the GAP program can be defined as the value obtained by dividing the total number of symbols in the shorter of the two sequences by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program may include (1) a binary comparison matrix (including identical values ​​of 1 and different values ​​of 0) and a weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix), disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and a penalty of 0.10 for each additional symbol for each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0059] As an example of the present disclosure, the variant of the present disclosure may have an activity of increasing L-lysine production capacity compared to a wild-type polypeptide.

[0060] In this disclosure, the term "corresponding to" refers to an amino acid residue at a listed position in a polypeptide, or an amino acid residue that is similar, identical, or homologous to a listed residue in a polypeptide. Identifying the amino acid at a corresponding position can determine the specific amino acid in a sequence with reference to a specific sequence. "Corresponding region" as used in this disclosure generally refers to a similar or corresponding position in a related protein or a reference protein.

[0061] For example, any amino acid sequence can be aligned with the following amino acid sequence, i.e., in the amino acid sequence, the amino acid at position 75 from the N-terminus in the amino acid sequence corresponding to SEQ ID NO: 105 is substituted with an amino acid different from the original amino acid (e.g., valine), or in the amino acid sequence, the amino acid at position 35 from the N-terminus in the amino acid sequence corresponding to SEQ ID NO: 106 is substituted with an amino acid different from the original amino acid (e.g., lysine); or the amino acid sequence is any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 to SEQ ID NO: 5, SEQ ID NO: 7 to SEQ ID NO: 16, and SEQ ID NO: 18 (e.g., the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15), and based on this, each amino acid residue in the amino acid sequence can be numbered with reference to the numbering position of the amino acid residue in the amino acid sequence in which the amino acid at position 75 from the N-terminus in the amino acid sequence corresponding to SEQ ID NO: 105 is substituted with an amino acid different from the original amino acid (e.g., valine); or with reference to the amino acid sequence corresponding to SEQ ID NO: 106. Each amino acid residue in the amino acid sequence is numbered by referring to the numbering position of the amino acid residue in the amino acid sequence in which the amino acid at position 35 from the N-terminus is substituted by an amino acid different from the original amino acid (e.g., lysine) in the amino acid sequence of NO: 106; or by referring to the numbering position of the amino acid residue in any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 to SEQ ID NO: 5, SEQ ID NO: 7 to SEQ ID NO: 16, and SEQ ID NO: 18 (e.g., the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15). For example, a sequence alignment algorithm as described in the present disclosure can be compared with a query sequence (also referred to as a "reference sequence") to confirm the position of an amino acid, or the position where a modification (such as a substitution, insertion, or deletion, etc.) occurs.

[0062] For this alignment, for example, the Needlean-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277), etc. can be used, but are not limited to these, and sequence alignment programs, pairwise sequence comparison algorithms, etc. known in the art can be appropriately used.

[0063] Another aspect of the present disclosure provides a polynucleotide encoding a protein variant of the present disclosure.

[0064] In the present disclosure, the term "polynucleotide" refers to a DNA or RNA chain above a certain length, which is a polymer of nucleotides in which nucleotide monomers are linked by covalent bonds into a long chain, and more specifically refers to a polynucleotide fragment encoding the variant.

[0065] In one embodiment, the polynucleotide may be a polynucleotide encoding a protein variant, wherein the amino acid sequence contained in the protein variant corresponds to the amino acid sequence of SEQ ID NO: 105 at the 75th amino acid from the N-terminus and is substituted by an amino acid different from the original amino acid (e.g., valine), or the amino acid sequence contained in the protein variant corresponds to the amino acid sequence of SEQ ID NO: 106 at the 35th amino acid from the N-terminus and is substituted by an amino acid different from the original amino acid (e.g., lysine).

[0066] In one embodiment, the polynucleotide may comprise a polynucleotide comprising any one nucleotide sequence selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 25 to SEQ ID NO: 27, SEQ ID NO: 29 to SEQ ID NO: 38 and SEQ ID NO: 40, for example, the nucleotide sequence of SEQ ID NO: 37.

[0067] The polynucleotide encoding the protein variant of the present disclosure may have or include any one of the nucleotide sequences selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 25 to SEQ ID NO: 27, SEQ ID NO: 29 to SEQ ID NO: 38 and SEQ ID NO: 40, for example, the nucleotide sequence of SEQ ID NO: 37. In another embodiment, the polynucleotide may include a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity with the nucleotide sequence. In addition, it is obvious that even if some sequences are deleted, modified, substituted, conservatively substituted or added in the nucleotide sequence included in the polynucleotide, the polynucleotide is also included in the scope of the present disclosure, as long as the polynucleotide is a sequence having such homology or identity and exhibits the efficacy corresponding to the variant of the present disclosure (efficacy of increasing L-lysine production).

[0068] In one embodiment, the nucleotide sequence or amino acid sequence provided in this specification may include a nucleotide sequence or amino acid sequence modified by a conventional mutagenesis method (e.g., direct evolution and / or site-directed mutagenesis, etc.) within the scope of maintaining its original function or desired function. In one embodiment, a polynucleotide or polypeptide "comprising a specific nucleotide or amino acid sequence" may refer to a polynucleotide or polypeptide (i) consisting of a specific nucleotide sequence or amino acid sequence, or substantially comprising the specific nucleotide sequence or amino acid sequence, or (ii) consisting of an amino acid sequence having a homology of more than 60%, more than 70%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, more than 99.5% or more than 99.9% with the specific nucleotide sequence or amino acid sequence, or substantially comprising the specific nucleotide sequence or amino acid sequence and maintaining the original function and / or desired function.

[0069] Taking into account the degeneracy of the code or the codons preferred in the organism expressing the variant of the present disclosure, the polynucleotides of the present disclosure may have various modifications in the coding region without changing the amino acid sequence of the variant of the present disclosure. Specifically, the polynucleotides of the present disclosure may have or contain a nucleotide sequence having 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, and less than 100% homology or identity with the nucleotide sequence of a polynucleotide encoding a protein variant, wherein the protein variant comprises: an amino acid sequence in which the amino acid at position 75 from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 105 is substituted by an amino acid different from the original amino acid (e.g., valine), or an amino acid sequence in which the amino acid at position 35 from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 106 is substituted by an amino acid different from the original amino acid (e.g., lysine), or any one nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO: 23, SEQ ID NO: 25 to SEQ ID NO: 27, SEQ ID NO: 29 to SEQ ID NO: 38, and SEQ ID NO: 40 (e.g., SEQ ID NO: 51 to SEQ ID NO: 52). NO:37), or consisting of or consisting essentially of a nucleotide sequence having more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% and less than 100% homology or identity with the nucleotide sequence, but is not limited thereto.

[0070] In addition, the polynucleotides of the present invention may include probes that can be prepared from known gene sequences, such as any sequence that can hybridize with all or part of the complementary sequence of the polynucleotide sequence of the present invention under stringent conditions, without limitation. "Stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. For example, hybridization with polynucleotides with high homology or identity (polynucleotides with homology or identity of more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99%), and conditions that do not hybridize with polynucleotides with homology or identity lower than it, or refers to conditions corresponding to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS salt concentration and temperature (which are common Southern hybridization washing conditions) washing once, specifically washing twice to three times.

[0071] Hybridization requires that the two nucleotides have complementary sequences, but mismatches between bases can be allowed depending on the stringency of the hybridization. The term "complementary" can be used to describe the relationship between nucleotide bases that can hybridize to each other. For example, in the case of DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of the present disclosure may also include not only substantially similar nucleotide sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0072] Specifically, polynucleotides having homology or identity with the polynucleotides disclosed herein can be detected using the above conditions using hybridization conditions including a hybridization step at a Tm value of 55° C. In addition, the Tm value may be 60° C., 63° C. or 65° C., but is not limited thereto, and can be appropriately adjusted by those skilled in the art according to the purpose.

[0073] The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementation, and variables are well known in the art.

[0074] Another aspect of the present disclosure provides a vector comprising the polynucleotide of the present disclosure. The vector may be an expression vector for expressing the polynucleotide in a host cell, but is not limited thereto.

[0075] The vector of the present disclosure may comprise a DNA product comprising a nucleotide sequence of a polynucleotide encoding a target polypeptide, which is operably linked to an expression control region (or expression control sequence) suitable for expressing the target polypeptide in a suitable host. The expression control region may comprise a promoter that can initiate transcription, any operator sequence that regulates such transcription, a sequence encoding a suitable mRNA ribosome binding site, and / or a nucleic acid sequence that regulates transcription and translation termination. The vector may be transformed into a suitable host cell, and then replicate or function independently of the host genome, and be integrated into the genome itself.

[0076] The vector used in the present disclosure is not particularly limited, and any vector known in the art can be used. Examples of common vectors may include plasmids, cosmids, viruses and phages in a natural state or a recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A and Charon21A etc. can be used as phage vectors or cosmid vectors, and pDZ-based, pBR-based, pUC-based, pBluescript II-based, pGEM-based, pTZ-based, pCL-based and pET-based vectors can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC or pDC24 vectors etc. can be used.

[0077] As an example, the polynucleotide encoding the target polypeptide can be inserted into the chromosome by a vector for inserting the chromosome into the cell. The polynucleotide insertion into the chromosome can be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. A selection marker may be further included to confirm whether the chromosome is inserted. The selection marker is used to select the cells transformed by the vector, that is, to confirm whether the target nucleic acid molecule is inserted, and a marker that confers the expression of a selectable phenotype such as drug resistance, auxotrophy, cytotoxic agent resistance or surface protein can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or show other phenotypes, so transformed cells can be selected.

[0078] In the present disclosure, the term "transformation" refers to the introduction of a vector comprising a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide is expressed in the host cell. As long as the converted polynucleotides can be expressed in the host cell, they can be included, regardless of whether they are inserted into the chromosome of the host cell or are located outside the chromosome. In addition, the polynucleotides include DNA and / or RNA encoding the target polypeptide. The polynucleotides can be introduced in any form, as long as they can be introduced into the host cell and expressed. For example, the polynucleotides can be introduced into the host cell in the form of an expression cassette, which is a gene structure comprising all elements required for self-expression. The expression cassette can generally include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal that are operably connected to the polynucleotides. The expression cassette can be in the form of an expression vector that can replicate itself. In addition, the polynucleotides can be introduced into the host cell in the form of themselves and can be operably connected to the sequence required for expression in the host cell, but are not limited thereto.

[0079] Furthermore, in the above description, the term "operably linked" means that a polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a target variant of the present disclosure.

[0080] Another aspect of the present invention provides a Corynebacterium microorganism comprising at least one selected from the group consisting of the protein variants disclosed herein and the polynucleotides disclosed herein.

[0081] The microorganism of the present disclosure may comprise the protein variant of the present disclosure, a polynucleotide encoding the protein variant, or a vector of the present disclosure comprising the polynucleotide.

[0082] In the present disclosure, the term "microorganism (or strain)" includes all wild-type microorganisms, naturally or artificially genetically modified microorganisms, and may be microorganisms in which a specific mechanism is weakened or strengthened due to reasons such as the insertion of exogenous genes or the enhancement or inactivation of the activity of endogenous genes, and the microorganism is a genetically modified microorganism for producing a target polypeptide, protein or product.

[0083] The microorganism of the present disclosure may be a microorganism comprising any one or more of the protein variant of the present disclosure, the polynucleotide of the present disclosure, and a vector comprising the polynucleotide of the present disclosure; a microorganism modified to express the variant of the present disclosure or the polynucleotide of the present disclosure; a microorganism (e.g., a recombinant microorganism) expressing the variant of the present disclosure or the polynucleotide of the present disclosure; or a microorganism (e.g., a recombinant microorganism) having the activity of the variant of the present disclosure, but is not limited thereto.

[0084] The microorganism of the present disclosure may be a microorganism having an L-lysine-producing ability.

[0085] The microorganism of the present disclosure may be a microorganism naturally having the ability to produce L-lysine, or a microorganism in which a variant of the present disclosure or a polynucleotide encoding the variant (or a vector comprising the polynucleotide) is introduced into a parent strain that does not have the ability to produce L-lysine and / or imparts the ability to produce L-lysine to the parent strain, but is not limited thereto.

[0086] As an example, the microorganism of the present disclosure is a cell or microorganism expressing a protein variant of the present disclosure, which is transformed with a vector comprising a polynucleotide of the present disclosure or a polynucleotide encoding a variant of the present disclosure, and for the purpose of the present disclosure, the microorganism of the present disclosure may include all microorganisms capable of producing L-Lysine by comprising a variant of the present disclosure. For example, the microorganism of the present disclosure may be a natural wild-type microorganism or a recombinant microorganism that improves L-Lysine production capacity by introducing a polynucleotide encoding a variant of the present disclosure into a microorganism producing L-Lysine.

[0087] In a specific embodiment, the microorganism of the present invention may have an increased L-lysine production capacity compared to a Corynebacterium microorganism that does not contain any one of the aforementioned protein variants and polynucleotides encoding the protein variants selected from the present invention (i.e., a microorganism that does not express any one of the protein variants comprising the following amino acid sequences: the amino acid sequence corresponding to SEQ ID NO: 105 in which the 75th amino acid from the N-terminus is substituted by an amino acid different from the original amino acid (e.g., valine), or the amino acid sequence corresponding to SEQ ID NO: 106 in which the 35th amino acid from the N-terminus is substituted by an amino acid different from the original amino acid (e.g., lysine); and a microorganism that does not express any one of the protein variants comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 to SEQ ID NO: 5, SEQ ID NO: 7 to SEQ ID NO: 16 and SEQ ID NO: 18, for example, an amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15; or a microorganism that expresses a wild-type protein corresponding to the protein variant).

[0088] The Corynebacterium microorganism that does not comprise any one of the group consisting of the aforementioned protein variants and polynucleotides encoding protein variants selected from the disclosure can be a natural wild-type microorganism or an unmodified microorganism, and can also be expressed as a host cell or a parent strain. The Corynebacterium microorganism that does not comprise any one of the group consisting of the aforementioned protein variants and polynucleotides encoding protein variants selected from the disclosure can be specifically ATCC13032 strain and / or CJ3P (US 9556463 ​​B2), but is not limited thereto.

[0089] The recombinant microorganism of the L-Lysine productivity with increase disclosed herein can be compared with natural wild-type microorganism or unmodified microorganism, the microorganism with the L-Lysine productivity of increase, but is not limited to this.As an example, the unmodified microorganism as the target strain for comparing the increase of L-Lysine productivity can be ATCC13032 bacterial strain and / or CJ3P (US 9556463 ​​B2), but is not limited to this.

[0090] In one embodiment, the microorganism with increased L-Lysine productivity (production volume) disclosed herein can increase the L-Lysine productivity (production volume) by about 1% or more, about 3% or more, about 5% or more, about 7.5% or more, about 10% or more, about 12.5% ​​or more, about 15% or more, about 15.25% or more (the upper limit is not particularly limited, and for example, it can be about 200% or less, about 150% or less, about 100% or less, or about 50% or less, about 30% or less) compared to the L-Lysine productivity of the parent strain or unmodified microorganism before mutation, but is not limited thereto. In another embodiment, compared with the L-Lysine productivity of the parent strain or unmodified microorganism before mutation, the microorganism with increased L-Lysine productivity (production volume) of the present disclosure can increase the L-Lysine productivity (production volume) by about 1.01 times or more, about 1.03 times or more, about 1.05 times or more, about 1.075 times or more, about 1.1 times or more, about 1.125 times or more, about 1.15 times or more, about 1.1525 times or more (the upper limit is not particularly limited, and for example, it can be about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less), but is not limited thereto.

[0091] The term "about" includes all ranges of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values ​​equal to or similar to the value appearing after the term "about", but is not limited thereto.

[0092] In the present invention, the term "unmodified microorganism" does not exclude microorganisms comprising mutations that can occur naturally in microorganisms, and may refer to wild-type microorganisms or natural microorganisms themselves, or to microorganisms before the properties are changed due to the genetic mutations caused by natural or artificial factors. For example, an unmodified microorganism may refer to a microorganism before the protein variants described in this specification are not introduced or introduced." unmodified microorganism" may be used interchangeably with "strain before modification", "microorganism before modification", "unmutated strain", "unmodified strain", "unmutated microorganism" or "reference microorganism".

[0093] In one embodiment, the microorganism of the present disclosure can be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium spp. flavescens), more specifically, it may be Corynebacterium glutamicum.

[0094] In the present disclosure, the term "attenuation" of a polypeptide is a concept including both reduced activity or no activity compared to endogenous activity. Attenuation can be used interchangeably with terms such as inactivation, deletion, downregulation, reduction, decrease, attenuation, etc.

[0095] Weakening can also include: the situation where the activity of the polypeptide itself is reduced or eliminated due to mutations in the polynucleotide encoding the polypeptide, etc., compared with the activity of the polypeptide possessed by the original microorganism; the situation where the overall polypeptide activity level and / or concentration (expression level) in the cell is lower than that of the natural strain due to the inhibition of expression of the gene encoding the polynucleotide or the inhibition of translation into the polypeptide; the situation where the polynucleotide is not expressed at all; and / or the situation where there is no polypeptide activity even if the polynucleotide is expressed. "Endogenous activity" refers to the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before the trait is changed when the trait is changed due to genetic mutations caused by natural or artificial factors. Endogenous activity can be used interchangeably with "activity before modification". Compared with endogenous activity, the activity of a polypeptide is "inactivated, missing, reduced, downregulated, reduced or attenuated" means that the polypeptide activity is reduced compared with the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before the trait is changed.

[0096] The weakening of the activity of this polypeptide can be carried out by any method known in the art, but is not limited thereto, and can be achieved by applying various methods well known in the art (e.g., 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.).

[0097] Specifically, the attenuation of the polypeptide may be:

[0098] 1) Deletion of all or part of the gene encoding the polypeptide;

[0099] 2) modifying the expression regulatory region (or expression regulatory sequence) to reduce the expression of the gene encoding the polypeptide;

[0100] 3) modifying (e.g., deletion / substitution / addition of at least one amino acid in the amino acid sequence) the amino acid sequence constituting the polypeptide to eliminate or weaken the activity of the polypeptide;

[0101] 4) modifying (e.g., adding / replacing / adding at least one nucleic acid base in the nucleic acid base sequence of a polypeptide gene encoding a polypeptide modified to eliminate or weaken the activity of the polypeptide) a gene sequence encoding a polypeptide to eliminate or weaken the activity of the polypeptide;

[0102] 5) modifying the base sequence of the start codon or 5'-UTR region of the gene transcriptome encoding the polypeptide;

[0103] 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcriptome of the gene encoding the polypeptide;

[0104] 7) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding the polypeptide to form a secondary structure that cannot bind to the ribosome;

[0105] 8) adding a promoter that transcribes in the opposite direction to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE); or

[0106] 9) Regulate the cellular localization of proteins (peptides); or

[0107] 10) A combination of at least two selected from 1) to 9), but not particularly limited thereto.

[0108] For example,

[0109] The above 1) deletion of part or all of the gene encoding the polypeptide may be performed by removing the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, replacing it with a polypeptide lacking some nucleotides, or replacing it with a marker gene.

[0110] In addition, the above 2) modification of the expression control region (or expression control sequence) may be a mutation of the expression control region (or expression control sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or replacement with a sequence having weaker activity. The expression control region contains a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating transcription and translation termination, but is not limited thereto.

[0111] Furthermore, the above 3) modification of the base sequence of the start codon or 5'-UTR region of the gene transcriptome encoding the polypeptide may be, for example, replacement with a base sequence encoding another start codon having a lower polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0112] In addition, the modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above may be mutation in the sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof, 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 not limited thereto. For example, by introducing a mutation into a polynucleotide sequence to form a stop codon, the expression of a gene may be inhibited or weakened, but not limited thereto.

[0113] Regarding the above 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcriptome of a gene encoding a polypeptide, reference can be made to, for example, the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews-Trends in Genetics, Vol. 1(1) 1986].

[0114] The above 7) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding the polypeptide to form a secondary structure that cannot bind to the ribosome can make the mRNA unable to be translated or inhibit the mRNA translation rate.

[0115] The above 8) adding a promoter transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE) can produce antisense nucleotides complementary to the transcriptome of the gene encoding the polypeptide to weaken the activity.

[0116] The above-mentioned 9) regulation of the cellular localization of proteins (polypeptides) can target proteins (polypeptides) to specific organelles or specific intracellular spaces in cells. For example, by adding or removing a leader sequence that plays a role in targeting proteins (polypeptides), they can be targeted to the periplasm or cytoplasm, but are not limited thereto.

[0117] Such attenuation of polypeptide activity can be based on the activity or concentration of the expressed polypeptide in the wild type or pre-transformed microbial strain, attenuating the activity or concentration expression level of the corresponding polypeptide, or increasing the amount of the product produced by the corresponding polypeptide, but is not limited thereto.

[0118] In the present disclosure, the term "enhancement" of polypeptide activity refers to an increase in the activity of a polypeptide compared to an endogenous activity. Enhancement can be used interchangeably with terms such as activation, upregulation, overexpression, increase, etc. Herein, activation, upregulation, overexpression and increase can include an activity that does not exist originally, or an activity that is improved compared to an endogenous activity or the activity before modification. "Endogenous activity" refers to the activity of a specific polypeptide that a parent strain or an unmodified microorganism originally had before a property change when a property change is caused by a genetic mutation caused by natural or artificial factors. This can be used interchangeably with "activity before modification". Compared to an endogenous activity, the activity "enhancement", "upregulation", "overexpression" or "increase" of a polypeptide refers to an activity and / or concentration (expression level) of a specific polypeptide that a parent strain or an unmodified microorganism originally had before a property change is compared to improve the activity of a polypeptide.

[0119] Enhancement can be achieved by introducing exogenous polypeptides or enhancing the activity and / or concentration (expression level) of endogenous polypeptides. The activity of the polypeptide can be confirmed by an increase in the activity degree, expression level or amount of product released from the corresponding polypeptide.

[0120] For the enhancement of polypeptide activity, various methods well known in the art can be applied, and there is no restriction, as long as the activity of the target polypeptide can be enhanced compared with the activity of the microorganism before modification. Specifically, genetic engineering and / or protein engineering well known to those skilled in the art can be used, which are conventional methods of molecular biology, but are not limited thereto (e.g., Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2.1-16, Sambrook et al., Molecular Cloning 2012, etc.).

[0121] Specifically, the enhancement of the polypeptide of the present disclosure can be:

[0122] 1) The cell copy number of the polynucleotide encoding the polypeptide increases;

[0123] 2) Replace the gene expression regulatory region on the chromosome encoding the polypeptide with a sequence with strong activity;

[0124] 3) modifying the base sequence of the start codon or 5'-UTR region of the gene transcriptome encoding the polypeptide;

[0125] 4) Modify the amino acid sequence of the polypeptide to enhance the activity of the polypeptide;

[0126] 5) modifying a polynucleotide sequence encoding a polypeptide to enhance the activity of the polypeptide (e.g., modifying a polynucleotide of a polypeptide gene to encode a polypeptide modified to enhance the activity of the polypeptide);

[0127] 6) introducing an exogenous polypeptide exhibiting polypeptide activity or an exogenous polynucleotide encoding a polypeptide;

[0128] 7) codon-optimized polynucleotides encoding polypeptides;

[0129] 8) Modification or chemical modification by analyzing the tertiary structure of the polypeptide and selecting the exposed sites;

[0130] 9) Regulate the cellular localization of proteins (peptides); or

[0131] 10) A combination of at least two selected from the above 1) to 9), but not limited thereto.

[0132] The increase in the number of copies of the polynucleotide encoding the polypeptide in the cell mentioned above 1) can be achieved by introducing a vector, which can replicate and function independently of the host, wherein the polynucleotide encoding the corresponding polypeptide is operably connected. In addition, it can be achieved by introducing one copy or two or more copies of the polynucleotide encoding the corresponding polypeptide into the chromosome in the host cell. The introduction into the chromosome can be carried out by introducing a vector capable of inserting the polynucleotide into the chromosome of the host cell, but is not limited thereto. The vector is as described above.

[0133] The above 2) replaces the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide with a sequence having strong activity, which can be, for example, a sequence mutation caused by deletion, insertion, non-conservative or conservative substitution or a combination thereof, to further enhance the activity of the expression control region, or replace it with a sequence having 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, a sequence regulating transcription and translation termination, etc. As an example, the original promoter can be replaced with a strong promoter, but is not limited thereto.

[0134] Examples of known strong promoters include CJ1 to CJ7 promoters (U.S. Patent No. 7,662,943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (U.S. Patent No. 1,058,4338 B2), O2 promoter (U.S. Patent No. 1,027,3491 B2), tkt promoter, ycA promoter, etc., but are not limited to these.

[0135] The above 3) modification of the base sequence of the start codon or 5'-UTR region of the gene transcriptome encoding the polypeptide may be, for example, replacement with a base sequence encoding another start codon having a higher polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0136] The modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above can be mutation in the sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof, or replacement with an amino acid sequence or polynucleotide sequence that is improved to have stronger activity on the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, thereby enhancing the activity of the polypeptide, but is not limited thereto. The polynucleotide can be specifically inserted into the chromosome for replacement by homologous recombination, but is not limited thereto. The vector used then can also include a selection marker for confirming chromosome insertion. The selection marker is as described above.

[0137] The above 6) introduction of an exogenous polypeptide showing polypeptide activity can be the introduction of an exogenous polynucleotide encoding a polypeptide showing the same / similar activity as the polypeptide into a host cell. The source or sequence of the exogenous polynucleotide is not limited, as long as it shows the same / similar activity as the polypeptide. The method for introduction can be carried out by those skilled in the art by appropriately selecting a known transformation method, and by expressing the introduced polynucleotide in a host cell, a polypeptide can be produced and its activity can be increased.

[0138] The above-mentioned 7) codon optimization polynucleotide encoding a polypeptide may be codon optimized so as to increase the transcription or translation of an endogenous polynucleotide in a host cell, or codon optimized so as to optimize the transcription and translation of an exogenous polynucleotide in a host cell.

[0139] The above 8) modification or chemical modification by analyzing the tertiary structure of the polypeptide and selecting the exposed site can be carried out by comparing the sequence information of the polypeptide to be analyzed, determining the template protein candidate according to the degree of sequence similarity, determining the structure based on this and selecting the exposed site to be modified or chemically modified, and transforming or modifying it.

[0140] The above 9) regulation of the cellular localization of proteins (polypeptides) can target proteins (polypeptides) to specific organelles or specific intracellular spaces in cells. For example, by adding or removing a leader sequence that plays a role in targeting proteins (polypeptides), they can be targeted to the periplasm or cytoplasm, but are not limited thereto.

[0141] Such enhancement of polypeptide activity refers to increasing the activity or concentration expression level of the corresponding polypeptide based on the activity or concentration of the polypeptide expressed in the wild type or pre-transformed microbial strain, or increasing the amount of the product produced by the corresponding polypeptide, but is not limited thereto.

[0142] In the microorganisms disclosed herein, partial or complete modification of a polypeptide (e.g., modification encoding the above-mentioned protein variant) can be induced by (a) homologous recombination using a vector for insertion into a chromosome in a microorganism or genome editing using gene scissors (engineered nucleases, e.g., CRISPR-Cas9) and / or (b) treatment with light such as ultraviolet rays and radioactive rays and / or chemicals, but not limited thereto. Methods for modifying part or all of a gene may include methods of DNA recombination technology. For example, partial or complete deletion of a gene may be achieved by injecting a nucleotide sequence or vector comprising a nucleotide sequence homologous to a target gene into a microorganism to cause homologous recombination. The injected nucleotide sequence or vector may include a dominant selection marker, but not limited thereto.

[0143] In the microorganism of the present disclosure, the protein variant, polynucleotide, L-lysine, etc. are as described in other aspects above.

[0144] Other aspects of the present disclosure provide a method for producing L-lysine, comprising culturing a microorganism of the genus Corynebacterium including at least one selected from the group consisting of the protein variant of the present disclosure and a polynucleotide encoding the protein variant in a culture medium.

[0145] The method for producing L-lysine of the present disclosure may include culturing a microorganism of the genus Corynebacterium containing the protein variant of the present disclosure, the polynucleotide of the present disclosure, or the vector of the present disclosure in a culture medium.

[0146] In the present disclosure, "culture" refers to the growth of the coryneform microorganisms of the present disclosure under appropriately adjusted environmental conditions. The culture process of the present disclosure can be carried out according to appropriate culture media and culture conditions known in the art. Depending on the selected strain, those skilled in the art can easily adjust and use the culture process. Specifically, the culture can be batch, continuous and / or batch fed, but is not limited thereto.

[0147] In the present invention, the term "culture medium" refers to a material that mixes the nutrients required for culturing Corynebacterium microorganisms as main components, and supplies nutrients and growth factors, etc., including water that is essential for survival and growth. Specifically, a culture medium and other culture conditions for culturing Corynebacterium microorganisms of the present disclosure can be used, as long as it is a culture medium for culturing common microorganisms without particular limitation, but Corynebacterium microorganisms of the present disclosure can be cultured in a common culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and / or vitamin, etc., by adjusting temperature, pH, etc. under aerobic conditions.

[0148] Specifically, the culture medium of Corynebacterium microorganisms can be found in the document ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D.C., USA, 1981)].

[0149] In the present disclosure, as a carbon source, it can include: carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrates, molasses, blackstrap molasses, rice bran, cassava, sugarcane residues and corn steep liquor can be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted with reducing sugars) can be used, and other carbon sources of appropriate amounts can be used differently without limitation. These carbon sources can be used alone or in combination of at least two, but are not limited thereto.

[0150] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium citrate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc., organic nitrogen sources such as amino acids including glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrate, fish or its degradation product, defatted soybean cake or its degradation product, etc. can be used alone or in combination of at least two, but are not limited thereto.

[0151] As a phosphorus source, potassium hydrogen phosphate, potassium dihydrogen phosphate or a sodium salt corresponding thereto may be included. As an inorganic compound, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. may be used. In addition to these, amino acids, vitamins and / or appropriate precursors may be included. These components or precursors may be added in batches or in a continuous manner. However, it is not limited thereto.

[0152] In addition, during the cultivation of the Corynebacterium microorganism of the present disclosure by an appropriate method, by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. in the substratum, the pH of the substratum can be regulated. In addition, during the incubation period, defoamers such as fatty acid polyethylene glycol esters can be used to suppress foaming. In addition, in order to maintain the aerobic state of the medium, oxygen or oxygen-containing gas can be injected into the substratum, or in order to keep anaerobic and microaerobic state, gas can not be injected or nitrogen, hydrogen or carbon dioxide can be injected, but is not limited thereto. In one embodiment, the production method of L-lysine can be carried out at pH 6 to pH 9, more specifically, it can be carried out at pH 6 to pH 9, pH 6 to pH 8.5, pH 6 to pH 8, pH 6 to pH 7.5, pH 6 to pH 7.25, pH 6 to pH 7, pH 6.5 to pH 9, pH 6.5 to pH 8.5, pH 6.5 to pH 8, pH 6.5 to pH 7.5, pH 6.5 to pH 7.25, pH 6.5 to pH 7, pH 6.75 to pH 9, pH 6.75 to pH 8.5, pH 6.75 to pH 8, pH 6.75 to pH 7.5, pH 6.75 to pH 7.25, or pH 6.75 to pH 7, but is not limited thereto.

[0153] The culture temperature in the culture of the present disclosure can be maintained at 20°C-45°C, specifically, at 25°C-40°C, 25°C-40°C, 25°C-37°C, 25°C-35°C, 27°C-40°C, 27°C-37°C, 27°C-35°C, 30°C-40°C, 30°C-37°C or 30°C-35°C, but not limited thereto. In the culture of the present disclosure, the culture time can be about 10-160 hours, but not limited thereto.

[0154] L-lysine produced by the culture of the present disclosure may be released into the medium or retained in the cells.

[0155] The method for producing L-lysine of the present disclosure may further include, for example, preparing the Corynebacterium microorganism of the present disclosure, preparing a culture medium for culturing the strain, or a combination thereof (in any order) before culturing.

[0156] The method for producing L-lysine of the present disclosure may further include recovering L-lysine from the culture medium according to the culture (culture medium) or the Corynebacterium microorganism. The recovery may further include after the culture.

[0157] Recovery can be based on the culture method of the disclosed microorganism, for example, in batches, continuously or in batches, using appropriate methods known in the art to collect target L-Methionine. For example, centrifugal, filtration, processing, extraction, ultrasonic crushing, ultrafiltration, dialysis, various chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.), HPLC or the combination of these methods can be used, and using appropriate methods known in the art, target L-amino acid can be recovered from culture medium or microorganism.

[0158] In addition, the production method of L-Lysine of the present disclosure may further include a purification step. Suitable methods known in the art may be used for purification. In one embodiment, when the production method of L-Lysine of the present disclosure includes a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously without regard to order, or may be performed by being combined into one step, but is not limited thereto.

[0159] In the methods of the present disclosure, variants, polynucleotides, vectors, strains, etc. are described in other aspects above.

[0160] Another aspect of the present disclosure provides a composition for producing L-lysine, the composition comprising at least one selected from the group consisting of: a protein variant of the present disclosure, a polynucleotide encoding the protein variant, a vector comprising the polynucleotide, the aforementioned microorganism of the present disclosure, and a culture medium in which the aforementioned microorganism is cultured.

[0161] The composition of the present disclosure may further comprise any suitable excipients commonly used in the production of amino acid compositions, and such excipients may be, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers or tonicifying agents, etc., but are not limited thereto.

[0162] In the compositions of the present disclosure, variants, polynucleotides, vectors, strains, culture media, L-amino acids, etc. are as described above in other aspects.

[0163] According to another aspect of the present disclosure, the present disclosure may provide a method for increasing the L-lysine production capacity of a microorganism, a method for imparting L-lysine production capacity to a microorganism, and / or a method for preparing a microorganism with increased L-lysine production capacity, comprising introducing (e.g., transforming) the aforementioned protein variant of the present disclosure, a polynucleotide encoding the variant, and / or a vector comprising the polynucleotide.

[0164] In the method for increasing the L-lysine productivity of a microorganism, the method for imparting L-lysine productivity to a microorganism and / or the method for preparing a microorganism having increased L-lysine productivity, the polynucleotide, the recombinant vector, the microorganism and the like are as described above.

[0165] According to another aspect of the present disclosure, the present disclosure provides at least one use selected from the following group consisting of for producing L-lysine, and / or preparing a microorganism for producing L-lysine, and / or imparting and / or increasing the L-lysine production capacity of a microorganism: the aforementioned protein variant; a polynucleotide encoding the protein variant; a recombinant vector comprising the polynucleotide; and a microorganism comprising the protein variant, a polynucleotide encoding the protein variant and / or a recombinant vector comprising the polynucleotide.

[0166] In the use of producing L-lysine, and / or preparing a microorganism producing L-lysine, and / or imparting and / or increasing the L-lysine production capacity of a microorganism, the protein variant, polynucleotide, recombinant vector, microorganism, etc. are as described above.

[0167] According to another aspect of the present disclosure, the present disclosure provides a composition, method, product, process or use characterized by at least one element disclosed in the present disclosure.

[0168] Beneficial Effects

[0169] When a microorganism of the genus Corynebacterium including the variant of the present disclosure is cultured, a high yield of L-lysine can be produced compared to a microorganism having a conventional unmodified polypeptide. DETAILED DESCRIPTION

[0170] Hereinafter, the present disclosure will be described in more detail by way of examples. These examples are only intended to describe the present disclosure in more detail, and it is obvious to those skilled in the art that, according to the spirit of the present disclosure, the scope of the present disclosure is not limited by these examples.

[0171] Example

[0172] (Throughout this specification, "%" used to express the concentration of a particular substance is (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid, unless otherwise specified.)

[0173] Example 1: Selection of mutant strains with increased lysine production capacity by artificial mutation

[0174] Example 1-1: Random mutagenesis by UV irradiation

[0175] In order to select mutant strains with increased lysine production capacity, the lysine production strain CJ3P (US9556463B2) was spread on a nutrient medium containing agar and cultured at 30°C for 16 hours. The colonies obtained in this way were irradiated with UV (ultraviolet mutagenesis) at room temperature, and random mutations were induced on the genome of the strain. The composition of the nutrient medium is as follows.

[0176] <Nutrient medium (pH 7.2)>

[0177] Glucose 10g, meat extract 5g, polypeptone 10g, sodium chloride 2.5g, yeast extract 5g, agar 20g, urea 2g (based on 1L distilled water)

[0178] Example 1-2: Selection of strains with improved L-lysine production capacity

[0179] About 3,000 fixed colonies were inoculated in 300 μL of selection medium respectively, and in 96-deep well plates, at 32 ° C, 1000rpm cultured for about 24 hours. In order to analyze the output of L-amino acid produced in culture fluid, ninhydrin method (J.Biol.Chem.1948.176:367-388) was used. After completing the culture, 10 μL of culture supernatant and 190 μL of ninhydrin reaction solution were reacted at 65 ° C for 30 minutes, then absorbance was measured at a wavelength of 570nm with a spectrophotometer, and about 30 colonies of mutant strains showing an absorbance increase of more than 10% were selected compared with the absorbance of the control CJ3P strain. Compared with the control group, other colonies showed similar or reduced absorbance. The composition of the selection medium is as follows.

[0180] <Selection medium (pH 8.0)>

[0181] Glucose 10 g, ammonium sulfate 5.5 g, magnesium sulfate heptahydrate 1.2 g, potassium dihydrogen phosphate 0.8 g, dipotassium hydrogen phosphate 16.4 g, biotin 100 μg, thiamine-HCl 1000 μg, calcium pantothenate 2000 μg, nicotinamide 2000 μg (based on 1 liter of distilled water).

[0182] For the selected 30 strains, the ninhydrin reaction was repeated by the same method, and the top 10 strains having improved L-lysine production ability compared with the CJ3P strain were selected.

[0183] Example 1-3: Analysis of L-lysine production capacity of selected mutant strains

[0184] In order to finally select a strain having improved L-lysine productivity among the 10 strains selected in Example 1-2, a reproducibility test was performed in a flask using the following medium.

[0185] The 10 strains and the parent strain were inoculated into 250 ml baffled flasks containing 25 ml seed medium, and cultured at 30°C with shaking at 200 rpm for 20 hours. 1 ml of the seed culture was inoculated into a 250 ml baffled flask containing 24 ml of production medium, and cultured at 30°C with shaking at 200 rpm for 72 hours. The composition of the seed medium and production medium is as follows.

[0186] <Seed culture medium (pH 7.0)>

[0187] Glucose 20g, peptone 10g, yeast extract 5g, urea 1.5g, KH 2 PO 4 4g, K 2 HPO 4 8g, MgSO 4 7H 2 O 0.5g, biotin 0.1mg, thiamine HCl 1mg, calcium pantothenate 2mg, niacinamide 2mg (based on 1 liter of distilled water)

[0188] <Production medium (pH 7.0)>

[0189] Glucose 100g, (NH 4 ) 2 SO 4 40g, soy protein 2.5g, corn steep solid 5g, urea 3g, KH 2 PO 4 1g, MgSO 4 7H 2 O 0.5g, biotin 100μg, thiamine hydrochloride 1000μg, calcium pantothenate 2000μg, nicotinamide 3000μg, CaCO 3 30g (based on 1 liter of distilled water)

[0190] After the cultivation was completed, the L-lysine concentration in the culture solution was analyzed using high performance liquid chromatography (HPLC), and the L-lysine production concentration of each mutant strain is shown in Table 1 below.

[0191] [Table 1]

[0192] strain L-Lysine(g / L) CJ3P 7.2 CJ3P_mt1 7.8 CJ3P_mt2 7.9 CJ3P_mt3 7.5 CJ3P_mt4 8.1 CJ3P_mt5 8.8 CJ3P_mt6 8.0 CJ3P_mt7 7.9 CJ3P_mt8 7.8 CJ3P_mt9 7.8 CJ3P_mt10 7.6

[0193] Among the 10 selected mutant strains, CJ3P_mt5 was finally selected as a strain with significantly improved L-lysine production capacity.

[0194] Example 2. Confirmation of mutations by whole genome sequencing (WGS)

[0195] Whole genome sequencing (WGS) was performed on the CJ3P_mt5 strain selected in Example 1 and compared with the parent strain CJ3P to confirm the genes whose protein sequences changed due to nucleotide sequence mutations, and the amino acid sequences of the protein variants and the nucleotide sequences of the genes encoding the protein variants are described in Tables 2 and 3.

[0196] [Table 2]

[0197]

[0198]

[0199] [Table 3]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220] Example 3: Construction of L-lysine production strain with introduced mutant gene

[0221] Example 3-1: Construction of a recombinant vector for introducing a mutant gene

[0222] In order to confirm the effect of some of the variants identified in Example 2, vectors capable of introduction into the chromosome were constructed.

[0223] Specifically, in order to insert each of the NCgl0030, NCgl0288, NCgl0337, NCgl0458, NCgl0686, NCgl0706, NCgl0799, NCgl165, NCgl1262, NCgl1432, NCgl1531, NCgl1588, NCgl1859, NCgl1874, and NCgl2356 gene mutations into CJ3P, a vector containing the target mutation was constructed.

[0224] Specifically, the genomic DNA of the CJ3P_mt5 strain was extracted using a G-spin Total DNA extraction kit (Intron company, catalog number 17045) according to the protocol provided in the kit, and PCR was performed using the genomic DNA as a template. For polymerase, Solg TM Pful-X DNA polymerase was used, and the PCR conditions were: denaturation at 95°C for 4 minutes; then denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 50 seconds, repeated 27 times; then polymerization at 72°C for 5 minutes. The primer sequences used in the above experiment are shown in Table 4 below.

[0225] [Table 4]

[0226]

[0227]

[0228] The obtained mutation-introduced fragment and the pDC24 vector treated with the restriction enzyme smaI (SEQ ID NO: 107, Table 5) were assembled using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL. 6 NO. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) was cloned to obtain recombinant plasmids, and named vectors PDC24-NCgl0030*, PDC24-NCgl0288*, PDC24-NCgl0337*, PDC24-NCgl0458*, PDC24-NCgl0686*, PDC24-NCgl0706*, PDC24-NCgl0799*, PDC24-NCgl1165*, PDC24-NCgl1262*, PDC24-NCgl1432*, PDC24-NCgl1531*, PDC24-NCgl1588*, PDC24-NCgl1859*, PDC24-NCgl1874* and PDC24-NCgl2356*, respectively, which included each fragment into which mutations were introduced.

[0229] [Table 5]

[0230]

[0231]

[0232]

[0233]

[0234] Example 3-2: Construction of L-lysine production strain with introduced mutant gene

[0235] The 15 vectors constructed in Example 3-1 were transformed into the lysine production strain CJ3P, in which the strains were selected by kanamycin medium to insert the vector into the chromosome by recombinant homologous sequences. Thereafter, for the transformed strains that completed the secondary recombination, the primer pairs in Table 6 below were used to confirm the strains into which the mutant gene was introduced by PCR.

[0236] [Table 6]

[0237]

[0238]

[0239] The recombinant strains were named CJ3P△NCgl0030::NCgl0030*, CJ3P△NCgl0288::NCgl0288*, CJ3P△NCgl0337::NCgl0337*, CJ3P△NCgl0458::NCgl0458*, CJ3P△NCgl0686::NCgl0686*, CJ3P△NCgl0706::NCgl0706*, CJ3P△NCgl0799::NCgl0799*, and CJ3P△NCgl0801::NCgl801*. l1165::NCgl1165*, CJ3P△NCgl1262::NCgl1262*, CJ3P△NCgl1432::NCgl1432*, CJ3P△NCgl1531::NCgl1531*, CJ3P△NCgl1588::NCgl1588*, CJ3P△NCgl1859::NCgl1859*, CJ3P△NCgl1874::NCgl1874* and CJ3P△NCgl2356::NCgl2356*.

[0240] Example 4. Evaluation of lysine production capacity of L-lysine producing strains into which mutant genes were introduced

[0241] In order to confirm the L-lysine-producing ability of the strains constructed in Example 3-2, they were cultured and evaluated by the following method.

[0242] Each strain was inoculated into a 250 ml baffled flask containing 25 ml of seed medium and cultured with shaking at 200 rpm for 20 hours at 30°C. 1 ml of the seed culture was inoculated into a 250 ml baffled flask containing 24 ml of production medium and cultured with shaking at 200 rpm for 72 hours at 30°C.

[0243] <Seed culture medium (pH 7.0)>

[0244] Glucose 20g, peptone 10g, yeast extract 5g, urea 1.5g, KH 2 PO 4 4g, K 2 HPO 4 8g, MgSO 4 7H 2 O 0.5g, biotin 0.1mg, thiamine HCl 1mg, calcium pantothenate 2mg, niacinamide 2mg (based on 1 liter of distilled water)

[0245] <Production medium (pH 7.0)>

[0246] Glucose 100g, (NH 4 ) 2SO 4 40g, soy protein 2.5g, corn steep solid 5g, urea 3g, KH 2 PO 4 1g, MgSO 4 7H 2 O 0.5g, biotin 100μg, thiamine hydrochloride 1000μg, calcium pantothenate 2000μg, nicotinamide 3000μg, CaCO 3 30g (based on 1 liter of distilled water)

[0247] After the cultivation was completed, the production of L-lysine was determined using HPLC. The analysis results are shown in Table 7 below.

[0248] [Table 7]

[0249] strain L-Lysine(g / L) CJ3P 7.2 CJ3P△NCgl0030::NCgl0030* 7.7 CJ3P△NCgl0288::NCgl0288* 7.4 CJ3P△NCgl0337::NCgl0337* 7.4 CJ3P△NCgl0458::NCgl0458* 7.6 CJ3P△NCgl0686::NCgl0686* 7.9 CJ3P△NCgl0706::NCgl0706* 7.4 CJ3P△NCgl0799::NCgl0799* 7.9 CJ3P△NCgl1165::NCgl1165* 7.5 CJ3P△NCgl1262::NCgl1262* 7.4 CJ3P△NCgl1432::NCgl1432* 7.4 CJ3P△NCgl1531::NCgl1531* 7.6 CJ3P△NCgl1588::NCgl1588* 7.7 CJ3P△NCgl1859::NCgl1859* 8.3 CJ3P△NCgl1874::NCgl1874* 7.6 CJ3P△NCgl2356::NCgl2356* 7.5

[0250] The results are shown in Table 7 above, confirming that most of the lysine-producing strains into which the gene variants were introduced have lysine production capacity equal to or greater than that of the parent strain. In particular, it was confirmed that CJ3P△NCgl1859::NCgl1859* had a significantly increased lysine production capacity compared to the parent strain CJ3P. Thus, it can be confirmed that by introducing a variant in which the 75th amino acid of the NCgl1859 gene is replaced from glycine to valine, L-lysine can be produced more efficiently.

[0251] According to the above description, those skilled in the art to which the present disclosure belongs will be able to understand that the present invention can be implemented in other specific forms without changing its technical spirit or essential characteristics. In this regard, the above-mentioned embodiments should be understood to be illustrative and not restrictive in all aspects. The scope of the present disclosure should be interpreted as including all changes or modifications derived from the meaning and scope of the claims described later, rather than the above detailed description and its equivalent concepts, which are included in the scope of the present disclosure.

Claims

1. A protein variant comprising an amino acid sequence in which the 75th amino acid from the N-terminus in the amino acid sequence corresponding to SEQ ID NO: 105 is substituted with valine.

2. The protein variant according to claim 1, wherein The protein variant has an amino acid sequence identity of 95% or more and less than 100% with the amino acid sequence of SEQ ID NO:

105.

3. The protein variant according to claim 1, wherein The protein variant comprises the amino acid sequence of SEQ ID NO:

15.

4. A polynucleotide encoding the protein variant according to claim 1. 5 . A Corynebacterium microorganism comprising at least one selected from the group consisting of the protein variant according to claim 1 and a polynucleotide encoding the protein variant.

6. The microorganism according to claim 5, wherein The Corynebacterium microorganism is Corynebacterium glutamicum.

7. The microorganism according to claim 5, wherein The microorganism has increased L-lysine-producing ability compared to a microorganism of the genus Corynebacterium that does not contain any one selected from the group consisting of the protein variant and the polynucleotide encoding the protein variant.

8. A composition for producing L-lysine, comprising the Corynebacterium microorganism, the Corynebacterium microorganism comprising at least one selected from the group consisting of a protein variant and a polynucleotide encoding the protein variant, the protein variant comprising the following amino acid sequence, in which the amino acid at position 75 from the N-terminus in the amino acid sequence corresponding to SEQ ID NO: 105 is substituted with valine.

9. The composition for producing L-Lysine according to claim 8, wherein The protein variant comprises the amino acid sequence of SEQ ID NO:

15.

10. The composition for producing L-lysine according to claim 8, wherein The Corynebacterium microorganism is Corynebacterium glutamicum.

11. A method for producing L-lysine, comprising culturing the microorganism according to claim 5 in a culture medium.

12. The method for producing L-lysine according to claim 11, wherein The Corynebacterium microorganism is Corynebacterium glutamicum.

13. The method for producing L-lysine according to claim 11, wherein The method further comprises recovering L-lysine from the culture medium or the microorganism.

14. The method for producing L-lysine according to claim 11, wherein The method is carried out at pH 6 to pH 9.

15. Use of the microorganism according to any one of claims 5 to 7 for producing L-lysine.

16. A composition, method, product, process or use, characterized in that At least one element disclosed in the present disclosure.

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