Protein variant and method for producing l-lysine using same

Abstract

The present disclosure provides a protein variant, a Corynebacterium sp. microorganism including the protein variant, and a method for producing L-lysine using the microorganism.

Description

Protein mutant and method for producing L-lysine using the same

[0001] Cross-citation with related application(s)

[0002] This disclosure claims the benefit of priority to Republic of Korea Patent Application No. 10-2023-0096086, dated July 24, 2023, the entire contents of which are incorporated herein by reference.

[0003] Numerous papers and patents are referenced and cited throughout this disclosure. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the present invention.

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

[0005]

[0006] To produce L-amino acids and other useful substances, various studies are being conducted to develop high-efficiency production microorganisms and fermentation process technologies. For example, target-substance-specific approaches, such as increasing the expression of genes encoding enzymes involved in L-lysine biosynthesis or removing genes unnecessary for biosynthesis, are primarily being utilized (WO2008-082181 A1).

[0007] However, due to the increasing demand for L-lysine, research is still needed to increase the effective production capacity of L-lysine.

[0008]

[0009] One object of the present disclosure is to provide protein variants.

[0010] Another object of the present disclosure is to provide a polynucleotide encoding the protein variant.

[0011] 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 the protein variants and polynucleotides encoding the same.

[0012] Another object of the present disclosure is to provide a composition for producing L-lysine, comprising the microorganism of the genus Corynebacterium.

[0013] Another object of the present disclosure is to provide a method for producing L-lysine, comprising the step of culturing the above-described microorganism of the genus Corynebacterium in a medium.

[0014] Another object of the present disclosure is to provide a use of the microorganism for producing L-lysine.

[0015]

[0016]

[0017] This is specifically described as follows. Meanwhile, each description and embodiment disclosed in this disclosure can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this disclosure fall within the scope of this disclosure. Furthermore, the scope of this disclosure is not limited by the specific descriptions set forth below. Furthermore, those skilled in the art will recognize or be able to identify numerous equivalents to the specific embodiments of this application described herein using only routine experimentation. Furthermore, such equivalents are intended to be encompassed by this application.

[0018]

[0019] One aspect of the present disclosure provides a protein variant comprising an amino acid sequence in which the amino acid corresponding to the 75th position from the N-terminus in the amino acid sequence of SEQ ID NO: 105 is replaced with a different amino acid, or an amino acid sequence in which the amino acid corresponding to the 35th position from the N-terminus in the amino acid sequence of SEQ ID NO: 106 is replaced with a different amino acid.

[0020] In the present disclosure, the sequence of SEQ ID NO: 105 is as follows: MVSQTERQHAIASLLAPTGAVSVGDLAEHFHVTTETVRRDLRIMESLGLLQRVHGGAISPEPMGTSPPRLKPALGKGMPPEPRVLELAETAVSLITPLARSIFLDSGLACTAIATVLGDPPEDARWTVVTSS PGAVIALSATDATSTVVLHGQVHGNCSSIIGSTAVDMISQLRADIAFVEVDAIQSDTSLCTFFPETIPIKQAMIKNAAFTVAVLSPRSPQDQELQLLKHPFSTLADFDALVTDDHTLDFPVLPDHNFQVVTP.

[0021] In the present disclosure, the sequence of SEQ ID NO: 106 is as follows: MTTNAPDGATNNINNAHSGAVGKPKVQLSDAEIQEYTAAFAGTTTTKPWELEVTTKFLKKIAWVAVVVIMAVHIFMGAVVDVDFTGAAVTFVDTLAFPALGIIFSVLVFLGLTRPRVRANEDGVEVRNFIGTRFYPWVVIYGMSFPKGSSVARLELPDFEFVPMWAFQSRDGEDVVRAVATFRDLENKYMPED.

[0022] In one embodiment, the protein variant of the present disclosure described above may essentially consist of an amino acid sequence in which the amino acid corresponding to the 75th position from the N-terminus in the amino acid sequence of SEQ ID NO: 105 is substituted with a different amino acid, or an amino acid sequence in which the amino acid corresponding to the 35th position from the N-terminus in the amino acid sequence of SEQ ID NO: 106 is substituted with a different amino acid. In another embodiment, the protein variant of the present disclosure described above may consist of an amino acid sequence in which the amino acid corresponding to the 75th position from the N-terminus in the amino acid sequence of SEQ ID NO: 105 is substituted with a different amino acid, or an amino acid sequence in which the amino acid corresponding to the 35th position from the N-terminus in the amino acid sequence of SEQ ID NO: 106 is substituted with a different amino acid.

[0023] In the present disclosure, the amino acid sequence of SEQ ID NO: 105 may be the amino acid sequence of the FruR (transcriptional regulator of sugar metabolism) protein. In one embodiment, the protein may be a protein encoded by the NCgl1859 gene.

[0024] 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 NCgl1531 gene.

[0025] In one embodiment, the amino acid corresponding to the 75th position from the N-terminus in the amino acid sequence of SEQ ID NO: 105 may be, but is not limited to, glycine (Gly, G).

[0026] In one embodiment, the amino acid corresponding to the 35th position from the N-terminus in the amino acid sequence of SEQ ID NO: 106 may be, but is not limited to, glutamic acid (Glu, E).

[0027] In one embodiment, the amino acid corresponding to the 75th position from the N-terminus in the amino acid sequence of SEQ ID NO: 105 may be substituted with valine (Val, V), but is not limited thereto.

[0028] In one embodiment, the amino acid corresponding to the 35th position from the N-terminus in the amino acid sequence of SEQ ID NO: 106 may be substituted with lysine (Lys, K), but is not limited thereto.

[0029] The protein variant of the present disclosure may have an activity that increases L-lysine production capacity compared to the wild type protein (polypeptide).

[0030] The protein variant of the present disclosure may comprise an amino acid sequence in which the amino acid corresponding to the 75th position from the N-terminus in the amino acid sequence of SEQ ID NO: 105 is substituted with a different amino acid (e.g., valine), or an amino acid sequence in which the amino acid corresponding to the 35th position from the N-terminus in the amino acid sequence of SEQ ID NO: 106 is substituted with a different 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% homology or identity with said amino acid sequence. In addition, it is obvious that a variant having an amino acid sequence in which some of the sequences are deleted, modified, substituted, conservatively substituted or added is also included within the scope of the present disclosure, provided that the amino acid sequence has such homology or identity and exhibits an effect corresponding to the effect of the variant of the present disclosure (effect of increasing L-lysine production).

[0031] For example, if the amino acid sequence has sequence additions or deletions, naturally occurring mutations, silent mutations or conservative substitutions that do not alter the function of the variant of the present disclosure at the N-terminus, C-terminus and / or within the amino acid sequence.

[0032] The term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be based on similarities 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 a protein or polypeptide.

[0033] As used herein, the term "variant" refers to a polypeptide in which one or more amino acids are conservatively substituted and / or modified, thereby differing from the amino acid sequence of the variant before the mutation, but retaining functions or properties. 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. Additionally, some variants may include variants in which one or more portions, such as the N-terminal leader sequence or the transmembrane domain, are deleted. Other variants may include variants in which portions are deleted from the N- and / or C-terminus of the mature protein. The above term "variant" may be used interchangeably with terms such as variant, modification, variant polypeptide, variant protein, variant and variant (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), and is not limited thereto if the term is used in the meaning of variant.

[0034] Additionally, variants may include deletions or additions of amino acids that have minimal impact on the properties and secondary structure of the polypeptide. For example, the N-terminus of the variant may be conjugated with a signal (or leader) sequence involved in co-translational or post-translational protein translocation. Furthermore, the variant may be conjugated to other sequences or linkers to facilitate identification, purification, or synthesis.

[0035] In one embodiment, the protein variant of the present disclosure described above may have at least 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but less than 100%, homology or identity with the amino acid sequence of SEQ ID NO: 105, or may have at least 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but less than 100%, homology or identity with the amino acid sequence of SEQ ID NO: 106, but is not limited thereto. That is, the protein variant of the present disclosure described above may have an amino acid sequence identity of 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more but less than 100% with the amino acid sequence of SEQ ID NO: 105 or SEQ ID NO: 106, respectively, but is not limited thereto.

[0036]

[0037] Another aspect of the present disclosure provides a protein variant comprising any one amino acid sequence selected from the group consisting of 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 essentially consist of the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15. In yet another embodiment, the protein variant may consist of the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15.

[0038] The protein variant of the present disclosure may comprise any one amino acid sequence selected from the group consisting of 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, or may comprise 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 said amino acid sequence. In addition, it is obvious that a variant having an amino acid sequence in which a part of the sequence is deleted, modified, substituted, conservatively substituted, or added is also included within the scope of the present disclosure, as long as it has such homology or identity and exhibits an efficacy corresponding to the variant of the present disclosure (efficacy of increasing L-lysine production).

[0039] 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 ABC transporter permease is substituted from asparagine (Asn, N) to isoleucine (Ile, I). The ABC transporter permease may be a protein encoded by the NCgl0030 gene.

[0040] 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 (EndoIII-related endonuclease) protein is substituted from alanine (Ala, A) to valine (Val, V). The nth protein may be a protein encoded by the NCgl0288 gene.

[0041] 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 partitioning 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.

[0042] 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 antitermination 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.

[0043] In the present disclosure, the amino acid sequence of sequence number 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).

[0044] 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 subunits protein is substituted from leucine (Leu, L) to phenylalanine (Phe, F). The Type II restriction enzyme, methylase subunits protein may be a protein encoded by the NCgl0706 gene.

[0045] 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+ / panthothenate 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 NCgl0799 gene.

[0046] 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 beta) protein is substituted from aspartic acid (Asp, D) to glutamic acid (Glu, E). The atpD protein may be a protein encoded by the NCgl1165 gene.

[0047] In the present disclosure, the amino acid sequence of SEQ ID NO: 11 may be a sequence of a protein variant in which the 456th amino acid 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.

[0048] 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.

[0049] In the present disclosure, the amino acid sequence of SEQ ID NO: 13 may be a sequence of a protein variant in which the 35th amino acid of a protein (hypothetical protein) encoded by the NCgl1531 gene is substituted from glutamic acid (Glu, E) to lysine (Lys, K).

[0050] 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 (hypothetical protein) encoded by the NCgl1588 gene is substituted from valine (Val, V) to isoleucine (Ile, I).

[0051] 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 protein may be a protein encoded by the NCgl1859 gene.

[0052] 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 methylthiotransferase) protein is substituted from serine (Ser, S) to proline (Pro, P). The miaB protein may be a protein encoded by the NCgl1874 gene.

[0053] In the present disclosure, the amino acid sequence of SEQ ID NO: 18 may be a sequence of a protein variant in which the 456th amino acid of the protein (hypothetical protein) encoded by the NCgl2356 gene is substituted from valine (Val, V) to alanine (Ala, A), the 464th amino acid is substituted from serine (Ser, S) to proline (Pro, P), the 465th amino acid is substituted from alanine (Ala, A) to valine (Val, V), the 466th amino acid is substituted from serine (Ser, S) to glycine (Gly, G), the 496th amino acid is substituted from glutamic acid (Glu, E) to threonine (Thr, T), and the 499th amino acid is substituted from serine (Ser, S) to aspartic acid (Asp, D).

[0054] The protein variant of the present disclosure may comprise any one amino acid sequence selected from the group consisting of 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 (e.g., the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15), or may comprise 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 said amino acid sequence. In addition, it is obvious that a variant having an amino acid sequence in which a part of the sequence is deleted, modified, substituted, conservatively substituted, or added is also included within the scope of the present disclosure, as long as it has such homology or identity and exhibits an efficacy corresponding to the variant of the present disclosure (efficacy of increasing L-lysine production).

[0055] For example, if the amino acid sequence has sequence additions or deletions, naturally occurring mutations, silent mutations or conservative substitutions that do not alter the function of the variant of the present disclosure at the N-terminus, C-terminus and / or within the amino acid sequence.

[0056] In the above protein variants, conservative substitutions, variants, etc. are as described above.

[0057]

[0058] As used herein, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid sequences or base sequences, which may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0059] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and may be combined with default gap penalties established by the program being used. In practice, homologous or identical sequences can generally hybridize with all or part of the sequence under moderate or high stringency conditions. It should be appreciated that hybridization also includes hybridization with polynucleotides containing common codons or codons that take codon degeneracy into account.

[0060] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program using default parameters, for example, as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as 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) can be determined using the 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, homology, similarity, or identity can be determined using BLAST or ClustalW of the National Center for Biotechnology Information Database.

[0061] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information, for example, using a GAP computer program such as that of Needleman et al. (1970), J Mol Biol. 48:443, as disclosed, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In brief, the GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and (2) a comparison matrix as disclosed by Gribskov et al. (1986) Nucl. Acids Res. 48:443, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979). 14: 6745 weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) permutation matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0062] As an example of the present disclosure, a variant of the present disclosure may have an activity that increases L-lysine production compared to a wild-type polypeptide.

[0063] 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 that is similar, identical, or homologous to the residue listed in the polypeptide. Identifying an amino acid at a corresponding position may be determining a specific amino acid in a sequence that references a particular sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0064] For example, an amino acid sequence in which the amino acid corresponding to the 75th position from the N-terminus in the amino acid sequence of SEQ ID NO: 105 is replaced with a different amino acid (e.g., valine), or an amino acid sequence in which the amino acid corresponding to the 35th position from the N-terminus in the amino acid sequence of SEQ ID NO: 106 is replaced with a different amino acid (e.g., lysine); Or, any one amino acid sequence selected from the group consisting of 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 (e.g., amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15) is aligned, and based on this, each amino acid residue of the amino acid sequence is an amino acid sequence in which the amino acid corresponding to the 75th position from the N-terminus in the amino acid sequence of SEQ ID NO: 105 is substituted with a different amino acid (e.g., valine), or an amino acid sequence in which the amino acid corresponding to the 35th position from the N-terminus in the amino acid sequence of SEQ ID NO: 106 is substituted with a different amino acid (e.g., lysine); Alternatively, the numbering may be done by referring to the numerical positions of amino acid residues corresponding to amino acid residues in any one amino acid sequence selected from the group consisting of 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 (e.g., amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15). For example, a sequence alignment algorithm such as that described in the present disclosure can identify the positions of amino acids, or positions where modifications such as substitutions, insertions, or deletions occur, by comparing them to a query sequence (also referred to as a "reference sequence").

[0065] For such alignment, for example, the Needleman-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) can be used, but is not limited thereto, and any sequence alignment program known in the art, pairwise sequence comparison algorithm, etc. can be appropriately used.

[0066] Another aspect of the present disclosure provides a polynucleotide encoding a protein variant of the present disclosure.

[0067] In the present disclosure, the term "polynucleotide" means a polymer of nucleotides in which nucleotide units (monomers) are covalently bonded to form a long chain, a DNA or RNA strand of a certain length or longer, and more specifically, a polynucleotide fragment encoding the variant.

[0068] In one example, the polynucleotide may be a polynucleotide encoding a protein variant comprising an amino acid sequence in which the amino acid corresponding to the 75th position from the N-terminus in the amino acid sequence of SEQ ID NO: 105 is substituted with a different amino acid (e.g., valine) than the original, or an amino acid sequence in which the amino acid corresponding to the 35th position from the N-terminus in the amino acid sequence of SEQ ID NO: 106 is substituted with a different amino acid (e.g., lysine).

[0069] In one example, the polynucleotide may include a polynucleotide comprising 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, for example, the nucleotide sequence of SEQ ID NO: 37.

[0070] A polynucleotide encoding a protein variant of the present disclosure may have or comprise 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, for example, the nucleotide sequence of SEQ ID NO: 37. In another example, the polynucleotide may comprise 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. Additionally, it is obvious that a polynucleotide having a nucleotide sequence in which some of the sequences are deleted, modified, substituted, conservatively substituted or added is also included within the scope of the present disclosure, provided that the sequence encodes a polypeptide or protein having such homology or identity and exhibiting an effect corresponding to the variant of the present disclosure (increased L-lysine production).

[0071] In one example, the nucleotide sequence or amino acid sequence provided herein may include a modification by conventional mutagenesis, such as directed evolution and / or site-directed mutagenesis, to the extent that it maintains its original function or desired function. In one example, a polynucleotide or polypeptide “comprising a particular nucleotide sequence or amino acid sequence” can mean that the polynucleotide or polypeptide (i) consists of or essentially comprises the particular nucleotide sequence or amino acid sequence, or (ii) consists of or essentially comprises an amino acid sequence that has at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% homology to the particular nucleotide sequence or amino acid sequence and retains its original function and / or desired function.

[0072] The polynucleotide of the present disclosure may have various modifications in the coding region within a range that does not change the amino acid sequence of the variant of the present disclosure, taking into account the degeneracy of the codon or the codon preferred in the organism that is intended to express the variant of the present disclosure. Specifically, the polynucleotide of the present disclosure is a polynucleotide encoding a protein variant comprising an amino acid sequence in which an amino acid corresponding to the 75th position from the N-terminus in the amino acid sequence of SEQ ID NO: 105 is substituted with a different amino acid (e.g., valine) or an amino acid sequence in which an amino acid corresponding to the 35th position from the N-terminus in the amino acid sequence of SEQ ID NO: 106 is substituted with a different amino acid (e.g., lysine), and has a homology or identity of 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 with 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., the nucleotide sequence of SEQ ID NO: 37). and may consist of or consist essentially of a nucleotide sequence having, but is not limited to, a base sequence having less than 100% homology or identity with said nucleotide sequence, or having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 100% homology or identity with said nucleotide sequence.

[0073] Additionally, the polynucleotides of the present disclosure may include, without limitation, probes that can be prepared from known genetic sequences, for example, sequences that can hybridize under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence of the present disclosure. The term "stringent conditions" refers to conditions that enable specific hybridization between polynucleotides. For example, conditions under which polynucleotides having high homology or identity hybridize with each other, polynucleotides 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, or 99% or more homology or identity hybridize with each other, and polynucleotides having lower homology or identity do not hybridize with each other, or conditions under which washing conditions of typical southern hybridization are performed once, specifically twice or three times, at a salt concentration and temperature equivalent to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, and more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0074] Hybridization requires that two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Accordingly, the polynucleotides of the present disclosure may also include isolated nucleic acid fragments that are complementary in their entirety, as well as substantially similar nucleotide sequences.

[0075] Specifically, a polynucleotide having homology or identity with the polynucleotide of the present disclosure can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the conditions described above. In addition, the Tm value may be, but is not limited to, 60°C, 63°C, or 65°C, and can be appropriately adjusted by a person skilled in the art depending on the purpose.

[0076] The appropriate stringency for hybridizing the above polynucleotides depends on the length and degree of complementarity of the polynucleotides, variables which are well known in the art.

[0077]

[0078] Another aspect of the present disclosure provides a vector comprising the polynucleotide of the present disclosure. The vector may be, but is not limited to, an expression vector for expressing the polynucleotide in a host cell.

[0079] The vector of the present disclosure may comprise a DNA construct comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the target polypeptide in a suitable host. The expression control region may comprise a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences regulating the termination of transcription and translation. The vector may be capable of replicating or functioning independently of the host genome after being transformed into a suitable host cell, or may be integrated into the genome itself.

[0080] The vector used in the present disclosure is not particularly limited, and any vector known in the art can 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 can be used as phage vectors or cosmid vectors, and pDZ series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, or pDC24 vectors can be used.

[0081] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a vector for intracellular chromosomal insertion. The insertion of the polynucleotide into the chromosome can be achieved by any method known in the art, such as, but not limited to, homologous recombination. A selection marker for confirming the chromosomal insertion can be additionally included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the insertion of the target nucleic acid molecule. Markers that confer a selectable phenotype, such as drug resistance, nutrient requirement, cytotoxic agent resistance, or expression of a surface polypeptide, can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypic traits, thereby allowing the selection of transformed cells.

[0082] As used herein, the term "transformation" refers to introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism, such that the polypeptide encoded by the polynucleotide can be expressed within the host cell. The transformed polynucleotide can be located within the chromosome of the host cell or located extrachromosomally, as long as it can be expressed within the host cell. In addition, the polynucleotide includes DNA and / or RNA encoding the target polypeptide. The polynucleotide can be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all the elements necessary for autonomous expression. The expression cassette can typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, which are operably linked to the polynucleotide. The above expression cassette may be in the form of a self-replicating expression vector. Furthermore, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0083] Additionally, the term "operably linked" as used herein means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target variant of the present disclosure.

[0084]

[0085] Another aspect of the present disclosure provides a microorganism of the genus Corynebacterium comprising at least one member selected from the group consisting of a protein variant of the present disclosure and a polynucleotide of the present disclosure.

[0086] The microorganism of the present disclosure may comprise a protein variant of the present disclosure, a polynucleotide encoding the protein variant, or a vector comprising the polynucleotide of the present disclosure.

[0087] In the present disclosure, the term "microorganism (or strain)" includes both wild-type microorganisms and microorganisms that have undergone genetic modification naturally or artificially, and may be microorganisms that have had a specific mechanism weakened or strengthened due to causes such as insertion of an external gene or enhanced or inactivated activity of an endogenous gene, and may be microorganisms that include genetic modification for the production of a desired polypeptide, protein or product.

[0088] The microorganism of the present disclosure may be, but is not limited to, a microorganism comprising at least one of a protein variant of the present disclosure, a polynucleotide of the present disclosure, and a vector comprising a polynucleotide of the present disclosure; a microorganism modified to express a variant of the present disclosure or a polynucleotide of the present disclosure; a microorganism (e.g., a recombinant microorganism) expressing a variant of the present disclosure or a polynucleotide of the present disclosure; or a microorganism having the activity of a variant of the present disclosure (e.g., a recombinant microorganism).

[0089] The microorganism of the present disclosure may be a microorganism having L-lysine production ability.

[0090] The microorganism of the present disclosure may be a microorganism that naturally has L-lysine production ability, or a parent strain that does not have L-lysine production ability into which a variant of the present disclosure or a polynucleotide encoding the same (or a vector including the polynucleotide) is introduced and / or a microorganism that is endowed with L-lysine production ability, but is not limited thereto.

[0091] For example, the microorganism of the present disclosure is a cell or microorganism that is transformed with a vector containing a polynucleotide encoding the polynucleotide of the present disclosure or a variant of the present disclosure, and expresses a protein variant of the present disclosure. For the purposes of the present disclosure, the strain of the present disclosure may include any microorganism capable of producing L-lysine, including the variant of the present disclosure. For example, the microorganism of the present disclosure may be a recombinant microorganism whose L-lysine production ability is increased by introducing a polynucleotide encoding the variant of the present disclosure into a naturally occurring wild-type microorganism or a microorganism that produces L-lysine.

[0092] In one specific example, the microorganism of the present disclosure may have an increased L-lysine productivity compared to a microorganism of the genus Corynebacterium that does not include at least one selected from the group consisting of the protein variant of the present disclosure and a polynucleotide encoding the protein variant (i.e., an amino acid sequence in which the amino acid corresponding to the 75th position from the N-terminus in the amino acid sequence of SEQ ID NO: 105 is substituted with another amino acid (e.g., valine), or an amino acid sequence in which the amino acid corresponding to the 35th position from the N-terminus in the amino acid sequence of SEQ ID NO: 106 is substituted with a different amino acid (e.g., lysine); or a microorganism that does not express a protein variant comprising any one amino acid sequence selected from the group consisting of 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, 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).

[0093] The Corynebacterium genus microorganism that does not comprise at least one member selected from the group consisting of the protein variant of the present disclosure and the polynucleotide encoding the protein variant may be a natural wild-type microorganism or an unmodified microorganism, and may also be expressed as a host cell or a parent strain. The Corynebacterium genus microorganism that does not comprise at least one member selected from the group consisting of the protein variant of the present disclosure and the polynucleotide encoding the protein variant may be, but is not limited to, ATCC13032 strain and / or CJ3P (US 9556463 ​​B2).

[0094] The recombinant microorganism with increased L-lysine productivity of the present disclosure may be a microorganism with increased L-lysine productivity compared to a natural wild-type microorganism or an unmodified microorganism, but is not limited thereto. For example, the unmodified microorganism, which is the target strain for comparison of increased L-lysine productivity, may be, but is not limited to, ATCC13032 strain and / or CJ3P (US 9556463 ​​B2).

[0095] In one embodiment, the microorganism with increased L-lysine productivity (production amount) of the present disclosure may have an L-lysine productivity (production amount) increased 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, or about 15.25% or more (the upper limit is not particularly limited and may be, for example, about 200% or less, about 150% or less, about 100% or less, or about 50% or less, or about 30% or less) compared to the parent strain before mutation or the unmodified microorganism, but is not limited thereto. In another embodiment, the microorganism of the present disclosure having increased L-lysine productivity (production amount) may have an L-lysine productivity (production amount) increased 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, or about 1.1525 times or more (the upper limit is not particularly limited and may be, for example, 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.

[0096] The term “about” above includes all ranges including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all ranges of values ​​equal to or similar to the value following the term “about,” but is not limited thereto.

[0097]

[0098] The term "unmodified microorganism" in this disclosure does not exclude microorganisms that contain mutations that may occur naturally in microorganisms, and may refer to wild-type microorganisms or natural microorganisms themselves, or microorganisms before their traits are changed by genetic mutations caused by natural or artificial factors. For example, the unmodified microorganism may refer to a microorganism that has not been introduced or before the protein variants described herein are introduced. The "unmodified microorganism" may be used interchangeably with "pre-modified strain," "pre-modified microorganism," "unmutated strain," "unmodified microorganism," or "reference microorganism."

[0099] In one embodiment, the microorganism of the present disclosure is Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium It may be Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens, or more specifically, Corynebacterium glutamicum.

[0100]

[0101] As used herein, the term "attenuation" of a polypeptide encompasses a reduction in activity or absence of activity compared to its intrinsic activity. The term "attenuation" may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation.

[0102] The above weakening may also include cases where the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide originally possessed by the microorganism due to mutation of the polynucleotide encoding the polypeptide, etc., cases where the overall polypeptide activity level and / or concentration (expression amount) within the cell is lower than that of the natural strain due to inhibition of expression of the gene of the polynucleotide encoding the polypeptide or inhibition of translation into the polypeptide, cases where the polynucleotide is not expressed at all, and / or cases where the polypeptide has no activity even if the polynucleotide is expressed. The above "intrinsic activity" refers to the activity of a specific polypeptide originally possessed by the parent strain, wild type, or unmodified microorganism before the change in phenotype when the phenotype is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before modification." The term "inactivation, deficiency, reduction, downregulation, deterioration, attenuation" of the activity of a polypeptide relative to its intrinsic activity means that the activity of a particular polypeptide is lowered compared to the activity that the parent strain or unmodified microorganism originally had prior to the transformation.

[0103] Attenuation of the activity of such polypeptides can be accomplished by any method known in the art, including but not limited to, and can be achieved by application of 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.).

[0104] Specifically, the weakening of the polypeptide of the present disclosure is

[0105] 1) Deletion of all or part of a gene encoding a polypeptide;

[0106] 2) Modification of the expression control region (or expression control sequence) so as to reduce the expression of the gene encoding the polypeptide;

[0107] 3) Modification of the amino acid sequence constituting the polypeptide (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence) so as to eliminate or weaken the activity of the polypeptide;

[0108] 4) Modification of the gene sequence encoding the polypeptide such that the activity of the polypeptide is eliminated or weakened (e.g., deletion / substitution / addition of one or more nucleotide bases in the nucleotide sequence of the polypeptide gene such that the polypeptide is modified such that the activity of the polypeptide is eliminated or weakened);

[0109] 5) Modification of the base sequence encoding the initiation codon or 5'-UTR region of a gene transcript encoding a polypeptide;

[0110] 6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to a transcript of the gene encoding the polypeptide;

[0111] 7) Addition of a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of a gene encoding a polypeptide to form a secondary structure to which ribosome attachment is impossible;

[0112] 8) Addition of a promoter that is 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

[0113] 9) Control of cellular localization of proteins (polypeptides); or

[0114] 10) It may be a combination of two or more selected from 1) to 9), but is not particularly limited thereto.

[0115] for example,

[0116] The above 1) deletion of 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 having some nucleotides deleted, or replacement with a marker gene.

[0117] In addition, the above 2) modification of the expression control region (or expression control sequence) may be a mutation in 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 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.

[0118] In addition, the above 3) modification of the base sequence encoding the initiation codon or 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, a substitution with a base sequence encoding another initiation codon having a lower polypeptide expression rate than the endogenous initiation codon, but is not limited thereto.

[0119] In addition, the modification of the amino acid sequence or polynucleotide sequence of the above 4) and 5) may be, but is not limited to, a mutation in the sequence of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, such as deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so as to weaken the activity of the polypeptide, 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. For example, the expression of a gene may be inhibited or weakened by introducing a mutation in the polynucleotide sequence to form a stop codon, but is not limited thereto.

[0120] 6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that complementarily binds to the transcript of the gene encoding the polypeptide may be described, for example, in the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

[0121] 7) Addition of a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of a gene encoding a polypeptide to form a secondary structure to which ribosome attachment is impossible may render mRNA translation impossible or slow it down.

[0122] 8) The addition of 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) may weaken the activity by creating an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide.

[0123] 9) Control of the intracellular location of a protein (polypeptide) above may be to target the protein (polypeptide) to a specific organelle or specific intracellular space within the cell. For example, it may be to target the protein (polypeptide) to the periplasm or cytoplasm by adding or removing a leader sequence that functions to target the protein (polypeptide), but is not limited thereto.

[0124] Such attenuation of polypeptide activity may be, but is not limited to, attenuation of the activity or concentration or expression level of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild type or pre-transformed microbial strain, or an increase in the amount of a product produced from the polypeptide.

[0125]

[0126] As used herein, the term "enhancement" of polypeptide activity means that the activity of the polypeptide is increased compared to the intrinsic activity. The enhancement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Here, activation, enhancement, up-regulation, overexpression, and increase may all include exhibiting an activity that was not originally present, or exhibiting an activity that is enhanced compared to the intrinsic activity or the activity before modification. The "intrinsic activity" refers to the activity of a specific polypeptide that a parent strain or an unmodified microorganism originally possessed before the trait change, when the trait change is caused by genetic mutation due to natural or artificial factors. This may be used interchangeably with "pre-modification activity." "Enhanced," "upregulated," "overexpressed," or "increased" the activity of a polypeptide relative to its intrinsic activity means that the activity and / or concentration (expression amount) of the specific polypeptide is improved compared to the activity and / or concentration (expression amount) that the parent strain or unmodified microorganism originally had prior to the transformation.

[0127] The above enhancement can be achieved by introducing an exogenous polypeptide, or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. Whether the activity of the polypeptide is enhanced can be determined by an increase in the level of activity, expression level, or amount of product excreted from the polypeptide.

[0128] Enhancement of the activity of the above polypeptide can be achieved by applying various methods well known in the art, and is not limited as long as the activity of the target polypeptide can be enhanced compared to the microorganism before modification. Specifically, it may be achieved by using genetic engineering and / or protein engineering, which are routine methods of molecular biology and are well known to those skilled in the art, 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.).

[0129] Specifically, the enhancement of the polypeptide of the present disclosure is

[0130] 1) Increase in the intracellular copy number of a polynucleotide encoding a polypeptide;

[0131] 2) Replacing the gene expression control region on the chromosome that codes for a polypeptide with a highly active sequence;

[0132] 3) Modification of the base sequence encoding the initiation codon or 5'-UTR region of a gene transcript encoding a polypeptide;

[0133] 4) Modification of the amino acid sequence of the polypeptide so as to enhance polypeptide activity;

[0134] 5) Modification of the polynucleotide sequence encoding the polypeptide so as to enhance the activity of the polypeptide (e.g., modification of the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the activity of the polypeptide);

[0135] 6) Introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same;

[0136] 7) Codon optimization of polynucleotides encoding polypeptides;

[0137] 8) Analyzing the tertiary structure of the polypeptide and selecting the exposed portion to modify or chemically modify; or

[0138] 9) Control of cellular localization of proteins (polypeptides); or

[0139] 10) It may be a combination of two or more selected from 1) to 9), but is not particularly limited thereto.

[0140] The increase in the intracellular copy number of the polynucleotide encoding the polypeptide described above may be achieved by introducing into the host cell a vector capable of replicating and functioning independently of the host, to which the polynucleotide encoding the polypeptide is operably linked. Alternatively, the polynucleotide encoding the polypeptide may be achieved by introducing one copy or two or more copies into the chromosome of the host cell. The introduction into the chromosome may be performed by introducing into the host cell 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.

[0141] 2) Replacing the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide with a sequence having strong activity may be, for example, a mutation in the sequence such as deletion, insertion, non-conservative or conservative substitution, or a combination thereof to further enhance the activity of the expression control region, or replacement with a sequence having stronger activity. The expression control region may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. As an example, it may be, but is not limited to, replacing the original promoter with a strong promoter.

[0142] Examples of known strong promoters include, but are not limited to, the CJ1 to CJ7 promoters (US Patent No. US 7662943 B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (US Patent No. US 10584338 B2), the O2 promoter (US Patent No. US 10273491 B2), the tkt promoter, and the yccA promoter.

[0143] The above 3) modification of the base sequence encoding the initiation codon or 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, a substitution with a base sequence encoding another initiation codon having a higher polypeptide expression rate than the endogenous initiation codon, but is not limited thereto.

[0144] The modification of the amino acid sequence or polynucleotide sequence of the above 4) and 5) may be, but is not limited to, a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide, 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 have increased activity. The replacement may be specifically performed by inserting the polynucleotide into a chromosome by homologous recombination, but is not limited thereto. The vector used at this time may additionally include a selection marker to confirm whether or not chromosomal insertion has occurred. The selection marker is as described above.

[0145] The introduction of the foreign polynucleotide exhibiting the activity of the polypeptide as described above 6) may be the introduction into the host cell of a foreign polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide. The foreign polynucleotide is not limited in its 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 a person skilled in the art appropriately selecting a known transformation method, and the polypeptide may be produced by expressing the introduced polynucleotide in the host cell, thereby increasing its activity.

[0146] The above 7) codon optimization of a polynucleotide encoding a polypeptide may be codon optimization of an endogenous polynucleotide to increase transcription or translation within a host cell, or codon optimization of a foreign polynucleotide to achieve optimized transcription or translation within a host cell.

[0147] The above 8) analyzing the tertiary structure of a polypeptide and selecting an exposed portion to modify or chemically modify may be done by, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing the sequence information of known proteins, determining a template protein candidate based on the degree of sequence similarity, confirming the structure based on this, and selecting an exposed portion to modify or chemically modify, and modifying or modifying it.

[0148] 9) Control of the intracellular location of a protein (polypeptide) above may be to target the protein (polypeptide) to a specific organelle or specific intracellular space within the cell. For example, it may be to target the protein (polypeptide) to the periplasm or cytoplasm by adding or removing a leader sequence that functions to target the protein (polypeptide), but is not limited thereto.

[0149] Such enhancement of polypeptide activity may be, but is not limited to, an increase in the activity or concentration of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild type or pre-transformed microbial strain, or an increase in the amount of a product produced from the polypeptide.

[0150]

[0151] In the microorganism of the present disclosure, modification of part or all of a polynucleotide (e.g., modification to encode the protein variant described above) may be induced by, but is not limited to, (a) homologous recombination using a vector for chromosomal integration into the microorganism or genome editing using engineered nucleases (e.g., CRISPR-Cas9) and / or (b) treatment with light and / or chemicals such as ultraviolet rays and radiation. The method for modifying part or all of the gene may include a method using DNA recombination technology. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene may be injected into the microorganism to cause homologous recombination, thereby causing deletion of part or all of the gene. The injected nucleotide sequence or vector may include, but is not limited to, a dominant selection marker.

[0152] In the microorganism of the present disclosure, the protein variant, polynucleotide, L-lysine, etc. are as described in the other aspects above.

[0153]

[0154] Another aspect of the present disclosure provides a method for producing L-lysine, comprising the step of culturing a microorganism of the genus Corynebacterium in a medium, the microorganism comprising at least one member selected from the group consisting of a protein variant of the present disclosure and a polynucleotide encoding the protein variant.

[0155] The method for producing L-lysine of the present disclosure may include a step of culturing a microorganism of the genus Corynebacterium comprising a protein variant of the present disclosure, a polynucleotide of the present disclosure, or a vector of the present disclosure in a medium.

[0156] In this disclosure, the term "cultivation" refers to growing a Corynebacterium microorganism of the present disclosure under appropriately controlled environmental conditions. The culturing process of the present disclosure can be performed using a suitable medium and culture conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing process may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0157] In the present disclosure, the term "medium" refers to a material containing nutrients as a main component necessary for culturing a Corynebacterium microorganism of the present disclosure, and supplies nutrients and growth factors, including water essential for survival and growth. Specifically, the medium and other culture conditions used for culturing a Corynebacterium microorganism of the present disclosure may be any medium used for culturing a typical microorganism without particular limitation, but the Corynebacterium microorganism of the present disclosure may be cultured under aerobic conditions while controlling temperature, pH, etc. in a typical medium containing an appropriate carbon source, nitrogen source, phosphorus, inorganic compound, amino acid, and / or vitamin.

[0158] Specifically, culture media for strains of the genus Corynebacterium can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0159] In the present disclosure, the carbon source may include carbohydrates such as glucose, saccharose, 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 hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane bagasse, and corn steep liquor may be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugar) may be used, and other appropriate amounts of carbon sources may be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0160] The nitrogen source may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc.; peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0161] The above-mentioned components may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added to the medium in batch or continuous manner, but are not limited thereto.

[0162] In addition, during the cultivation of the Corynebacterium genus microorganism of the present disclosure, 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 the cultivation, 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 an oxygen-containing gas may be injected into the medium, or in order to maintain the anaerobic and microaerobic state, nitrogen, hydrogen, or carbon dioxide gas may be injected without gas injection, but is not limited thereto. In one embodiment, the L-lysine production method can be performed at a pH of from 6 to pH 9, and more specifically, at a pH of from 6 to pH 9, from pH 6 to pH 8.5, from pH 6 to pH 8, from pH 6 to pH 7.5, from pH 6 to pH 7.25, from pH 6 to pH 7, from pH 6.5 to pH 9, from pH 6.5 to pH 8.5, from pH 6.5 to pH 8, from pH 6.5 to pH 7.5, from pH 6.5 to pH 7.25, from pH 6.5 to pH 7, from pH 6.75 to pH 9, from pH 6.75 to pH 8.5, from pH 6.75 to pH 8, from pH 6.75 to pH 7.5, from pH 6.75 to pH 7.25, or from pH 6.75 to pH 7, but is not limited thereto.

[0163] In the culture of the present disclosure, the culture temperature may be maintained at 20°C to 45°C, specifically, 25°C to 40°C, 25°C to 40°C, 25°C to 37°C, 25°C to 35°C, 27°C to 40°C, 27°C to 37°C, 27°C to 35°C, 30°C to 40°C, 30°C to 37°C, or 30°C to 35°C, but is not limited thereto. In the culture of the present disclosure, the culture time may be about 10 to 160 hours, but is not limited thereto.

[0164] L-lysine produced by the culture of the present disclosure may be secreted into the medium or remain within the cells.

[0165] The method for producing L-lysine of the present disclosure may additionally include, for example, a step of preparing a microorganism of the genus Corynebacterium of the present disclosure, a step of preparing a medium for culturing the strain, or a combination thereof (in any order), prior to the culturing step.

[0166] The L-lysine production method of the present disclosure may further include a step of recovering L-lysine from a culture medium (a medium in which culture is performed) or a Corynebacterium genus microorganism according to the above-described cultivation. The recovering step may be additionally included after the culturing step.

[0167] The above recovery may be performed by collecting the desired L-amino acid using a suitable method known in the art according to the culture method of the microorganism of the present disclosure, such as a batch, continuous or fed-batch culture method. For example, various chromatographies such as centrifugation, filtration, treatment with a crystallizing protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC or a combination thereof may be used, and the desired L-amino acid may be recovered from the medium or microorganism using a suitable method known in the art.

[0168] Additionally, the L-lysine production method of the present disclosure may additionally include a purification step. The purification may be performed using any suitable method known in the art. In one example, when the L-lysine production method of the present disclosure includes both a recovery step and a purification step, the recovery step and the purification step may be performed sequentially or discontinuously, regardless of order, or may be performed simultaneously or integrated into a single step, but is not limited thereto.

[0169] In the method of the present disclosure, the variants, polynucleotides, vectors, strains, etc. are as described in the other aspects above.

[0170]

[0171] Another aspect of the present disclosure provides a composition for producing L-lysine, 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 microorganism of the present disclosure described above, and a medium for culturing the same.

[0172] The composition of the present disclosure may further comprise any suitable excipient commonly used in compositions for producing amino acids, including, but not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, or isotonic agents.

[0173] In the composition of the present disclosure, the variant, polynucleotide, vector, strain, medium, and L-amino acid, etc. are as described in the other aspects above.

[0174]

[0175] According to another aspect of the present disclosure, the present disclosure may provide a method for increasing L-lysine production ability of a microorganism, a method for imparting L-lysine production ability to a microorganism, and / or a method for producing a microorganism with increased L-lysine production ability, the method comprising a step of introducing (e.g., transforming) into a microorganism a protein variant of the present disclosure described above, a polynucleotide encoding the variant, and / or a vector comprising the polynucleotide.

[0176] In the method for increasing L-lysine production ability of a microorganism of the present disclosure, the method for imparting L-lysine production ability to a microorganism, and / or the method for producing a microorganism with increased L-lysine production ability, the polynucleotide, the recombinant vector, and the microorganism are as described above.

[0177]

[0178] According to another aspect of the present disclosure, the present disclosure provides a method for producing L-lysine, and / or preparing an L-lysine-producing microorganism, and / or imparting and / or increasing L-lysine production ability of a microorganism, wherein the method comprises at least one selected from the group consisting of a protein variant of the present disclosure described above; 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.

[0179] In the above L-lysine production, and / or L-lysine production microorganism production, and / or use for imparting and / or increasing L-lysine production ability of microorganisms, the protein variant, polynucleotide, recombinant vector, and microorganism are as described above.

[0180]

[0181] In another aspect of the present disclosure, the present disclosure provides a composition, method, product, process, or use characterized by one or more elements disclosed in the present disclosure.

[0182]

[0183]

[0184] When culturing a Corynebacterium genus microorganism containing a protein variant of the present disclosure, high yield L-lysine production is possible compared to a microorganism having an existing unmodified polypeptide.

[0185]

[0186] Hereinafter, the present disclosure will be described in more detail through examples. These examples are intended solely to more specifically illustrate the present disclosure, and it will be apparent to those skilled in the art that the scope of the present disclosure is not limited by these examples, in accordance with the gist of the present disclosure.

[0187]

[0188] Example

[0189]

[0190] (Throughout this specification, "%" used to indicate 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 stated.)

[0191]

[0192] Example 1: Selection of a mutant strain with increased lysine production through artificial mutation.

[0193] Example 1-1: Random mutagenesis through UV irradiation

[0194] To select mutant strains with increased lysine production, the lysine-producing strain CJ3P (US 9556463 ​​B2) was spread on agar-containing nutrient medium and cultured at 30°C for 16 hours. The resulting colonies were then irradiated with UV (ultraviolet mutation) at room temperature to induce random mutations in the strain genome. The composition of the nutrient medium is as follows.

[0195] Nutrient medium (pH 7.2)

[0196] 10g glucose, 5g meat extract, 10g polypeptone, 2.5g sodium chloride, 5g yeast extract, 20g agar, 2g urea (per 1 liter of distilled water)

[0197]

[0198] Example 1-2: Selection of strains with improved L-lysine production

[0199] Approximately 3,000 colonies were each inoculated into 300 μl of selective medium and cultured in a 96-deep well plate at 32°C and 1000 rpm for approximately 24 hours. The ninhydrin method was used to analyze the production of L-amino acids produced in the culture medium (J. Biol. Chem. 1948. 176:367-388). After the culture was completed, 10 μl of the culture supernatant and 190 μl of the ninhydrin reaction solution were reacted at 65°C for 30 minutes, and the absorbance was measured using a spectrophotometer at a wavelength of 570 nm. Approximately 30 colonies of mutant strains showing an absorbance increased by 10% or more compared to the absorbance of the control CJ3P strain were selected. The other colonies showed similar or decreased absorbance compared to the control. The composition of the selective medium is as follows.

[0200] <Selection medium (pH 8.0)>

[0201] Glucose 10 g, ammonium sulfate 5.5 g, magnesium sulfate heptahydrate 1.2 g, monobasic potassium phosphate 0.8 g, dibasic potassium 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)

[0202] The 30 selected strains were repeatedly subjected to the ninhydrin reaction in the same manner as described above, and the top 10 strains with improved L-lysine production compared to the CJ3P strain were selected.

[0203]

[0204] Example 1-3: Analysis of L-lysine production of selected mutant strains

[0205] In order to finally select strains with increased L-lysine production ability among the 10 strains selected in the above examples 1-2, a reproducibility test was conducted in a flask using the following medium.

[0206] The above 10 strains and the parent strain were each inoculated into a 250 ml corner-baffle flask containing 25 ml of seed medium and cultured at 30°C for 20 hours with shaking at 200 rpm. 1 ml of the seed culture was inoculated into a 250 ml corner-baffle flask containing 24 ml of production medium and cultured at 30°C for 72 hours with shaking at 200 rpm. The compositions of the seed medium and production medium are as follows.

[0207] <Seed medium (pH 7.0)>

[0208] Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH2PO4 4 g, K2HPO4 8 g, MgSO4·7H2O 0.5 g, biotin 0.1 mg, thiamine HCl 1 mg, calcium-pantothenic acid 2 mg, nicotinamide 2 mg (per 1 liter of distilled water)

[0209] <Production medium (pH 7.0)>

[0210] Glucose 100 g, (NH4)2SO4 40 g, soy protein 2.5 g, corn steep solids 5 g, urea 3 g, KH2PO4 1 g, MgSO4·7H2O 0.5 g, biotin 100 μg, thiamine hydrochloride 1000 μg, calcium pantothenic acid 2000 μg, nicotinamide 3000 μg, CaCO3 30 g (based on 1 liter of distilled water)

[0211] After the culture 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.

[0212] Strain name L-lysine (g / L) CJ3P7.2 CJ3P_mt17.8 CJ3P_mt27.9 CJ3P_mt37.5 CJ3P_mt48.1 CJ3P_mt58.8 CJ3P_mt68.0 CJ3P_mt77.9 CJ3P_mt87.8 CJ3P_mt97.8 CJ3P_mt107.6

[0213] Among the 10 selected mutant strains, CJ3P_mt5 was finally selected as the strain with significantly improved L-lysine production ability.

[0214]

[0215] Example 2. Mutation identification through whole-genome sequencing (WGS)

[0216] Whole-genome sequencing (WGS) was performed on the CJ3P_mt5 strain selected in Example 1 above, and compared with the parent strain CJ3P to identify genes with changes in protein sequence due to nucleotide sequence mutations. The amino acid sequences of the protein variants and the nucleotide sequences of the genes encoding the protein variants are listed in Tables 2 and 3.

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235] Example 3: Production of an L-lysine-producing strain with a mutant gene introduced.

[0236] Example 3-1: Production of a recombinant vector for introducing a mutant gene

[0237] In order to confirm the effects of some of the mutants identified in Example 2 above, a vector capable of introducing them onto a chromosome was constructed.

[0238] Specifically, vectors containing target mutations were constructed to insert each of the NCgl0030, NCgl0288, NCgl0337, NCgl0458, NCgl0686, NCgl0706, NCgl0799, NCgl1165, NCgl1262, NCgl1432, NCgl1531, NCgl1588, NCgl1859, NCgl1874, and NCgl2356 gene mutations into CJ3P.

[0239] Specifically, the genomic DNA of the CJ3P_mt5 strain was extracted using a G-spin Total DNA extraction mini kit (Intron, Cat. No. 17045) according to the protocol provided in the kit, and PCR was performed using the genomic DNA as a template. The polymerase was Solg TM Pfu-X DNA polymerase was used, and the PCR conditions were as follows: denaturation at 95°C for 4 minutes; 27 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 50 seconds; and polymerization reaction was performed at 72°C for 5 minutes. The primer sequences used in the above experiment are shown in Table 4 below.

[0240]

[0241] The obtained mutation introduction fragment and the pDC24 vector (SEQ ID NO: 107, Table 5) treated with the restriction enzyme smaI were cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) to obtain recombinant plasmids, and the vectors PDC24-NCgl0030*, PDC24-NCgl0288*, PDC24-NCgl0337*, PDC24-NCgl0458*, PDC24-NCgl0686*, PDC24-NCgl0706*, PDC24-NCgl0799*, PDC24-NCgl1165*, PDC24-NCgl1262*, They were named PDC24-NCgl1432*, PDC24-NCgl1531*, PDC24-NCgl1588*, PDC24-NCgl1859*, PDC24-NCgl1874*, and PDC24-NCgl2356*.

[0242]

[0243]

[0244] Example 3-2: Production of an L-lysine-producing strain with a mutant gene introduced

[0245] The 15 vectors produced in Example 3-1 were transformed into the lysine-producing strain CJ3P by electroporation, and strains in which the vectors were inserted into the chromosome by recombination of homologous sequences were selected on kanamycin medium. Thereafter, strains into which mutant genes had been introduced were identified by PCR using the primer pairs in Table 6 below for the transformants that had completed the secondary recombination.

[0246] 명칭서열 (5‘->3‘) 서열번호NCgl0030_FGGCGCCGTGCTTGTTTTTAC75NCgl0030_RATCGCTCCTTGTTGAAACGC76NCgl0288_FCCTGCACGTCACCGTCATTT77NCgl0288_RGCCTCAAATGGATCTGACGG78NCgl0337_FTTGCGCGATATTGAAGAGGT79NCgl0337_RGCCCCCGGAAGCTCAAGTTC80NCgl0458_FCTGGAAGAAGAGACCCTAGA81NCgl0458_RCTGATCTTTACGTAACCGGG82NCgl0686_FCCCCAGAAGCACCCGTTGTG83NCgl0686_RCGCCATTGGCCACATCCAAG84NCgl0706_FGATCTGCTTGCTGAACATGAT85NCgl0706_RTCATAAGTGGGCTTTGTTTG86NCgl0799_FGTCTACGTTCTGGTTGCATC87NCgl0799_RAGCTGAGTGGTCAGAGTTGC88NCgl1165_FTTACCAGCCAACCCTGGCTG89NCgl1165_RACTACTTCAGCTAAACGGAG90NCgl1262_FAATCGATGGCTCCGCACTGA91NCgl1262_RACAGAAGTTCGATGTCGGAC92NCgl1432_FGCGTGAAGATCAAGACGCAG93NCgl1432_RCTGGTGGATCGCGGTGGCAA94NCgl1531_FGTAGTGCAAGAACTGGCACG95NCgl1531_RCGCACCACATCTTCTCCATC96NCgl1588_FGCAGGCAATAGGTAGTAAAT97NCgl1588_RCTTAGGTTTAAATATTCGAA98NCgl1859_FAAGCGATCACCATATTTCAT99NCgl1859_RGGGCATCAAAATCAGCCAAG100NCgl1874_FATAGGCGTTTATGTCAGCTC101NCgl1874_RGGGATCTTCGCACGGACCTC102NCgl2356_FCAACCCCATCACCGCCATGA103NCgl2356_RCACCGGTGAAAAGGAGAGAA104

[0247] The above recombinant strains were CJ3P△NCgl0030::NCgl0030*, CJ3P△NCgl0288::NCgl0288*, CJ3P△NCgl0337::NCgl0337*, CJ3P△NCgl0458::NCgl0458*, CJ3P△NCgl0686::NCgl0686*, CJ3P△NCgl0706::NCgl0706*, CJ3P△NCgl0799::NCgl0799*, CJ3P△NCgl1165::NCgl1165*, CJ3P△NCgl1262::NCgl1262*, CJ3P△NCgl1432::NCgl1432*, respectively. They were named CJ3P△NCgl1531::NCgl1531*, CJ3P△NCgl1588::NCgl1588*, CJ3P△NCgl1859::NCgl1859*, CJ3P△NCgl1874::NCgl1874*, and CJ3P△NCgl2356::NCgl2356*.

[0248]

[0249] Example 4. Evaluation of lysine production capacity of L-lysine producing strains into which mutant genes have been introduced.

[0250] In order to confirm the L-lysine production ability of the strain produced in Example 3-2 above, it was cultured and evaluated using the following method.

[0251] Each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of seed medium and cultured at 30°C for 20 hours with shaking at 200 rpm. 1 ml of seed culture was inoculated into a 250 ml corner-baffle flask containing 24 ml of production medium and cultured at 30°C for 72 hours with shaking at 200 rpm.

[0252] <Seed medium (pH 7.0)>

[0253] Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH2PO4 4 g, K2HPO4 8 g, MgSO4·7H2O 0.5 g, biotin 0.1 mg, thiamine HCl 1 mg, calcium-pantothenic acid 2 mg, nicotinamide 2 mg (per 1 liter of distilled water)

[0254] <Production medium (pH 7.0)>

[0255] Glucose 100 g, (NH4)2SO4 40 g, soy protein 2.5 g, corn steep solids 5 g, urea 3 g, KH2PO4 1 g, MgSO4·7H2O 0.5 g, biotin 100 μg, thiamine hydrochloride 1000 μg, calcium pantothenic acid 2000 μg, nicotinamide 3000 μg, CaCO3 30 g (based on 1 liter of distilled water)

[0256] After the culture was completed, the amount of L-lysine produced was measured using HPLC, and the analysis results are shown in Table 7 below.

[0257] Strain name L-lysine concentration (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 △N Cgl1165::NCgl1165*7.5CJ3P△NCgl1262::NCgl1262*7.4CJ3P△NCgl1432::NCgl1432*7.4CJ3P△NCgl1531::NCgl1531*7.6CJ3 P△NCgl1588::NCgl1588*7.7CJ3P△NCgl1859::NCgl1859*8.3CJ3P△NCgl1874::NCgl1874*7.6CJ3P△NCgl2356::NCgl2356*7.5

[0258] As a result, as shown in Table 7 above, it was confirmed that most of the lysine-producing strains into which genetic variants were introduced had lysine production capabilities equivalent to or greater than those of the parent strain. In particular, it was confirmed that CJ3P△NCgl1859::NCgl1859* had a significantly increased lysine production capability compared to the parent strain CJ3P. Through this, it was confirmed that L-lysine could be produced more efficiently by introducing a variant in which the 75th amino acid of the NCgl1859 gene was substituted from glycine to valine.

[0259] From the above description, those skilled in the art will understand that the present disclosure can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present disclosure should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

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

2. In the first paragraph, 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. In the first paragraph, the protein variant comprises an amino acid sequence of SEQ ID NO:

15.

4. A polynucleotide encoding the protein variant of paragraph 1.

5. A microorganism of the genus Corynebacterium, comprising at least one selected from the group consisting of a protein variant of paragraph 1 and a polynucleotide encoding the protein variant.

6. In the fifth paragraph, the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.

7. In the fifth paragraph, the microorganism has increased L-lysine production ability compared to a microorganism of the genus Corynebacterium that does not include at least one member selected from the group consisting of the protein variant and a polynucleotide encoding the protein variant.

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

9. A composition for producing L-lysine, wherein the protein variant comprises an amino acid sequence of SEQ ID NO:

15.

10. A composition for producing L-lysine in claim 8, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.

11. A method for producing L-lysine, comprising a step of culturing the microorganism of clause 5 in a medium.

12. A method for producing L-lysine in claim 11, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.

13. A method for producing L-lysine, wherein the method further comprises a step of recovering L-lysine from a cultured medium or microorganism in accordance with claim 11.

14. A method for producing L-lysine, wherein the method is performed at pH 6 to pH 9 in the 11th paragraph.

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

16. A composition, method, product, process, or use characterized by one or more elements disclosed herein.

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