Variant of protein and method for producing l-isoleucine by using same

Abstract

The present disclosure relates to: a variant of a protein; a polynucleotide encoding the variant of a protein; a microorganism of the genus Corynebacterium, comprising at least one selected from the group consisting of the variant of a protein and the polynucleotide; and a method for producing L-isoleucine by using the microorganism.

Description

Protein variant and method for producing L-isoleucine using the same

[0001] This disclosure claims the benefit of priority to Republic of Korea Patent Application No. 10-2024-0066104, dated May 21, 2024, and Republic of Korea Patent Application No. 10-2024-0133999, dated October 2, 2024, the entire contents of which are incorporated herein by reference.

[0002] 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.

[0003] The present disclosure relates to a microorganism of the genus Corynebacterium comprising at least one selected from the group consisting of a novel protein variant, a polynucleotide encoding the protein variant, the protein variant and a polynucleotide encoding the same, and a method for producing L-isoleucine using the microorganism.

[0004] L-isoleucine (Ile, I) is classified as an essential amino acid and is a type of branched-chain amino acid among the 20 essential amino acids. It is used in the manufacture of various products such as animal feed, food additives, and pharmaceuticals. Because L-isoleucine functions in post-metabolism energy production, hemoglobin production, blood sugar regulation, muscle building and repair, etc., its use is increasing not only in fluids, nutritional supplements, and sports nutrition, but also in animal feed.

[0005] Based on this trend, various attempts are being made to improve productivity in the production of L-isoleucine using various microorganisms, including microorganisms of the genus Corynebacterium (US6072083A).

[0006] Despite these efforts, the development of technologies to improve the production of L-isoleucine is still required.

[0007] One object of the present disclosure is to provide novel protein variants.

[0008] Another object of the present disclosure is to provide a polynucleotide encoding the protein variant.

[0009] 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.

[0010] Another object of the present disclosure is to provide a method for producing L-isoleucine, comprising a step of culturing the microorganism in a medium.

[0011] Another object of the present disclosure is to provide a use of the microorganism for producing L-isoleucine.

[0012] 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 ascertain, through routine experimentation alone, numerous equivalents to the specific embodiments of the present disclosure described herein. Furthermore, such equivalents are intended to be encompassed by this disclosure.

[0013] Additionally, numerous papers and patents are referenced and cited throughout this specification. 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.

[0014]

[0015] One aspect of the present disclosure provides a protein variant comprising an amino acid sequence in which histidine (His, H), glutamic acid (Glu, E) and glycine (Gly, G) are inserted between the amino acid corresponding to the 15th position from the N-terminus and the amino acid corresponding to the 16th position in the amino acid sequence of SEQ ID NO: 52.

[0016] In the present disclosure, the sequence of sequence number 52 is as shown in Table 1 below.

[0017] Protein name (gene ID, gene name) Sequence (N-terminal → C-terminal) Sequence number Transporter (NCgl2828) MMNSMSQAIDSKVEAHEGHEGHEGIERGTRNYKRAVFAMLAAGLAAFNGLYCTQALLPTMTEELGITPTESALTVSATTGMLALCIVPASILSEKFGRGRVLTISLTLAIIVGLILPLVPNITALILLRGLQGALLAGTPAVAMTWLSEEIHPKDIGHAMGIYIAGNTVGGLTGRMIPAGLLEVTHWQNALLGS SIAALIFGVIMVVLLPKQRKFQPKNINLRHEISAMAAHWRNPRLALLFGTAFLGMGTFVSLYNYLGFRMIDQFGLSEVLVGAVFIMYLAGTWSSTQAGALREKIGNGSTVIF LSLTMIASMALMGINNLWVTLVALFVFTAAFFALHSSASGWIGIIATKDRAEASSMYLFCYYVGSSVIGWVSGFAFTHLPWLAFIGWLILLLCGVLAICVTLARLARNAN52

[0018] In one embodiment, the protein variant of the present disclosure described above may include an amino acid sequence in which histidine (His, H), glutamic acid (Glu, E) and glycine (Gly, G) are inserted between the amino acid corresponding to the 15th position from the N-terminus and the amino acid corresponding to the 16th position in the amino acid sequence of SEQ ID NO: 52. The protein variant of the present disclosure described above may essentially consist of an amino acid sequence in which histidine (His, H), glutamic acid (Glu, E) and glycine (Gly, G) are inserted between the amino acid corresponding to the 15th position from the N-terminus and the amino acid corresponding to the 16th position in the amino acid sequence of SEQ ID NO: 52. In another embodiment, the protein variant of the present disclosure described above may consist of an amino acid sequence in which histidine (His, H), glutamic acid (Glu, E) and glycine (Gly, G) are inserted between the amino acid corresponding to the 15th position from the N-terminus and the amino acid corresponding to the 16th position in the amino acid sequence of SEQ ID NO: 52.

[0019] The above histidine (His, H), glutamic acid (Glu, E), and glycine (Gly, G) may be inserted in the order of histidine-glutamic acid-glycine.

[0020] In the present disclosure, the amino acid sequence of SEQ ID NO: 52 may be a sequence of a transporter protein. In one embodiment, the transporter protein may be a protein encoded by the NCgl2828 gene.

[0021] In one embodiment, the amino acid corresponding to the 15th position from the N-terminus in the amino acid sequence of SEQ ID NO: 52 may be, but is not limited to, alanine (Ala, A).

[0022] In one embodiment, the amino acid corresponding to the 16th position from the N-terminus in the amino acid sequence of SEQ ID NO: 52 may be, but is not limited to, histidine (His, H).

[0023] The protein variant of the present disclosure may have an activity that increases L-isoleucine production compared to the wild type protein (polypeptide).

[0024] As used herein, the term "variant" refers to a polypeptide in which one or more amino acids or nucleotides are conservatively substituted and / or modified, thereby differing from the amino acid sequence or polypeptide 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 or one or more nucleotides in the nucleotide sequence of the polynucleotide, and evaluating the properties of the modified polypeptide or polynucleotide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the polypeptide or polynucleotide before the mutation. In addition, some variants may include variants in which one or more portions, such as the N-terminal leader sequence or transmembrane domain, or the nucleotide sequence portion encoding the leader sequence or transmembrane domain, are deleted. Other variants may include variants in which a portion or a portion of the nucleotide sequence encoding the same is deleted from the N- and / or C-terminus of the mature protein. The term "variant" may be used interchangeably with terms such as variant, modification, variant polypeptide, modified protein, variant (mutant) polynucleotide, variant, and variant (in English, modification, modified polypeptide, modified protein, modified polynucleotide, mutant, mutein, divergent, etc.), and is not limited thereto as long as the term is used in the meaning of variant.

[0025] Additionally, the variant may comprise deletions or additions of amino acids or nucleotides (in the case of a polynucleotide encoding said polypeptide) 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 or a nucleotide sequence encoding the same that is involved in co-translational or post-translational protein translocation. Furthermore, the variant may be conjugated with other sequences or linkers to enable identification, purification, or synthesis.

[0026] In one embodiment, the protein variant of the present disclosure described above may have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.25%, 99.275%, or 99.28% homology or identity with the amino acid sequence of SEQ ID NO: 52; and less than 100% homology or identity.

[0027] More specifically, the protein variant of the present disclosure described above may have histidine (His, H), glutamic acid (Glu, E) and glycine (Gly, G) inserted between the amino acid corresponding to the 15th position from the N-terminus and the amino acid corresponding to the 16th position in the amino acid sequence of SEQ ID NO: 52, and may have a homology or identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, at least 99.1%, at least 99.2%, at least 99.25%, at least 99.275%, or at least 99.28%; and less than 100% with the amino acid sequence of SEQ ID NO: 52.

[0028] 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-isoleucine production).

[0029] 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.

[0030] 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.

[0031]

[0032] 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 to SEQ ID NO: 5. In one embodiment, the protein variant may be a protein variant comprising the amino acid sequence of SEQ ID NO: 4. In another embodiment, the protein variant of the present disclosure may essentially consist of the amino acid sequence of SEQ ID NO: 4. In yet another embodiment, the variant of the present disclosure may consist of the amino acid sequence of SEQ ID NO: 4.

[0033] In the present disclosure, the amino acid sequence of sequence number 1 may be a sequence of a protein variant in which the 118th amino acid of a protein (membrane protein) encoded by the NCgl0214 gene is substituted from alanine (Ala, A) to valine (Val, V).

[0034] In the present disclosure, the amino acid sequence of sequence number 2 may be a sequence of a protein variant in which the 38th amino acid of a protein (hypothetical protein) encoded by the NCgl0216 gene is substituted from alanine (Ala, A) to threonine (Thr, T).

[0035] In the present disclosure, the amino acid sequence of SEQ ID NO: 3 may be a sequence of a protein variant in which the 303rd amino acid of a protein (ABC-type transporter, duplicated ATPase subunit) encoded by the NCgl2176 gene is substituted from valine (Val, V) to methionine (Met, M).

[0036] In the present disclosure, the amino acid sequence of SEQ ID NO: 4 may be a sequence of a protein variant in which histidine (His, H), glutamic acid (Glu, E), and glycine (Gly, G) are inserted between the 15th amino acid, alanine (Ala, A), and the 16th amino acid, histidine (His, H), of the protein (transporter) encoded by the NCgl2828 gene.

[0037] In the present disclosure, the amino acid sequence of sequence number 5 is a sequence of a mutant of a protein (chorismate mutase) encoded by the NCgl0819 gene (csm), and may be a sequence of a mutant in which the first nucleotide of the NCgl0819 gene (csm) is substituted from A to G.

[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 to SEQ ID NO: 5 (e.g., the amino acid sequence of SEQ ID NO: 4), 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-isoleucine production).

[0039] 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.

[0040] In the above protein variants, conservative substitutions, variants, etc. are as described above.

[0041]

[0042] Another aspect of the present disclosure provides a polynucleotide variant comprising any one nucleotide sequence selected from the group consisting of polynucleotide sequences of SEQ ID NO: 11 to SEQ ID NO: 14. The polynucleotide variant may be a variant encoding rRNA. The rRNA encoded in the polynucleotide variant may have improved L-isoleucine production ability (e.g., increased production amount, increased production rate, increased production yield, etc.) compared to a pre-mutation RNA (e.g., a corresponding wild-type RNA).

[0043] In one embodiment, the polynucleotide variant of the present disclosure may consist essentially of any one nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO: 11 to SEQ ID NO: 14. In another embodiment, the polynucleotide variant of the present disclosure may consist of any one nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO: 11 to SEQ ID NO: 14.

[0044] The polynucleotide variant of the present disclosure may comprise any one nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO: 11 to SEQ ID NO: 14, or 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 said nucleotide sequence. In addition, it is obvious that a variant 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, as long as it is a nucleotide sequence having such homology or identity and exhibits an efficacy corresponding to the variant of the present disclosure (efficacy of increasing L-isoleucine production).

[0045] For example, if the nucleotide 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 5'-end, 3'-end and / or within the nucleotide sequence.

[0046] In the above nucleotide variants, conservative substitutions, variants, etc. are as described above.

[0047]

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] As an example of the present disclosure, the variant of the present disclosure described above may have an activity that increases L-isoleucine production ability compared to a wild type protein (polypeptide).

[0053]

[0054] As used herein, the term "corresponding to" refers to an amino acid residue or nucleotide at a position listed in a polypeptide or polynucleotide, or an amino acid residue or nucleotide that is similar, identical, or homologous to the residue listed in the polypeptide or polynucleotide. Identifying an amino acid or nucleotide at a corresponding position may determine a specific amino acid or nucleotide in a sequence that references a particular sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related protein, nucleic acid molecule, or reference protein or nucleic acid molecule.

[0055] For example, any amino acid sequence; or an amino acid sequence in which histidine (His, H), glutamic acid (Glu, E) and glycine (Gly, G) are inserted between the amino acid corresponding to the 15th position from the N-terminus and the amino acid corresponding to the 16th position in the amino acid sequence of SEQ ID NO: 52, or any amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 5 (e.g., the amino acid sequence of SEQ ID NO: 4); Or, the nucleotide sequence of SEQ ID NO: 6 to SEQ ID NO: 14 is aligned, and based on this, each amino acid residue of the amino acid sequence is numbered by referring to the numerical position of the amino acid residue or nucleotide selected from the group consisting of an amino acid sequence in which histidine (His, H), glutamic acid (Glu, E) and glycine (Gly, G) is inserted between the amino acid corresponding to the 15th position from the N-terminus in the amino acid sequence of SEQ ID NO: 52 and the amino acid sequence corresponding to the 16th position; or an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 5 (e.g., an amino acid sequence of SEQ ID NO: 4); or a nucleotide of the nucleotide sequence of SEQ ID NO: 6 to SEQ ID NO: 14. For example, a sequence alignment algorithm such as that described in the present disclosure can identify the position of an amino acid or nucleotide, or a position where a modification such as a substitution, insertion or deletion occurs, by comparing it with a query sequence (also referred to as a “reference sequence”).

[0056] 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.

[0057]

[0058] Another aspect of the present disclosure provides a polynucleotide encoding a protein variant of the present disclosure.

[0059] 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.

[0060] In one example, the polynucleotide may be a polynucleotide encoding a protein variant comprising an amino acid sequence in which histidine (His, H), glutamic acid (Glu, E) and glycine (Gly, G) are inserted between the amino acid corresponding to the 15th position from the N-terminus and the amino acid corresponding to the 16th position in the amino acid sequence of SEQ ID NO: 52.

[0061] In one example, the polynucleotide may comprise a polynucleotide comprising any one nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO: 6 to SEQ ID NO: 10, for example, the nucleotide sequence of SEQ ID NO: 9.

[0062] A polynucleotide encoding a protein variant of the present disclosure may have or comprise any one nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NO: 6 to SEQ ID NO: 10, for example, the nucleotide sequence of SEQ ID NO: 9. 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-isoleucine production).

[0063]

[0064] 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.

[0065] 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 has or includes a base sequence 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 less than 100% homology or identity with any one nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO: 6 to SEQ ID NO: 10 (e.g., the nucleotide sequence of SEQ ID NO: 9), or may consist of or consist essentially of a base sequence 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 less than 100% homology or identity with said nucleotide sequence, but is not limited thereto.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070]

[0071] Another aspect of the present disclosure provides a vector comprising a polynucleotide or polynucleotide variant of the present disclosure. The vector may be, but is not limited to, an expression vector for expressing the polynucleotide or polynucleotide variant in a host cell.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077]

[0078] 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, a polynucleotide of the present disclosure, and a polynucleotide variant of the present disclosure.

[0079] The microorganism of the present disclosure may comprise a protein variant of the present disclosure, a polynucleotide encoding the protein variant, a polynucleotide of the present disclosure, or a vector comprising a polynucleotide variant of the present disclosure.

[0080] 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.

[0081] 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, a polynucleotide of the present disclosure, and a vector comprising a polynucleotide variant of the present disclosure; a microorganism modified to express a protein variant of the present disclosure, a polynucleotide of the present disclosure, or a polynucleotide variant of the present disclosure; a microorganism (e.g., a recombinant microorganism) expressing a variant of the present disclosure, a polynucleotide of the present disclosure, or a polynucleotide variant of the present disclosure; or a microorganism (e.g., a recombinant microorganism) having a protein variant activity or a polynucleotide variant activity of the present disclosure.

[0082] The microorganism of the present disclosure may be a microorganism having the ability to produce L-isoleucine.

[0083] The microorganism of the present disclosure may have an increased ability to produce L-isoleucine 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 described above, the polynucleotide encoding the protein variant, and the polynucleotide variant of the present disclosure (i.e., an amino acid sequence in which histidine (His, H), glutamic acid (Glu, E) and glycine (Gly, G) are inserted between the amino acid corresponding to the 15th position from the N-terminus in the amino acid sequence of SEQ ID NO: 52 and the amino acid corresponding to the 16th position; or a microorganism that does not express a protein variant including any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 5, for example, the amino acid sequence of SEQ ID NO: 4, a microorganism including the nucleotide sequence of SEQ ID NO: 6 to 14, a microorganism expressing a wild-type protein corresponding to the protein variant, or a microorganism expressing a wild-type polynucleotide corresponding to the polynucleotide variant). there is.

[0084] The microorganism of the present disclosure may be a microorganism that naturally has the ability to produce L-isoleucine, or a microorganism that has been introduced with a protein variant of the present disclosure, a polynucleotide encoding the same, or a polynucleotide variant of the present disclosure (or a vector including the polynucleotide or polynucleotide variant) and / or has been endowed with the ability to produce L-isoleucine, but is not limited thereto.

[0085] For example, the microorganism of the present disclosure is a cell or microorganism that is transformed with a polynucleotide of the present disclosure, a polynucleotide encoding a variant of the present disclosure, or a vector comprising a polynucleotide variant of the present disclosure, thereby expressing 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-isoleucine, including the variant of the present disclosure. For example, the microorganism of the present disclosure may be a recombinant microorganism whose L-isoleucine production ability is increased by introducing a polynucleotide encoding a protein variant of the present disclosure or a polynucleotide variant of the present disclosure into a naturally occurring wild-type microorganism or a microorganism that produces L-isoleucine.

[0086] In one specific example, the microorganism of the present disclosure may have increased L-isoleucine production ability compared to a microorganism of the genus Corynebacterium that does not comprise at least one member selected from the group consisting of a protein variant of the present disclosure, a polynucleotide encoding the protein variant, and a polynucleotide variant of the present disclosure. The microorganism of the genus Corynebacterium that does not comprise at least one member selected from the group consisting of a protein variant of the present disclosure, a polynucleotide encoding the protein variant, and a polynucleotide variant of the present disclosure may be a natural wild-type microorganism or an unmodified microorganism, and may also be referred to as a parent strain.

[0087] In the present application, the microorganism (unmodified microorganism) as a host cell may be (1) a microorganism that naturally has L-isoleucine production ability, (2) a microorganism that naturally has no or significantly low L-isoleucine production ability, and / or (3) a microorganism that has a mutation introduced (transformed) into the microorganism that naturally has L-isoleucine production ability or the microorganism that has no or significantly low L-isoleucine production ability, so that the microorganism has L-isoleucine production ability or has improved L-isoleucine production ability. In one specific example, the microorganism as a host cell may be, but is not limited to, Corynebacterium glutamicum KCCM12739P or Corynebacterium glutamicum KCCM11248P (Korean Patent No. 10-1335789).

[0088] In one specific example, the microorganism (mutant microorganism or microorganism as a host cell) may be, but is not limited to, a microorganism whose biosynthetic pathway of L-isoleucine has been additionally strengthened to increase the production of L-isoleucine.

[0089] In one example, the microorganism may be a microorganism in which feedback inhibition of L-threonine dehydratase is relieved and / or feedback inhibition of homoserine dehydrogenase is relieved.

[0090] In one specific example, the microorganism may be a microorganism in which feedback inhibition of L-threonine dehydratase is released.

[0091] The term “threonine dehydratase (EC 4.3.1.19)” in this application refers to an enzyme that produces 2-ketobutyrate from threonine, which is encoded by the ilvA gene and is known to be subject to feedback inhibition by L-isoleucine. Microorganisms of the genus Corynebacterium synthesize L-isoleucine through three intermediate metabolites using pyruvate and 2-ketobutyrate as precursors. The amino acid sequence of the threonine dehydratase can be obtained from the known database, NCBI’s GenBank or US 2023-0098971 A1 and US 10982244 B2.

[0092] In another embodiment, the microorganism may be one in which feedback inhibition of homoserine dehydrogenase is released.

[0093] The term "homoserine dehydrogenase (EC: 1.1.1.3)" used in this application refers to an enzyme encoded by the hom gene that catalyzes the synthesis of homoserine. Homoserine dehydrogenase is known to be subject to feedback inhibition by isoleucine. The amino acid sequence of homoserine dehydrogenase can be obtained from the known databases, such as NCBI's GenBank or US 10982244 B2.

[0094] In one specific example, the microorganism may be one in which feedback inhibition of aspartate kinase is released. The term “aspartate kinase (EC: 2.7.2.4)” in the present application is encoded by the lysC gene, and the amino acid sequence of the aspartate kinase can be obtained from a known database such as NCBI’s GenBank or US 10662450 B2.

[0095] As used herein, the term, release of feedback inhibition, may mean that the activity of a polypeptide, protein or enzyme is increased or enhanced compared to its intrinsic activity, or that the activity is not inhibited compared to its intrinsic activity. In one specific example, the microorganism may be a microorganism that further includes, but is not limited to, any one or more mutations selected from the group consisting of additional introduction of a genetic mutation (R407H) into the hom gene (US 10982244 B2), additional introduction of a genetic mutation (T381A, F383A and / or V323A) into the ilvA gene (US 2023-0098971 A1, US 10982244 B2), and additional introduction of a genetic mutation (L377K) into the lysC gene encoding aspartokinase (US Registered Publication No. US 10662450 B2).

[0096] In one embodiment, the microorganism with increased L-isoleucine production of the present disclosure may have an L-isoleucine production ability increased by about 1% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 42.5% or more, about 42.75% or more, or about 42.8% 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) compared to the parent strain before mutation or the unmodified microorganism, but is not limited thereto. In another embodiment, the microorganism with increased L-isoleucine production of the present disclosure may have an L-isoleucine production ability increased by about 1.01 times or more, about 1.1 times or more, about 1.2 times or more, about 1.3 times or more, about 1.4 times or more, about 1.425 times or more, about 1.4275 times or more, or about 1.428 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.

[0097] 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.

[0098] 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.

[0099]

[0100] 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.

[0101] 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.

[0102] 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.).

[0103] Specifically, the weakening of the polypeptide of the present disclosure is

[0104] 1) Deletion of all or part of a gene encoding a polypeptide;

[0105] 2) Modification of the expression control region (or expression control sequence) so as to reduce the expression of the gene encoding the polypeptide;

[0106] 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;

[0107] 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);

[0108] 5) Modification of the base sequence encoding the initiation codon or 5'-UTR region of a gene transcript encoding a polypeptide;

[0109] 6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to a transcript of the gene encoding the polypeptide;

[0110] 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;

[0111] 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

[0112] 9) Control of cellular localization of proteins (polypeptides); or

[0113] 10) It may be a combination of two or more selected from 1) to 9), but is not particularly limited thereto.

[0114] for example,

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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].

[0120] 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.

[0121] 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.

[0122] 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.

[0123] Such attenuation of polypeptide activity may include, but is not limited to, a decrease in 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 a decrease in the amount of a product produced from the polypeptide.

[0124]

[0125] 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.

[0126] 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.

[0127] 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 is 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.).

[0128] Specifically, the enhancement of the polypeptide of the present disclosure is

[0129] 1) Increase in the intracellular copy number of a polynucleotide encoding a polypeptide;

[0130] 2) Replacing the gene expression control region on the chromosome that codes for a polypeptide with a highly active sequence;

[0131] 3) Modification of the base sequence encoding the initiation codon or 5'-UTR region of a gene transcript encoding a polypeptide;

[0132] 4) Modification of the amino acid sequence of the polypeptide so as to enhance polypeptide activity;

[0133] 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);

[0134] 6) Introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same;

[0135] 7) Codon optimization of polynucleotides encoding polypeptides;

[0136] 8) Analyzing the tertiary structure of the polypeptide and selecting the exposed portion to modify or chemically modify; or

[0137] 9) Control of cellular localization of proteins (polypeptides); or

[0138] 10) It may be a combination of two or more selected from 1) to 9), but is not particularly limited thereto.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149]

[0150] 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.

[0151] In the microorganism of the present disclosure, the protein variant, polynucleotide, L-isoleucine, etc. are as described in the other aspects above.

[0152]

[0153] Another aspect of the present disclosure provides a method for producing L-isoleucine, comprising the 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 polynucleotide variant of the present disclosure in a medium.

[0154] The method for producing L-isoleucine 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, a polynucleotide variant of the present disclosure, or a vector of the present disclosure in a medium.

[0155] 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.

[0156] 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.

[0157] 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)].

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] In the culture of the present disclosure, the culture temperature may be 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.

[0163] L-amino acids produced by the culture of the present invention may be secreted into the medium or remain within the cells.

[0164] The method for producing L-amino acids 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.

[0165] The method for producing L-amino acids of the present disclosure may further include a step of recovering L-amino acids from a culture medium (a medium in which culture is performed) or a Corynebacterium genus microorganism according to the above-described culturing step. The recovering step may be additionally included after the culturing step.

[0166] 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.

[0167] Additionally, the L-amino acid 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-amino acid 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.

[0168] In the method of the present disclosure, the variants, polynucleotides, polynucleotide variants, vectors, strains, etc. are as described in the other aspects above.

[0169]

[0170] Another aspect of the present disclosure provides a composition for producing L-isoleucine, comprising a microorganism of the genus Corynebacterium, comprising at least one selected from the group consisting of a protein variant of the present disclosure, a polynucleotide encoding the protein variant, a polynucleotide variant of the present disclosure, and a vector comprising the polynucleotide or the polynucleotide variant; a medium for culturing the microorganism; or a combination of two or more thereof.

[0171] 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.

[0172] In the composition of the present disclosure, the variant, polynucleotide, vector, strain, medium, and L-isoleucine, etc. are as described in the other embodiments above.

[0173]

[0174] According to another aspect of the present disclosure, the present disclosure may provide a method for increasing L-isoleucine production ability of a microorganism, a method for imparting L-isoleucine production ability to a microorganism, or a method for producing a microorganism with increased L-isoleucine production ability, comprising a step of introducing (e.g., transforming) into a microorganism a novel protein variant of the present disclosure described above, a polynucleotide encoding the variant, a polynucleotide variant of the present disclosure, and / or a recombinant vector comprising the polynucleotide or polynucleotide variant.

[0175] In the method for producing a microorganism of the present disclosure, the protein variant, the polynucleotide encoding the same, the polynucleotide variant, the recombinant vector, and the microorganism are as described above.

[0176]

[0177] According to another aspect of the present disclosure, the present disclosure provides a method for producing L-isoleucine, and / or preparing an L-isoleucine-producing microorganism, and / or imparting and / or increasing L-isoleucine production ability to 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, the polynucleotide encoding the protein variant, and / or the recombinant vector comprising the polynucleotide.

[0178] In the use for producing L-isoleucine, and / or manufacturing an L-isoleucine-producing microorganism, and / or imparting and / or increasing the L-isoleucine production ability of a microorganism, the protein variant, polynucleotide, recombinant vector, and microorganism are as described above.

[0179]

[0180] 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.

[0181] When culturing a microorganism of the genus Corynebacterium comprising the novel protein variant of the present disclosure, high yields of L-isoleucine can be produced compared to microorganisms having conventional unmodified polypeptides.

[0182] The present disclosure is described in more detail below through examples. However, the following examples are merely illustrative embodiments of the present disclosure and are therefore not intended to limit the scope of the present disclosure. Furthermore, technical details not described in this specification can be readily understood and implemented by those skilled in the technical field of the present disclosure or similar technical fields.

[0183] 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 noted.

[0184]

[0185] Example

[0186] Example 1. Selection of mutant strains with increased isoleucine production through artificial mutation.

[0187] Example 1-1. Random mutation induction through UV irradiation

[0188] To select mutant strains with increased isoleucine production, the isoleucine-producing strain, Corynebacterium glutamicum KCCM12739P, was spread on a nutrient medium containing agar and cultured at 30°C for 16 hours. The colonies thus obtained were irradiated with UV (Ultraviolet mutation) at room temperature to induce random mutations in the genome of the strain.

[0189] Nutrient medium (pH 7.2)

[0190] 10g glucose, 5g meat extract, 10g polypeptone, 2.5g sodium chloride, 5g yeast extract, 20g agar, 2g urea (per 1 liter of distilled water)

[0191]

[0192] Example 1-2: Selection of strains with improved L-isoleucine production

[0193] In order to select mutant strains with increased isoleucine production compared to the parent strain KCCM12739P, the KCCM12739P strain and the mutant strains in which random mutations were induced were cultured using the following method.

[0194] The above strains were each inoculated into a 96-Deep Well Plate-Dome (Bioneer) containing 400 μl of seed medium and cultured in a plate shaking incubator (TAITEC) at 32°C and 1200 rpm for approximately 48 hr. The isoleucine concentrations of approximately 3,000 cultured strains were individually confirmed by NIR (near-infrared spectroscopy), and the top five mutant strains with enhanced isoleucine production compared to the parent strain KCCM12739P were selected.

[0195] <Seed medium (pH 7.0)>

[0196] 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 100 μg, thiamine HCl 1000 μg, calcium-pantothenic acid 2000 μg, nicotinamide 2000 μg (based on 1 liter of distilled water)

[0197]

[0198] To select the strains with reproducibly increased L-isoleucine production ability among the five selected mutant strains, they were cultured and evaluated using the following method.

[0199] The parent strain and the mutant strains were inoculated into a 250 mL corner-bottom flask containing 25 mL of isoleucine production medium, and then cultured with shaking at 200 rpm at 32°C for 60 hours. After culture was completed, the L-isoleucine concentration in the culture solution was analyzed using HPLC, and the L-isoleucine production concentration of each mutant strain is shown in Table 2 below.

[0200] <Production medium (pH 7.2)>

[0201] Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, potassium phosphate monobasic 0.1%, magnesium sulfate heptahydrate 0.1%, iron sulfate heptahydrate 10 mg / ℓ, manganese sulfate monohydrate 10 mg / ℓ, biotin 200 μg / ℓ (based on 1L of distilled water)

[0202] Strain nameL-isoleucine (g / L)KCCM12739P2.0KCCM12739P_mt12.8KCCM12739P_mt22.6KCCM12739P_mt32.7KCCM12739P_mt43.6KCCM12739P_mt52.5

[0203] Among the five selected mutant strains, KCCM12739P_mt4 was finally selected as the strain with significantly improved L-isoleucine production ability.

[0204]

[0205] Example 2: Mutation Identification through Whole-Genome Sequencing (WGS)

[0206] Whole-genome sequencing (WGS) was performed on the KCCM12739P_mt4 strain selected in the above Example 1-2 to analyze the sequence, and by comparing it with the parent strain KCCM12739P, the location where mutations occurred in the base sequence within the CDS region or rRNA region was identified, and each substitution form is indicated in Tables 3 to 5 below.

[0207] Mutation in CDS (Coding Sequence) region Wild strain Amino acid Protein Mutation position Mutation strain Amino acid gene ID Gene Name Expressed protein Mutant amino acid sequence Sequence number Mutant nucleic acid sequence Sequence number A118VNCgl0214 - membrane protein 16 A38TNCgl0216 - hypothetical protein 27 V303MNCgl2176 - ABC-type transporter, duplicated ATPase subunit 38 Insertion of HEG between amino acids at positions 15 and 16 (mutation due to insertion of "CACGAAGGC" between nucleotides at positions 45 and 46 of the gene sequence) NCgl28 28 - transporter 49 M (atg) 1 M (gtg) NCgl0819 csm Chorismate mutase 5 10 (mutation due to substitution of nucleotide 1 of the gene sequence with G)

[0208] rRNA region mutation gene ID variant nucleic acid sequence sequence number NCg1r0511 NCg1r0912 NCg1r1613 NCg1r1914

[0209]

[0210]

[0211]

[0212]

[0213]

[0214] In the following examples, the effect of each mutant listed in Tables 3 to 5 on the L-isoleucine production ability of a Corynebacterium genus microorganism was evaluated to identify effective factors affecting the L-isoleucine production ability.

[0215]

[0216] Example 3: Production of an L-isoleucine-producing strain with a mutant gene introduced.

[0217] Example 3-1: Production of a recombinant vector for introducing a mutant gene

[0218] In order to confirm the effect of the mutant identified in Example 2 above, a vector capable of introducing it onto a chromosome was created.

[0219]

[0220] Specifically, vectors containing target mutations were constructed to insert each of the NCgl0214, NCgl0216, NCgl2176, NCgl2828, NCgl0819, NCg1r05, NCg1r09, NCg1r16, and NCg1r19 gene mutations into KCCM12739P.

[0221] Specifically, the genomic DNA of the KCCM12739P_mt4 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 2 minutes; and polymerization at 72°C for 5 minutes. Each PCR result was obtained using primers of SEQ ID NO: 16 and SEQ ID NO: 17; or SEQ ID NO: 18 and SEQ ID NO: 19; or SEQ ID NO: 20 and SEQ ID NO: 21; or SEQ ID NO: 22 and SEQ ID NO: 23; or SEQ ID NO: 24 and SEQ ID NO: 25; or SEQ ID NO: 26 and SEQ ID NO: 27; or SEQ ID NO: 28 and SEQ ID NO: 29; or SEQ ID NO: 30 and SEQ ID NO: 31; or SEQ ID NO: 32 and SEQ ID NO: 33. The primer sequences used in the above experiment are as shown in Table 7 below.

[0222] The obtained mutation introduction fragment and the pDC24 vector (SEQ ID NO: 15, WO2024-242409 A1, Table 6) 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-NCgl0214*, pDC24-NCgl0216*, pDC24-NCgl2176*, pDC24-NCgl2828*, pDC24-NCgl0819*, pDC24-NCg1r05*, pDC24-NCg1r09*, pDC24-NCg1r16* and pDC24-NCg1r19* containing each mutation introduction fragment were cloned. Named.

[0223]

[0224]

[0225] Sequence number name sequence (5'->3')16NCgl0214_FtgaattcgagctcggtacccATGCCACGTTTTCCC17NCgl0214_RgtcgactctagaggatccccGCTGGGTAGTCTTTG18N Cgl0216_FtgaattcgagctcggtacccTTAGGACAGGGACGC19NCgl0216_RgtcgactctagaggatccccAGAACCATTAGATTT20NCgl2176_Ftga attcgagctcggtacccGCTTCTACGAAGAAC21NCgl2176_RgtcgactctagaggatccccATCGGTTTTAAGAGC22NCgl2828_Ftgaattcgagctcgg tacccTACGTTGGGGTGTTC23NCgl2828_RgtcgactctagaggatccccGCCGGAATCATACGT24NCgl0819_FtgaattcgagctcggtacccGCCCTGG TCAGGGTAT25NCgl0819_RgtcgactctagaggatccccCCACCCACATGCGCT26NCg1r05_FtgaattcgagctcggtacccACCTGCCGTAGAAGG27NC g1r05_RgtcgactctagaggatccccAGCCGGGCGATCTGGG28NCg1r09_FtgaattcgagctcggtacccAGGCGTGATGGCGGAG29NCg1r09_Rgtcgact ctagaggatccccGCCCCAGTTAAACTA30NCg1r16_FtgaattcgagctcggtacccTAATAAAGCTCCTTA31NCg1r16_RgtcgactctagaggatccccA CTGTCACCTTTGTG32NCg1r19_FtgaattcgagctcggtacccCTACGAGCTCTTTAC33NCg1r19_RgtcgactctagaggatccccTTTTTTAGAATCATT

[0226]

[0227] Example 3-2: Production of an L-isoleucine-producing strain with a mutant gene introduced

[0228] The nine vectors produced in Example 3-1 were transformed into KCCM12739P, an isoleucine-producing strain, by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), and strains in which the vectors were inserted into the chromosome by recombination of homologous sequences were selected through kanamycin medium. Thereafter, transformants in which secondary recombination was completed were selected for the vectors of SEQ ID NO: 34 and SEQ ID NO: 35; or SEQ ID NO: 36 and SEQ ID NO: 37; or SEQ ID NO: 38 and SEQ ID NO: 39; or SEQ ID NO: 40 and SEQ ID NO: 41; or SEQ ID NO: 42 and SEQ ID NO: 43; or SEQ ID NO: 44 and SEQ ID NO: 45; or SEQ ID NO: 46 and SEQ ID NO: 47; or SEQ ID NO: 48 and SEQ ID NO: 49; Alternatively, the strain into which the mutant gene was introduced was confirmed through PCR using primers of sequence numbers 50 and 51. PCR was performed in the same manner as in Example 3-1, and the primer pairs used for the confirmation are as shown in Table 8 below.

[0229] Sequence number name sequence (5'->3') 34NCgl0214_SEQ_FCGGAGAGTTCGGTAATTTTC35NCgl0214_SEQ_RTGGCGGCTAAATTGGCCGAG36NCgl0216_SEQ_FCTGACTACGGATCAGAAGGT37NCgl0216_SEQ_RACATGCATGGGAGGTTCAAC38NCgl2176_SEQ_F GACTCATCTCCGACATCGAC39NCgl2176_SEQ_RAGTTAGTGCAAAAATATGTT40NCgl2828_SEQ_FCGCCCACCCTAAAGAGGTAC41NCgl2828_SEQ_RTTCTTCGGCTGGAATTTCCG42NCgl0819_SEQ_FACCTCCATTCGGGATT CTGC43NCgl0819_SEQ_RCGATCATCGTGAGCAAACGC44NCg1r05_SEQ_FCTAAAACTTATGTGTGTGAT45NCg1r05_SEQ_RGGAATCACTCGACCAGTGAG46NCg1r09_SEQ_FGGACCTGATCTGGTAGTAGC47NCg1r09_SEQ_R GTCCCTGCTCCACCTGTCAG48NCg1r16_SEQ_FGTTTGCTGTGTTGTTGTCAC49NCg1r16_SEQ_RTCTGCTTGTTCCCCGTCAAG50NCg1r19_SEQ_FCTGCGCATTTCACCGCTACA51NCg1r19_SEQ_RTTATCCATCACTGTAAACAA

[0230] The above recombinant strains were KCCM12739P△NCgl0214:: NCgl0214*, KCCM12739P△NCgl0216:: NCgl0216*, KCCM12739P△NCgl2176:: NCgl2176*, KCCM12739P△NCgl2828:: NCgl2828*, KCCM12739P△NCgl0819:: NCgl0819*, KCCM12739P△NCg1r05:: NCgl1r05*, KCCM12739P△NCg1r09:: NCgl1r09*, KCCM12739P△NCg1r16:: NCgl1r16*, respectively. It was named KCCM12739P△NCg1r19:: NCgl1r19*.

[0231]

[0232] Example 4. Evaluation of isoleucine production capacity of L-isoleucine producing strains into which mutant genes have been introduced.

[0233] In order to confirm the L-isoleucine production ability of the strain produced in Example 3-2 above at the flask level, it was cultured and evaluated using the following method.

[0234] The parent strain and the mutant strain were inoculated into a 250 mL corner-bottom flask containing 25 mL of isoleucine production medium, and then cultured at 32°C with shaking at 200 rpm for 60 hours. After completion of culture, the amount of L-isoleucine produced was measured using high-performance liquid chromatography (HPLC), and the analysis results are shown in Table 9 below.

[0235] <Production medium (pH 7.2)>

[0236] Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, potassium phosphate monobasic 0.1%, magnesium sulfate heptahydrate 0.1%, iron sulfate heptahydrate 10 mg / ℓ, manganese sulfate monohydrate 10 mg / ℓ, biotin 200 μg / ℓ (based on 1L of distilled water)

[0237] Strain name L-isoleucine concentration (g / L) KCCM12739P 2.1 KCCM12739P △NCgl0214:: NCgl0214*2.3 KCCM12739P △NCgl0216:: NCgl0216*2.3 KCCM12739P △NCgl2176:: NCgl2176*2.2 KCCM12739P △NCgl2828:: NCgl2828*3.0 KCCM12739P △NCgl0819:: NCgl0819*2.1 KCCM12739P △NCg1r05:: NCgl1r05*2.1 KCCM12739P △NCg1r09:: NCgl1r09*2.2KCCM12739P△NCg1r16:: NCgl1r16*2.2KCCM12739P△NCg1r19:: NCgl1r19*2.3

[0238] As a result, as shown in Table 9 above, some mutants (KCCM12739P△NCgl0819:: NCgl0819*, KCCM12739P△NCg1r05:: NCgl1r05*) showed a level of productivity equivalent to that of the parent strain, and the remaining mutants were all confirmed to have superior isoleucine productivity compared to the parent strain. In particular, it was confirmed that KCCM12739P△NCgl2828:: NCgl2828* showed a significantly increased isoleucine productivity compared to the parent strain KCCM12739P.

[0239] Through this, it was confirmed that L-isoleucine could be produced more efficiently by introducing a protein variant in which histidine (His, H), glutamic acid (Glu, E), and glycine (Gly, G) were inserted between the 15th and 16th amino acids of the protein (transporter) encoded by the NCgl2828 gene.

[0240]

[0241] 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 equivalents, rather than the detailed description above.

Claims

A protein variant comprising an amino acid sequence in which histidine (His, H), glutamic acid (Glu, E) and glycine (Gly, G) are inserted between the amino acid corresponding to the 15th position from the N-terminus and the amino acid corresponding to the 16th position in the amino acid sequence of SEQ ID NO:

52. In claim 1, the protein variant has an amino acid sequence identity of 80% or more and less than 100% with the amino acid sequence of SEQ ID NO:

52. In claim 1, the protein variant comprises an amino acid sequence of SEQ ID NO:

4. A polynucleotide encoding a protein variant of the first clause. A microorganism of the genus Corynebacterium, comprising at least one member selected from the group consisting of a protein variant of claim 1 and a polynucleotide encoding the protein variant. In claim 5, the microorganism of the genus Corynebacterium is Corynebacterium glutamicum. In claim 5, the microorganism has increased L-isoleucine 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. A method for producing L-isoleucine, comprising the step of culturing the microorganism of claim 5 in a medium. A method for producing L-isoleucine, wherein the method further comprises a step of recovering L-isoleucine from a cultured medium or cultured microorganism. Use of a microorganism according to any one of claims 5 to 7 for producing L-isoleucine.

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