RU2824530C1 - Version of isopropyl malate synthase and method of producing l-leucine using same - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention represents a polypeptide variant having isopropyl malate synthase activity, containing one or more substitutions selected from a group consisting of 1) replacing the amino acid residue corresponding to position 138 with another amino acid residue, 2) replacing the amino acid residue corresponding to position 162 with another amino acid residue, 3) replacing the amino acid residue corresponding to position 211 with another amino acid residue, 4) replacing the amino acid residue corresponding to position 245 with another amino acid residue and 5) replacing the amino acid residue corresponding to position 588 with another amino acid residue in the amino acid sequence SEQ ID NO: 1. Invention also relates to a method of producing L-leucine, comprising a stage of culturing in a medium of a microorganism of the genus Corynebacterium, which produces L-leucine, wherein the microorganism contains said version of the polypeptide, a polynucleotide encoding it or a vector comprising it.

EFFECT: invention enables to obtain L-isoleucine with high efficiency.

14 cl, 13 tbl, 4 ex

Description

Field of invention

The present invention relates to a variant of isopropylmalate synthase and a method for producing L-leucine using it.

Prior art

L-Leucine is an essential amino acid, and is a high-value amino acid widely used in medicine, food, feed additives, industrial chemicals, etc. It is mainly obtained using microorganisms. The production of branched-chain amino acids including L-leucine by fermentation is mainly carried out using Escherichia microorganisms or Corynebacterium microorganisms, and branched-chain amino acids are known to be biosynthesized from pyruvic acid in several steps using 2-ketoisocaproate as a precursor (Korean Patent No. 10-0220018, Korean Patent No. 10-0438146).

Isopropylmalate synthase, which is an enzyme involved in the biosynthesis of L-leucine, is the first step enzyme in the biosynthesis of leucine, which converts 2-ketoisovalerate produced in the biosynthesis pathway of valine into isopropylmalate, which is necessary in the biosynthesis of leucine instead of valine, and isopropylmalate synthase is an important enzyme in the biosynthesis of leucine. However, isopropylmalate synthase is subject to feedback inhibition by L-leucine, which is the final product, or its derivatives. Accordingly, although there are many prior art documents related to isopropylmalate synthase variants that alleviate feedback inhibition, research is still ongoing to find better variants for the purpose of obtaining high leucine concentration (US Patent Publication No. 2015-0079641 and US Patent No. 6,403,342).

Description of the invention

Technical problem

The present inventors endeavored to develop a variant of isopropylmalate synthase that can be used to produce L-leucine in a high concentration, and as a result, they developed a new variant of isopropylmalate synthase and confirmed that L-leucine can be produced in a high yield from a microorganism comprising it, thereby completing the present invention.

Technical solution

The aim of the present invention is to provide a polypeptide variant having isopropylmalate synthase activity.

Another object of the present invention is to provide a polynucleotide encoding a variant of the polypeptide of the present invention.

Another object of the present invention is to provide a vector comprising a polynucleotide of the present invention.

Another object of the present invention is to provide a microorganism of the genus Corynebacterium producing L-leucine, wherein the microorganism comprises a variant of the polypeptide of the present invention; a polynucleotide encoding it; or a vector comprising it.

Another object of the present invention is to provide a method for producing L-leucine, comprising the step of culturing in a medium a microorganism of the genus Corynebacterium producing L-leucine, wherein the microorganism comprises a variant of the polypeptide of the present invention; a polynucleotide encoding it; or a vector comprising it.

Another object of the present invention is to provide a composition for producing L-leucine, wherein the composition comprises a strain of Corynebacterium glutamicum comprising a variant of a polypeptide of the present invention or a polynucleotide of the present invention; or a medium in which the strain is cultured.

Beneficial effects

In the present invention, a variant polypeptide having isopropylmalate synthase activity has an increased activity compared to wild-type isopropylmalate synthase, and it can be used for large-scale production of L-leucine with a high yield.

Detailed Description of Preferred Embodiments

The present invention will be described in detail below. Meanwhile, each description and embodiment disclosed in this specification can also be applied to other descriptions and embodiments. That is, all combinations of various elements disclosed in this specification are included in the scope of the present invention. In addition, the scope of the present invention is not limited to the specific description provided below. In addition, those skilled in the art will understand or can determine, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. In addition, these equivalents should be interpreted as being included in the scope of the present invention.

To achieve these objectives, in one aspect of the present invention, a polypeptide variant is provided that has isopropylmalate synthase activity.

In particular, the polypeptide variant may comprise one or more substitutions selected from the group consisting of 1) a substitution of the amino acid residue corresponding to position 138 with another amino acid residue, 2) a substitution of the amino acid residue corresponding to position 162 with another amino acid residue, 3) a substitution of the amino acid residue corresponding to position 211 with another amino acid residue, 4) a substitution of the amino acid residue corresponding to position 245 with another amino acid residue, and 5) a substitution of the amino acid residue corresponding to position 588 with another amino acid residue, in the amino acid sequence of SEQ ID NO: 1.

As used herein, the term "isopropylmalate synthase (IPMS)" refers to an enzyme that converts 2-ketoisovalerate to isopropylmalate, a precursor of L-leucine, by reacting with acetyl-CoA. In the present invention, the term isopropylmalate synthase may be used interchangeably with the terms isopropylmalate synthesizing enzyme, IPMS, LeuA protein, or LeuA.

In the present invention, the sequence of LeuA can be obtained from GenBank in NCBI (National Center for Biotechnology Information), which is a known database, and in particular, LeuA may be a protein having isopropylmalate synthase activity, which is encoded by the leuA gene, but is not limited thereto.

LeuA may be an enzyme derived from a microorganism of the genus Corynebacterium. Specifically, LeuA may be isopropylmalate synthase derived from Corynebacterium glutamicum.

LeuA of the present invention may include the amino acid sequence of SEQ ID NO: 1, but is not limited thereto. In addition, LeuA may include a polypeptide having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% homology with the amino acid sequence of SEQ ID NO: 1. In addition, it is obvious that an amino acid sequence having such homology or identity and demonstrating an activity corresponding to the activity of isopropyl malate synthase may be included in the scope of the present invention, even though it has an amino acid sequence in which some sequences are deleted, modified, replaced or added.

For example, LeuA may include sequences having an insertion or deletion of a sequence that does not alter the function of the protein of the present invention, at the N-terminus, C-terminus and/or within the amino acid sequence, or a natural mutation, a silent mutation or a conservative substitution.

"Conservative substitution" means the replacement of one amino acid with another amino acid having similar structural and/or chemical properties. Such amino acid substitution may typically be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues. Typically, a conservative substitution may have little or no effect on the activity of proteins or polypeptides.

LeuA of the present invention may have the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 90% or more identity thereto, or may consist of this amino acid sequence, or may consist essentially of this amino acid sequence.

As used herein, the term "polypeptide variant" refers to a polypeptide having an amino acid sequence that differs from the amino acid sequence of the polypeptide variant prior to modification by conservative substitution and/or modification of one or more amino acids, but with retention of function or properties. Such polypeptide variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and assessing the properties of the modified polypeptide. In other words, the ability of the polypeptide variant may be increased, unchanged, or decreased compared to the native polypeptide prior to the modification. In addition, some polypeptide variants may include polypeptide variants in which one or more moieties, such as the N-terminal leader sequence or the transmembrane domain, are deleted. Other polypeptide variants may include polypeptide variants in which a portion of the N- and/or C-terminus of the mature protein is deleted. The term "polypeptide variant" may be used interchangeably with, but is not limited to, terms such as modification, modified polypeptide, modified protein, mutant, mutein, and divergent, provided that the term is used in the sense of variation.

In addition, a polypeptide variant may also include deletions or insertions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence that is co- or post-translationally involved in protein translocation may be conjugated to the N-terminus of a polypeptide variant. In addition, a polypeptide variant may be conjugated to other sequences or linkers for identification, purification, or synthesis.

A variant of a polypeptide of the present invention may have isopropyl malate synthase activity. In addition, a variant of a polypeptide of the present invention may have enhanced isopropyl malate synthase activity compared to a wild-type polypeptide having isopropyl malate synthase activity.

A polypeptide variant of the present invention may comprise one or more substitutions selected from the group consisting of 1) a substitution of the amino acid residue corresponding to position 138 with a different amino acid residue, 2) a substitution of the amino acid residue corresponding to position 162 with a different amino acid residue, 3) a substitution of the amino acid residue corresponding to position 211 with a different amino acid residue, 4) a substitution of the amino acid residue corresponding to position 245 with a different amino acid residue, and 5) a substitution of the amino acid residue corresponding to position 588 with a different amino acid residue in the amino acid sequence of SEQ ID NO: 1, in particular one or more substitutions selected from the group consisting of 1) a substitution of leucine, which is the amino acid residue corresponding to position 138, with a different amino acid residue other than leucine, 2) a substitution of histidine, which is the amino acid residue corresponding to position 162, with different amino acid residues a residue other than histidine, 3) a substitution of serine, which is the amino acid residue corresponding to position 211, with another amino acid residue other than serine, 4) a substitution of asparagine, which is the amino acid residue corresponding to position 245, with another amino acid residue other than asparagine, and 5) a substitution of isoleucine, which is the amino acid residue corresponding to position 588, with another amino acid residue other than isoleucine, in the amino acid sequence of SEQ ID NO: 1, and more particularly, one or more substitutions selected from the group consisting of 1) a substitution of leucine, which is the amino acid residue corresponding to position 138, with glycine, 2) a substitution of histidine, which is the amino acid residue corresponding to position 162, with glutamate, 3) a substitution of serine, which is the amino acid residue corresponding to position 211, with leucine, 4) a substitution asparagine, which is the amino acid residue corresponding to position 245, in serine, and 5) a substitution of isoleucine, which is the amino acid residue corresponding to position 588, with proline in the amino acid sequence of SEQ ID NO: 1, and even more particularly, one or more than one, two or more than two, three or more than three, four or more than four, and five substitutions. The two or more substitutions may be, but are not limited to, a combination of (1) and (5); a combination of (2) and (5); a combination of (3) and (5); or a combination of (4) and (5). Four or more substitutions may be, but are not limited to, a combination of (1), (2), (3), and (4). Five or more substitutions may be, but are not limited to, a combination of (1), (2), (3), (4), and (5).

A variant polypeptide of the present invention may have/include the amino acid sequence of SEQ ID NO: 6, or SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12, or SEQ ID NO: 14, or may consist of/may consist essentially of the amino acid sequence of SEQ ID NO: 6, or SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12, or SEQ ID NO: 14. A variant polypeptide of the present invention may comprise a polypeptide having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99%, or more than 99%, and less than 100% identity or homology to the amino acid sequence of SEQ ID NO: 6, or SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12, or SEQ ID NO: 14, wherein 1) the amino acid residue corresponding to position 138 is glycine, 2) the amino acid residue corresponding to position 162 is glutamate, 3) the amino acid residue corresponding to position 211 is leucine, 4) the amino acid residue corresponding to position 245 is serine, or 5) the amino acid residue corresponding to position 588 is proline, in the amino acid sequence of SEQ ID NO: 6, or SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12, or SEQ ID NO: 14. In particular, SEQ ID NO: 6 may be an amino acid sequence in which leucine, which is the amino acid residue corresponding to position 138 in the amino acid sequence of SEQ ID NO: 1, is replaced with glycine, SEQ ID NO: 8 may be an amino acid sequence in which histidine, which is the amino acid residue corresponding to position 162, is replaced with glutamate, SEQ ID NO: 10 may be an amino acid sequence in which serine, which is the amino acid residue corresponding to position 211, is replaced with leucine, SEQ ID NO: 12 may be an amino acid sequence in which isoleucine, which is the amino acid residue corresponding to position 588, is replaced with proline, and SEQ ID NO: 14 may be an amino acid sequence in which asparagine, which is the amino acid residue corresponding to position 245, is replaced with serine.

It is further evident that a polypeptide variant having an amino acid sequence in which certain sequences are deleted, modified, substituted, conservatively substituted or added, in addition to 1) position 138, 2) position 162, 3) position 211, 4) position 245 or 5) position 588 in the amino acid sequence of SEQ ID NO: 6, or SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12, or SEQ ID NO: 14, is also included within the scope of the present invention, provided that the amino acid sequence has such identity or homology and exhibits an efficacy corresponding to the efficacy of the polypeptide variant of the present invention. In particular, the substitution may include any one or more of (1) a variant (R558H) substituting histidine for arginine, which is the amino acid corresponding to position 558 in the LeuA protein, by substituting A for G, which is the nucleotide at position 1673 of the leuA gene encoding isopropylmalate synthase, (2) a variant (G561D) substituting aspartic acid for glycine, which is the amino acid corresponding to position 561, by substituting AT for GC, which are the nucleotides at positions 1682 and 1683 of the leuA gene, or (3) a variant (P247C) substituting cysteine for proline, which is the amino acid at position 247, by substituting TG for CC, which are the nucleotides at positions 739 and 740 of the leuA gene, and the descriptions thereof provided above.

More particularly, a polypeptide variant may comprise a polypeptide comprising variants (SEQ ID NO: 38) at positions 247, 558 and 561 in addition to 1) a variant at position 138; or variants (SEQ ID NO: 40) at positions 247, 558 and 561 in addition to 2) a variant at position 162; or variants (SEQ ID NO: 42) at positions 247, 558 and 561 in addition to 3) a variant at position 211; variants (SEQ ID NO: 44) at positions 247, 558 and 561 in addition to 4) a variant at position 245; variants (SEQ ID NO: 46) at positions 247, 558 and 561 in addition to 5) a variant at position 588; variants (SEQ ID NO: 48) at positions 247, 558 and 561 in addition to 3) the variant at position 211 and 5) the variant at position 588; variants (SEQ ID NO: 50) at positions 247, 558 and 561 in addition to 1) the variant at position 138, 2) the variant at position 162, 3) the variant at position 211 and 4) the variant at position 245; or variants (SEQ ID NO: 52) at positions 247, 558 and 561 in addition to 1) the variant at position 138, 2) the variant at position 162, 3) the variant at position 211, 4) the variant at position 245 and 5) the variant at position 588, but is not limited thereto.

As used herein, the term "corresponding" refers to amino acid residues at positions listed in a polypeptide, or to amino acid residues that are similar, identical, or homologous to those listed in a polypeptide. Identification of an amino acid at a corresponding position may be the determination of a particular amino acid in a sequence that relates to a particular sequence. As used herein, the term "corresponding region" generally refers to an analogous or corresponding position in a related protein or reference protein.

For example, an arbitrary amino acid sequence is aligned with SEQ ID NO: 1 and based on this, each amino acid residue of the amino acid sequence can be numbered with reference to the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, the sequence alignment algorithm described in the description of the present invention can determine the position of an amino acid or the position at which a modification, such as a substitution, insertion or deletion, is made by comparison with a sequence of interest (also called a "reference sequence").

For such alignments, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277) and the like, without limitation, can be used, and a sequence alignment program, a pairwise sequence comparison algorithm, etc. known in the art can be suitably used.

As used herein, the term "homology" or "identity" means the degree of similarity between two given amino acid sequences or nucleotide sequences, and may be expressed as a percentage. Often, the terms "homology" and "identity" may be used interchangeably.

Homology or sequence identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, and may use a penalty for a gap in the sequence specified in the program used. As such, homologous or identical sequences are typically capable of hybridizing to the entire sequence or part of the sequence under moderately stringent or very stringent conditions. It is understood that hybridization also includes hybridization of a polynucleotide to a polynucleotide containing a common codon or a codon with degeneracy of codons.

Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the FASTA program, for example, using default parameters such as in Pearson et al., (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, homology, similarity, or identity can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), which is performed using 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) (which includes the GCG software 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 ET AL/.](1988) SIAM J Applied Math 48: 1073). For example, BLAST from the National Center for Biotechnology Information or ClustalW can be used to determine homology, similarity, or identity.

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

In another aspect of the present invention, there is provided a polynucleotide encoding a variant of the polypeptide of the present invention.

The term "polynucleotide" as used herein is a DNA or RNA chain of a certain length or more in the form of a polymer of nucleotides in which the nucleotide monomers are linked into a long chain by covalent bonds, and more specifically, it means a polynucleotide fragment encoding a protein variant.

A polynucleotide encoding a variant of a polypeptide of the present invention may include a nucleotide sequence encoding an amino acid sequence described in SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO: 10 or SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 38 or SEQ ID NO: 40 or SEQ ID NO: 42 or SEQ ID NO: 44 or SEQ ID NO: 46 or SEQ ID NO: 48 or SEQ ID NO: 50 or SEQ ID NO: 52, but is not limited thereto. In particular, a polynucleotide of the present invention may have or include the nucleotide sequence of SEQ ID NO: 7, or SEQ ID NO: 9, or SEQ ID NO: 11, or SEQ ID NO: 13, or SEQ ID NO: 15, or SEQ ID NO: 39, or SEQ ID NO: 41, or SEQ ID NO: 43, or SEQ ID NO: 45, or SEQ ID NO: 47, or SEQ ID NO: 49, or SEQ ID NO: 51, or SEQ ID NO: 53.

In a polynucleotide, various modifications may be made in the coding region as long as the amino acid sequence of the polypeptide does not change, taking into account the degeneracy of the codons or codons preferred in the organisms in which the polypeptide is intended to be expressed. In particular, the polynucleotide may consist of a nucleotide sequence having a homology or identity of 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% with SEQ ID NO: 7, or SEQ ID NO: 9, or SEQ ID NO: 11, or SEQ ID NO: 13, or SEQ ID NO: 15, or SEQ ID NO: 39, or SEQ ID NO: 41, or SEQ ID NO: 43, or SEQ ID NO: 45, or SEQ ID NO: 47, or SEQ ID NO: 49, or SEQ ID NO: 51, or SEQ ID NO: 53, but is not limited thereto.

In addition, the polynucleotide of the present invention may include a probe that can be derived from a known gene sequence, such as a sequence without limitation, as long as it is a sequence that can hybridize with a complementary sequence to all or part of the polynucleotide sequence of the present invention under stringent conditions. "Stringent conditions" mean conditions that ensure specific hybridization of polynucleotides. These conditions are specifically described in the documents (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). Examples include conditions in which polynucleotides having higher homology or identity, namely 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 to each other while polynucleotides having less homology or identity do not hybridize to each other, or washing conditions for conventional Southern hybridization wherein washing is performed once, in particular two or three times, at a salt concentration and temperature equivalent to 60°C, 1×SSC (sodium citrate chloride solution), 0.1% SDS (sodium dodecyl sulfate), in particular 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1 × SSC, 0.1% SDS.

Hybridization requires that the two nucleic acids have complementary sequences, although base pairing errors are tolerated depending on the stringency of hybridization. The term "complementary" is used to describe the interaction between nucleotide bases that are capable of hybridizing to each other. For example, in the case of DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Thus, a polynucleotide of the present invention may also include substantially similar nucleic acid sequences, as well as isolated nucleic acid fragments that are complementary to the full-length sequence.

In particular, a polynucleotide having homology or identity with the polynucleotide of the present invention can be detected using hybridization conditions comprising a hybridization step at a _Tm value of 55°C under the above-described conditions. The _Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by a person skilled in the art depending on the task.

The appropriate stringency of conditions for polynucleotide hybridization depends on the length and degree of complementarity of the polynucleotides, and these variables are well known in the art (e.g., J. Sambrook et al., supra).

For example, a polynucleotide of the present invention may include any sequence without limitation as long as it encodes the amino acid sequence of SEQ ID NO: 6, or SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12, or SEQ ID NO: 14, or SEQ ID NO: 38, or SEQ ID NO: 40, or SEQ ID NO: 42, or SEQ ID NO: 44, or SEQ ID NO: 46, or SEQ ID NO: 48, or SEQ ID NO: 50, or SEQ ID NO: 52.

In a polynucleotide of the present invention, the polypeptide variant is as described in other aspects.

In another aspect of the present invention, there is provided a vector comprising a polynucleotide of the present invention.

The vector of the present invention refers to a DNA construct comprising a polynucleotide sequence encoding a polypeptide of interest, operably linked to a suitable expression control region (expression control sequence), such that the polypeptide of interest can be expressed in a suitable host. The expression control region can comprise a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding a suitable mRNA binding site for the ribosome, and a sequence regulating the termination of transcription and translation. The vector can be transformed into a suitable host cell and then replicated or functioned independently of the host genome or can be integrated into its genome.

The vector used in the present invention is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors may include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc. can be used as the phage vector or the cosmid vector. As the plasmid vector, pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, pET system, etc. can be used. In particular, the vector pDCM2 (WO 2021-187781 A1), pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, etc. can be used.

For example, a polynucleotide encoding a polypeptide of interest can be inserted into a chromosome using a vector for intracellular insertion into a chromosome. The polynucleotide can be inserted into a chromosome using any method known in the art, such as, but not limited to, homologous recombination. The vector can further comprise a selectable marker for identifying the insertion into the chromosome. The selectable marker is designed to select cells transformed by the vectors, i.e., to identify the insertion of the nucleic acid molecule of interest, and markers conferring selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface polypeptides can be used. In a medium treated with a selective agent, only cells expressing the selectable markers survive or exhibit other phenotypic traits, and thus the transformed cells can be selected.

As used herein, the term "transformation" means that a vector containing a polynucleotide encoding a target polypeptide is introduced into a host cell or microorganism such that the protein encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide can be localized by insertion into a chromosome of the host cell or localized extrachromosomally, as long as it can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding the protein of interest. The polynucleotide can be introduced in any form, as long as it can be introduced into the host cell and then expressed. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a genetic construct that includes all the elements necessary for its self-expression. Typically, the expression cassette can include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, the polynucleotide may be introduced into the host cell in its own form and operably linked to a sequence required for expression in the host cell, without being limited thereto.

Additionally, the term "operably linked" as used herein means that the polynucleotide sequence is operably linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the protein variant of interest of the present invention.

In the vector of the present invention, the polynucleotide is as described in other aspects.

In another aspect of the description of the present invention, a microorganism of the genus Corynebacterium producing L-leucine is provided, wherein the microorganism comprises a variant of the polypeptide of the present invention; a polynucleotide encoding it; or a vector comprising it.

The term "microorganism" as used herein includes all of the wild-type microorganisms or naturally occurring or artificially genetically modified microorganisms, and may be a microorganism in which a specific mechanism is weakened or strengthened due to the insertion of a foreign gene, or the enhancement or inactivation of an endogenous gene, and may be a microorganism containing a genetic modification to produce a polypeptide, protein or product of interest.

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

The microorganism of the present invention may be a microorganism naturally possessing isopropylmalate synthase activity or the ability to produce L-leucine, or a microorganism obtained by expressing a variant of the polypeptide of the present invention in a parent strain lacking isopropylmalate synthase activity or the ability to produce L-leucine, or by imparting the ability to produce L-leucine to the parent strain, but is not limited thereto.

In particular, the microorganism of the present invention may be a cell or a microorganism expressing a variant polypeptide of the present invention by transformation with a polynucleotide of the present invention or a vector comprising a gene encoding a variant polypeptide of the present invention, and, with respect to the objects of the present invention, the microorganism of the present invention may include all microorganisms capable of producing L-leucine by incorporating a variant polypeptide of the present invention. For example, the microorganism of the present invention may be a recombinant microorganism having an enhanced ability to produce L-leucine, in which the variant polypeptide of the present invention is expressed by introducing a polynucleotide encoding a variant polypeptide of the present invention into a natural wild-type microorganism or into a microorganism producing L-leucine. The recombinant microorganism having an enhanced ability to produce L-leucine may be a microorganism having an enhanced ability to produce L-leucine, as compared to a natural wild-type microorganism or an unmodified microorganism, but is not limited thereto.

The term "unmodified microorganism" as used herein does not exclude strains comprising mutations that may occur naturally in microorganisms, and may be a wild-type strain or a natural strain itself, or may be a strain before characteristics are changed by genetic variation due to natural or artificial factors. For example, an unmodified microorganism may be a strain into which a variant protein described in the description of the present invention has not been introduced or has not yet been introduced. The term "unmodified microorganism" may be used interchangeably with "strain before modification", "microorganism before modification", "unmodified strain", "unmodified strain", "unmodified microorganism" or "reference microorganism".

In particular, the microorganism of the present invention may be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans , Corynebacterium testudinoris or Corynebacterium flavescens.

The microorganism of the present invention may be a microorganism comprising a nucleotide sequence encoding isopropyl malate synthase in which one or more amino acid residues except for the amino acid residue 1) at position 138, 2) at position 162, 3) at position 211, 4) at position 245 or 5) at position 588 in the amino acid sequence of SEQ ID NO: 1 constituting the isopropyl malate synthase of the present invention are replaced with another amino acid. In particular, the substitution may include any one or more of (1) a variant (R558H) substituting histidine for arginine, which is the amino acid at position 558 of the LeuA protein, by substituting A for G, which is the nucleotide at position 1673 of the leuA gene encoding isopropylmalate synthase, (2) a variant (G561D) substituting aspartic acid for glycine, which is the amino acid at position 561, by substituting AT for GC, which are the nucleotides at positions 1682 and 1683 of the leuA gene, or (3) a variant (P247C) substituting cysteine for proline, which is the amino acid at position 247, by substituting TG for CC, which are the nucleotides at positions 739 and 740 of the leuA gene, and their descriptions are as described above.

In particular, the microorganism producing L-leucine according to the present invention may be a microorganism having enhanced isopropylmalate synthase activity by expressing isopropylmalate synthase including such a variation.

As used herein, the term "enhancement" of a polypeptide activity means that the activity of the polypeptide is increased compared to its own activity. Enhancement may be used interchangeably with terms such as up-regulation, overexpression, increase, etc. Thus, an increase may include both the demonstration of an activity that the polypeptide originally did not possess and the demonstration of an improved activity compared to its own activity or the activity before modification. "Inherent activity" means the activity of a particular polypeptide originally displayed by the parent strain before the alteration of characteristics or by an unmodified microorganism when the characteristics are altered by genetic variation due to natural or artificial factors. This term may be used interchangeably with "activity before modification". The fact that the activity of a polypeptide is "enhanced" or "increased" compared to its own activity means that the activity of the polypeptide is improved compared to the activity of the particular polypeptide originally possessed by the parent strain before the alteration of characteristics or by an unmodified microorganism.

Enhancement may be achieved by introducing a foreign polypeptide or enhancing the polypeptide's own activity. Enhancement of the polypeptide's activity may be confirmed by increasing the degree of activity and the expression level of the corresponding polypeptide or the amount of product produced from the corresponding polypeptide.

To enhance the activity of a polypeptide, various methods well known in the art can be used, and the method is not limited as long as the activity of the polypeptide of interest can be enhanced compared to the activity of the microorganism before modification. In particular, genetic engineering and/or protein engineering well known to those skilled in the art, which are conventional methods for molecular biology, can be used, but the method is not limited thereto (for example, Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

In particular, enhancing the activity of the polypeptide of the present invention can be accomplished by:

1) an increase in the number of copies of the polynucleotide encoding the polypeptide in cells;

2) replacement of the region regulating gene expression in the chromosome encoding the polypeptide with a sequence possessing strong activity;

3) modifications of the start codon of the gene encoding the polypeptide, or the base sequence of the 5'-UTR region;

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

5) modifications of the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide;

6) introduction of a foreign polynucleotide demonstrating polypeptide activity;

7) optimization of codons of the polynucleotide encoding the polypeptide;

8) analyzing the tertiary structure of the polypeptide to select the exposed site and to perform modification or chemical modification of the exposed site; or

9) combinations (1)-(8), but not specifically limited to them.

More specifically, (1) an increase in the number of copies of a polynucleotide encoding a polypeptide in a cell can be accomplished by introducing a vector that replicates and functions independently of the host cell and is operably linked to a polynucleotide encoding the corresponding polypeptide in the host cell. Alternatively, this increase can be achieved by introducing one copy, or two or more than two copies of a polynucleotide encoding the corresponding polypeptide into a chromosome of the host cell. Introduction into the chromosome can be accomplished by introducing a vector capable of integrating the polynucleotide into the chromosome of the host cell into the host cell, but is not limited to this. The vector is as described above.

2) The replacement of a gene expression control region (or expression control sequence) in a chromosome encoding a polypeptide with a sequence exhibiting strong activity may be, for example, the occurrence of a variant in the sequence due to a deletion, an insertion, a non-conservative or conservative substitution, or a combination thereof, or a replacement with a sequence exhibiting stronger activity, so that the activity of the expression control region is further enhanced. The expression control region is not particularly limited to this, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence controlling the termination of transcription and translation, and the like. For example, the replacement may be the replacement of an original promoter with a strong promoter, but is not limited to this.

Examples of known strong promoters include, but are not limited to, the cj1-cj7 promoters (U.S. Patent No. 7,662,943 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 (U.S. Patent No. 1,0584,338 B2), the O2 promoter (U.S. Patent No. 1,0273,491 B2), the tkt promoter, the yccA promoter, etc.

3) Modification of the start codon of a gene encoding a polypeptide or the base sequence of the 5'-UTR region may be, for example, a replacement with another start codon that has a higher level of expression of the polypeptide compared to the endogenous start codon, but is not limited to this.

4) and 5) The modification of the amino acid sequence or the polynucleotide sequence may be, but is not limited to, the occurrence of a variation in the sequence due to a deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, or a combination thereof, or a substitution with an amino acid sequence or a polynucleotide sequence modified to have a stronger activity, or an amino acid sequence or a polynucleotide sequence modified to be more active, so that the activity of the polypeptide is enhanced. The substitution may be, in particular, carried out by integrating the polynucleotide into the chromosome by homologous recombination. The vector used herein may further comprise a selectable marker for confirming the integration into the chromosome. The selectable marker is as described above.

6) Introduction of a foreign polynucleotide exhibiting the activity of a polypeptide may be the introduction into a host cell of a foreign polynucleotide encoding a polypeptide exhibiting an activity identical/similar to the activity of the polypeptide. The foreign polynucleotide is not limited in its origin or sequence as long as it exhibits an activity identical/similar to the activity of the polypeptide. The introduction can be carried out by appropriately selecting a transformation method known to those skilled in the art. Since the introduced polynucleotide is expressed in the host cell, the polypeptide can be obtained and its activity can be increased.

7) Optimization of codons of a polynucleotide encoding a polypeptide may be optimization of codons of an endogenous polynucleotide in such a way as to increase transcription or translation in the host cell, or optimization of codons of a foreign polynucleotide in such a way as to achieve optimized transcription and translation in the host cell.

8) Analysis of the tertiary structure of a polypeptide for selecting an exposed site and for performing modification or chemical modification of the exposed site can be performed, for example, to determine a candidate template protein according to the degree of sequence similarity by comparing the sequence information of the analyzed polypeptide with a database in which sequence information of known proteins is stored, to identify the structure on this basis and to select and modify or chemically modify the exposed fragment that is supposed to be modified or chemically modified.

Such improvement in polypeptide activity may be, but is not limited to, an increase in the activity or concentration of the corresponding polypeptide based on the activity or concentration of the polypeptide expressed in the wild-type strain or in the microbial strain before modification, or an increase in the amount of product produced from the corresponding polypeptide.

In the microorganism of the present invention, a partial or complete modification of a polynucleotide (for example, a modification to encode the above-described protein variant) can be caused by (a) homologous recombination using a vector for insertion into a chromosome in the microorganism or genome editing using an engineered nuclease (for example, CRTSPR-Cas9) and/or (b) treatment with light such as ultraviolet rays and radiation and/or chemicals, but is not limited thereto. The method for modifying a part or the whole of a gene can include a method using recombinant DNA technology. For example, by introducing a nucleotide sequence or a vector containing a nucleotide sequence homologous to a gene of interest into a microorganism to cause homologous recombination, a part or the whole of a gene can be deleted. The introduced nucleotide sequence or vector may include a dominant selectable marker, but is not limited thereto.

More specifically, the L-leucine-producing microorganism of the present invention may be a microorganism further comprising a polypeptide comprising SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO: 10 or SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 38 or SEQ ID NO: 40 or SEQ ID NO: 42 or SEQ ID NO: 44 or SEQ ID NO: 46 or SEQ ID NO: 48 or SEQ ID NO: 50 or SEQ ID NO: 52, a polynucleotide encoding a polypeptide comprising SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO: 10 or SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 38 or SEQ ID NO: 40 or SEQ ID NO: 42, or SEQ ID NO: 44, or SEQ ID NO: 46, or SEQ ID NO: 48, or SEQ ID NO: 50, or SEQ ID NO: 52, or a polynucleotide comprising SEQ ID NO: 7 or SEQ ID NO: 9, or SEQ ID NO: 11, or SEQ ID NO: 13, or SEQ ID NO: 15, or SEQ ID NO: 39, or SEQ ID NO: 41, or SEQ ID NO: 43, or SEQ ID NO: 45, or SEQ ID NO: 47, or SEQ ID NO: 49, or SEQ ID NO: 51, or SEQ ID NO: 53.

In the microorganism of the present invention, the polypeptide variant, polynucleotide, vector, L-leucine, etc. are as described in other aspects.

In another aspect of the present invention, a method for producing L-leucine is provided, comprising the step of culturing in a medium a microorganism of the genus Corynebacterium producing L-leucine, wherein the microorganism comprises a variant of the polypeptide of the present invention; a polynucleotide encoding it; or a vector comprising it.

The term "cultivation" as used herein means culturing the genus Corynebacterium of the present invention under suitably controlled environmental conditions. The culturing process of the present invention can be carried out using a suitable medium and culturing conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art according to the selected strain. Specifically, the culturing may be a batch culturing, a continuous culturing, and a fed-batch culturing, but is not limited to these.

The term "medium" used herein means a mixed substance containing nutrients necessary for culturing the microorganism of the genus Corynebacterium of the present invention as a main component, and the medium provides nutrients and growth factors including water, which are necessary for survival and development. In particular, any medium without particular limitation can be used as the medium and other culture conditions used for culturing the microorganism of the genus Corynebacterium of the present invention as long as it is a medium used for conventional microorganism culturing. The Corynebacterium glutamicum strain of the present invention can be cultured in a conventional medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and/or vitamins, etc., while controlling temperature, pH, etc. under aerobic conditions. In particular, the culture medium for the strain of the genus Corynebacterium can be found in the document ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D.C., USA, 1981)].

In the present invention, carbon sources include carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. Natural organic nutrients such as starch hydrolysate, molasses, black molasses, rice bran, cassava, sugar cane bagasse, and corn steep liquor can be used. In particular, carbohydrates such as glucose and sterile pre-treated molasses (i.e., molasses converted into reduced sugar) can be used, and suitable amounts of other carbon sources can be variously used without limitation. These carbon sources may be used alone or in combination of two or more of them, but are not limited to this.

As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc. can be used; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, liquid corn steep liquor, casein hydrolysate, fish or its degradation products, and defatted soybean meal or its degradation products. These nitrogen sources can be used alone or in combination of at least two or more of them, but are not limited thereto.

Phosphorus sources may include monobasic potassium phosphate, dibasic potassium phosphate or the corresponding sodium-containing salts. Inorganic compounds that may be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition to these compounds, amino acids, vitamins and/or suitable precursors may be included. These components or precursors may be added to the medium in batches or continuously, but are not limited to this.

In addition, during the cultivation of the Corynebacterium glutamicum strain of the present invention, the pH of the medium can be adjusted by appropriately adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, orthophosphoric acid and sulfuric acid to the medium. During the cultivation, the formation of foam can be suppressed by using an antifoam such as a fatty acid polyglycol ester. Oxygen or an oxygen-containing gas can be introduced into the medium to maintain an aerobic state of the medium, or the gas may not be introduced, or nitrogen, hydrogen or carbon dioxide can be introduced to maintain anaerobic and microaerobic states, without being limited thereto.

In the culture of the present invention, the culture temperature can be maintained at 20°C to 45°C, particularly 25°C to 40°C, and the strain can be cultured for about 10 to 160 hours, but is not limited thereto.

L-leucine produced by the culturing according to the present invention may be secreted into the medium or may be retained in the cells.

The method for producing L-leucine according to the present invention may further include a step of preparing the Corynebacterium glutamicum strain according to the present invention, or a step of preparing a medium for culturing the strain.

The method for producing L-leucine according to the present invention may further include a step of isolating L-leucine from a medium according to the culture or from the Corynebacterium glutamicum strain according to the present invention.

The isolation can be carried out to collect the L-leucine of interest by a suitable method known in the art according to the method for culturing the microorganism of the present invention, for example, by a batch culture method, a continuous culture method or a fed-batch culture. For example, centrifugation, filtration, treatment with an agent precipitating crystallized protein (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various forms of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography and affinity chromatography, HPLC (high performance liquid chromatography) or a combination thereof can be used. The L-leucine of interest can be isolated from the medium or the microorganism by a suitable method known in the art.

In addition, the method for producing L-leucine according to the present invention may further comprise a purification step. The purification may be carried out using a suitable method known in the art. As an example, when the method for producing L-leucine according to the present invention comprises both an isolation step and a purification step, then the isolation step and the purification step may be carried out continuously or intermittently regardless of the sequence, or may be carried out simultaneously or by combining into one step, but is not limited thereto.

In the method of the present invention, the polypeptide variant, polynucleotide, L-leucine, etc. are as described in other aspects.

In another aspect of the present invention, there is provided a composition for producing L-leucine, wherein the composition comprises a strain of Corynebacterium glutamicum comprising a variant of a polypeptide of the present invention or a polynucleotide of the present invention; or a medium in which the strain is cultured.

The composition of the present invention may further comprise arbitrary suitable excipients commonly used in compositions for the preparation of amino acids. Such excipients may be, for example, a preservative, a humectant, a dispersing agent, a suspending agent, a buffering agent, a stabilizer, or an isotonic agent, but are not limited thereto.

In the composition of the present invention, the polypeptide variant, polynucleotide, L-leucine, etc. are as described in other aspects.

Method of carrying out the invention

Hereinafter, the present description will be described in more detail with reference to embodiment examples. However, the following embodiment examples are only preferred embodiments for illustrating the description of the present invention, and therefore are not intended to limit the scope of the present invention. Meanwhile, technical issues not described in the description of the present invention can be sufficiently understood and easily implemented by those skilled in the art of the present invention or similar technical fields.

Example 1. Construction of a DNA library encoding a mutant from oppilm lat synthase

1-1. Construction of a vector containing leuA

To construct a library of leuA mutants possessing isopropylmalate synthase activity, a recombinant vector containing leuA was first constructed. To amplify the leuA gene (SEQ ID NO: 2) encoding the LeuA protein (SEQ ID NO: 1, Uniprot accession code: P42455) originating from the wild-type Corynebacterium glutamicum, PCR was performed using the chromosome of the wild-type Corynebacterium glutamicum strain ATCC13032 as a template and primers SEQ ID NOs: 3 and 4, repeating 25 cycles consisting of denaturation at 94°C for 1 minute, annealing at 58°C for 30 seconds, and polymerization at 72°C for 1 minute using Pfu DNA polymerase. The sequences of the primers used are as shown in Table 1 below.

The PCR product was cloned into the E. coli pCR2.1 vector using the TOPO cloning kit (Invitrogen) to yield 'pCR-leuA'.

1-2. Construction of a leuA mutant library

Based on the vector obtained in Example 1-1, a leuA mutant library was prepared using a reduced fidelity PCR kit (clontech ^Diversify® PCR Random Mutagenesis Kit). The PCR reaction was carried out using the primers SEQ ID NO: 3 and SEQ ID NO: 4 described in Table 1 under conditions in which 0 to 3 mutations occurred per 1000 bp.

Specifically, PCR was performed by preheating at 94°C for 30 seconds, followed by 25 cycles of denaturation at 94°C for 30 seconds and polymerization at 68°C for 1 minute 30 seconds. The PCR product obtained at this time point was subjected to 25 cycles of denaturation at 95°C for 50 seconds, annealing at 60°C for 50 seconds and polymerization at 68°C for 12 minutes using megaprimer (50 ng to 125 ng), followed by DpnI treatment, and then transformed into E. coli DH5α by the heat shock method and plated on LB solid medium containing 25 mg/L kanamycin. After selecting 20 types of transformed colonies, plasmids were obtained and sequenced. As a result, it was confirmed that mutations were introduced at different positions with a frequency of 2 mutations/kb. Approximately 20,000 transformed E. coli colonies were collected and plasmids were extracted, which were named 'pTOPO-leuA-library'.

Example 2. Evaluation of the constructed library and selection of mutants

2-1. Selection of mutant strains with increased production of L-leucine

The pTOPO-leuA-library obtained in Example 1-2 was transformed into wild-type Corynebacterium glutamicum ATCC13032 by electroporation, and then spread on a nutrient medium (Table 2) containing 25 mg/L kanamycin to select 10,000 colonies of the strain into which the mutant genes were inserted. Each selected colony was named ATCC13032/pTOPO_leuA(mt)1 - ATCC 13032/pTOPO_leuA(mt) 10000.

To identify colonies in which L-leucine production increased and L-phenylalanine production among aromatic amino acids increased or decreased, among the 10,000 colonies obtained, the fermentation titer for each colony was estimated using the following method.

Each colony was inoculated using a platinum loop into a 250 ml baffled flask containing 25 μg/ml kanamycin in 25 ml of the production medium according to Table 2, and then cultured at 30°C for 60 hours with shaking at 200 rpm. After completion of the culture, the production of L-leucine was measured by a high performance liquid chromatography (HPLC, SHIMAZDU LC20A) method.

As a result, 5 kinds of strains (ATCC13032/pTOPO_leuA(mt)3847, ATCC13032/pTOPO_leuA(mt)4708, ATCC13032/pTOPO_leuA(mt)5109, ATCC13032/pTOPO_leuA(mt)7563, ATCC13032/pTOPO_leuA(mt)8459) demonstrating the most improved ability to produce L-leucine, compared with the wild-type Corynebacterium glutamicum ATCC13032, were selected from 10,000 colonies. The concentrations of L-leucine obtained in the selected strains are presented in Table 3 below.

As shown in Table 3, it was confirmed that Corynebacterium glutamicum ATCC13032/pTORO_leuA(mt)3847, which has a mutation in the leuA gene, showed approximately 1.41-fold improvement in L-leucine production compared with the parent strain Corynebacterium glutamicum ATCC13032. It was also confirmed that ATCC13032/pTOPO_leuA(mt)4708, ATCC13032/pTOPO_leuA(mt)5109, ATCC13032/pTOPO_leuA(mt)7563, and ATCC13032/pTOPO_leuA(mt)8459 exhibited approximately 1.45-, 1.59-, 1.36-, and 1.38-fold improvement, respectively, in L-leucine production compared with the parental strain.

2-2. Identification of mutations in mutant strains with increased production of L-leucine

To identify the mutation of the leuA gene in the selected 5 mutant strains, PCR was performed using the DNA of each mutant strain as a template and primers with SEQ ID NO: 3 and SEQ ID NO: 4 described in Table 1, under conditions of denaturation at 94°C for 5 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds and polymerization at 72°C for 1 minute and 30 seconds, and then polymerization at 72°C for 5 minutes, and DNA sequencing was performed.

According to the sequencing results, in the strain ATCC13032/pTORO_leuA(mt)3847, both C and T at positions 412 and 413 of the leuA gene of SEQ ID NO: 2 were replaced by G, indicating that it encodes a variant (hereinafter referred to as L138G) having a glycine-to-leucine substitution, which is the amino acid at position 138 (at position 103 based on literature data in which the translation start codon is read 35 in the upstream direction and the LeuA protein consists of 581 amino acids (SEQ ID NO: 5); hereinafter referred to only as at position 138) of the LeuA protein. The amino acid sequence of the LeuA variant (L138G) and the nucleotide sequence of the leuA variant encoding it are as in SEQ ID NO: 6 and SEQ ID NO: 7.

Strain ATCC 13032/pTOPO_leuA(mt)4708 was confirmed to have a G to C substitution, which are nucleotides at positions 484 and 486 of the leuA gene, indicating that it encodes a variant (hereinafter referred to as H162E) having a glutamate to histidine substitution, which is an amino acid at position 162 (at position 127 based on literature data in which the translation start codon is read 35 backwards and the LeuA protein consists of 581 amino acids (SEQ ID NO: 5); hereinafter referred to only as position 162) of the LeuA protein. The amino acid sequence of the LeuA variant (H162E) and the base sequence of the leuA variant encoding it are as in SEQ ID NO: 8 and SEQ ID NO: 9.

It was confirmed that the strain ATCC13032/pTOPO_leuA(mt)5109 had a substitution of CTT to TCC, which are nucleotides at positions 631-633 of the leuA gene, indicating that it encodes a variant (hereinafter referred to as S211L) having a substitution of leucine to serine, which is an amino acid at position 211 (at position 176 based on literature data in which the translation start codon is read 35 in the upstream direction and the LeuA protein consists of 581 amino acids (SEQ ID NO: 5); hereinafter referred to only as position 211) of the LeuA protein. The amino acid sequence of the LeuA variant (S211L) and the nucleotide sequence of the leuA variant encoding it are as in SEQ ID NO: 10 and SEQ ID NO: 11.

The strain ATCC13032/pTOPO_leuA(mt)7563 was confirmed to have a substitution of CC for AT, which are nucleotides at positions 1762-1763 of the leuA gene, indicating that it encodes a variant (hereinafter referred to as I588P) having a substitution of proline for isoleucine, which is the amino acid at position 588 (at position 553 based on literature data in which the translation start codon is read 35 in the upstream direction and the LeuA protein consists of 581 amino acids (SEQ ID NO: 5); hereinafter referred to only as at position 553) of the LeuA protein. The amino acid sequence of the LeuA variant (I588P) and the nucleotide sequence of the leuA variant encoding it are as in SEQ ID NO: 12 and SEQ ID NO: 13.

It was also confirmed that the strain ATCC13032/pTOPO_leuA(mt)8459 has a TC to AA substitution, which are nucleotides at positions 733-734 of the leuA gene, indicating that it encodes a variant (hereinafter referred to as N245S) having a serine to asparagine substitution, which is the amino acid at position 245 (at position 210 based on literature data in which the translation start codon is read 35 in the upstream direction and the LeuA protein consists of 581 amino acids (SEQ ID NO: 5); hereinafter referred to only as position 245) of the LeuA protein. The amino acid sequence of the LeuA variant (N245 S) and the nucleotide sequence of the leuA variant encoding it are as in SEQ ID NO: 14 and SEQ ID NO: 15.

In the following examples, it was tested whether the variants (L138G, H162E, S211L, N245S, I588P) affect L-leucine production by microorganisms of the genus Corynebacterium.

Example 3. Testing the ability of selected mutant strains to produce L-leucine

3-1. Construction of an embedding vector containing the leuA variant

In order to introduce the variants selected in Example 2 into the strain, it was intended to construct insertion vectors. Site-directed mutagenesis was used to construct a vector for introducing the leuA variants (L138G, H162E, S211L, N245S, I588P). Specifically, PCR was performed using the chromosome of the wild-type Corynebacterium glutamicum strain ATCC13032 as a template, and using the primer pair with SEQ ID NO: 16 and SEQ ID NO: 17, the pair with SEQ ID NO: 18 and SEQ ID NO: 19 to create the L138G variant, and using the primer pair with SEQ ID NO: 16 and SEQ ID NO: 20 and the primer pair with SEQ ID NO: 19 and SEQ ID NO: 21 to create the H162E variant. PCR was performed using the primer pair with SEQ ID NO: 16 and SEQ ID NO: 22, the primer pair with SEQ ID NO: 19 and SEQ ID NO: 23 to create the S211L variant, and using the primer pair with SEQ ID NO: 16 and SEQ ID NO: 24, the primer pair with SEQ ID NO: 19 and SEQ ID NO: 25 to create the N245S variant. PCR was performed using the primer pair with SEQ ID NO: 16 and SEQ ID NO: 26, the primer pair with SEQ ID NO: 19 and SEQ ID NO: 27 to create the I588P variant. More specifically, PCR was performed by denaturation at 94°C for 5 minutes followed by 30 cycles consisting of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute and 30 seconds, and then polymerization at 72°C for 5 minutes. The specific sequences of the primers used are presented in Table 4 below.

Cloning was performed by fusing the PCR product with the linear pDCM2 vector digested with the restriction enzyme SmaI using In-Fusion enzyme through the homologous sequence of the terminal 15 bases of the DNA fragments, thus constructing 'pDCM2-leuA(L138G)', 'pDCM2-leuA(H162E)', 'pDCM2-leuA(S211L)', 'pDCM2-leuA(N245S)' and 'pDCM2-leuA(I588P)', which are vectors for amino acid replacement in LeuA. In addition, 'pDCM2-leuA(S211L, I588P), pDCM2-leuA(L138G, H162E, S211L, N245S)', 'pDCM2-leuA(L138G, H162E, S211L, N245S, I588P)', which are vectors for amino acid replacement in LeuA, were constructed according to the combination of variants.

3-2. Introduction of the variant into the Corynebacterium glutamicum strain ATCC13032 and evaluation

The vectors pDCM2-leuA(L138G), pDCM2-leuA(H162E), pDCM2-leuA(S211L), pDCM2-leuA(N245S), pDCM2-leuA(I588P), pDCM2-leuA(S211L, I588P), pDCM2-leuA(L138G, H162E, S211L, N245S), _P DCM2-leuA(L138G, H162E, S211L, N245S, I588P) obtained in Example 3-1 were respectively transformed into the Corynebacterium glutamicum ATCC 13032 strain by electroporation, and the strains in which each vector was inserted into the chromosome by recombination of the homologous sequence were selected in a medium containing 25 mg/L kanamycin. The selected primary strains were again subjected to secondary crossing over, and the strains into which the variant of the target gene was introduced were selected. Finally, it was confirmed whether or not the variant of the leuA gene was introduced into the transformed strain by performing PCR using the primers of SEQ ID NO: 3 and SEQ ID NO: 4, and then analyzing the nucleotide sequence, thereby identifying that the variant was introduced into the strain. A total of 8 strains were prepared and named 'ATCC13032_leuA_L138G', 'ATCC13032_leuA_H162E', 'ATCC13032_leuA_S211L', 'ATCC13032_leuA_N245S', 'ATCC13032_leuA_I588P', 'ATCC13032_leuA_(S211L, I588P)''ATCC13032_leuA_(L138G, H162E, S211L, N245S)', 'ATCC13032_leuA_(L138G, H162E, S211L, N245S, I588P)', respectively.

To evaluate the L-leucine producing capacity, a total of 8 strains obtained in this way were evaluated for fermentation titer in a flask. Each one platinum loop of the parent strain Corynebacterium glutamicum ATCC13032 and the resulting ATCC13032_leuA_L138G, ATCC13032_leuA_H162E, ATCC13032_leuA_S211L, ATCC13032_leuA_N245S, ATCC13032_leuA_I588P, ATCC13032_leuA_(S211L, I588P), ATCC13032_leuA_(L138G, H162E, S211L, N245S), ATCC13032 leuA (L138G, H162E, S211L, N245S, I588P) were inoculated into a flask with with 250 ml angular partitions containing 25 ml of production medium, and then cultured at 30°C for 60 hours with shaking at 200 rpm to obtain L-leucine. After completion of the culture, the production of L-leucine was measured by HPLC. The concentration of leucine in the culture medium of each test strain is shown in Table 5 below.

As shown in Table 5, ATCC13032_leuA_L138G with the L138G variant in the leuA gene showed approximately 1.45-fold improvement in L-leucine yield compared with the parental Corynebacterium glutamicum strain ATCC13032, ATCC13032_leuA_H162E with the H162E variant showed approximately 1.49-fold improvement in L-leucine yield compared with the parental Corynebacterium glutamicum strain ATCC13032, ATCC13032_leuA_S211L with the S211L variant showed approximately 1.58-fold improvement in L-leucine yield compared with the parental Corynebacterium glutamicum strain ATCC13032, ATCC13032_leuA_N245S with the N245S variant showed approximately 1.40-fold improvement in L-leucine yield compared to the parent strain Corynebacterium glutamicum ATCC13032, ATCC 13032_leuA_I588P with the I588P variant showed approximately 1.37-fold improvement in L-leucine yield compared to the parent strain Corynebacterium glutamicum ATCC13032, and ATCC13032_leuA_(S211L, I588P) showed approximately 1.51-fold improvement in L-leucine yield compared to the parent strain. ATCC13032_leuA_(L138G, H162E, S211L, N245S) and ATCC13032_leuA_(L138G, H162E, S211L, N245S, I588P) showed approximately 1.56-fold improvement in L-leucine yield compared to the parent strain Corynebacterium glutamicum.

Example 4. Testing the ability of selected leuA variants to produce leucine in leucine-producing strains

The wild-type strain of the genus Corynebacterium produces only trace amounts of leucine, although it produces leucine. Accordingly, a leucine-producing strain derived from the wild type of Corynebacterium glutamicum ATCC 13032 was prepared, and the selected variants were introduced to perform an experiment to test the ability to produce leucine. The detailed experimental method and results are as follows.

4-1. Preparation of L-leucine-producing strain CJL-8109

As strains for producing high concentrations of L-leucine, strains derived from the wild-type Corynebacterium glutamicum ATCC 13032 were obtained, each comprising (1) a variant (R558H) in which histidine is replaced with arginine, which is the amino acid at position 558 of the LeuA protein, by replacing A with G, which is the nucleotide at position 1673 of the leuA gene, (2) a variant (G561D) in which aspartic acid is replaced with glycine, which is the amino acid at position 561 of the LeuA protein, by replacing AT with GC, which are the nucleotides at positions 1682 and 1683 of the leuA gene, or (3) a variant (P247C) in which cysteine is replaced with proline, which is the amino acid at position 247 of the LeuA protein, by replacing TG with CC, which are the nucleotides at positions 739 and 740 of the leuA gene.

Specifically, the pDCM2-leuA(R558H, G561D) vector (US Patent Publication No. 2021-0254111) comprising the leuA gene variants (R558H, G561D) was transformed into Corynebacterium glutamicum ATCC13032 by electroporation, and the strains in which the vector was inserted into the chromosome by recombination of the homologous sequence were selected in a medium containing 25 mg/L kanamycin. The selected primary strains were again subjected to secondary crossing over, and the strains in which the leuA gene variant was introduced were selected. Finally, whether the variant was introduced into the transformed strain was confirmed by performing PCR (94°C for 5 minutes, followed by 30 cycles of 94°C for 30 seconds/55°C for 30 seconds/72°C for 90 seconds and 72°C for 5 minutes) using primers SEQ ID NOs: 28 and 55, and then analyzing the nucleotide sequence, thus identifying the introduction of the R558H, G561D variants. The specific sequences of the primers used are shown in Table 6 below. The strain ATCC13032_leuA_(R558H, G561D) transformed with the pDCM2-leuA(R558H, G561D) vector was named 'CJL-8100'.

To insert the variant (P247C) into the L-leucine-producing strain, a CJL-8100 insertion vector was constructed.

More specifically, PCR was performed using the chromosome of strain CJL-8100 as a template and primer pairs of SEQ ID NOs: 28 and 29 and SEQ ID NOs: 54 and 55. PCR was performed as follows: denaturation at 94°C for 5 minutes, 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute 30 seconds, followed by polymerization at 72°C for 5 minutes. The resulting PCR product was cloned into the linear pDCM2 vector digested with the restriction enzyme SmaI using the In-Fusion enzyme by fusing the homologous sequence of the terminal 15 bases of the DNA fragments, thereby constructing the pDCM2-leuA(P247C, R558H, G561D) vector comprising the leuA variant encoding the LeuA variant in which histidine is replaced by arginine, which is the amino acid at position 558 of the amino acid sequence of LeuA of the wild-type strain, and aspartic acid is replaced by glycine, which is the amino acid at position 561, and cysteine (Cys) is replaced by proline (Pro), which is the amino acid at position 247 of LeuA.

The pDCM2-leuA(P247C, R558H, G561D) vector was transformed into wild-type Corynebacterium glutamicum ATCC 13032 by electroporation, and strains in which the vector was inserted into the chromosome by recombination of the homologous sequence were selected in medium containing 25 mg/L kanamycin. The selected primary strains were again subjected to secondary crossing over, and strains in which the leuA gene variants were introduced were selected. Finally, whether or not the variants were introduced into the transformed strain was confirmed by performing PCR (94°C for 5 minutes, 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds and polymerization at 72°C for 90 seconds, followed by polymerization at 72°C for 5 minutes) using the primers of SEQ ID NOs: 3 and 4, and then analyzing the nucleotide sequence, thus identifying the introduction of the variants P247C, R558H and G561D. The strain ATCC13032_leuA_(P247C, R558H, G561D) transformed with the vector pDCM2-leuA(P247C, R558H, G561D) was named 'CA13-8105'.

CA13-8105 was deposited with the Korea Microbial Culture Center, an international depository organization under the Budapest Treaty, on April 29, 2020, and assigned Accession No. KSCM12709P.

In order to increase the production of L-leucine in the obtained strain CA13-8105, a strain into which the ilvE variant (V156A) encoding branched-chain amino acid transferase (WO 2021-112469 A1) was introduced was obtained. Specifically, the pDCM2-ilvE(V156A) vector containing the ilvE gene variant was transformed into Corynebacterium glutamicum CJL-8100 by electroporation, and the strains in which the vector was inserted into the chromosome by recombination of the homologous sequence were selected in a medium containing 25 mg/L kanamycin. The selected primary strains were again subjected to secondary crossing over, and the strains into which the ilvE gene variant was introduced were selected. Finally, whether or not the variant was introduced into the transformed strain was confirmed by performing PCR (94°C for 5 minutes, 30 cycles of 94°C for 30 seconds/55°C for 30 seconds/72°C for 90 seconds, then 72°C for 5 minutes) using primers SEQ ID NOs: 30 and 31 according to Table 7 below, and then analyzing the nucleotide sequence, thereby identifying the introduction of the V156A variant. The strain transformed with the pDCM2-ilvE(V156A) vector was named 'CJL-8108'.

To increase the production of L-leucine in the obtained strain CJL-8108, a strain was obtained into which a gltA variant (M3121) with weakened citrate synthase activity was introduced.

Specifically, site-directed mutagenesis was used to construct a vector to introduce the gltA(M3121) variant. PCR was performed using the wild-type Corynebacterium glutamicum ATCC13032 chromosome as a template and primers according to Table 8 below. PCR was performed under denaturing conditions of 94°C for 5 minutes, 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute and 30 seconds, followed by polymerization at 72°C for 5 minutes. The resulting gene fragment was cloned into the linear pDCM2 vector digested with the restriction enzyme SmaI using the In-Fusion enzyme by fusing the homologous sequence of the terminal 15 bases of the DNA fragments, thus constructing the pDCM2-gltA(M3121) vector to replace methionine at position 312 with isoleucine.

The pDCM2-gltA(M3121) vector containing the gltA gene variant was transformed into Corynebacterium glutamicum CJL-8108 by electroporation, and strains in which the vector was inserted into the chromosome by recombination of the homologous sequence were selected in a medium containing 25 mg/L kanamycin. The selected primary strains were again subjected to secondary crossing over, and strains in which the gltA gene variant was introduced were selected. Finally, whether the variant was introduced into the transformed strain was confirmed by performing PCR (94°C for 5 minutes, 30 cycles of 94°C for 30 seconds/55°C for 30 seconds/72°C for 90 seconds, followed by 72°C for 5 minutes) using primers SEQ ID NOs: 36 and 37 according to Table 9 below, and then analyzing the nucleotide sequence, thereby identifying the introduction of the M312I variant. The strain transformed with the pDCM2-gltA(M3121) vector was named 'CJL-8109'.

4-2. Construction of an embedding vector containing the leuA variant

In order to introduce the variants (L138G, H162E, S211L, N245S, I588P) selected in Example 2 into the L-leucine-producing strain CJL-8109 obtained in Example 4-1, it was intended to construct an insertion vector.

PCR was performed using the chromosome of strain CJL-8109 as a template and the primer pairs according to Table 4. PCR was performed under conditions of denaturation at 94°C for 5 minutes, 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute and 30 seconds, followed by polymerization at 72°C for 5 minutes. The obtained PCR product was cloned into the linear pDCM2 vector digested with the restriction enzyme SmaI using the In-Fusion enzyme by fusing the homologous sequence of the terminal 15 bases of the DNA fragments, thus constructing a total of 8 vectors 'pDCM2-leuA(L138G, P247C, R558H, G561D)', 'pDCM2-leuA(H162E, P247C, R558H, G561D)', 'pDCM2-leuA(S211L, P247C, R558H, G561D)', 'pDCM2-leuA(N245S, P247C, R558H, G561D)', 'pDCM2-leuA(P247C, R558H, G561D, I588P)', 'pDCM2-leuA(S211L, P247C, I588P)', 'pDCM2-leuA(L138G, H162E, S211L, N245S, P247C, R558H, G561D)' and 'pDCM2-leuA(L138G, H162E, 1L, N245S, P247C, R558H, G561D, I588P)'.

4-3. Introduction of the leuA variant into strain CJL-8109 and evaluation

The L-leucine-producing strain CJL-8109 was transformed with each of the vectors obtained in Example 4-2, and the strains in which each vector was inserted into the chromosome by recombination of a homologous sequence were selected in a medium containing 25 mg/L of kanamycin. The selected primary strains were again subjected to secondary crossing over, and the strains in which the variant of the target gene was introduced were selected. Finally, whether or not the variant of the leuA gene was introduced into the transformed strain was confirmed by performing PCR using the primers of SEQ ID NO: 3 and SEQ ID NO: 4, and then analyzing the nucleotide sequence, thereby identifying that the variant of leuA was introduced into the strain. A total of 8 strains thus obtained were named as in Table 11 below, and the amino acid sequence of the variant including the variation and the nucleotide sequence of the leuA variant encoding it are presented in Table 10 below.

Then, the L-leucine-producing ability of the wild-type Corynebacterium glutamicum ATCC13032 and the derived strains CJL-8109, CJL-8117, CJL-8118, CA13-8119, CJL-8120, CJL-8121, CJL-8122, CJL-8123, and CJL-8125 was evaluated. More specifically, flask culture was performed by the method according to Example 2-1. After completion of the culture, the L-leucine production of the parent strain and the variant strains was measured by HPLC, and the results are shown in Table 11 below.

According to Table 11, CJL-8117, CJL-8118, CA13-8119, CJL-8120, CJL-8121, CJL-8122, CJL-8124 and CJL-8125, which are L-leucine-producing strains having an additional variant of L138G, H162E, S211L, N245S, I588P, S211L/I588P, L138G/H162E/S211L/N245S or L138G/H162E/S211L/N245S/I588P in the leuA gene, showed approximately 4-5-fold improvement in L-leucine productivity compared with the parent strain Corynebacterium glutamicum ATCC 13032 wild type. It was also confirmed that L-leucine-producing Corynebacterium glutamicum strains CJL-8117, CJL-8118, CA13-8119, CJL-8120, CJL-8121, CJL-8122, CJL-8123 and CJL-8125 showed approximately 1.2-1.6-fold improvement in L-leucine productivity compared with the parent strain Corynebacterium glutamicum CJL-8109.

These results indicate that amino acids at positions 138, 162, 211, 245 and 588 of the LeuA protein amino acid sequence represent important sites for L-leucine production.

4-4. Measurement of isopropylmalate synthase activity in a strain with an introduced LeuA variant

To measure the activity of isopropylmalate synthase in CJL-8109 and CJL-8117, CJL-8118, CA13-8119, CJL-8120, CJL-8121, CJL-8122, CJL-8123, and CJL-8125, which are the L-leucine-producing strains obtained in Example 4-3, an experiment was carried out as follows.

Each one platinum loop of the strains (CJL-8109, CJL-8117, CJL-8118, CA13-8119, CJL-8120, CJL-8121, CJL-8122, CJL-8123, CJL-8125) and wild-type Corynebacterium glutamicum ATCC13032 were inoculated into a 250-mL baffled flask containing 25 mL of the production medium according to Table 2, and then cultured at 30°C for 16 hours with shaking at 200 rpm. After completion of the culture, each culture medium was centrifuged and the supernatant was removed. The pellet was washed and suspended with lysis buffer and destroyed. Quantification of protein in the lysate was performed according to the Bradford assay, and a lysate containing 100 μg/ml protein was used. At this time point, the change in absorbance at 412 nm due to thionitrobenzoate (TNB) formed from DTNB (5,5'-dithiobis-(2-nitrobenzoic acid), Ellman's reagent) by reduction using the produced CoA was measured to determine the activity of isopropylmalate synthase enzyme. The results of isopropylmalate synthase activity measurement in each strain are presented in Table 12 below.

Next, to test the degree to which leucine alleviates the feedback inhibition of the enzyme, the isopropylmalate synthase activity was determined by measuring the Co A formed when a lysate containing 100 μg/ml protein was used under conditions in which 2 g/l leucine was added. The results of the isopropylmalate synthase activity measurements in each strain are presented in Table 13 below.

As shown in Tables 12 and 13, CJL-8109 and CJL-8117, CJL-8118, CA13-8119, CJL-8120, CJL-8121, CJL-8122, CJL-8123, and CJL-8125, which are L-leucine-producing strains transformed with a vector expressing the LeuA variant, exhibited approximately 1.18-1.38-fold improvement in isopropylmalate synthase activity compared to the wild-type control Corynebacterium glutamicum ATCC 13032. It was also confirmed that L-leucine-producing strains retained 83% to 93% of isopropylmalate synthase activity even under conditions where 2 g/L leucine was added, indicating a weakening of the feedback inhibition by leucine.

CA13-8119 was deposited with the Korean Culture Center of Microorganisms, an international depository organization under the Budapest Treaty, on February 8, 2021, and assigned the accession number KCCM12949P.

Based on the above description of the invention, it should be clear to those skilled in the art that the present invention can be implemented in another specific form without changing its technical essence or essential characteristics. It should be understood that the above embodiment is not limiting, but illustrative in all respects. The scope of the invention is defined by the appended claims, and not by the preceding description of the invention, and therefore it is intended that all changes and modifications that are within the scope of the claims or equivalents of such scope are covered by the claims.

Claims (14)

1. A polypeptide variant having isopropyl malate synthase activity comprising one or more substitutions selected from the group consisting of 1) a substitution of the amino acid residue corresponding to position 138 with another amino acid residue, 2) a substitution of the amino acid residue corresponding to position 162 with another amino acid residue, 3) a substitution of the amino acid residue corresponding to position 211 with another amino acid residue, 4) a substitution of the amino acid residue corresponding to position 245 with another amino acid residue, and 5) a substitution of the amino acid residue corresponding to position 588 with another amino acid residue, in the amino acid sequence of SEQ ID NO: 1. 2. A variant of the polypeptide according to claim 1, wherein 1) leucine, which is the amino acid residue corresponding to position 138, is replaced by glycine. 3. A polypeptide variant according to claim 1, wherein 2) histidine, which is an amino acid residue corresponding to position 162, is replaced by glutamate. 4. A variant of the polypeptide according to claim 1, wherein 3) serine, which is the amino acid residue corresponding to position 211, is replaced by leucine. 5. A variant of the polypeptide according to claim 1, wherein 4) asparagine, which is the amino acid residue corresponding to position 245, is replaced by serine. 6. A variant of the polypeptide according to claim 1, wherein 5) isoleucine, which is the amino acid residue corresponding to position 588, is replaced by proline. 7. A polypeptide variant according to claim 1, comprising one amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14. 8. A polynucleotide encoding a variant of the polypeptide according to any one of claims 1-7. 9. An expression vector containing the polynucleotide according to claim 8. 10. A microorganism of the genus Corynebacterium that produces L-leucine, wherein the microorganism contains a variant of the polypeptide according to claim 1, encoding its polynucleotide, or including its vector. 11. A microorganism of the genus Corynebacterium according to claim 10, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum . 12. A method for producing L-leucine, comprising the step of culturing in a medium a microorganism of the genus Corynebacterium that produces L-leucine, wherein the microorganism contains a variant of the polypeptide according to claim 1, a polynucleotide encoding it or a vector including it. 13. The method according to claim 12, further comprising the step of isolating L-leucine from the medium or from the microorganism after the culturing step. 14. A composition for producing L-leucine, comprising (i) a microorganism of the genus Corynebacterium producing L-leucine, containing a variant of the polypeptide according to claim 1, encoding its polynucleotide or including its vector; or a medium in which said microorganism is cultured, and (ii) an excipient.