Ketol-acid reductoisomerase mutant and method for producing L-valine using the same

Abstract

The present application relates to ketol-acid reductoisomerase mutants, polynucleotides encoding the mutants of the present application, L-valine-producing microorganisms containing the ketol-acid reductoisomerase mutants of the present application or polynucleotides encoding them, and methods for producing L-valine using the microorganisms of the present application.

Description

[Technical Field]

[0001] The present application relates to ketol-acid reductoisomerase mutants, polynucleotides encoding the mutants of the present application, L-valine-producing microorganisms containing the ketol-acid reductoisomerase mutants of the present application or polynucleotides encoding them, and methods for producing L-valine using the microorganisms of the present application. [Background technology]

[0002] L-amino acids are the basic building blocks of proteins and are used as important ingredients in pharmaceuticals, food additives, animal feed, nutrients, insecticides, fungicides, etc. In particular, branched-chain amino acids (BCAAs) are a collective term for the essential amino acids L-valine, L-leucine, and L-isoleucine, and these branched-chain amino acids have antioxidant effects and promote protein synthesis in muscle cells.

[0003] Branched-chain amino acids are produced using microorganisms, primarily Corynebacterium species, and are known to be biosynthesized from pyruvate in several steps [APPLIED AND ENVIRONMENTAL MICROBIOLOGY, December 2010, pp. 8053-8061]. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] US 8465962 B [Patent Document 2] US Patent US 7662943 B2 [Patent Document 3] US Patent US 10584338 B2 [Patent Document 4] US Patent US 10273491 B2 [Patent Document 5] Republic of Korea Publication No. 10-2020-0136813

Patent document 6

Non-licensed literature

[0005] [Non-licensed document 1] APPLIED AND ENVIRONMENTAL MICROBIOLOGY,Dec.2010,p.8053-8061 [Non-licensed document 2] Pearson et al(1988)[Proc.Natl.Acad.Sci.USA 85]:2444 [Non-licensed document 3] Rice et al., 2000, Trends Genet.16:276-277

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[0006] The production of branched-chain amino acids using microorganisms has the problem that it is not easy to mass-produce them industrially. [Means for solving the problem]

[0007] One object of the present application is to provide a ketol-acid reductoisomerase mutant in which the amino acid corresponding to position 87 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine (V) or aspartate (D).

[0008] Another object of the present application is to provide polynucleotides encoding the variants of the present application.

[0009] Another object of the present application is to provide a microorganism comprising a variant of the present application or a polynucleotide encoding the variant of the present application.

[0010] Another object of the present application is to provide a method for producing L-valine, which includes culturing the microorganism of the present application in a medium.

[0011] Another object of the present application is to provide a composition for producing L-valine, comprising the microorganism of the present application, a medium in which the microorganism of the present application has been cultured, or a combination of two or more thereof. [Effects of the Invention]

[0012] The mutant of the present application can be used to produce L-valine in high yields. DETAILED DESCRIPTION OF THE INVENTION

[0013] This will be explained in more detail as follows: Meanwhile, each description and embodiment disclosed in this application also applies to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the specific descriptions set forth below are not deemed to limit the category of this application. Furthermore, throughout this specification, numerous papers and patent documents are referenced and citations thereof are displayed. The disclosure contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the state of the art to which this application pertains and the contents of this application.

[0014] One aspect of the present application provides a ketol-acid reductoisomerase mutant, in which the amino acid corresponding to position 87 of SEQ ID NO: 1 has been substituted with another amino acid.

[0015] In the present application, the term "ketol-acid reductoisomerase mutant" refers to any polypeptide having ketol-acid reductoisomerase activity or a mutant of ketol-acid reductoisomerase comprising a substitution of the amino acid corresponding to the 87th position from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0016] The mutants of the present application may also be referred to as "ketol-acid reductoisomerase mutants," "(mutant) polypeptides having ketol-acid reductoisomerase activity," or "IlvC mutants."

[0017] In the present application, the term "ketol-acid reductoisomerase" refers to an enzyme also known as "acetohydroxy acid isomeroreductase," "KARI," or "AHAIR," which is involved in the biosynthesis of L-branched-chain amino acids. The ketol-acid reductoisomerase can be classified as EC 1.1.1.86.

[0018] The biosynthetic pathway of L-branched-chain amino acids is characterized by the following: acetohydroxy acid synthase catalyzes the decarboxylation of pyruvate and its condensation with another pyruvate molecule to produce acetolactate, a precursor to valine; and ketol acid reductoisomerase catalyzes the condensation of pyruvate with 2-ketobutyrate to produce acetohydroxybutyrate, a precursor to isoleucine. Ketol acid reductoisomerase then uses the resulting acetolactate or acetohydroxybutyrate as substrates to produce L-valine, L-leucine, and L-isoleucine.

[0019] Specifically, ketol-acid reductoisomerase can convert 2-aceto-2-hydroxybutyrate to 2,3-dihydroxy-3-methylvalerate and 2-acetolactate to 2,3-dihydroxyisovalerate.

[0020] When 2,3-dihydroxy-3-methylvalerate undergoes a reaction catalyzed by dihydroxy acid dehydratase and transaminase B, L-isoleucine is produced. When 2,3-dihydroxyisovalerate undergoes a reaction mediated by dihydroxy acid dehydratase, 2-ketoisovalerate is produced. 2-ketoisovalerate can be converted to L-valine by transaminase B or to 2-ketoisocaproate, which can then be converted to L-leucine through enzymatic conversion. Therefore, ketol-acid reductoisomerase is an important enzyme for the production of branched-chain amino acids, including L-valine, L-leucine, and L-isoleucine.

[0021] The amino acid sequence of the ketol-acid reductoisomerase of the present application may be an amino acid sequence encoded by the ilvC gene, which may also be referred to as an "IlvC protein." The amino acid sequence of the ketol-acid reductoisomerase of the present application can be obtained from the publicly known database, NCBI GenBank. The ketol-acid reductoisomerase may be, but is not limited to, a protein comprising the amino acid sequence of SEQ ID NO: 1. In another example, the ketol-acid reductoisomerase may be derived from a microorganism of the genus Corynebacterium, such as Corynebacterium glutamicum. Examples include WP_003854117.1, 6JX2_A, HJE10081.1, WP_059289140.1, WP_060564426.1, WP_006286981.1, WP_096455581.1, WP_066565326.1, WP_015651057.1, WP_053544709.1, WP_006769331.1, and BAC181 77.1, WP_156227806.1, WP_191733749.1, WP_042621277.1, WP_126119396.1, NLZ56857.1, or WP_015400720.1, but is not limited to these, and may include, without limitation, sequences having the same ketol-acid reductoisomerase activity as the amino acid sequence.

[0022] As a specific example, the ketol-acid reductoisomerase of the present application may be a protein comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or more, or 99% or more homology or identity thereto. Furthermore, it is clear that proteins having an amino acid sequence with such homology or identity and with partial deletion, modification, substitution, or addition of the sequence are also included within the scope of proteins to be mutated in the present application, as long as the amino acid sequence has the same effect as the protein.

[0023] Furthermore, although a protein containing the amino acid sequence of SEQ ID NO: 1 is defined as an example of a ketol-acid reductoisomerase to be mutated in the present application, this does not exclude the addition of meaningless sequences before or after the amino acid sequence of SEQ ID NO: 1, or naturally occurring mutations, or silent mutations thereof, and it will be obvious to those skilled in the art that any protein having the same or corresponding activity as a protein consisting of the amino acid sequence of SEQ ID NO: 1 falls under the ketol-acid reductoisomerase of the present application.

[0024] In other words, even if the present application describes a "protein or polypeptide having an amino acid sequence described in a specific SEQ ID NO," or a "protein or polypeptide comprising an amino acid sequence described in a specific SEQ ID NO," it is obvious that a protein having an amino acid sequence in which part of the sequence has been deleted, modified, substituted, or added is also used in the present application, as long as it has the same or equivalent activity as a polypeptide consisting of the amino acid sequence of the SEQ ID NO.

[0025] As used herein, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid or nucleotide sequences, which can be expressed as a percentage. The terms homology and identity can often be used interchangeably.

[0026] Homology or identity of conserved polynucleotide or polypeptide sequences can be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences are generally capable of hybridizing to all or a portion of the sequence under moderately or highly stringent conditions. Hybridization obviously includes hybridization to polynucleotides containing common codons in polynucleotides or codons that take codon degeneracy into account.

[0027] 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, it can be determined using 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), as implemented in 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 et al. (1988) SIAM J Applied Math 48:1073. For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0028] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, e.g., as known in Smith and Waterman, Adv. Appl. Math (1981) 2:482, or as described in, e.g., Needleman et al. (1970), J. Mol. Biol. 48:443. Briefly, the GAP program defines a match as the total number of symbols in the shorter of the two sequences divided by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include: (1) a unitary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by 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 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps. Thus, the terms "homology" or "identity," as used herein, indicate the relevance between sequences.

[0029] As used herein, the term "variant" or "modified polypeptide" refers to a protein that differs from the recited sequence by conservative substitutions and / or modifications in one or more amino acids, but that maintains the functions or properties of the protein.

[0030] A variant differs from the identified sequence by the substitution, deletion, or addition of a few amino acids. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the protein and evaluating the properties of the modified protein. That is, the ability of the variant may be increased, unchanged, or decreased compared to the native protein. Some variants may also include mutant polypeptides in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, have been deleted. Other variants may include variants in which portions have been deleted from the N- and / or C-termini of the mature protein. The term "variant" or "variant polypeptide" may be interchangeable with terms such as "mutation," "modification," "mutated protein," "mutant," "mutein," "divergent," and "variant," and are not limited to these terms as long as they are used to mean "mutated."

[0031] As used herein, the term "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Typically, a conservative substitution has little or no effect on the activity of the resulting polypeptide.

[0032] Such variants may, for example, have one or more conservative substitutions while retaining one or more biological activities. Such amino acid substitutions are generally made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues.

[0033] Examples of such classifications include positively charged (basic) amino acids such as arginine, lysine, and histidine; negatively charged (acidic) amino acids such as glutamic acid and aspartate; amino acids with nonpolar side chains (nonpolar amino acids) such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids) such as serine, threonine, cysteine, tyrosine, asparagine, and glutamine. As another example, amino acids can be classified into electrically charged amino acids (arginine, lysine, histidine, glutamic acid, and aspartate) and uncharged amino acids (glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine). Phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. Valine, leucine, and isoleucine can be classified as branched-chain amino acids. As another example, the 20 amino acids can be classified by size, and the amino acids can be divided into five groups, starting from the group with the smallest volume: glycine, alanine, serine; cysteine, proline, threonine, aspartate, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, and tyrosine, but are not necessarily limited thereto.

[0034] Variants can also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide can be conjugated to an N-terminal signal (or leader) sequence of a protein involved in co- or post-translational protein transfer. The polypeptide can also be conjugated to other sequences or linkers that allow the polypeptide to be identified, purified, or synthesized.

[0035] In one embodiment, the mutant of the present application may be a ketol-acid reductoisomerase mutant in which the amino acid corresponding to the 87th position from the N-terminus of SEQ ID NO: 1 in the aforementioned ketol-acid reductoisomerase is substituted with another amino acid, or a mutant polypeptide having ketol-acid reductoisomerase activity.

[0036] In any one of the above embodiments, the mutant may increase L-valine production compared to the pre-mutation protein, wild-type protein, native polypeptide, or unmutated polypeptide.

[0037] In the present application, "substitution with another amino acid" is not limited as long as the amino acid is different from the amino acid before substitution. On the other hand, when the expression "a specific amino acid is substituted" is used in the present application, it is obvious that the amino acid is substituted with an amino acid different from the amino acid before substitution, even if it is not specifically stated that the amino acid is substituted with another amino acid.

[0038] In any one of the above embodiments, the "other amino acids" may be amino acids other than glutamine (Q). Specifically, the other amino acids may be selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, aspartate, glutamic acid, lysine, arginine, and histidine.

[0039] In any one of the above embodiments, the mutant may be a ketol-acid reductoisomerase mutant in which the amino acid corresponding to position 87 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine (V) or aspartate (D).

[0040] The "Nth position" in the present application may include the Nth position and an amino acid position corresponding to the Nth position. For example, it may include an amino acid position corresponding to any amino acid residue in a mature polypeptide disclosed in a specific amino acid sequence. The specific amino acid sequence may be the amino acid sequence of SEQ ID NO: 1.

[0041] As used herein, the term "corresponding to" refers to the amino acid residue at the recited position in the polypeptide, or an amino acid residue that is similar, identical, or homologous to the recited residue in the polypeptide. Identifying the amino acid at the corresponding position can also determine the specific amino acid of the sequence to which the particular sequence refers. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0042] In the present application, a specific numbering system may be used for the positions of amino acid residues in the proteins used in the present application. For example, by aligning the polypeptide sequences of the subject protein to be compared with the protein of the present application, it is possible to renumber the positions corresponding to the positions of the amino acid residues in the protein of the present application.

[0043] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, based on which each amino acid residue in the amino acid sequence can be numbered with reference to the numeric position of the corresponding amino acid residue in SEQ ID NO: 1. For example, a sequence alignment algorithm such as that described herein can identify amino acid positions or positions where variations such as substitutions, insertions, or deletions occur compared to a query sequence (also referred to as a "reference sequence").

[0044] For such alignment, 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), etc. can be used, but are not limited to these, and sequence alignment programs, pairwise sequence comparison algorithms, etc. known in the art can be appropriately used.

[0045] In one embodiment, the ketol-acid reductoisomerase variant of the present application may be a variant in which the amino acid corresponding to position 87 of SEQ ID NO: 1 is substituted with valine (V) or aspartate (D) and has a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity to SEQ ID NO: 1. The ketol-acid reductoisomerase variant may also be a variant having less than 100% homology or identity to SEQ ID NO: 1.

[0046] In any one of the above-described embodiments, the ketol-acid reductoisomerase variant of the present application may be a polypeptide in which the amino acid corresponding to position 87 of SEQ ID NO: 1 is substituted with valine (V) or aspartate (D), which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity to SEQ ID NO: 1, and which has ketol-acid reductoisomerase activity.

[0047] In any one of the above-described embodiments, the ketol-acid reductoisomerase variant of the present application may be a polypeptide in which the amino acid corresponding to position 87 of SEQ ID NO: 1 is valine (V) or aspartate (D), which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity to SEQ ID NO: 1, and which has ketol-acid reductoisomerase activity.

[0048] Furthermore, it is obvious that variants having amino acid sequences in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application, as long as the variant has such homology or identity and exhibits efficacy corresponding to the variant of the present application.

[0049] In any one of the above-mentioned embodiments, the variant of the present application may have, comprise, or consist essentially of or consist of the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 25.

[0050] Although the present application refers to a "protein having an amino acid sequence set forth in a particular SEQ ID NO," it is clear that the present application also refers to a protein having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added, so long as it has the same or equivalent activity as a protein consisting of the amino acid sequence of the SEQ ID NO. For example, if it has the same or equivalent activity as the mutant protein, this does not exclude addition of sequences before or after the amino acid sequence that do not change the function of the protein, naturally occurring mutations, silent mutations, or conservative substitutions, and it is clear that even if it has such additions or mutations, it falls within the scope of the present application.

[0051] In any one of the above embodiments, the variant of the present application may comprise or consist of an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or more homology or identity to SEQ ID NO: 3 or SEQ ID NO: 25. In the variant, the amino acid corresponding to position 87 of SEQ ID NO: 3 or 25 may be valine (V) or aspartate (D).

[0052] In any one of the above-mentioned embodiments, the ketol-acid reductoisomerase variant of the present application may have enhanced ketol-acid reductoisomerase activity, but is not limited thereto.

[0053] In any one of the above-described embodiments, the ketol-acid reductoisomerase variant of the present application may have an activity that increases L-valine production ability compared to a wild-type, native, or unmodified polypeptide having ketol-acid reductoisomerase activity, but is not limited thereto.

[0054] Another aspect of the present application provides a polynucleotide encoding a variant of the present application, said variant being as described in the other aspect above.

[0055] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide units are linked in a long chain by covalent bonds, and is a DNA or RNA chain of a certain length or more, and more specifically, refers to a polynucleotide fragment encoding the above-mentioned variant.

[0056] The polynucleotide of the present application may be modified in various ways in the coding region within the scope that does not change the amino acid sequence of the variant of the present application, taking into consideration codon degeneracy or codons preferred in the organism in which the variant of the present application is to be expressed. Therefore, it is obvious that the polynucleotide also includes a polynucleotide that, due to codon degeneracy, is translated into a polypeptide consisting of the amino acid sequence of the variant of the present application or a polypeptide having homology or identity thereto.

[0057] For example, the polynucleotide of the present application may have, comprise, or consist essentially of a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more, but less than 100%, homologous or identical to the polynucleotide sequence of SEQ ID NO: 66, or may consist of or consist essentially of a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homologous or identical to the sequence of SEQ ID NO: 2. Alternatively, in the polynucleotide sequence having the homology or identity, the codon encoding the amino acid corresponding to position 87 of SEQ ID NO: 1 may be, but is not limited to, one of the codons encoding valine (V) or aspartate (D).

[0058] Furthermore, the polynucleotides of the present application may include, without limitation, probes that can be prepared from known gene sequences, for example, sequences that can hybridize under stringent conditions to complementary sequences to all or part of the polynucleotide sequences of the present application. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (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.A. Usubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, pp. 9.50-9.51, 11.7-11.8). For example, conditions include conditions under which polynucleotides with high homology or identity, such as polynucleotides with a homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, hybridize with each other, but polynucleotides with lower homology or identity do not hybridize with each other; and conditions under which washing is performed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions for conventional Southern hybridization: 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0059] Hybridization requires that two nucleic acids have complementary sequences, even though mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to one another. For example, for DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application can include isolated nucleic acid fragments that are complementary to entire sequences, as well as substantially similar nucleic acid sequences.

[0060] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the above-mentioned hybridization conditions, including a hybridization step at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and may be appropriately adjusted by those skilled in the art depending on the purpose.

[0061] The appropriate stringency for hybridizing such polynucleotides depends on the length and degree of complementation of the polynucleotides, variables well known in the art (eg, J. Sambrook et al., supra).

[0062] Another aspect of the present application provides a vector comprising a polynucleotide encoding a variant of the present application, the variant and polynucleotide being as described in the other aspects above.

[0063] The vector may be, but is not limited to, an expression vector for expressing the polynucleotide in a microorganism.

[0064] In this application, the term "vector" may include a DNA construct containing a base sequence of a polynucleotide encoding a polypeptide of interest operably linked to a suitable expression control region (or expression control sequence) so as to express the polypeptide of interest in a suitable host. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating the termination of transcription and translation. After being transformed into a suitable microorganism, the vector can replicate or function independently of the host genome, or it can be integrated into the genome itself.

[0065] The vectors used in this application are not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0066] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a vector for chromosomal integration in a cell. The polynucleotide can be inserted into a chromosome by any method known in the art, including, but not limited to, homologous recombination. A selection marker for confirming the presence or absence of the insertion into the chromosome may be further included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of the insertion of the target nucleic acid molecule. Markers that confer selectable phenotypes, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface polypeptides, can be used. In an environment treated with a selective agent, only cells expressing the selection marker will survive or exhibit other phenotypes, allowing the selection of transformed cells.

[0067] The term "transformation" as used herein refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a microorganism or microorganisms, thereby enabling the expression of the polypeptide encoded by the polynucleotide in the microorganism. A transformed polynucleotide may include any polynucleotide, whether it is inserted into the chromosome of the microorganism or located extrachromosomally, as long as it is expressible in the microorganism. The polynucleotide may also include DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form that can be introduced and expressed in the microorganism. For example, the polynucleotide may be introduced into the microorganism in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression. The expression cassette typically includes a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may also be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the microorganism in its own form and operably linked to sequences necessary for its expression in the microorganism, but is not limited thereto.

[0068] In addition, the term "operably linked" 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 application.

[0069] As used herein, the term "transfection" refers to a method for transferring a polynucleotide encoding a variant of the present application or a vector containing the same into a host cell. Such introduction can be easily achieved by methods commonly used in the art. Common methods include the CaCl precipitation method, the Hanahan method, which uses a reducing agent, DMSO (dimethyl sulfoxide), to enhance the efficiency of the CaCl method, electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicon carbide fibers, PEG-based transformation, dextran sulfate, lipofectamine, and desiccation / repression-mediated transformation. The method for transforming the vector is not limited to the above examples, and any transformation or transfection method commonly used in the art can be used without limitation.

[0070] Another aspect of the present application provides a microorganism comprising a ketol-acid reductoisomerase variant of the present application or a polynucleotide encoding said variant.

[0071] In one embodiment, the microorganism may comprise a vector comprising a polynucleotide encoding the variant.

[0072] In one embodiment, the microorganism of the present application may be a microorganism capable of producing L-valine.

[0073] In this application, the term "L-valine" refers to an L-amino acid with the chemical formula (CH3)2CHCH(NH2)COOH, which is one of the essential amino acids and structurally belongs to the branched-chain amino acids along with L-leucine and L-isoleucine.

[0074] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have been genetically modified naturally or artificially, and may be microorganisms in which a specific mechanism has been weakened or strengthened by inserting an exogenous gene or by enhancing or inactivating the activity of an endogenous gene, and may also be microorganisms that have been genetically modified to produce a desired polypeptide, protein, or product. In this application, the terms "microorganism," "strain," and "microorganism" may be used interchangeably and without limitation.

[0075] In this application, the term "microorganism capable of producing L-valine" refers to a prokaryotic or eukaryotic strain of microorganism capable of producing L-valine in vivo, and includes both microorganisms in which the ability to produce L-valine has been imparted to a parent strain that does not have the ability to produce L-valine, and microorganisms that have the ability to produce L-valine endogenously. The ability to produce L-valine can be imparted or enhanced by species improvement.

[0076] In one embodiment, the microorganism of the present application may be, but is not limited to, a microorganism that naturally has the ability to produce ketol-acid reductoisomerase or L-valine; or a microorganism into which a mutant of the present application or a polynucleotide encoding the mutant (or a vector containing the polynucleotide) has been introduced into a parent strain that does not have the ability to produce ketol-acid reductoisomerase or L-valine, and / or into which the ability to produce L-valine has been imparted.

[0077] In one embodiment, the microorganism of the present application includes, but is not limited to, a microorganism in which a chromosomal gene encoding a ketol-acid reductoisomerase is mutated to contain the ketol-acid reductoisomerase variant sequence of the present application and / or a microorganism in which a vector containing a polynucleotide encoding the ketol-acid reductoisomerase variant of the present application is introduced to contain the ketol-acid reductoisomerase variant of the present application.

[0078] In any one of the above embodiments, the microorganism provided herein may be a microorganism that has been genetically modified to express the ketol-acid reductoisomerase of the present application.

[0079] In the present application, the term "unmodified microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, but refers to a wild-type or naturally occurring strain itself, or a strain before its traits are changed by genetic mutation due to natural or artificial factors. For example, the unmodified microorganism refers to a strain into which a ketol-acid reductoisomerase mutant described herein has not been introduced or before it has been introduced. The term "unmodified microorganism" may be used interchangeably with "pre-modified strain," "pre-modified microorganism," "non-mutated strain," "non-modified strain," "non-mutated microorganism," or "reference microorganism."

[0080] The microorganism capable of producing L-valine of the present application may be, but is not limited to, a microorganism comprising one or more of the variant of the present application, the polynucleotide of the present application, and the vector comprising the polynucleotide of the present application; a microorganism that has been modified to express the variant of the present application or the polynucleotide of the present application; a microorganism (e.g., a recombinant strain) that expresses the variant of the present application or the polynucleotide of the present application; or a microorganism (e.g., a recombinant strain) that has the activity of the variant of the present application.

[0081] For example, the strain of the present application is a cell or microorganism that has been transformed with a vector containing the polynucleotide of the present application or a polynucleotide encoding the variant of the present application and expresses the variant of the present application, and the strain of the present application may include all microorganisms that contain the variant of the present application and are capable of producing L-valine. For example, the microorganism of the present application may be a naturally occurring wild-type microorganism, or a recombinant strain in which a polynucleotide encoding the variant of the present application has been introduced into a microorganism capable of producing L-valine, thereby expressing a ketol-acid reductoisomerase variant and increasing L-valine-producing ability.

[0082] The strain having increased L-valine production ability may be, but is not limited to, a microorganism having increased L-valine production ability compared to a natural wild-type microorganism or a microorganism not expressing a ketol-acid reductoisomerase variant (e.g., a microorganism expressing a wild-type ketol-acid reductoisomerase or a microorganism not expressing the mutant of the present application).

[0083] In one embodiment, the microorganism of the present application having increased L-valine-producing ability may be, but is not limited to, a microorganism having increased L-valine-producing ability compared to a microorganism containing the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding the same.

[0084] For example, the microorganism with increased L-valine production ability may have an increase of about 1% or more, about 2% or more, about 2.5% or more, or about 3% or more (there is no particular upper limit, and it may be, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, or about 20% or less) compared to the L-valine production ability of the parent strain or unmodified microorganism before mutation, but is not limited thereto as long as there is an increase in the + value compared to the production ability of the parent strain or unmodified microorganism before mutation. In another example, the recombinant strain with increased L-valine production ability may have an increased L-valine production ability of about 1.01-fold or more, about 1.02-fold or more, or about 1.03-fold or more (there is no particular upper limit, and the increase may be, for example, about 10-fold or less, about 5-fold or less, about 3-fold or less, about 2-fold or less, or about 1.5-fold or less) compared to the parent strain or untransformed microorganism before mutation, but is not limited thereto.

[0085] Examples of parent strains or untransformed microorganisms before mutation for comparison with the increase in L-valine production ability include microorganisms containing the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding the same. Other examples include, but are not limited to, Corynebacterium glutamicum KCCM11201P (US 8465962 B), Corynebacterium glutamicum ATCC13869, and Corynebacterium glutamicum ATCC14067.

[0086] The term "about" refers to a range that includes, but is not limited to, ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values in a range that is equal to or similar to the numerical value following the term "about."

[0087] The microorganism of the present application may include any microorganism that can express the ketol-acid reductoisomerase mutant of the present application by various known methods other than the introduction of the nucleic acid or vector.

[0088] The microorganism of the present application may be a Corynebacterium microorganism.

[0089] In one embodiment, the microorganism of the present application is Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium In any one of the above embodiments, the microorganism may be Corynebacterium glutamicum.

[0090] The L-valine-producing microorganisms of the genus Corynebacterium of the present application include all of: natural wild-type microorganisms themselves; microorganisms of the genus Corynebacterium that have improved L-valine-producing ability by strengthening or weakening the activity of genes related to the L-valine production mechanism; and microorganisms of the genus Corynebacterium that have improved L-valine-producing ability by introducing or strengthening the activity of an exogenous gene.

[0091] In one embodiment, the microorganism of the present application may comprise an acetohydroxy acid synthase modified to have enhanced L-valine production. In any one of the above-described embodiments, the microorganism of the present application may comprise a modified acetohydroxy acid synthase subunit (ilvN). The acetohydroxy acid synthase subunit may comprise a mutation in which amino acid 42 is substituted with valine (A42V). However, the present application is not limited thereto.

[0092] The microorganism of the present application may have enhanced activity of the ketol-acid reductoisomerase variant of the present application.

[0093] As used herein, the term "enhancement" of polypeptide activity means that the activity of a polypeptide is increased compared to its endogenous activity. The term "enhancement" may be used interchangeably with terms such as "activation," "up-regulation," "overexpression," and "increase." Here, activation, enhancement, up-regulation, overexpression, and increase can all encompass the display of an activity not inherently possessed, or the display of an activity that is improved compared to the endogenous activity or the activity before modification. The term "endogenous activity" refers to the activity of a specific polypeptide inherently possessed by a parent strain or an unaltered microorganism before the transformation, in cases where a trait is altered by genetic mutation due to natural or artificial factors. This term may be used interchangeably with "activity before modification." "Enhancement," "up-regulation," "overexpression," or "increase" of a polypeptide activity compared to its endogenous activity means that the activity and / or concentration (expression level) of a specific polypeptide is improved compared to the activity and / or concentration (expression level) of a specific polypeptide inherently possessed by a parent strain or an unaltered microorganism before the transformation.

[0094] The enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. The enhancement of the activity of the polypeptide can be confirmed by an increase in the activity level, expression level, or amount of a product secreted from the polypeptide.

[0095] The activity of the polypeptide can be enhanced by various methods well known in the art, and is not limited thereto, as long as the activity of the polypeptide of interest can be enhanced compared to that of the microorganism before transformation. Specifically, the enhancement may be achieved by using genetic engineering and / or protein engineering, which are routine methods in molecular biology and 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.).

[0096] Specifically, the enhancement of the polypeptide of the present application is 1) an increase in the intracellular copy number of the polynucleotide encoding the polypeptide; 2) replacing the expression regulatory region of a gene on a chromosome that encodes a polypeptide with a sequence with strong activity; 3) a modification of the nucleotide sequence encoding the initiation codon or 5'UTR region of a gene transcript encoding a polypeptide; 4) modifying the amino acid sequence of the polypeptide so that the polypeptide activity is enhanced; 5) modifying a polynucleotide sequence encoding the polypeptide so as to enhance the polypeptide's activity (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the polypeptide's activity); 6) introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same; 7) codon optimization of the polynucleotide encoding the polypeptide; 8) analyzing the tertiary structure of the polypeptide and selectively altering or chemically modifying exposed sites; or 9) It may be a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0097] More specifically, The 1) increase in the intracellular copy number of a polynucleotide encoding a polypeptide may be achieved by introducing into a host cell a vector capable of replicating and functioning independently of the host, to which a polynucleotide encoding the polypeptide is operably linked. Alternatively, the increase may be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide into a chromosome in the host cell. The introduction into a chromosome can be achieved by, but is not limited to, introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome in the host cell. The vector is as described above.

[0098] The replacement of the expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide with a sequence with stronger activity may involve, for example, mutation of the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further enhance the activity of the expression regulatory region, or replacement with a sequence with stronger activity. The expression regulatory region may include, 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. For example, the original promoter may be replaced with a strong promoter, but this is not limited thereto.

[0099] Examples of known strong promoters include, but are not limited to, the CJ1 to 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. 10,584,338 B2), the O2 promoter (U.S. Patent No. 10,273,491 B2), the tkt promoter, and the yccA promoter.

[0100] The modification of the nucleotide sequence encoding the start codon or 5'UTR region of the gene transcript encoding the polypeptide (3) may be, for example, but is not limited to, substituting a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate than the endogenous start codon.

[0101] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) above may be, but is not limited to, a sequence mutation such as 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 an amino acid sequence or polynucleotide sequence modified to have stronger activity or to have increased activity. Specifically, the replacement can be achieved by, but is not limited to, inserting the polynucleotide into a chromosome by homologous recombination. The vector used in this case may further contain a selection marker for detecting the presence or absence of insertion into the chromosome. The selection marker is as described above.

[0102] The introduction of an exogenous polynucleotide that exhibits the activity of a polypeptide (6) may be the introduction of an exogenous polynucleotide that encodes a polypeptide that exhibits the same or similar activity as the polypeptide into a host cell. The exogenous polynucleotide is not limited in its origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The method used for the introduction can be any known transformation method appropriately selected by those skilled in the art, and the introduced polynucleotide may be expressed in a host cell to produce a polypeptide, and its activity may be increased.

[0103] The codon optimization of the polynucleotide encoding the polypeptide (7) may be that of an endogenous polynucleotide that has been codon-optimized to increase transcription or translation in a host cell, or that of an exogenous polynucleotide that has been codon-optimized to optimize transcription or translation in a host cell.

[0104] 8) Analyzing the tertiary structure of a polypeptide and selecting and deforming or chemically modifying exposed sites may involve, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, determining candidate template proteins according to the degree of sequence similarity, confirming the structure based on the candidate template proteins, and selecting and deforming or chemically modifying exposed sites.

[0105] Such enhancement of polypeptide activity may be, but is not limited to, an increase in the activity or expression level of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in a wild-type or untransformed microbial strain, or an increase in the amount of a product produced from the polypeptide.

[0106] Modification of a portion or all of a polynucleotide in the microorganism of the present application may be induced by, but is not limited to, (a) homologous recombination using a vector for chromosomal insertion in the microorganism or genome editing using engineered nucleases (e.g., CRISPR-Cas9) and / or (b) treatment with light and / or chemicals, such as ultraviolet light and radiation. Methods for modifying a portion or all of the gene include methods using DNA recombination technology. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene can be injected into the microorganism to cause homologous recombination, thereby deleting a portion or all of the gene. The injected nucleotide sequence or vector may contain, but is not limited to, a dominant selectable marker.

[0107] In the microorganism of the present application, the mutant, polynucleotide, L-valine, etc. are as described in the other aspects above.

[0108] Another aspect of the present application provides a method for producing L-valine, comprising culturing the microorganism of the present application in a medium.

[0109] Specifically, the method for producing L-valine of the present application may include, but is not limited to, culturing a microorganism containing a variant of the present application, a polynucleotide of the present application, or a vector of the present application in a medium.

[0110] In the present application, the term "culturing" means growing the microorganism of the present application under appropriately controlled environmental conditions. The culturing process of the present application can be carried out using appropriate media 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 may be, but is not limited to, a batch, continuous, and / or fed-batch culture.

[0111] In the present application, the term "culture medium" refers to a substance containing a mixture of nutrients, primarily as components, required for culturing the microorganism of the present application, and provides nutrients such as water essential for survival and growth, as well as growth factors, etc. Specifically, the culture medium and other culture conditions used for culturing the microorganism of the present application can be any medium used for culturing conventional microorganisms without any particular limitations. The microorganism of the present application can be cultured in a conventional culture medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins, etc., under aerobic conditions while controlling the temperature, pH, etc.

[0112] For example, culture media for Corynebacterium strains can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0113] In the present application, 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.; and amino acids such as glutamic acid, methionine, lysine, etc. Natural organic nutrient sources such as starch hydrolysate, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid may also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used. A variety of other suitable carbon sources may also be used without limitation. These carbon sources may be used alone or in combination, and are not limited thereto.

[0114] Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its hydrolyzate, defatted soybean cake or its hydrolyzate, etc. These nitrogen sources may be used alone or in combination of two or more, and are not limited thereto.

[0115] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or the corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and other compounds, including amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the culture medium in a batch or continuous manner. However, the present invention is not limited to these.

[0116] During the cultivation of the microorganism of the present application, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the medium in an appropriate manner. Also, during cultivation, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. To maintain an aerobic state in the medium, oxygen or an oxygen-containing gas can be injected into the medium, or to maintain an anaerobic or microaerobic state, no gas can be injected or nitrogen, hydrogen, or carbon dioxide gas can be injected, but this is not limiting.

[0117] In the present invention, the culture temperature can be maintained at 20 to 45°C, specifically 25 to 40°C, and the culture can be carried out for about 10 to 160 hours, but is not limited thereto.

[0118] The L-valine produced by the culture of the present application is either secreted into the medium or remains intracellularly.

[0119] The method for producing L-valine of the present application may further include, for example, before the culturing step, a step of preparing the microorganism of the present application, a step of preparing a medium for culturing the strain, or a combination thereof (in any order).

[0120] The method for producing L-valine of the present application may further include a step of recovering L-valine from the culture medium (culture medium) or the microorganism of the present application. The recovery step may be performed after the culturing step.

[0121] The recovery may involve collecting L-valine using a suitable method known in the art based on the microbial culture method of the present application, such as a batch, continuous, or fed-batch culture method. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination of these methods can be used to recover L-valine from the medium or the microorganism using a suitable method known in the art.

[0122] The method for producing L-valine of the present application may further include a purification step. The purification can be performed using an appropriate method known in the art. For example, when the method for producing L-valine of the present application includes both a recovery step and a purification step, the recovery step and the purification step can be performed continuously or discontinuously in any order, or simultaneously or integrated into one step, but are not limited thereto.

[0123] In the method of the present application, the mutant, polynucleotide, microorganism, L-valine, etc. are as described above in other aspects.

[0124] Another aspect of the present application is to provide a composition for producing L-valine, comprising: a microorganism containing a ketol-acid reductoisomerase variant of the present application or a polynucleotide encoding the variant; a medium in which the microorganism is cultured; or a combination of two or more of them.

[0125] The composition of the present application may further contain any suitable excipient commonly used in compositions for producing amino acids, and such excipients may be, for example, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, or an isotonic agent, but are not limited thereto.

[0126] In the composition of the present application, the mutant, polynucleotide, microorganism, L-valine, etc. are as described above in other aspects.

[0127] Another aspect of the present application is to provide use of the ketol-acid reductoisomerase mutant of the present application for producing L-valine. The mutant is as described above.

[0128] The present application will be described in more detail below through experimental examples. However, the following examples are merely preferred embodiments for illustrating the present application, and are not intended to limit the scope of the present application. Meanwhile, technical matters not described in this specification can be fully understood and easily performed by those of ordinary skill in the technical field of the present application or a similar technical field.

[0129] Example 1-1. Induction of artificial mutations through UV irradiation To select mutants with increased valine production, the valine-producing strain Corynebacterium glutamicum KCCM11201P (US 8465962 B) was spread onto a nutrient medium containing agar and cultured at 30°C for 36 hours. Several hundred colonies thus obtained were irradiated with UV light at room temperature to induce random mutations in the genome of the strain.

[0130] Example 1-2. Evaluation of fermentation titer of mutagenized strains and selection of strains To select mutants with increased L-valine production compared to the parent strain, Corynebacterium glutamicum KCCM11201P, fermentation titer experiments were performed on randomly mutated strains. Each colony was subcultured in nutrient medium, and each strain was inoculated into a 250ml corner-baffled flask containing 25ml of production medium. The strains were then cultured at 30°C for 72 hours with shaking at 200 rpm. The L-valine concentrations were then analyzed using HPLC, and the analyzed L-valine concentrations are shown in Table 1.

[0131] [Nutrient medium (pH 7.2)] Glucose 10g, meat extract 5g, polypeptone 10g, sodium chloride 2.5g, yeast extract 5g, agar 20g, urea 2g (based on 1 liter of distilled water)

[0132] [Production medium (pH 7.0)] Glucose 100g, ammonium sulfate 40g, soy protein 2.5g, corn steep solids 5g, urea 3g, dipotassium phosphate 1g, magnesium sulfate heptahydrate 0.5g, biotin 100μg, thiamine-HCl 1mg, calcium pantothenate 2mg, nicotinamide 3mg, calcium carbonate 30g (based on 1 liter of distilled water)

[0133] [Table 1] JPEG2025526824000002.jpg46149

[0134] Referring to Table 1, the C12 strain was selected, which showed the greatest increase in valine production compared to the control strain KCCM11201P.

[0135] Example 2. Confirmation of mutations through gene sequencing The major genes of these strains were sequenced and compared with those of the KCCM11201P strain and the wild-type Corynebacterium glutamicum ATCC14067 strain. The results confirmed that the strains with increased valine production contained nucleotide sequence mutations at specific positions in the ORF (open reading frame) region of the ilvC gene. Specifically, the C12 strain, which showed the greatest increase in valine production, contained three mutations in the nucleotide sequence located 259-261 bp downstream from the initiation codon of the ilvC gene, changing the existing CAG (SEQ ID NO: 66) to GTT (SEQ ID NO: 2) and substituting the 87th amino acid, glutamine, with valine (SEQ ID NO: 3).

[0136] Analysis of the mutated region of SEQ ID NO: 2 confirmed that it affects the effector binding domain of the valine biosynthetic enzyme, and predicted that the activity of the protein would be enhanced. In the following example, we investigated whether application of the Q87V mutation inserted at a specific position in the ORF of the ilvC gene affects the ability of Corynebacterium to produce the branched-chain amino acid valine. We also investigated whether substitution of an amino acid other than valine with a mutation at the 87th amino acid glutamine position affects the ability of Corynebacterium to produce the branched-chain amino acid valine.

[0137] Example 3. Construction of KCCM11201P strain with ilvC mutation and confirmation of valine production ability Example 3-1. Construction of a Corynebacterium glutamicum KCCM11201P strain with an ilvC mutation and evaluation of its L-valine production ability A vector containing the target mutation was constructed to insert the ilvC(Q87V) mutant shown in SEQ ID NO:2 into S. glutamicum KCCM11201P. Specifically, genomic DNA from the C12 strain was extracted using a G-spin Total DNA Extraction Mini Kit (Intron, Cat. No. 17045) according to the protocol provided with the kit, and PCR was performed using the genomic DNA as a template. PCR conditions included denaturation at 94°C for 5 minutes, followed by 25 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 150 seconds, followed by polymerization at 72°C for 7 minutes. A 1010 bp PCR product (hereafter referred to as "mutagenesis fragment 1") was obtained using SEQ ID NO:4 and SEQ ID NO:5.

[0138] The resulting mutagenized fragment 1 was ligated to pDCM2 vector (Korea Publication No. 10-2020-0136813) treated with the restriction enzyme SmaI (New England Biolabs, Beverly, MA) using an Infusion Cloning Kit (Takara Bio Inc., Otsu, Japan), and then transformed into E. coli DH5α. The constructed gene was transformed into E. coli DH5α, which was then selected in LB medium containing kanamycin. DNA was then isolated using a DNA-spin Plasmid DNA Purification Kit (iNtRON) to prepare vector pDCM2-ilvC(Q87V) containing mutagenized fragment 1.

[0139] [Table 2]

[0140] The pDCM2-ilvC(Q87V) was transformed into Corynebacterium glutamicum KCCM11201P by homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which the vector had been integrated into the chromosome through recombination of the homologous sequences were selected on a medium containing 25 mg / L kanamycin. After the secondary recombination, the Corynebacterium glutamicum transformants were subjected to PCR using SEQ ID NOs: 4 and 5 to identify strains in which glutamine was replaced with valine at amino acid position 87 of SEQ ID NO: 1 in the ilvC gene ORF on the chromosome. The recombinant strain was designated Corynebacterium glutamicum KCCM11201P::ilvC(Q87V). A flask assay was performed to compare the valine-producing abilities of the valine-producing strains Corynebacterium glutamicum KCCM11201P and KCCM11201P::ilvC(Q87V). After subculture in nutrient medium, each strain was inoculated into a 250-ml corner-baffled flask containing 25 ml of production medium and cultured at 30°C for 72 hours with shaking at 200 rpm. The L-valine concentrations were then analyzed using HPLC, and the analyzed L-valine concentrations are shown in Table 3.

[0141] [Nutrient medium (pH 7.2)] Glucose 10g, meat extract 5g, polypeptone 10g, sodium chloride 2.5g, yeast extract 5g, agar 20g, urea 2g (based on 1 liter of distilled water)

[0142] [Production medium (pH 7.0)] Glucose 100g, ammonium sulfate 40g, soy protein 2.5g, corn steep solids 5g, urea 3g, dipotassium phosphate 1g, magnesium sulfate heptahydrate 0.5g, biotin 100μg, thiamine-HCl 1mg, calcium pantothenate 2mg, nicotinamide 3mg, calcium carbonate 30g (based on 1 liter of distilled water)

[0143] [Table 3]

[0144] As a result, it was confirmed that the L-valine productivity of the KCCM11201P::ilvC(Q87V) strain was increased by 16% compared to KCCM11201P.

[0145] Example 3-2: Construction of a Corynebacterium glutamicum CJ7V strain with an ilvC mutation and evaluation of its L-valine production ability To confirm whether the increased L-valine productivity effect also exists in other strains of Corynebacterium glutamicum that produce L-valine, a single mutation [ilvN(A42V); Biotechnology and Bioprocess Engineering, June 2014, Volume 19, Issue 3, pp. 456-467] was introduced into the wild-type Corynebacterium glutamicum ATCC14067 to create a strain with improved L-valine productivity.

[0146] Specifically, genomic DNA from the wild-type Corynebacterium glutamicum strain ATCC14067 was extracted using a G-spin Total DNA Extraction Mini Kit (Intron, Cat. No. 17045) according to the protocol provided with the kit. PCR was performed using the genomic DNA as a template. To construct a vector for introducing the A42V mutation into the ilvN gene, gene fragments (A and B) were obtained using the primer pair SEQ ID NOs: 6 and 7 and 8 and 9, respectively. PCR conditions were as follows: denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 60 seconds, repeated 25 times; polymerization at 72°C for 7 minutes.

[0147] As a result, polynucleotides of 528 bp and 509 bp were obtained from fragments A and B, respectively. Using these fragments as templates, overlapping PCR was performed using SEQ ID NO: 6 and SEQ ID NO: 9 to obtain a 1010 bp PCR product (hereinafter referred to as "mutagenesis fragment 2").

[0148] The resulting mutated fragment 2 was digested with the restriction enzyme SmaI (New England Biolabs, Beverly, MA) and then ligated with pDCM2 vector digested with the same restriction enzyme using T4 ligase (New England Biolabs, Beverly, MA). The resulting gene was transformed into Escherichia coli DH5α, which was then selected on LB medium containing kanamycin, and DNA was isolated using a DNA-spin plasmid DNA purification kit (iNtRON). The vector used to introduce the A42V mutation into the ilvN gene was designated pDCM2-ilvN(A42V).

[0149] [Table 4]

[0150] The pDCM2-ilvN(A42V) was then transformed into wild-type Corynebacterium glutamicum ATCC14067 by homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which the vector had been integrated into the chromosome through recombination of the homologous sequences were selected on a medium containing 25 mg / L kanamycin. After the secondary recombination, the Corynebacterium glutamicum transformants were subjected to PCR amplification of gene fragments using SEQ ID NO: 6 and SEQ ID NO: 9, and the mutant-inserted strains were identified through gene sequence analysis. The recombinant strain was designated Corynebacterium glutamicum CJ7V. Finally, the Corynebacterium glutamicum CJ7V was transformed with a vector in the same manner as in Example 3-1 to prepare a strain named Corynebacterium glutamicum CJ7V::ilvC(Q87V). To compare the L-valine production ability of the prepared strains, they were cultured in the same manner as in Example 3-1 and the L-valine concentration was analyzed. The analyzed L-valine concentrations are shown in Table 5 below.

[0151] [Table 5]

[0152] As a result, it was confirmed that the L-valine productivity of the CJ7V::ilvC(Q87V) strain was increased by 18% compared to CJ7V.

[0153] Example 3-3: Construction of a strain containing the ilvC mutation introduced into Corynebacterium glutamicum CJ8V strain and evaluation of its L-valine production ability To confirm whether the increased L-valine productivity effect also applies to other strains of Corynebacterium glutamicum that produce L-valine, a single mutation [ilvN(A42V); Biotechnology and Bioprocess Engineering, June 2014, Volume 19, Issue 3, pp. 456-467] was introduced into Corynebacterium glutamicum ATCC13869 to create a strain with improved L-valine productivity (KR 10-1947945 B1).

[0154] Specifically, the wild-type Corynebacterium glutamicum ATCC13869 strain was transformed with the vector pDCM2-ilvN(A42V) constructed in Example 3-2, and homologous recombination was induced in the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which the vector had been integrated into the chromosome via homologous recombination were selected in a medium containing 25 mg / L kanamycin. The selected Corynebacterium glutamicum transformants were subjected to PCR amplification of gene fragments using a primer pair of SEQ ID NOS: 10 and 11, and the gene sequence was analyzed to confirm that the mutation had been successfully introduced. The recombinant strain was designated Corynebacterium glutamicum CJ8V. The primer sequences used in this example are listed in Table 6 below.

[0155] [Table 6]

[0156] Finally, the Corynebacterium glutamicum CJ8V was transformed with a vector in the same manner as in Example 3-1 to prepare a strain named Corynebacterium glutamicum CJ8V::ilvC(Q87V). To compare the L-valine production ability of the prepared strains, they were cultured in the same manner as in Example 3-1 and the L-valine concentration was analyzed. The analyzed L-valine concentrations are shown in Table 7 below.

[0157] [Table 7]

[0158] As a result, it was confirmed that the L-valine producing ability of the CJ8V::ilvC(Q87V) strain was increased by 21% compared to CJ8V.

[0159] Example 4: Construction of KCCM11201P strain with Q87X mutation and confirmation of valine production ability To mutate the 87th amino acid of ilvC, glutamine, to an amino acid other than valine, site-directed mutagenesis was performed using pDCM2-ilvC(Q87V) used in Example 3-1 as a template. Site-directed mutagenesis was performed as follows.

[0160] [Table 8]

[0161] [Table 9]

[0162] The 87th amino acid of ilvC, glutamine, was replaced with other amino acids other than valine, such as alanine (A) (SEQ ID NO: 12), valine (V) (SEQ ID NO: 3), isoleucine (I) (SEQ ID NO: 13), glycine (G) (SEQ ID NO: 14), phenylalanine (F) (SEQ ID NO: 15), methionine (M) (SEQ ID NO: 16), serine (S) (SEQ ID NO: 17), proline (P) (SEQ ID NO: 18), threonine (T) (SEQ ID NO: 19), and tyrosine (Y) (SEQ ID NO: 20). To replace the amino acids with nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,

[0163] [Table 10] JPEG2025526824000012.jpg202149 JPEG2025526824000013.jpg103149

[0164] pDCM2_ilvC(Q87A), pDCM2_ilvC(Q87I), pDCM2_ilvC(Q87G), pDCM2_ilvC(Q87F), pDCM2_ilvC(Q87M), pDCM2_ilvC(Q87S), pDCM2_ilvC(Q87P), pDCM2_ilvC(Q87T), pDCM2_ilvC(Q87Y), pDCM2_ilvC(Q87H), and pDCM2_ilvC(Q87M) were constructed as shown in Table 10. KCCM11201P was transformed with the pDCM2_ilvC(Q87Q), pDCM2_ilvC(Q87N), pDCM2_ilvC(Q87K), pDCM2_ilvC(Q87D), pDCM2_ilvC(Q87C), pDCM2_ilvC(Q87W), pDCM2_ilvC(Q87R), and pDCM2_ilvC(Q87E) vectors by electroporation, and 18 strains carrying the mutant ilvC gene inserted into the chromosome were obtained through a second crossover process. The genetic manipulation was confirmed by PCR using primers (SEQ ID NO: 4 and SEQ ID NO: 5) that can amplify the upstream and downstream regions of the inserted homologous recombination region, respectively, and by genome sequencing.

[0165] The transformed strains thus obtained were designated KCCM11201P::ilvC(Q87A), KCCM11201P::ilvC(Q87I), KCCM11201P::ilvC(Q87G), KCCM11201P::ilvC(Q87F), KCCM11201P::ilvC(Q87M), KCCM11201P::ilvC(Q87S), KCCM11201P::ilvC(Q87P), KCCM11201P::ilvC(Q87T), and KCCM11201P::ilvC(Q87T). They were named vC(Q87Y), KCCM11201P::ilvC(Q87H), KCCM11201P::ilvC(Q87Q), KCCM11201P::ilvC(Q87N), KCCM11201P::ilvC(Q87K), KCCM11201P::ilvC(Q87D), KCCM11201P::ilvC(Q87C), KCCM11201P::ilvC(Q87W), KCCM11201P::ilvC(Q87R), and KCCM11201P::ilvC(Q87E).

[0166] KCCM11201P::ilvC(Q87V), KCCM11201P::ilvC(Q87A), KCCM11201P::ilvC(Q87I), KCCM11201P::ilvC(Q87G), KCCM11201P::ilvC(Q87F), KCCM11201P::ilvC(Q87M), KCCM11201P::ilvC(Q87S), KCCM11201P::ilvC(Q87P), KCCM11201P::ilvC(Q87T), KCCM11201P::ilvC(Q87Y), KCCM11201P::ilvC(Q To confirm the valine production amounts of the KCCM11201P::ilvC(Q87H), KCCM11201P::ilvC(Q87Q), KCCM11201P::ilvC(Q87N), KCCM11201P::ilvC(Q87K), KCCM11201P::ilvC(Q87D), KCCM11201P::ilvC(Q87C), KCCM11201P::ilvC(Q87W), KCCM11201P::ilvC(Q87R), and KCCM11201P::ilvC(Q87E), the strains were cultured in the same manner as in Example 3-1 and the L-valine concentrations were analyzed. The analyzed L-valine concentrations are shown in Table 11 below.

[0167] [Table 11] JPEG2025526824000015.jpg39149

[0168] As a result, the L-valine productivity of the KCCM11201P::ilvC(Q87V) strain was increased by 16% compared to KCCM11201P, and the L-valine productivity of the KCCM11201P::ilvC(Q87D) strain was also confirmed to be increased compared to KCCM11201P. Strains with other mutations introduced showed productivity equivalent to that of KCCM11201P or had only a slight effect.

[0169] This confirmed that the mutant of the present application increases L-valine production in microorganisms.

[0170] From the above description, those skilled in the art to which the present application pertains will understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present application should be interpreted as including within the meaning and scope of the following claims and any modifications or variations derived from the equivalent concepts thereof, rather than the above detailed description.

Claims

1. A ketol-acid reductoisomerase mutant in which the amino acid corresponding to position 87 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine (V) or aspartate (D).

2. The variant of claim 1, wherein the variant comprises SEQ ID NO: 3 or SEQ ID NO: 25; or an amino acid sequence having 80% or more sequence identity thereto.

3. A polynucleotide encoding the variant of claim 1 or 2.

4. A microorganism comprising a ketol-acid reductoisomerase mutant in which the amino acid corresponding to position 87 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine (V) or aspartate (D), or a polynucleotide encoding said mutant.

5. The microorganism according to claim 4 , wherein the microorganism is a Corynebacterium microorganism.

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

7. A method for producing L-valine, comprising the step of culturing in a medium a microorganism containing a ketol-acid reductoisomerase mutant in which the amino acid corresponding to position 87 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine (V) or aspartate (D), or a polynucleotide encoding said mutant.

8. 8. The method for producing L-valine according to claim 7, further comprising recovering L-valine.

9. A composition for producing L-valine, comprising: a microorganism containing a ketol-acid reductoisomerase mutant in which the amino acid corresponding to position 87 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine (V) or aspartate (D), or a polynucleotide encoding said mutant; a culture medium in which said microorganism is cultured; or a combination of two or more of them.

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