Novel acetohydroxy acid synthase subunit variant and method for producing L-valine using the same

Abstract

The present application relates to novel acetohydroxyacid synthase subunit (ilvN) mutants, polynucleotides encoding the mutants of the present application, L-valine-producing microorganisms containing the acetohydroxyacid synthase subunit (ilvN) mutants of the present application, and methods for producing L-valine using the microorganisms of the present application.

Description

Technical Field

[0001] This application relates to a novel acetohydroxy acid synthase subunit (ilvN) variant, a polynucleotide encoding the variant of the present application, an L-valine-producing microorganism comprising the acetohydroxy acid synthase subunit (ilvN) variant of the present application, and an L-valine production method using the microorganism of the present application.

Background Art

[0002] L-amino acids are the basic building units of proteins and are used as important materials such as pharmaceutical raw materials, food additives, animal feeds, nutritional agents, insecticides, and bactericides. In particular, branched-chain amino acids (BCAAs) are a general term for the essential amino acids L-valine, L-leucine, and L-isoleucine, and these branched-chain amino acids have an antioxidant effect and an effect of promoting protein synthesis in muscle cells.

[0003] The production of branched-chain amino acids using microorganisms is mainly carried out by microorganisms of the genus Corynebacterium, and it is known that 2-ketoisocaproate is biosynthesized from pyruvic acid through various steps as a precursor (Patent Documents 1, 2). However, the production of branched-chain amino acids by said microorganisms has the problem that industrial mass production is not easy.

[0004] Under such circumstances, the present applicants confirmed that when a mutant with enhanced activity of the gene ilvN (acetohydroxy acid synthase small subunit), which encodes an enzyme involved in L-valine biosynthesis in microorganisms, was introduced for the purpose of improving the ability to produce branched-chain amino acids using microorganisms, the ability to produce branched-chain amino acids was improved.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

[0006] [Non-licensed Document 1] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-licensed Document 2] Rice et al., 2000, Trends Genet.16: 276-277 [Non-licensed Document 3] J. Sambrook et al., MolecularCloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989 [Non-licensed Document 4] FM Ausubel et al.,CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc., New York,9.50-9.51, 11.7-11.8 [Non-licensed Document 5] Pearson et al (1988) [Proc. Natl.Acad. Sci. USA 85]: 2444 [Non-licensed Document 6] Devereux, J., et al, Nucleic AcidsResearch 12: 387(1984) [Non-Patent Document 7] Atschul, [S.] [F.,] [ET AL, JMOLEC BIOL 215]: 403 (1990) [Non-Patent Document 8] Guide to Huge Computers, Martin J.Bishop, [ED.,] Academic Press, San Diego,1994 [Non-Patent Document 9] [CARILLO ETA / .](1988) SIAM JApplied Math 48: 1073 [Non-Patent Document 10] Smith and Waterman, Adv. Appl.Math(1981) 2:482 [Non-Patent Document 11] Schwartz and Dayhoff, eds., AtlasOf Protein Sequence And Structure, National Biomedical Research Foundation, pp.353-358(1979) [Non-Patent Document 12] Gribskov et al (1986) Nucl. Acids Res. 14: 6745 [Non-Patent Document 13] "Manual of Methods for General Bacteriology" by the American Society for Bacteriology (WashingtonD.C., USA, 1981) [Non-Patent Document 14] van der Rest et al., Appl MicrobiolBiotechnol 52:541-545, 1999 [Overview of the project] [Problems that the invention aims to solve]

[0007] This application aims to provide a novel acetohydroxy acid synthase subunit (ilvN) variant, a polynucleotide encoding the variant of this application, an L-valine-producing microorganism containing the acetohydroxy acid synthase subunit (ilvN) variant of this application, and an L-valine production method using the microorganism of this application.

Means for Solving the Problems

[0008] This application aims to provide an acetohydroxy acid synthase subunit (ilvN) variant in which the amino acid corresponding to the 159th position in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.

[0009] Moreover, this application aims to provide a polynucleotide encoding the variant of this application.

[0010] Furthermore, this application aims to provide a microorganism containing the variant of this application or a polynucleotide encoding the variant of this application.

[0011] Furthermore, this application aims to provide an L-valine production method including the step of culturing the microorganism of this application in a medium.

[0012] Furthermore, this application aims to provide a composition for L-valine production containing the microorganism of this application, the medium in which the microorganism of this application is cultured, or a combination of at least two of them.

Effects of the Invention

[0013] When culturing a microorganism containing the acetohydroxy acid synthase subunit variant of this application, L-valine can be produced in a high yield compared to a microorganism having a conventional unmodified polypeptide.

Modes for Carrying Out the Invention

[0014] These will be explained in detail below. Note that each description and embodiment disclosed in this application applies to other descriptions and embodiments. That is, any combination of the various elements disclosed in this application is included. Furthermore, this application is not limited to the following specific descriptions. In addition, numerous papers and patent documents are referenced throughout this specification, and their citations are indicated. The disclosures of the cited papers and patent documents are incorporated in their entirety as references in this specification, thereby more clearly explaining the level of the art to which this application belongs and the content of this application.

[0015] One aspect of this application provides an acetohydroxy acid synthase subunit (ilvN) variant in which the amino acid corresponding to position 159 in the amino acid sequence of Sequence ID No. 1 is substituted with another amino acid.

[0016] In this application, "acetohydroxy acid synthase subunit (ilvN) variant" means an acetohydroxy acid synthase subunit (ilvN) variant that includes at least one amino acid substitution in the amino acid sequence of a polypeptide having acetohydroxy acid synthase subunit (ilvN) activity.

[0017] In this application, "acetohydroxy acid synthase" refers to the first enzyme in L-valine biosynthesis, also known as acetolactic acid synthase. Acetohydroxy acid synthase catalyzes the decarboxylation of pyruvate and its condensation with other pyruvate molecules to produce acetolactic acid, a precursor of valine, or catalyzes the decarboxylation of pyruvate and its condensation with 2-ketobutyrate to produce acetohydroxybutyrate, a precursor of isoleucine.

[0018] Acetohydroxy acid synthase is encoded by two genes, ilvB and ilvN. The ilvB gene encodes the large subunit of acetohydroxy acid synthase, and the ilvN gene encodes the small subunit. Of these, the small subunit encoded by the ilvN gene is thought to be importantly involved in feedback inhibition.

[0019] In this application, "acetohydroxy acid synthase subunit (ilvN)" refers to the acetohydroxy acid synthase subunit (ilvN) encoded by the ilvN gene, but is not limited to this. Furthermore, the acetohydroxy acid synthase subunit (ilvN) protein or its variants derived from a Corynebacterium microorganism, specifically Corynebacterium glutamicum, is not limited to these. Specifically, the acetohydroxy acid synthase subunit (ilvN) protein includes, for example, the amino acid sequence of Sequence ID No. 1, or an amino acid sequence having 70% or more homology or identity thereto, but is not limited to these as long as it has acetohydroxy acid synthase subunit (ilvN) activity. Specifically, the amino acid sequence may include SEQ ID NO: 1, or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with SEQ ID NO: 1. The sequence of SEQ ID NO: 1 can be obtained from known databases such as NCBI's GenBank or KEGG (Kyoto Encyclopedia of Genes and Genomes). For example, it may be derived from the genus Corynebacterium or Corynebacterium glutamicum, and more specifically, a polypeptide / protein containing the amino acid sequence represented by SEQ ID NO: 1, but is not limited to these. Furthermore, it goes without saying that auxiliary proteins having amino acid sequences in which some sequences are deleted, modified, substituted, or added are also included in this application, as long as they have such homology or identity and exhibit efficacy equivalent to the aforementioned protein.

[0020] Furthermore, an acetohydroxy acid synthase subunit (ilvN) protein having the amino acid sequence of SEQ ID NO: 1 is encoded by a polynucleotide having, or containing, or consisting of, or substantially composed of, the sequence of SEQ ID NO: 2, or a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or less than 100% homology or identity with the sequence of SEQ ID NO: 2, or a polynucleotide substantially composed of the said nucleotide sequence.

[0021] In this application, "variant" refers to a polypeptide whose amino acid sequence differs from that of the original polypeptide due to a conservative substitution and / or modification of at least one amino acid, but whose functions or properties are maintained. Such variants can generally be identified by modifying at least one amino acid in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the capabilities of the variant are improved, unchanged, or decreased compared to the original polypeptide. Some variants also include those in which at least one part, such as the N-terminal leader sequence or transmembrane domain, has been removed. Other variants include those in which a portion of the N and / or C-terminus of a mature protein has been removed. The term "variant" is used interchangeably with terms such as mutant, modified, mutant polypeptide, mutated protein, and mutation (in English, these include modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), but any term that means mutation is acceptable.

[0022] Furthermore, the variants may include the deletion or addition of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide may be bound to the N-terminal signal (or leader) sequence of a protein involved in protein transfer, either co-translationally or post-translationally. Alternatively, the polypeptide may be bound to other sequences or linkers so that the polypeptide can be identified, purified, or synthesized.

[0023] The acetohydroxy acid synthase subunit (ilvN) variant of this application may be, but is not limited to, an acetohydroxy acid synthase subunit (ilvN) variant in which the amino acid corresponding to position 159 in the amino acid sequence of Sequence ID No. 1 is substituted with another amino acid.

[0024] In one embodiment, the acetohydroxy acid synthase subunit (ilvN) variant of this application may be, but is not limited to, a variant in which the amino acid corresponding to the 159th position in the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid, and further, the amino acid corresponding to the 42nd position in the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid.

[0025] The aforementioned "other amino acid" may be any amino acid different from the amino acid before substitution. It goes without saying that in this application, "a specific amino acid has been substituted" means that the amino acid has been substituted with an amino acid different from the amino acid before substitution, even if it is not explicitly stated that it has been substituted with another amino acid.

[0026] Amino acids are generally classified based on the similarities of their residues in terms of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity.

[0027] As an example of this classification, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; amino acids with nonpolar side chains (nonpolar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Other examples include amino acids with electrically charged side chains (arginine, lysine, histidine, glutamic acid, and aspartic acid) and amino acids with uncharged side chains (also called neutral amino acids) (glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine). Further examples include phenylalanine, tryptophan, and tyrosine, which are classified as aromatic amino acids. Still further examples include valine, leucine, and isoleucine, which are classified as branched amino acids. As another example, the 20 amino acids are classified by size and, in order of increasing volume, into five groups: glycine, alanine, and serine; cysteine, proline, threonine, aspartic acid, and asparagine; valine, histidine, glutamic acid, and glutamine; isoleucine, leucine, methionine, lysine, and arginine; and phenylalanine, tryptophan, and tyrosine. However, the classification is not necessarily limited to these groups.

[0028] For example, "the amino acid corresponding to position 159 in Sequence ID No. 1 has been substituted with another amino acid" means that, excluding alanine, glutamate, phenylalanine, glycine, arginine, aspartate, cysteine, asparagine, glutamine, histidine, proline, serine, tyrosine, isoleucine, lysine, and This means that the amino acid is substituted with tryptophan, valine, methionine, threonine, or leucine. "The amino acid corresponding to the 42nd position in SEQ ID NO: 1 is substituted with another amino acid" means, but is not limited to, substitution with valine, asparagine, glycine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, proline, serine, tyrosine, isoleucine, leucine, lysine, phenylalanine, tryptophan, methionine, or threonine, excluding alanine.

[0029] Even if this application describes "a protein having an amino acid sequence represented by a specific sequence number," it goes without saying that any protein having the same or equivalent activity as the protein consisting of the amino acid sequence of said sequence number, even if it has an amino acid sequence in which some of the sequence is deleted, modified, substituted, conserved substituted, or added, can be used in this application. For example, if it has the same or equivalent activity as the mutant protein, this does not exclude the addition of sequences that do not change the function of the protein before or after the amino acid sequence, naturally occurring mutations, silent mutations, or conserved substitutions, and it goes without saying that any protein having such sequence additions or mutations is also included in this application.

[0030] In this application, "position N" includes the Nth position and the amino acid position corresponding to (correspoding) the Nth position. Specifically, it includes the amino acid position corresponding to any amino acid residue in a mature polypeptide represented by a specific amino acid sequence. The specific amino acid sequence may be the amino acid sequence of Sequence ID No. 1.

[0031] In this application, "corresponding to" means an amino acid residue at a position listed in the polypeptide, or an amino acid residue that is similar, identical, or homologous to a residue listed in the polypeptide. Identifying the amino acid at the corresponding position will determine the specific amino acid in the sequence referencing the particular sequence. In this application, "corresponding region" generally refers to a similar or corresponding position in the related protein or reference protein.

[0032] For example, by aligning any amino acid sequence with Sequence ID No. 1, each amino acid residue in the sequence can be numbered based on the number and position of amino acid residues corresponding to the amino acid residues in Sequence ID No. 1. For example, the sequence alignment algorithm in this application can be used to identify the positions of amino acids, or the positions where modifications such as substitutions, insertions, or deletions occur, by comparing it with a query sequence (also called a "reference sequence").

[0033] For such alignment, for example, the Needleman-Wunsch algorithm (Non-Patent Literature 1) and the Needle program from the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Literature 2) can be used, but are not limited to these. Sequence alignment programs and pairwise sequence comparison algorithms known in the art can be used as appropriate.

[0034] In one embodiment, the acetohydroxy acid synthase subunit (ilvN) variant of this application may include an amino acid sequence in which one or two amino acids at positions corresponding to the 42nd and 159th positions from the N-terminus of Sequence ID No. 1 are substituted with other amino acids.

[0035] In any of the examples described above, the acetohydroxy acid synthase subunit (ilvN) variant of this application is a polypeptide in which alanine, the amino acid corresponding to position 159 of SEQ ID NO: 1, is substituted with glutamic acid, but is not limited to this.

[0036] In any of the examples described above, the acetohydroxy acid synthase subunit (ilvN) variant of this application is a polypeptide in which the amino acid alanine, corresponding to the 159th position of SEQ ID NO: 1, is replaced with glutamic acid, and the amino acid alanine, corresponding to the 42nd position, is replaced with valine, but is not limited to this.

[0037] In any of the embodiments described above, the variant provided in this application may include a substitution of the amino acid corresponding to the 159th position from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0038] In any of the above-described examples, the variant provided by this application may be one in which the amino acid corresponding to the 159th position from the N-terminus of SEQ ID NO: 1 is substituted with an amino acid selected from arginine, lysine, histidine, glutamic acid, and aspartic acid, which are amino acids having a charged side chain. For example, the amino acid may be an amino acid selected from the acidic amino acids glutamic acid and aspartic acid. In any of the above-described examples, the variant may be one in which the amino acid corresponding to the 159th position from the N-terminus of SEQ ID NO: 1 is substituted with glutamic acid (E).

[0039] In any of the embodiments described above, the variant provided in this application may include a substitution of the amino acid corresponding to the 42nd position from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0040] In any of the above-described examples, the mutant may be one in which the amino acid corresponding to the 42nd position from the N-terminus of SEQ ID NO: 1 is substituted with a nonpolar amino acid. For example, the amino acid may be an amino acid selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. In any of the above-described examples, the mutant may be one in which the amino acid corresponding to the 42nd position from the N-terminus of SEQ ID NO: 1 is substituted with valine (V).

[0041] On the other hand, a person skilled in the art can identify the amino acid corresponding to the 159th position and the amino acid corresponding to the 42nd position of the amino acid sequence of Sequence ID No. 1 of this application in any given amino acid sequence, using sequence alignment known in the art. In this application, it goes without saying that "amino acids at specific positions in a particular Sequence ID" include "amino acids at corresponding positions" in any given amino acid sequence.

[0042] Furthermore, the variants of this application are amino acid sequences that have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with the amino acid sequence represented by SEQ ID NO: 1, in which the amino acid at the 159th position from the N-terminus of SEQ ID NO: 1 may be substituted with another amino acid, or the amino acid corresponding to the 159th position from the N-terminus of SEQ ID NO: 1 may be substituted with another amino acid, and furthermore, the amino acid corresponding to the 42nd position of the amino acid sequence of SEQ ID NO: 1 may be substituted with another amino acid. Moreover, it goes without saying that variants having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted, or added are also included in this application, as long as they have such homology or identity and exhibit efficacy equivalent to the variants of this application.

[0043] For example, the variant of this application may have an amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 5, may contain the amino acid sequence, or may be substantially composed of the amino acid sequence. Alternatively, it may contain an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 5.

[0044] Examples include the addition or deletion of a sequence that does not alter the function of the variant of this application, a spontaneous mutation, a silent mutation, or a conserved substitution at the N-terminus, C-terminus, and / or within the amino acid sequence.

[0045] In this application, "conservative substitution" means that one amino acid is replaced by another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Furthermore, amino acids are classified into those with electrically charged side chains and those with uncharged side chains. Examples of amino acids with electrically charged side chains include aspartic acid, glutamic acid, lysine, arginine, and histidine. Amino acids with uncharged side chains are further classified into nonpolar amino acids and polar amino acids. Examples of nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Examples of polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Typically, conservative substitutions have little to no effect on the activity of the resulting protein or polypeptide.

[0046] In one example, the acetohydroxy acid synthase subunit (ilvN) mutant of this application has enhanced acetohydroxy acid synthase subunit (ilvN) activity, but is not limited thereto. Furthermore, the mutant of this application has the activity to improve L-valine production capacity compared to the wild-type polypeptide having acetohydroxy acid synthase subunit (ilvN) activity, but is not limited thereto.

[0047] Another aspect of this application provides a polynucleotide encoding a variant of the present application.

[0048] In this application, "polynucleotide" means a polymer of nucleotides in which nucleotide monomers are covalently linked together in a long chain, and refers to a DNA or RNA chain longer than a predetermined length, and more specifically, refers to a polynucleotide fragment that codes for the aforementioned variant.

[0049] The polynucleotide encoding the acetohydroxy acid synthase subunit (ilvN) variant of this application may be any polynucleotide sequence that encodes the acetohydroxy acid synthase subunit (ilvN) variant of this application. For example, the polynucleotide encoding the acetohydroxy acid synthase subunit (ilvN) variant of this application may be, but is not limited to, a polynucleotide sequence that encodes the amino acid sequence of the acetohydroxy acid synthase subunit (ilvN) variant of this application.

[0050] The polynucleotides of this application can be modified in various ways in the coding region, either through codon degeneracy or by considering the preferred codons in the organism that intends to express the variant of this application, as long as the amino acid sequence of the variant of this application does not change. Therefore, it goes without saying that polynucleotides that are translated by codon degeneracy into polypeptides consisting of the amino acid sequence of the variant of this application, or polypeptides homologous or identical thereto, are also included.

[0051] For example, the polynucleotides of this application have a base sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the sequence of SEQ ID NO: 4 or SEQ ID NO: 6, or include such a base sequence, or consist of a base sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the sequence of SEQ ID NO: 4 or SEQ ID NO: 6, or substantially consist of such a base sequence, but are not limited to these. Alternatively, in the homologous or identical sequences, the codon encoding the amino acid corresponding to position 159 of SEQ ID NO: 3 or SEQ ID NO: 5 is one of the codons encoding glutamic acid, and the codon encoding the amino acid corresponding to position 42 of SEQ ID NO: 5 is one of the codons encoding valine, but is not limited to these.

[0052] Furthermore, the polynucleotide of this application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a sequence complementary to all or part of the polynucleotide sequence of this application. The "stringent conditions" refer to conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Documents 3 and 4). For example, this could involve hybridizing polynucleotides with high homology or identity, such as polynucleotides with 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, while not hybridizing polynucleotides with lower homology or identity. Alternatively, it could involve washing once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of a typical Southern hybridization: 60°C, 1×SSC, 0.1%SDS, more specifically 60°C, 0.1×SSC, 0.1%SDS, or more specifically 68°C, 0.1×SSC, 0.1%SDS.

[0053] Hybridization requires that the two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of this application may include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0054] Specifically, polynucleotides homologous or identical to the polynucleotide of this application can be detected using hybridization conditions in which the hybridization step is performed at a Tm value of 55°C and the conditions described above. The Tm value may be 60°C, 63°C, or 65°C, but is not limited to these, and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0055] The appropriate stringency for hybridizing the polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are known in the art (e.g., Non-Patent Document 3).

[0056] In this application, "homology" or "identity" refers to the degree to which two given amino acid sequences or base sequences are similar, and is expressed as a percentage. Homology and identity are often used interchangeably.

[0057] The sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard sequence algorithms, and a default gap penalty established by the program used may also be applied. Substantially, homologous or identical sequences generally hybridize with all or part of the sequence under moderate to high stringent conditions. Needless to say, hybridization includes hybridization with polynucleotides that have common codons or codons considering codon degeneracy in the polynucleotide.

[0058] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using default parameters, such as those in Non-Patent Document 5, and known computer algorithms such as the "FASTA" program. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 1), as performed in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later) (including the GCG program package (Non-Patent Document 6), BLASTP, BLASTN, and FASTA (Non-Patent Documents 7, 8, and 9)). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information.

[0059] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as Non-Patent Document 1, as disclosed in Non-Patent Document 10, for example. In summary, the GAP program defines the number of similar sequence symbols (i.e., nucleotides or amino acids) as the total number of symbols in the shorter of two sequences divided by the number of similar sequence symbols. Default parameters for the GAP program include (1) a binary comparison matrix (where identity is 1 and non-identity is 0) and a weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix) as disclosed in Non-Patent Document 11, as in Non-Patent Document 12; (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extended penalty of 0.5); and (3) no penalty for terminal gaps. Thus, "homology" or "identity" in this application indicates the relevance between sequences.

[0060] A further aspect of this application provides a vector comprising the polynucleotide of this application, which is an expression vector for expressing the polynucleotide in a microorganism, but is not limited thereto.

[0061] In this application, “vector” means a DNA product comprising a polynucleotide sequence encoding a target polypeptide operably ligated to a suitable regulatory region (or regulatory sequence) so as to enable the expression of the target polypeptide in a suitable host. The regulatory region includes a promoter to initiate transcription, an optional operator sequence to regulate the transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences to regulate the termination of transcription and translation. When transformed into a suitable microorganism, the vector can replicate and function independently of the host genome and is integrated into the genome itself.

[0062] The vectors used in this application are not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state. For example, as phage vectors or cosmid vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used, and as plasmid vectors, pDZ series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, pET series, etc. can be used. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

[0063] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using an intracellular chromosome introduction vector. The insertion of the polynucleotide into the chromosome can be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. The vector may further include a selection marker to confirm whether or not the polynucleotide has been inserted into the chromosome. The selection marker is used to select cells transformed by the vector, that is, to confirm whether or not the target nucleic acid molecule has been inserted, and markers that confer selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, and expression of surface polypeptides are used. In an environment treated with a selective agent, only cells expressing the selection marker will survive or exhibit different phenotypes, thus allowing for the selection of transformed cells.

[0064] In this application, "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a microorganism or into a microorganism, thereby causing the polypeptide encoded by the polynucleotide to be expressed in the microorganism. The transformed polynucleotide may be any form, regardless of whether it is inserted into or outside the chromosome of the microorganism, as long as it is expressed in the microorganism. The polynucleotide also contains DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced into the microorganism in any form, as long as it is introduced into the microorganism and expressed. For example, the polynucleotide may be introduced into the microorganism in the form of an expression cassette, which is a gene structure containing all the elements necessary for its expression. Typically, the expression cassette 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 the sequence necessary for expression in the microorganism, but is not limited to this.

[0065] Furthermore, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the target variant of this application.

[0066] Further embodiments of this application provide microorganisms comprising an acetohydroxy acid synthase subunit (ilvN) variant of this application, a polynucleotide encoding an acetohydroxy acid synthase subunit (ilvN) variant of this application, or a vector comprising a polynucleotide of this application.

[0067] In one embodiment, the microorganism of this application may be a microorganism capable of producing L-valine.

[0068] In this application, "L-valine" refers to an L-amino acid that is one of the essential amino acids, structurally classified as a branched-chain amino acid along with L-leucine and L-isoleucine, and represented by the chemical formula (CH3)2CHCH(NH2)COOH.

[0069] In this application, "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have been genetically modified naturally or artificially. These are microorganisms in which a specific mechanism has been weakened or strengthened due to causes such as the insertion of external genes or the enhancement or inactivation of endogenous gene activity, and which have been genetically modified for the production of a target polypeptide, protein, or product. In this application, "microorganism" and "strain" are used interchangeably and are used together.

[0070] In this application, "L-valine-producing microorganism" refers to a prokaryotic or eukaryotic microbial strain that produces L-valine within its body, and includes microorganisms in which L-valine production ability has been conferred from a parent strain that lacks L-valine production ability, as well as microorganisms that inherently possess L-valine production ability. L-valine production ability can be conferred or improved through selective breeding.

[0071] In one embodiment, the microorganism of the present application may be a microorganism that naturally possesses an acetohydroxyacid synthase subunit (ilvN) mutant or L-valine production ability, or it may be a microorganism in which the mutant of the present application or a polynucleotide encoding it (or a vector containing the polynucleotide) has been introduced into a parent strain that lacks an acetohydroxyacid synthase subunit (ilvN) mutant or L-valine production ability, and / or has been conferred L-valine production ability.

[0072] In one embodiment, the microorganisms of the present application include, but are not limited to, microorganisms containing the acetohydroxy acid synthase subunit (ilvN) variant sequence of the present application due to a mutation in a chromosomal gene encoding the acetohydroxy acid synthase subunit (ilvN) variant, and / or microorganisms containing the acetohydroxy acid synthase subunit (ilvN) variant by introducing a vector containing a polynucleotide encoding the acetohydroxy acid synthase subunit (ilvN) variant of the present application.

[0073] In this application, "unmodified microorganism" does not exclude strains containing naturally occurring mutations in microorganisms, but rather means wild-type strains or natural strains themselves, or strains before they undergo genetic mutation and changes in trait due to natural or artificial factors. For example, the aforementioned unmodified microorganism means a strain in which the acetohydroxy acid synthase subunit (ilvN) mutant as defined herein has not been introduced, or before its introduction. The aforementioned "unmodified microorganism" is used interchangeably with "pre-modification strain," "pre-modification microorganism," "non-mutant strain," "unmodified strain," "non-mutant microorganism," or "reference microorganism."

[0074] The microorganisms having the ability to produce L-valine according to this application include, but are not limited to, the mutants of this application, the polynucleotides of this application, and microorganisms comprising at least one of the vectors comprising the polynucleotides of this application, the mutants of this application, or microorganisms modified to express the polynucleotides of this application, the mutants of this application, or microorganisms expressing the polynucleotides of this application (e.g., recombinant strains), or microorganisms having the activity of the mutants of this application (e.g., recombinant strains).

[0075] For example, the strain of this application is a cell or microorganism transformed with a vector containing the polynucleotide of this application or the variant of this application, and expressing the variant of this application. The strain of this application may be any microorganism that contains the variant of this application and produces L-valine. For example, the microorganism of this application may be a recombinant strain in which the acetohydroxyacid synthase subunit (ilvN) variant is expressed and L-valine production is improved by introducing the polynucleotide encoding the variant of this application into a naturally occurring wild-type microorganism or a microorganism capable of producing L-valine. The recombinant strain with improved L-valine production is a microorganism that has improved L-valine production compared to a naturally occurring wild-type microorganism or a microorganism that does not express the acetohydroxyacid synthase subunit (ilvN) (for example, a microorganism that expresses wild-type acetohydroxyacid synthase subunit (ilvN) or a microorganism that does not express the variant of this application), but is not limited to this. For example, the microorganisms with improved L-valine production capacity described in this application are, but are not limited to, microorganisms with improved L-valine production capacity compared to microorganisms containing the polypeptide of Sequence ID No. 1 or the polynucleotide encoding it.

[0076] The microorganisms of this application include all microorganisms that express the acetohydroxy acid synthase subunit (ilvN) variant of this application by various known methods other than the introduction of nucleic acids or vectors.

[0077] As an example, the microorganisms with improved L-valine production capacity show an improvement of approximately 1% or more compared to the L-valine production capacity of the parent strain before mutation or the unmodified microorganism. Specifically, this improvement is approximately 1% or more, approximately 2.5% or more, approximately 5% or more, approximately 6% or more, approximately 7% or more, approximately 8% or more, approximately 9% or more, approximately 10% or more, approximately 10.5% or more, approximately 11% or more, approximately 11.5% or more, approximately 12% or more, approximately 12.5% ​​or more, approximately 13% or more, approximately 13.5% or more, approximately 14% or more, and approximately 14.5% or more. The product is an improvement of approximately 14.6% or more, approximately 14.7% or more, or approximately 14.8% or more (there is no particular limit on the upper limit, for example, approximately 200% or less, approximately 150% or less, approximately 100% or less, approximately 50% or less, approximately 45% or less, approximately 40% or less, approximately 35% or less, approximately 30% or less, approximately 25% or less, approximately 20% or less, or approximately 15% or less), but any product is acceptable as long as it has a positive increase in productivity compared to the parent strain before mutation or the unmodified microorganism. As another example, the recombinant strain with improved L-valine production capacity is one in which the L-valine production capacity has improved by approximately 1.1 times or more, approximately 1.12 times or more, approximately 1.13 times or more, or approximately 1.14 times or more compared to the parent strain before mutation or the unmodified microorganism (there is no particular upper limit, for example, approximately 10 times or less, approximately 5 times or less, approximately 3 times or less, approximately 2 times or less, approximately 1.5 times or less, or approximately 1.2 times or less), but is not limited to these. The term "approximately" includes a range that encompasses ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and is not limited to any numerical value that is equivalent to or in a similar range to the numerical value following the term "approximately".

[0078] The microorganisms covered by this application are Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, and Corynebacterium ammoniagenes. The microorganisms may be Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens. Specifically, the microorganisms of this application are, but are not limited to, microorganisms of the genus Corynebacterium, more specifically Corynebacterium glutamicum.

[0079] On the other hand, while it is already known that microorganisms of the genus Corynebacterium produce L-valine, their production capacity is very low, and the genes and mechanisms involved in the production process have not been elucidated. Therefore, the Corynebacterium microorganisms possessing L-valine production capacity described in this application include the naturally occurring wild-type microorganisms themselves, Corynebacterium microorganisms whose L-valine production capacity has been improved by enhancing or weakening the activity of genes related to the L-valine production mechanism, and Corynebacterium microorganisms whose L-valine production capacity has been improved by introducing or enhancing the activity of external genes.

[0080] Furthermore, the microorganism of this application may have enhanced activity of the acetohydroxy acid synthase subunit (ilvN) compared to the parent strain.

[0081] In this application, "enhancement" of polypeptide activity means improving the polypeptide activity compared to its endogenous activity. This enhancement is used interchangeably with activation, upregulation, overexpression, and increase. Here, activation, enhancement, upregulation, overexpression, and increase all include exhibiting activity that was not originally present, or improving activity compared to endogenous activity or activity before modification. "Endogenous activity" refers to the activity of a specific polypeptide that was originally present in the parent strain or unmodified microorganism before the trait change, when a genetic mutation occurs due to natural or artificial factors and the trait changes. This is used interchangeably with "activity before modification." When polypeptide activity is "enhanced," "upregulated," "overexpressed," or "improved" compared to its endogenous activity, it means that the activity and / or concentration (expression level) of the specific polypeptide that was originally present in the parent strain or unmodified microorganism before the trait change is improved.

[0082] Various methods known in the field can be applied to enhance the activity of the polypeptide, including, but are not limited to, methods such as increasing the intracellular copy number of the gene encoding the mutant, introducing a mutation into the expression regulatory sequence of the gene encoding the mutant on the chromosome, replacing the expression regulatory sequence of the gene encoding the mutant on the chromosome with a more potent sequence, replacing the gene encoding the protein on the chromosome with a mutated gene that enhances the activity of the mutant, and introducing a mutation into the gene encoding the mutant protein on the chromosome that enhances the activity of the mutant.

[0083] In this application, "introduction" means a method of delivering a polynucleotide encoding the acetohydroxy acid synthase variant or a vector containing the same to a host cell. Such introduction can be easily carried out by conventional methods in the art. Generally, methods such as CaCl2 precipitation, the Hanahan method (which improves efficiency by using DMSO (dimethyl sulfoxide) as a reducing agent in addition to CaCl2), electroporation, calcium phosphate precipitation, plasmofusion, stirring with silicon carbide fibers, transformation using PEG, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation methods are used. The method of transforming the vector is not limited to these examples, and any transformation or transfection method commonly used in the art may be used. Furthermore, the delivered polynucleotide may be any form, regardless of whether it is inserted into or outside the chromosome of the host cell, as long as it is expressed in the host cell. In addition, the polynucleotide may be introduced in any form, as long as it is introduced into and expressed in the host cell. For example, the polynucleotide is introduced into a host cell in the form of an expression cassette, which is a polynucleotide structure containing all the elements necessary for its own expression, but is not limited to this. Typically, the expression cassette includes a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, all operably linked to the open reading frame (ORF) of the gene. The expression cassette may also be in the form of a self-replicating expression vector. Alternatively, the polynucleotide may be introduced into a host cell in its own form and operably linked to the sequences necessary for expression in the host cell, but is not limited to this.

[0084] A further aspect of this application provides a method for producing L-valine, comprising the step of culturing the microorganism of this application in a culture medium.

[0085] Specifically, the method for producing L-valine according to this application includes, but is not limited to, the step of culturing a microorganism containing the mutant, polynucleotide, or vector of this application in a culture medium.

[0086] In this application, "cultivation" means growing the microorganisms of this application under appropriately adjusted environmental conditions. The cultivation process of this application can be carried out using suitable culture media and cultivation conditions known in the art. Such a cultivation process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the cultivation is batch, continuous, and / or fed-batch culture, but is not limited to these.

[0087] In this application, "culture medium" refers to a substance that is a mixture mainly composed of nutrients necessary for culturing the microorganisms of this application, and supplies nutrients and growth factors, including water, which are essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the microorganisms of this application may be any that are normally used for culturing microorganisms, and the microorganisms of this application can be cultured in a normal culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, under aerobic conditions, with the temperature, pH, etc. adjusted.

[0088] For example, a culture medium for strains of the genus Corynebacterium is disclosed in Non-Patent Document 13.

[0089] In this application, the carbon source can be carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn maceration liquid can be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and any other carbon source in an appropriate amount may be used. These carbon sources can be used individually or in combination of two or more, but are not limited to these uses.

[0090] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate can be used, as well as organic nitrogen sources such as amino acids like glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn maceration liquid, casein hydrolysates, fish or their decomposition products, defatted soybean cake or its decomposition products. These nitrogen sources can be used individually or in combination of two or more, but are not limited to these uses.

[0091] As the phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or equivalent sodium-containing salts can be used. As inorganic compounds, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc., can be used, and in addition, amino acids, vitamins, and / or suitable precursors can be used. These components or precursors can be added to the culture medium in batches or continuously, but are not limited to these.

[0092] Furthermore, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture medium in a suitable manner during the cultivation of the microorganisms of this application. In addition, during cultivation, the formation of bubbles can be suppressed using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, oxygen or oxygen-containing gas may be injected into the culture medium to maintain an aerobic state, and to maintain an anaerobic and microaerobic state, it is not necessary to inject gas, but nitrogen, hydrogen, or carbon dioxide gas may be injected, but the invention is not limited to these.

[0093] In the culture described in this application, the culture temperature is maintained at 20-45°C, specifically 25-40°C, and the culture is performed for approximately 10-160 hours, but is not limited to these values.

[0094] The L-valine produced by the culture described in this application is either secreted into the culture medium or remains within the cells.

[0095] The method for producing L-valine according to this application may further include, for example, the steps of preparing the microorganism of this application, preparing a culture medium for culturing the strain, or a combination thereof (in any order) before the culturing step.

[0096] The method for producing L-valine according to this application may further include a step of recovering L-valine from the culture medium used for the culture (the culture medium in which the culture was performed) or from the microorganism of this application. The recovery step may further include a step after the culture step.

[0097] The aforementioned recovery may involve collecting L-valine using a suitable method known in the art, depending on the microorganism culture method of this application, such as batch, continuous, or fed-batch culture. For example, various chromatography methods such as centrifugation, filtration, crystallization, treatment with protein precipitants (salting-out method), extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof can be used, and L-valine can be recovered from the culture medium or microorganism using a suitable method known in the art.

[0098] Furthermore, the L-valine production method of this application may further include a purification step. The purification can be carried out by a preferred method known in the art. For example, if the L-valine production method of this application includes both a recovery step and a purification step, the recovery step and the purification step may be carried out sequentially or discontinuously, regardless of order, simultaneously or as a single integrated step, but are not limited thereto.

[0099] Further embodiments of this application provide a composition for L-valine production comprising an acetohydroxy acid synthase subunit (ilvN) variant of this application, a polynucleotide encoding the variant of this application, a vector comprising the polynucleotide of this application, or a microorganism comprising the polynucleotide of this application, a culture medium thereof, or a combination of at least two thereof.

[0100] The composition of this application may further contain any suitable excipients commonly used in compositions for amino acid production. Examples of such excipients include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents. [Examples]

[0101] The present application will be described in more detail below with reference to examples. However, these examples are merely preferred embodiments illustrating the present application, and the application is not limited thereto. Technical matters not described herein can be fully understood and readily implemented by a skilled technician in the art of this application or a similar art. [Examples]

[0102] Selection of mutant strains with improved valine production capacity using artificial mutation. Example 1-1. Induction of artificial mutations by UV irradiation To select mutant strains with improved valine production, the valine-producing strain Corynebacterium glutamicum KCCM11201P (Patent Document 3) was streaked onto a nutrient medium containing agar and cultured at 30°C for 36 hours. Several hundred colonies obtained in this way were irradiated with UV light at room temperature to induce random mutations in the strain's genome.

[0103] Examples 1-2. Evaluation of fermentation activity of mutagenic strains and selection of strains To select mutant strains with improved L-valine production compared to the parent strain Corynebacterium glutamicum KCCM11201P, fermentation titer experiments were conducted on strains induced by random mutation. Each colony was subcultured in nutrient medium, and then each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of production medium and cultured with shaking at 30°C and 200 rpm for 72 hours. Subsequently, the concentration of L-valine was analyzed using HPLC. The analyzed L-valine concentrations are shown in Table 1. [Nutrient medium (pH 7.2)] Glucose 10g, meat extract 5g, polypeptone 10g, sodium chloride 2.5g, yeast extract 5g, agar 20g, urea 2g (per liter of distilled water) [Production medium (pH 7.0)] 100g glucose, 40g ammonium sulfate, 2.5g soy protein, 5g corn steep solids, 3g urea, 1g dipotassium hydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 100μg biotin, 1mg thiamine HCl, 2mg calcium pantothenate, 3mg nicotinamide, 30g calcium carbonate (per 1 liter of distilled water)

[0104] [Table 1]

[0105] As shown in Table 1, we selected the C6 strain, which showed the greatest increase in valine production compared to the control group, the KCCM11201P strain. [Examples]

[0106] Confirmation of mutations by gene sequencing The major genes of the aforementioned strain were sequenced and compared with those of the KCCM11201P strain. As a result, it was confirmed that the C6 strain, which showed improved valine production, contained a nucleotide sequence mutation at a specific position in the ORF (open reading frame) region of the ilvN gene.

[0107] Specifically, the C6 strain, which showed the greatest increase in valine production, also contained the A42V mutation present in the parent strain KCCM11201P. In addition, it was confirmed that a mutation was introduced at a base located 476 bp upstream from the start codon of the ilvN gene, changing the previous GCA (SEQ ID NO: 2) to GAA (SEQ ID NO: 4), and that the 159th amino acid, alanine, was replaced with glutamic acid (SEQ ID NO: 3).

[0108] Analysis of the A159E mutation region revealed that it affects the effector binding domain of valine biosynthesis enzyme, and it was predicted that the activity of the protein would be enhanced. In the following examples, we will examine the effect of applying the A159E mutation inserted at a specific ORF position within the ilvN gene on the production capacity of valine, a branched-chain amino acid, in microorganisms of the genus Corynebacterium. [Examples]

[0109] Preparation of the KCCM11201P strain with the ilvN mutation and confirmation of its valine production capacity. Example 3-1. Preparation of a strain by introducing the ilvN mutation into the Corynebacterium glutamicum KCCM11201P strain and evaluation of its L-valine production capacity - 1 To insert the ilvN(A159E) mutant into Corynebacterium glutamicum KCCM11201P containing the A42V mutation, a vector containing the target mutation was prepared. Specifically, the genomic DNA of the C6 strain was extracted using the G-spin Total DNA Extraction Mini-Kit (Intron, Cat. No. 17045) according to the kit's protocol. PCR was performed using the genomic DNA as a template. The PCR conditions were denaturation at 94°C for 5 minutes, followed by denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 150 seconds, followed by polymerization at 72°C for 7 minutes, for 25 cycles. Using SEQ ID NOs. 7 and 8, a 1010 bp PCR product (hereinafter referred to as "mutation-introduced fragment 1") was obtained.

[0110] The mutant fragment 1 obtained as described above was ligated using a pDCM2 vector treated with restriction enzyme XbaI (New England Biolabs, Beverly, MA) (Patent Documents 4 and 5) and an Infusion Cloning Kit (Takara Bio Inc., Otsu, Japan). The gene prepared as described above was transformed into E. coli DH5α, and then selected from kanamycin-containing LB medium. DNA was obtained using a DNA-spin plasmid DNA purification kit (iNtRON), and the vector pDCM2-ilvN (A42V, A159E) containing mutant fragment 1 was prepared.

[0111] [Table 2]

[0112] The aforementioned pDCM2-ilvN(A42V,A159E) was transformed into Corynebacterium glutamicum KCCM11201P by homologous recombination on the chromosome (Non-Patent Literature 14). Strains in which the vector was inserted on the chromosome by homologous sequence recombination were selected from a medium containing 25 mg / L of kanamycin. Subsequently, PCR using SEQ ID NOs: 7 and SEQ ID NOs: 8 was performed on the Corynebacterium glutamicum transformed strains after secondary recombination was completed to confirm strains that similarly contained the A42V mutation found in the parental strain KCCM11201P, in which alanine was replaced with valine at the 42nd amino acid position of SEQ ID NO: 1 in the ORF of the ilvN gene, and also contained a substitution of glutamic acid with alanine at the 159th amino acid position of SEQ ID NO: 1 in the ORF of the ilvN gene. The recombinant strain mentioned above was named Corynebacterium glutamicum KCCM11201P::ilvN(A159E).

[0113] Flask evaluations were performed to compare the valine production capacity of the valine-producing strains Corynebacterium glutamicum KCCM11201P and KCCM11201P::ilvN(A159E). Each strain was subcultured in nutrient medium, then inoculated into a 250 ml corner-baffled flask containing 25 ml of production medium, and cultured with shaking at 30°C and 200 rpm for 72 hours. Subsequently, the concentration of L-valine was analyzed using HPLC. The analyzed L-valine concentrations are shown in Table 3. [Nutrient medium (pH 7.2)] Glucose 10g, meat extract 5g, polypeptone 10g, sodium chloride 2.5g, yeast extract 5g, agar 20g, urea 2g (per liter of distilled water) [Production medium (pH 7.0)] 100g glucose, 40g ammonium sulfate, 2.5g soy protein, 5g corn steep solids, 3g urea, 1g dipotassium hydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 100μg biotin, 1mg thiamine HCl, 2mg calcium pantothenate, 3mg nicotinamide, 30g calcium carbonate (per 1 liter of distilled water)

[0114] [Table 3]

[0115] As a result, it was confirmed that the L-valine production capacity of the KCCM11201P::ilvN(A159E) strain was increased by 14.8% compared to KCCM11201P.

[0116] Example 3-2. Preparation of a strain with the ilvN mutation introduced into the Corynebacterium glutamicum KCCM11201P strain and evaluation of its L-valine production capacity - 2 To confirm the effect of the ilvN(A159E) mutation alone in the wild-type ilvN enzyme, a strain was created in which alanine was replaced with glutamic acid at the 159th amino acid position of Sequence ID No. 1 in the ORF of the ilvN gene. Specifically, a vector containing the target mutation was prepared to insert ilvN(V42A,A159E) into glutamicum KCCM11201P. Specifically, the genomic DNA of the C6 strain was extracted using the G-spin Total DNA Extraction Mini-Kit (Intron, Cat. No. 17045) according to the kit's protocol. PCR was performed using the genomic DNA as a template. The PCR conditions were denaturation at 94°C for 5 minutes, followed by denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 150 seconds, followed by polymerization at 72°C for 7 minutes, for 25 cycles. Using sequence numbers 9 and 10, a 515 bp PCR product (hereinafter referred to as "mutation fragment 2") was obtained, and using sequence numbers 11 and 12, a 518 bp PCR product (hereinafter referred to as "mutation fragment 3") was obtained.

[0117] The mutant fragments 2 and 3 obtained as described above were ligated using a pDCM2 vector treated with restriction enzyme XbaI (New England Biolabs, Beverly, MA) (Patent Documents 4 and 5) and an Infusion Cloning Kit (Takara Bio Inc., Otsu, Japan). The genes prepared as described above were transformed into E. coli DH5α, and then selected from kanamycin-containing LB medium. DNA was obtained using a DNA-spin plasmid DNA purification kit (iNtRON), and the vector pDCM2-ilvN (V42A, A159E) containing mutant fragments 2 and 3 was constructed.

[0118] [Table 4]

[0119] The aforementioned pDCM2-ilvN(V42A,A159E) was transformed into Corynebacterium glutamicum KCCM11201P by homologous recombination on the chromosome (Non-Patent Literature 14). The A42V mutation was restored and the A159E mutation was introduced by the homologous recombination. Strains in which the vector was inserted on the chromosome by homologous sequence recombination were selected from a medium containing 25 mg / L of kanamycin. Subsequently, PCR using SEQ ID NOs: 9 and SEQ ID NOs: 12 was performed on the Corynebacterium glutamicum transformed strains in which valine was restored to alanine at the 42nd amino acid position of SEQ ID NO: 1 in the ORF of the ilvN gene on the chromosome, and alanine was substituted with glutamic acid at the 159th amino acid position. The aforementioned recombinant strain was named Corynebacterium glutamicum KCCM11201P::ilvN(V42A,A159E).

[0120] To compare the L-valine production capacity 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.

[0121] [Table 5]

[0122] As a result, it was confirmed that the L-valine production capacity of the KCCM11201P::ilvN(V42A,A159E) and KCCM11201P::ilvN(A159E) strains increased by 3.7% and 14.8%, respectively, compared to KCCM11201P.

[0123] From the above explanation, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. It should be understood that the above embodiments are merely illustrative and not limiting. This application should be interpreted as including all modified or altered forms derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. A variant of the acetohydroxy acid synthase subunit (ilvN) in which the amino acid corresponding to the 159th position in the amino acid sequence of SEQ ID NO: 1 is substituted with glutamic acid, wherein the variant has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

1.

2. The variant according to claim 1, wherein the variant is further characterized in that the amino acid corresponding to the 42nd position in the amino acid sequence of SEQ ID NO: 1 is replaced with valine.

3. The variant according to claim 1, wherein the variant consists of the amino acid sequence represented by Sequence ID No.

3.

4. The variant according to claim 2, wherein the variant consists of the amino acid sequence represented by Sequence ID No.

5.

5. A polynucleotide encoding a variant according to any one of claims 1 to 4.

6. A microorganism comprising a mutant according to any one of claims 1 to 4, or a polynucleotide encoding the mutant.

7. The microorganism according to claim 6, wherein the microorganism has improved L-valine production ability compared to a microorganism containing the polypeptide of Sequence ID No. 1 or a polynucleotide encoding it.

8. The microorganism according to claim 6, wherein the microorganism is a microorganism of the genus Corynebacterium.

9. The microorganism according to claim 8, wherein the Corynebacterium genus microorganism is Corynebacterium glutamicum.

10. A method for producing L-valine, comprising the step of culturing the microorganism described in claim 6 in a culture medium.

11. The method according to claim 10, further comprising the step of recovering a target substance from the culture medium.

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