Acetohydroxy acid synthase subunit variant and method for producing l-valine using the same

Abstract

Provided are a novel acetohydroxy acid synthase subunit (ilvN) variant, a polynucleotide encoding the variant, an expression vector including the polynucleotide, an L-valine-producing microorganism including the acetohydroxy acid synthase subunit (ilvN) variant, and a method of producing L-valine using the microorganism.SOLUTION: A subunit (ilvN) mutant of acetohydroxy acid synthase in which an amino acid corresponding to the 44th position in a specific amino acid sequence is substituted with another amino acid is provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present application relates to a novel acetohydroxyacid synthase subunit (ilvN) mutant, a polynucleotide encoding the mutant, an expression vector containing the polynucleotide, an L-valine-producing microorganism containing the acetohydroxyacid synthase subunit (ilvN) mutant, and a method for producing L-valine using the microorganism. [Background technology]

[0002] L-amino acids are the basic building blocks of proteins and are used as important raw materials for 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 are known to have antioxidant effects and to directly promote protein synthesis in muscle cells.

[0003] Meanwhile, production of branched-chain amino acids using microorganisms is mainly carried out using Escherichia or Corynebacterium microorganisms, and it is known that they are biosynthesized from pyruvate through various steps to ketoisocaproic acid (2-ketoisocaproate) as a precursor (Patent Documents 1 and 2). However, the production of L-branched-chain amino acids using these microorganisms presents a problem in that it is not easy to mass-produce them industrially.

[0004] Under these circumstances, the present inventors have demonstrated that the L-valine productivity can be significantly increased by introducing a mutant with enhanced activity of the ilvN (acetohydroxy acid synthase small subunit) gene, which encodes an enzyme involved in L-valine biosynthesis in microorganisms, in order to improve the L-valine productivity using microorganisms. [Prior art documents] [Patent documents]

[0005] [License 1] Korean Registration Patent No. 10-0220018 [License 2] Korean Registration Patent No. 10-0438146 [License 3] U.S. Patent No. 7662943 [License 4] U.S. Patent No. 10584338 [Patent Document 5] U.S. Patent No. 10273491 [License 6] Korean Registration Patent No. 10-1117022 [License 7] Korean Registration Patent No. 10-0924065 [License 8] International Publication No. 2008-033001 [Non-licensed literature]

[0006] [Non-licensed Document 1] Pearson et al(1988)[Proc.Natl.Acad.Sci.USA 85]:2444 [Non-licensed Document 2] Rice et al., 2000, Trends Genet.16:276-277 [Non-licensed Document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-licensed Document 4] Devereux,J.,et al,Nucleic Acids Research 12:387(1984) [Non-licensed Document 5] Atschul,[S.][F.,][ET AL,J MOLEC BIOL 215]:403 (1990) [Non-licensed Document 6] Guide to Huge Computers,Martin J.Bishop,[ED.,]Academic Press,San Diego, 1994

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

[0007] The present inventors have developed novel acetohydroxyacid synthase subunit mutants that enhance L-valine production, polynucleotides encoding the mutants, expression vectors containing the polynucleotides, L-valine-producing microorganisms containing the acetohydroxyacid synthase subunit mutants, and methods for producing L-valine using the microorganisms, and have completed the present application. [Means for solving the problem]

[0008] One object of the present application is to provide a mutant acetohydroxyacid synthase subunit (ilvN) in which the amino acid corresponding to position 44 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.

[0009] Another object of the present application is to provide polynucleotides encoding the variants of the present application and expression vectors containing said polynucleotides. Another object of the present application is to provide a microorganism comprising the variant of the present application or a polynucleotide encoding said variant.

[0010] Another object of the present application is to provide a method for producing L-valine, which comprises culturing the microorganism in a medium. Another object of the present application is to provide a composition for producing L-valine, comprising a variant of the present application, a polynucleotide encoding the variant, a vector containing the polynucleotide, or a microorganism containing the polynucleotide of the present application; a medium in which the same is cultured; or a combination of two or more of them.

[0011] Another object of the present application is to provide use of a mutant acetohydroxyacid synthase subunit in which the amino acid corresponding to position 44 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid for L-valine production. [Effects of the Invention]

[0012] When a microorganism containing the acetohydroxyacid synthase subunit mutant of the present application is cultured, it is possible to produce L-valine at a higher yield than a microorganism containing the existing unmodified polypeptide. DETAILED DESCRIPTION OF THE INVENTION

[0013] This will be explained in more detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied 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 intended to limit the category of this application. Furthermore, numerous papers and patent documents are referenced throughout this specification, and citations thereof are provided. The disclosures 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 content of the present invention.

[0014] In one aspect to achieve the above object, the present application provides a mutant acetohydroxyacid synthase subunit (ilvN) in which the amino acid corresponding to position 44 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.

[0015] 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.

[0016] In this application, the term "acetohydroxyacid synthase" refers to the first enzyme in L-valine biosynthesis, also known as acetolactate synthase. Acetohydroxyacid synthase catalyzes the decarboxylation of pyruvate and its condensation with another pyruvate molecule to produce acetolactate, a precursor of valine, and the decarboxylation of pyruvate and its condensation with 2-ketobutyrate to produce acetohydroxybutyrate, a precursor of isoleucine.

[0017] Acetohydroxyacid synthase is encoded by the ilvB and ilvN genes. The ilvB gene encodes the large subunit of acetohydroxyacid synthase, and the ilvN gene encodes the small subunit. Of these, the small subunit encoded by the ilvN gene is thought to be largely involved in feedback inhibition.

[0018] The acetohydroxy acid synthase encoded by the ilvN gene may have the amino acid sequence of SEQ ID NO:1, but is not particularly limited thereto. The mutant of the present application may be, but is not limited to, a mutant in which an amino acid at a specific position in the amino acid sequence of an existing acetohydroxyacid synthase subunit (ilvN) is substituted to enhance activity.

[0019] In one embodiment, the acetohydroxy acid synthase subunit (ilvN) mutant may be, but is not limited to, an acetohydroxy acid synthase subunit (ilvN) mutant containing one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 1.

[0020] Specifically, the variant of the present application may be, but is not limited to, a variant in which the amino acid corresponding to position 44 and / or position 42 of SEQ ID NO: 1 is substituted with another amino acid. More specifically, the variant may be, but is not limited to, a variant in which one or more, or both, of the aforementioned positions or the corresponding positions are substituted with another amino acid.

[0021] The "other amino acid" is not limited as long as it is different from the amino acid before substitution. For example, when describing "the amino acid corresponding to the 44th position in SEQ ID NO: 1 is substituted with another amino acid," the amino acid can be any of alanine (excluding threonine), phenylalanine, glycine, arginine, aspartate, cysteine, glutamic acid, asparagine, glutamine, histidine, proline, serine, tyrosine, and isoleucine. e), lysine, tryptophan, valine, methionine or leucine, and when it is stated that "the amino acid corresponding to the 42nd position in SEQ ID NO: 1 is substituted with another amino acid," it may mean that the amino acid is substituted with valine, asparagine, glycine, arginine, aspartate, cysteine, glutamic acid, glutamine, histidine, proline, serine, tyrosine, isoleucine, leucine, lysine, phenylalanine, tryptophan, methionine or threonine, excluding alanine, but is not limited thereto.

[0022] Meanwhile, a person skilled in the art can identify the amino acids corresponding to positions 44 and 42 of SEQ ID NO: 1 in any amino acid sequence through sequence alignment known in the art, and it is obvious that the term "amino acids at specific positions in a particular SEQ ID NO" in this application also includes "amino acids at corresponding positions" in any amino acid sequence, even if not otherwise specified. Therefore, amino acid sequences in which one or more amino acids selected from the group consisting of amino acids corresponding to positions 44 and 42 of SEQ ID NO: 1 are substituted with other amino acids are also included within the scope of this application.

[0023] For example, if one or more amino acids corresponding to positions 44 and 42 of SEQ ID NO: 1 are replaced with other amino acids, a mutant having higher activity than the unsubstituted (unaltered) amino acid sequence can be provided.

[0024] Specifically, the variant of the present application may be one in which the amino acids corresponding to positions 44 and / or 42 of SEQ ID NO: 1 are substituted with other amino acids, but is not limited thereto.

[0025] As a specific example, the variant of the present application may be, but is not limited to, one in which the threonine amino acid corresponding to position 44 of SEQ ID NO: 1 is replaced with alanine and / or the alanine amino acid corresponding to position 42 is replaced with valine.

[0026] As a more specific example, the variant of the present application may have the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, or may essentially consist of said amino acid sequence.

[0027] Furthermore, the variants of the present application are not limited to the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, but may also be variants having 80% or more, specifically 90% or more, more specifically 95% or more, and even more specifically 99% or more homology thereto, and may include variants in which the amino acid at the position corresponding to amino acid 44 and / or amino acid 42 from the N-terminus of SEQ ID NO: 1 is substituted with another amino acid. Furthermore, it is obvious that variants having an amino acid sequence 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 they have such homology or identity and exhibit efficacy corresponding to the variants of the present application.

[0028] For example, the amino acid sequence may have additions or deletions, naturally occurring mutations, silent mutations or conservative substitutions at the N-terminus, C-terminus and / or internally that do not alter the function of the variant of the present application.

[0029] 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. Such amino acid substitutions may generally be made on the basis of similarity in 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 aspartate; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Amino acids can also be classified as those with electrically charged side chains or those with uncharged side chains. Charged amino acids include aspartic acid, glutamic acid, lysine, arginine, and histidine. Uncharged amino acids can be further classified as nonpolar or polar amino acids. Nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Conservative substitutions typically have little or no effect on the activity of the resulting polypeptide. Conservative substitutions typically have little or no effect on the activity of the resulting protein or polypeptide.

[0030] 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.

[0031] As used herein, the term "variant" refers to a polypeptide in which one or more amino acids have been conservatively substituted and / or modified, resulting in a difference from the amino acid sequence of the variant but maintaining its functions or properties. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the variant. That is, the performance of the variant may be increased, unchanged, or decreased compared to the polypeptide before the mutation. Some variants may also include variants 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 a mature protein. The term "variant" may be interchangeable with other terms, such as "mutated type," "modified," "mutated polypeptide," "mutant protein," "mutant," "mutein," and "divergent," as long as the term is used in the sense of "mutated." These terms are not limited to these terms. For purposes of this application, the variant may be a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3, in which the amino acid threonine corresponding to position 44 of SEQ ID NO: 1 is substituted with alanine; or the amino acid sequence set forth in SEQ ID NO: 5, in which the amino acid threonine corresponding to position 44 of SEQ ID NO: 1 is substituted with alanine and the amino acid alanine corresponding to position 42 of SEQ ID NO: 1 is substituted with valine.

[0032] 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 are often used interchangeably.

[0033] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences can generally hybridize to all or part 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.

[0034] 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), the GCG program package (Devereux, J., et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.][F.,][ET AL, J MOLEC BIOL 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [Ed.,] Academic Press, San Diego, 1994, and [CARILLO ETA / .] (1988) SIAM J Applied Math 48:1073). For example, BLAST or ClustalW from the National Database Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0035] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as that described in Smith and Waterman, Adv. Appl. Math (1981) 2:482, or in 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 binary 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.

[0036] As an example of the present application, the mutant of the present application may be obtained by mutation of an L-valine-producing microorganism. Mutation of the microorganism can be carried out by various means widely known in the art, and either physical or chemical mutagenesis can be used. For example, chemical mutagens suitable for the present invention include, but are not limited to, N-methyl-N'-nitro-N-nitrosoguanidine (NTG), diepoxybutane, ethyl methanesulfonate, mustard compounds, hydrazine, and nitrous acid. Physical mutagens can include, but are not limited to, ultraviolet light and gamma radiation.

[0037] In the present application, the expression of the acetohydroxyacid synthase subunit (ilvN) may be enhanced, and the enhanced expression can result in an increase in L-valine productivity.

[0038] 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 may be determining the specific amino acid of a sequence that references a particular sequence. As used herein, a "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0039] 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 amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, a sequence alignment algorithm such as that described in this application 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").

[0040] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) or the Needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) can be used, but is not limited to these. Sequence alignment programs, pairwise sequence comparison algorithms, etc. known in the art can be used as appropriate.

[0041] Another aspect of the present application is to provide polynucleotides encoding the variants of the present application. In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and a DNA or RNA chain of a certain length or more, and more specifically, refers to a polynucleotide fragment encoding the above-mentioned variant.

[0042] A polynucleotide encoding a variant of the present application may comprise a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5. As an example of the present application, a polynucleotide of the present application may have or comprise the sequence of SEQ ID NO: 4 or SEQ ID NO: 6. Alternatively, a polynucleotide of the present application may consist of or consist essentially of the sequence of SEQ ID NO: 4 or SEQ ID NO: 6.

[0043] The polynucleotide of the present application may be modified in various ways in the coding region without changing the amino acid sequence of the variant of the present application, taking into consideration codon degeneracy or codons preferred in an organism in which the variant of the present application is to be expressed. Specifically, the polynucleotide of the present application may have or contain a nucleotide 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% homologous or identical to the sequence of SEQ ID NO: 4 or SEQ ID NO: 6, or may consist of or essentially consist of a nucleotide 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% homologous or identical to the sequence of SEQ ID NO: 4 or SEQ ID NO: 6, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 44th position of SEQ ID NO: 3 or SEQ ID NO: 5 may be one of the codons encoding alanine, and the codon encoding the amino acid corresponding to the 42nd position of SEQ ID NO: 5 may be one of the codons encoding valine.

[0044] Furthermore, the polynucleotides of the present application include, without limitation, sequences that can hybridize under stringent conditions with probes that can be prepared from known gene sequences, for example, sequences complementary 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, namely, 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.

[0045] 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 are capable of hybridizing to one another. For example, with respect to 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.

[0046] 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 also be 60° C., 63° C., or 65° C., but is not limited thereto, and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0047] 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).

[0048] Another aspect of the present application is to provide a vector comprising the polynucleotide of the present application, which may be, but is not limited to, an expression vector for expressing the polynucleotide in a microorganism.

[0049] 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 enable the expression of 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 can be integrated into the genome itself.

[0050] 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.

[0051] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome of a cell using a vector for chromosomal integration. 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 insertion into the chromosome may also be included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of 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 survive or exhibit other phenotypes, allowing the selection of transformed cells.

[0052] 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.

[0053] 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.

[0054] Another aspect of the present application is to provide a microorganism comprising a variant of the present application or a polynucleotide of the present application. The microorganism of the present application may comprise a mutant polypeptide of the present application, a polynucleotide encoding said polypeptide, or a vector comprising a polynucleotide of the present application.

[0055] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and naturally or artificially genetically modified microorganisms, including microorganisms in which a specific mechanism has been weakened or enhanced by inserting an exogenous gene or by enhancing or inactivating the activity of an endogenous gene, and may also be a microorganism that contains a genetic modification for producing a desired polypeptide, protein, or product. In this application, the terms "microorganism," "strain," and "microorganism" may be used interchangeably and without limitation.

[0056] Specifically, the microorganism may be a microorganism of the genus Corynebacterium, more specifically, Corynebacterium glutamicum, but is not limited thereto.

[0057] The strain of the present application may be, but is not limited to, a strain comprising one or more of a variant of the present application, a polynucleotide of the present application, and a vector comprising a polynucleotide of the present application; a strain that has been modified to express a variant of the present application or a polynucleotide of the present application; a strain (e.g., a recombinant strain) that expresses a variant of the present application or a polynucleotide of the present application; or a strain (e.g., a recombinant strain) that has activity of a variant of the present application.

[0058] The strain of the present application may be a microorganism that produces L-valine. In this application, the term "microorganism capable of producing L-valine" refers to a prokaryotic or eukaryotic microbial strain capable of producing L-valine in vivo. For the purposes of the present invention, the microorganism may be either a prokaryotic or eukaryotic cell, as long as it is capable of producing L-valine by incorporating the acetohydroxyacid synthase subunit mutant. Examples of such a microorganism include strains of microorganisms belonging to the genus Corynebacterium, such as Corynebacterium glutamicum.

[0059] The L-valine-producing microorganism containing the acetohydroxy acid synthase subunit mutant includes, but is not limited to, microorganisms in which the chromosomal gene encoding the acetohydroxy acid synthase is mutated to contain the acetohydroxy acid synthase subunit mutant sequence according to the present invention, and / or microorganisms into which a vector containing a polynucleotide encoding the acetohydroxy acid synthase subunit mutant has been introduced to contain the acetohydroxy acid synthase subunit mutant.

[0060] Furthermore, the L-valine-producing microorganism containing the mutant subunit of acetohydroxy acid synthase may be one in which the activity of the mutant subunit of acetohydroxy acid synthase is enhanced compared to that of the parent strain.

[0061] For example, the strain of the present application is a cell or microorganism transformed with a vector containing a polynucleotide encoding the polynucleotide of the present application or a polynucleotide encoding the variant of the present application and expressing the variant of the present application. For purposes of this application, the strain of the present application may include all microorganisms capable of producing L-valine, including the variant of the present application. For example, the strain 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 is introduced into an L-valine-producing microorganism to express a mutant acetohydroxy acid synthase subunit (ilvN) and thereby increase L-valine production ability. The recombinant strain with increased L-valine production ability may be, but is not limited to, a naturally occurring wild-type microorganism or a microorganism not modified with acetohydroxy acid synthase (i.e., a microorganism expressing a wild-type acetohydroxy acid synthase (SEQ ID NO: 1) or a microorganism not expressing a mutant (SEQ ID NO: 3 or SEQ ID NO: 5) protein).

[0062] For example, the recombinant strain with increased production ability has an L-valine production ability of about 1% or more, specifically about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 10.5% or more, about 11% or more, about 11.5% or more, about 12% or more, about 12.5% ​​or more, about 13% or more, about 13.5% or more, about 14% or more, about 14.5% or more, about 15% or more, about 15.5% or more, about 16% or more, about 16.5% or more, about 17% or more, or about 17% or more, compared to the L-valine production ability of the parent strain or untransformed microorganism before mutation. The increase may be 0.5% or more, about 18% or more to about 18.1% or more, about 18.2% or more, about 18.3% or more, about 18.4% or more, or about 18.5% 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, about 20% or less, or about 15% or less), but is not limited thereto as long as there is an increase in the + value compared to the productivity of the parent strain or untransformed microorganism before mutation. In another example, the recombinant strain with increased L-valine production ability may be, but is not limited to, an increase of about 1.1-fold or more, about 1.12-fold or more, about 1.13-fold or more, about 1.14-fold or more, about 1.15-fold or more, about 1.16-fold or more, about 1.17-fold or more, or about 1.18-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, about 1.5-fold or less, or about 1.2-fold or less) compared to the parent strain or unmodified microorganism before mutation. The term "about" refers to a range that includes ±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 value following the term "about," but is not limited thereto.

[0063] 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 due to genetic mutations caused by natural or artificial factors. For example, the unmodified microorganism refers to a strain into which a mutant of the acetohydroxyacid synthase subunit (ilvN) 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."

[0064] In yet another example of the present application, 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 striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens.

[0065] 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 prior to transformation. The term "endogenous activity" refers to the activity of a specific polypeptide inherently possessed by a parent strain or an unaltered microorganism prior to transformation, in cases where a trait has been altered by genetic mutation due to natural or artificial factors. This term may be used interchangeably with "activity prior to transformation." "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 prior to transformation.

[0066] 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.

[0067] 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.).

[0068] Specifically, the enhancement of the polypeptide activity of the present application is achieved by: 1) an increase in the intracellular copy number of the polynucleotide encoding the polypeptide; 2) replacing the expression regulatory region of the gene on the chromosome that encodes the 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 a polypeptide and selectively modifying 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.

[0069] More specifically, The 1) increase in the intracellular copy number of a polynucleotide encoding a polypeptide may be achieved by introducing into a microorganism a vector capable of replicating and functioning independently of a 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 of the microorganism. The introduction into a chromosome can be achieved by, but is not limited to, introducing into the microorganism a vector capable of inserting the polynucleotide into the chromosome of the microorganism. The vector is as described above.

[0070] 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.

[0071] Examples of known strong promoters include, but are not limited to, the CJ1 to CJ7 promoters (U.S. Pat. No. 7,662,943), 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. Pat. No. 10,584,338), the O2 promoter (U.S. Pat. No. 10,273,491), the tkt promoter, and the yccA promoter.

[0072] 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.

[0073] 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 modification can be achieved by, but is not limited to, inserting the polynucleotide into a chromosome via 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.

[0074] The introduction of an exogenous polynucleotide that exhibits the activity of a polypeptide (6) may be the introduction into a microorganism of an exogenous polynucleotide that encodes a polypeptide that exhibits the same or similar activity as the polypeptide. 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 increase its activity.

[0075] In the present application, the term "introduction" refers to a method for transferring a polynucleotide encoding the acetohydroxyacid synthase subunit mutant or a vector containing the same into a microorganism. Such introduction can be easily performed using 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. Methods for transforming the vector are not limited to the above examples, and any transformation or transfection method commonly used in the art can be used without limitation. The transferred polynucleotide can be inserted into the chromosome of the microorganism or extrachromosomally located, as long as it can be expressed in the host cell. The polynucleotide can be introduced in any form as long as it can be introduced and expressed in the host cell. For example, the polynucleotide may be introduced into a microorganism in the form of an expression cassette, which is a polynucleotide structure containing all elements necessary for its own expression, but is not limited thereto. The expression cassette typically contains a promoter operably linked to the open reading frame (ORF) of the gene, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of a self-replicating expression vector. Alternatively, the polynucleotide may be introduced into a microorganism in its own form and operably linked to sequences necessary for expression in the microorganism, but is not limited thereto.

[0076] The codon optimization of the polynucleotide encoding the polypeptide (7) may be an endogenous polynucleotide that has been codon-optimized to increase transcription or translation within the microorganism, or an exogenous polynucleotide that has been codon-optimized to optimize transcription and translation within the microorganism.

[0077] 8) The step of analyzing the tertiary structure of a polypeptide and selecting and modifying 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 modifying exposed sites to be modified or chemically modified.

[0078] Such enhancement of polypeptide activity may mean, 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.

[0079] Modification of a portion or the entire 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 the entire gene include DNA recombination techniques. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene may be injected into the microorganism to cause homologous recombination, thereby deleting a portion or the entire gene. The injected nucleotide sequence or vector may contain, but is not limited to, a dominant selectable marker.

[0080] In the microorganism of the present application, the mutant, polynucleotide, L-valine, etc. are as described above in other aspects. Another aspect of the present application provides a method for producing L-valine, comprising culturing the microorganism in a medium.

[0081] Specifically, the method for producing L-valine of the present application may include, but is not limited to, culturing a Corynebacterium glutamicum strain containing a mutant of the present application, a polynucleotide of the present application, or a vector of the present application in a medium.

[0082] 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.

[0083] In the present application, the term "culture medium" refers to a substance containing a mixture of nutrients, primarily those 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 ordinary microorganisms without any particular limitations. The microorganism of the present application can be cultured in an ordinary 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.

[0084] For example, the culture medium for Corynebacterium strains is described in the literature ["Manual of Methods for General Bacteriology” by the American Society for Bacteriology (Washington DC, USA, 1981).

[0085] 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 of two or more, and are not limited thereto.

[0086] 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 hydrolyzed products, defatted soybean cake or its hydrolyzed products, etc. These nitrogen sources may be used alone or in combination of two or more, and are not limited thereto.

[0087] 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, they are not limited thereto.

[0088] 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. Furthermore, during cultivation, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, to maintain an aerobic state in the medium, oxygen or an oxygen-containing gas can be injected into the medium, and 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.

[0089] In the culture of the present application, the culture temperature can be maintained at 20 to 45°C, specifically 25 to 40°C, and the culture can be performed for about 10 to 160 hours, but is not limited thereto.

[0090] The L-valine produced by the culture of the present application is either secreted into the medium or remains intracellularly. The method for producing L-valine of the present application may further include 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), for example, before the culturing step.

[0091] 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.

[0092] The recovery may involve collecting the desired 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 the desired L-valine from the medium or the microorganism using a suitable method known in the art.

[0093] Furthermore, 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, regardless of the order, or simultaneously or integrated into one step, but are not limited thereto.

[0094] In the methods of the present application, the mutant, polynucleotide, vector, microorganism, etc. are as described above in other aspects. Another aspect of the present application is to provide a composition for producing L-valine, comprising a variant of the present application, a polynucleotide encoding the variant, a vector comprising the polynucleotide, or a microorganism comprising the polynucleotide of the present application; a medium in which the variant or the polynucleotide has been cultured; or a combination of two or more of them.

[0095] 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, but are not limited to, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, or an isotonic agent.

[0096] In the composition of the present application, the mutant, polynucleotide, vector, strain, medium, L-valine, etc. are as described above in other aspects. Another aspect of the present application provides use of a mutant acetohydroxyacid synthase subunit in which the amino acid corresponding to position 44 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid for the production of L-valine.

[0097] For purposes of this application, the variants and L-valine etc. are as described above in other aspects. 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.

[0098] Example 1. Selection of mutants with increased valine productivity through artificial mutagenesis Example 1-1. Induction of artificial mutations through UV irradiation To select mutants with increased valine production, the valine-producing strain Corynebacterium glutamicum KCCM11201P (Korean Patent Registration No. 10-1117022) 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.

[0099] 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.

[0100] [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) [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)

[0101] [Table 1-1]

[0102] [Table 1-2]

[0103] Based on the results in Table 1, the C14 strain, which showed the greatest increase in valine production compared to the control strain KCCM11201P, was selected. 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 KCCM11201P strain and the C14 strain with increased valine production contained a nucleotide sequence mutation at a specific position in the ilvN gene open reading frame (ORF). Specifically, the KCCM11201P strain contained a single mutation at the 125th base from the start codon of the ilvN gene, changing the existing GCA to GTA and substituting the 42nd amino acid, alanine, with valine.

[0104] The C14 strain, which showed the greatest increase in valine production, contained the same A42V mutation as the parent strain KCCM11201P. In addition, a mutation was introduced into the base located 130th from the start codon of the ilvN gene, changing the existing ACC to GCC, and the 44th amino acid, threonine, was replaced with alanine.

[0105] Analysis of the mutated region confirmed that it affected the effector binding domain of the valine biosynthetic enzyme, predicting that the activity of the protein would be enhanced. In the following example, we investigated whether the individual effects of the A42V and T44A mutations inserted at specific positions in the ORF of the ilvN gene, when used together, affected the ability of Corynebacterium to produce the branched-chain amino acid valine. We also investigated whether the substitution of amino acids other than alanine for the 44th amino acid threonine mutation affected the ability of Corynebacterium to produce the branched-chain amino acids valine, isoleucine, and leucine.

[0106] Example 3. Construction of KCCM11201P strain with ilvN mutation and confirmation of valine production ability Example 3-1. Construction of a Corynebacterium glutamicum KCCM11201P strain with an ilvN mutation and evaluation of its L-valine productivity A vector containing the target mutation was constructed to insert the ilvN(A42V+T44A) mutant (SEQ ID NO: 6) into C. glutamicum KCCM11201P. Specifically, genomic DNA from the C14 strain 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 then performed using the genomic DNA as a template. The PCR conditions were 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 "mutagenized fragment 1") was obtained using SEQ ID NO: 7 and SEQ ID NO: 8.

[0107] The resulting mutagenized fragment 1 was ligated to a pDZ vector (Korean Patent Registration No. 10-0924065 and International Publication No. 2008-033001) treated with the restriction enzyme XbaI (New England Biolabs, Beverly, Massachusetts) 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), and vector pDZ-ilvN(A42V+T44A) containing the mutagenized fragment 1 was prepared.

[0108] [Table 2]

[0109] The pDZ-ilvN(A42V+T44A) 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. The Corynebacterium glutamicum transformants in which secondary recombination had been completed were then subjected to PCR using SEQ ID NOs: 7 and 8 to identify strains in which alanine was replaced with valine at amino acid position 42 and threonine was replaced with alanine at amino acid position 44 of SEQ ID NO: 1 in the ORF of the ilvN gene on the chromosome. The recombinant strain was named Corynebacterium glutamicum KCCM11201P::ilvN(A42V+T44A).

[0110] A flask assay was performed to compare the valine-producing strains Corynebacterium glutamicum KCCM11201P and KCCM11201P::ilvN(A42V+T44A). 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. The analyzed L-valine concentrations are shown in Table 3.

[0111] [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) [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)

[0112] [Table 3]

[0113] As a result, it was confirmed that the L-valine productivity of the KCCM11201P::ilvN(A42V+T44A) strain was increased by 18.5% compared to KCCM11201P. Example 3-2. Construction of a strain containing the ilvN mutation introduced into Corynebacterium glutamicum KCCM11201P strain and evaluation of its L-valine production ability A vector containing the target mutation was constructed to insert the ilvN(T44A) mutant (SEQ ID NO: 3) into Corynebacterium glutamicum KCCM11201P. 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. The PCR conditions were 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 515-bp PCR product (hereafter referred to as "mutagenized fragment 2") was obtained using SEQ ID NO: 9 and SEQ ID NO: 10, and a 518-bp PCR product (hereafter referred to as "mutagenized fragment 3") was obtained using SEQ ID NO: 11 and SEQ ID NO: 12.

[0114] The resulting mutagenized fragments 2 and 3 were ligated to a pDZ vector (Korean Patent Registration No. 10-0924065 and International Publication No. 2008-033001) treated with the restriction enzyme XbaI (New England Biolabs, Beverly, Massachusetts) 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 pDZ-ilvN(T44A) containing mutagenized fragments 2 and 3.

[0115] [Table 4]

[0116] The pDZ-ilvN(T44A) was transformed into Corynebacterium glutamicum KCCM11201P by homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). The A42V mutation was reversed and the T44A mutation was introduced by this homologous recombination. 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 NO: 9 and SEQ ID NO: 12 to identify strains in which threonine was replaced with alanine at amino acid position 44 of SEQ ID NO: 1 in the ORF of the ilvN gene on the chromosome. The recombinant strain was named Corynebacterium glutamicum KCCM11201P::ilvN(T44A).

[0117] To compare the L-valine productivity of the constructed 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.

[0118] [Table 5]

[0119] As a result, it was confirmed that the L-valine productivity of the KCCM11201P::ilvN(T44A) and KCCM11201P::ilvN(A42V+T44A) strains was increased by 7.4% and 18.5%, respectively, compared to KCCM11201P.

[0120] Example 3-3: Construction of a strain containing the ilvN mutation introduced into Corynebacterium glutamicum CJ7V strain 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.

[0121] 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 of SEQ ID NOs: 13 and 14 and the primer pair of SEQ ID NOs: 15 and 16, 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.

[0122] 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: 13 and SEQ ID NO: 16 to obtain a 1010 bp PCR product (hereinafter referred to as "mutagenesis fragment 4").

[0123] The resulting mutated fragment 4 was digested with the restriction enzyme XbaI (New England Biolabs, Beverly, MA) and then ligated with pDZ 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 pDZ-ilvN(A42V).

[0124] [Table 6]

[0125] The pDZ-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 NOs: 13 and 16, and the mutant-inserted strains were identified through gene sequence analysis. The recombinant strain was designated Corynebacterium glutamicum CJ7V.

[0126] Finally, the Corynebacterium glutamicum CJ7V was transformed with each vector in the same manner as in Examples 3-1 and 3-2 to prepare strains named Corynebacterium glutamicum CJ7V::ilvN(T44A) and CJ7V::ilvN(A42V+T44A), respectively. To compare the L-valine production abilities of the prepared strains, they were cultured in the same manner as in Example 3-1 and analyzed for L-valine concentration. The analyzed L-valine concentrations are shown in Table 7 below.

[0127] [Table 7]

[0128] As a result, it was confirmed that the L-valine productivity of the CJ7V::ilvN(T44A) and CJ7V::ilvN (A42V+T44A) strains was increased by 8.5% and 12.3%, respectively, compared to CJ7V.

[0129] 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 claims below, and any modifications or variations derived from the equivalent concepts thereof, rather than the above detailed description.

Claims

1. A mutant subunit (ilvN) of acetohydroxyacid synthase in which the amino acid corresponding to position 44 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.

2. The mutant according to claim 1, wherein the amino acid corresponding to position 42 in the amino acid sequence of SEQ ID NO: 1 is further substituted with another amino acid.

3. The mutant of claim 1 , wherein the amino acid corresponding to position 44 of SEQ ID NO: 1 is substituted with alanine.

4. The mutant of claim 2, wherein the amino acid corresponding to position 44 of SEQ ID NO: 1 is substituted with alanine and the amino acid corresponding to position 42 is substituted with valine.

5. The mutant of claim 1 , wherein the mutant consists of the amino acid sequence set forth in SEQ ID NO:

3.

6. The mutant of claim 2, wherein the mutant consists of the amino acid sequence set forth in SEQ ID NO:

5.

7. A polynucleotide encoding the variant according to any one of claims 1 to 6.

8. An expression vector comprising the polynucleotide of claim 7.

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

10. 10. The microorganism according to claim 9, wherein the microorganism has increased L-valine-producing ability compared to a microorganism containing the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding the same.

11. The microorganism according to claim 9 , wherein the microorganism is a Corynebacterium microorganism.

12. The microorganism according to claim 11, wherein the Corynebacterium microorganism is Corynebacterium glutamicum.

13. A method for producing L-valine, comprising culturing the microorganism of claim 9 in a medium.

14. The method of claim 13, further comprising recovering the target substance in the culture medium.

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