Novel acetohydroxyacid synthase subunit mutant 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] 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. [Background technology]

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

[0003] Branched-chain amino acids are produced using microorganisms, mainly Corynebacterium microorganisms, and are known to be biosynthesized from pyruvic acid through various steps to 2-ketoisocaproate, a precursor (Patent Documents 1 and 2). However, the production of branched-chain amino acids using microorganisms has the problem that it is not easy to mass-produce them industrially.

[0004] Against this background, the present applicants have confirmed that the ability to produce branched-chain amino acids is improved 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 ability to produce branched-chain amino acids using microorganisms. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Korean Patent Registration No. 10-0220018 [Patent Document 2] Korean Patent Registration No. 10-0438146 [Patent Document 3] Korean Patent Registration No. 10-1117022 [Patent Document 4] Korean Patent Publication No. 10-2020-0136813 [Patent Document 5] International Publication No. 2008 / 033001

Non-licensed literature

[0006]

Non-patent document 1

Non-patent document 2

Non-patent document 3

Non-patent document 4

Non-patented document 5

Non-patent document 6

[0007] An objective of the present application is to provide 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. [Means for solving the problem]

[0008] The present application aims to provide an acetohydroxyacid synthase subunit (ilvN) mutant in which the amino acid corresponding to position 159 in the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid.

[0009] The present application also aims to provide polynucleotides encoding the variants of the present application.

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

[0011] Furthermore, the present application aims to provide a method for producing L-valine, which includes a step of culturing the microorganism of the present application in a medium.

[0012] Furthermore, the present application aims to provide a composition for producing L-valine, comprising the microorganism of the present application, a medium in which the microorganism of the present application has been cultured, or a combination of at least two of them. [Effects of the Invention]

[0013] When a microorganism containing an acetohydroxyacid synthase subunit mutant of the present application is cultured, L-valine can be produced in a higher yield than when a microorganism containing a conventional unmodified polypeptide is cultured. DETAILED DESCRIPTION OF THE INVENTION

[0014] These will be described in detail below. Note that each description and embodiment disclosed in this application also applies to other descriptions and embodiments. In other words, all combinations of various elements disclosed in this application are included in this application. Furthermore, this application is not limited to the specific descriptions below. Furthermore, many 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, thereby more clearly explaining the state of the art to which this application pertains and the contents of this application.

[0015] One aspect of the present application provides an acetohydroxyacid synthase subunit (ilvN) mutant in which the amino acid corresponding to position 159 in the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid.

[0016] In the present application, the term "acetohydroxyacid synthase subunit (ilvN) mutant" refers to an acetohydroxyacid synthase subunit (ilvN) mutant that contains at least one amino acid substitution in the amino acid sequence of a polypeptide having acetohydroxyacid synthase subunit (ilvN) activity.

[0017] In this application, "acetohydroxyacid synthase" is the first enzyme in L-valine biosynthesis and is also referred to as acetolactate synthase. Acetohydroxyacid synthase catalyzes the decarboxylation of pyruvate and its condensation with another pyruvate molecule to produce acetolactate, a precursor of valine, or the decarboxylation of pyruvate and its condensation with 2-ketobutyrate to produce acetohydroxybutyrate, a precursor of isoleucine.

[0018] Acetohydroxyacid synthase is encoded by two genes, ilvB and ilvN. 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 important for feedback inhibition.

[0019] The "acetohydroxy acid synthase subunit (ilvN)" in the present application refers to, but is not limited to, the acetohydroxy acid synthase subunit (ilvN) encoded by the ilvN gene. The present application also refers to, but is not limited to, an acetohydroxy acid synthase subunit (ilvN) protein or a mutant thereof derived from a Corynebacterium microorganism, specifically Corynebacterium glutamicum. Specifically, the acetohydroxy acid synthase subunit (ilvN) protein includes, for example, the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 70% or more homology or identity thereto. However, the acetohydroxy acid synthase subunit (ilvN) protein 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% homology or identity to SEQ ID NO: 1. The sequence of SEQ ID NO: 1 can be obtained from publicly known databases such as NCBI's GenBank or KEGG (Kyoto Encyclopedia of Genes and Genomes). For example, the amino acid sequence may be derived from the genus Corynebacterium or Corynebacterium glutamicum, and more specifically, may be a polypeptide / protein containing the amino acid sequence represented by SEQ ID NO: 1, but is not limited thereto. Needless to say, the present application also includes auxiliary proteins having an amino acid sequence with partial deletion, modification, substitution, or addition, as long as the amino acid sequence has such homology or identity and exhibits efficacy equivalent to that of the protein.

[0020] Furthermore, the acetohydroxyacid synthase subunit (ilvN) protein having the amino acid sequence of SEQ ID NO: 1 has, contains, consists of, or is encoded by a polynucleotide that is substantially composed of the sequence of SEQ ID NO: 2 or a nucleotide sequence that has homology or identity to the sequence of SEQ ID NO: 2 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 less than 100%, but is not limited to these.

[0021] In this application, the term "variant" refers to a polypeptide that differs from the amino acid sequence of the variant by conservative substitution and / or modification of at least one amino acid, but maintains its functions or properties. 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 performance of the variant may be improved, unchanged, or decreased compared to the polypeptide of the variant. Some variants also include variants in which at least one portion, such as an N-terminal leader sequence or a transmembrane domain, has been deleted. Other variants include variants in which a portion has been deleted from the N- and / or C-termini of a mature protein. The term "variant" is often used interchangeably with terms such as "mutated type," "modification," "mutated polypeptide," "mutated protein," "mutant," "mutein," and "divergent" (in English), but any term meaning a mutation may be used.

[0022] Variants may also include deletions or additions of amino acids that minimally affect the properties and secondary structure of the polypeptide. For example, the polypeptide may be linked to an N-terminal signal (or leader) sequence of a protein involved in co- or post-translational protein transfer. The polypeptide may also be linked to other sequences or linkers that allow for identification, purification, or synthesis of the polypeptide.

[0023] The acetohydroxyacid synthase subunit (ilvN) mutant of the present application may be, but is not limited to, an acetohydroxyacid synthase subunit (ilvN) mutant in which the amino acid corresponding to position 159 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.

[0024] In one embodiment, the acetohydroxyacid synthase subunit (ilvN) mutant of the present application may have the amino acid corresponding to position 159 in the amino acid sequence of SEQ ID NO: 1 substituted with another amino acid, and may also have the amino acid corresponding to position 42 in the amino acid sequence of SEQ ID NO: 1 substituted with another amino acid, but is not limited to this.

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

[0026] Amino acids are commonly classified based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues.

[0027] As an example of such 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 electrically charged amino acids (arginine, lysine, histidine, glutamic acid, and aspartic acid) and uncharged amino acids (neutral amino acids) (glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine). Phenylalanine, tryptophan, and tyrosine are classified as aromatic amino acids. Valine, leucine, and isoleucine are classified as branched-chain amino acids. As another example, the 20 amino acids are classified by size and divided into five groups in order of decreasing relative volume: glycine, alanine, serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, tyrosine. However, the groups are not necessarily limited to these.

[0028] For example, "the amino acid corresponding to the 159th position in SEQ ID NO: 1 is substituted with another amino acid" means any of glutamic acid, phenylalanine, glycine, arginine, aspartic acid, cysteine, asparagine, glutamine, histidine, proline, serine, tyrosine, isoleucine, lysine, thiamin ... This means that the amino acid corresponding to position 42 in SEQ ID NO: 1 has been substituted with another amino acid, and the phrase "the amino acid corresponding to position 42 in SEQ ID NO: 1 has been substituted with another amino acid" means that the amino acid has been substituted with, but is not limited to, 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] Although the present application describes a "protein having an amino acid sequence represented by a specific SEQ ID NO," it goes without saying that proteins having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added are also included in the present application, as long as they have the same or corresponding activity as a protein consisting of the amino acid sequence of the SEQ ID NO. For example, addition of sequences before or after the amino acid sequence that do not change the function of the protein, naturally occurring mutations, silent mutations, or conservative substitutions are not excluded, and it goes without saying that proteins having such sequence additions or mutations are also included in the present application, as long as they have the same or corresponding activity as the mutant protein.

[0030] In the present application, "Nth position" includes the Nth position and an amino acid position corresponding to the Nth position. Specifically, it includes an 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 SEQ ID NO: 1.

[0031] "Corresponding to," as used herein, means the amino acid residue at the recited position in the polypeptide, or an amino acid residue similar, identical, or homologous to the recited residue in the polypeptide. Identifying the amino acid at the corresponding position will determine the specific amino acid of the sequence to which the particular sequence refers. "Corresponding region," as used herein, generally refers to a similar or corresponding position in a related or reference protein.

[0032] For example, when any amino acid sequence is aligned with SEQ ID NO: 1, each amino acid residue in the amino acid sequence can be numbered based on the number and position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, the sequence alignment algorithm in the present application can identify the amino acid positions or positions where modifications such as substitutions, insertions, deletions, etc. occur when compared with a query sequence (also referred to as a "reference sequence").

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

[0034] In one embodiment, the acetohydroxyacid synthase subunit (ilvN) mutant of the present application may include an amino acid sequence in which one or two amino acids at positions corresponding to positions 42 and 159 from the N-terminus of SEQ ID NO: 1 are substituted with other amino acids.

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

[0036] In any of the above-mentioned examples, the acetohydroxyacid synthase subunit (ilvN) mutant of the present application is a polypeptide in which the alanine corresponding to the 159th amino acid of SEQ ID NO: 1 is substituted with glutamic acid and the alanine corresponding to the 42nd amino acid of SEQ ID NO: 1 is substituted with valine, but is not limited thereto.

[0037] In any of the above-mentioned examples, the variants provided by the present 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-mentioned embodiments, the variant provided by the present application may be one in which the amino acid corresponding to position 159 from the N-terminus of SEQ ID NO: 1 is substituted with an amino acid having a charged side chain selected from arginine, lysine, histidine, glutamic acid, and aspartic acid. For example, the amino acid may be an amino acid selected from acidic amino acids glutamic acid and aspartic acid. In any of the above-mentioned embodiments, the variant may be one in which the amino acid corresponding to position 159 from the N-terminus of SEQ ID NO: 1 is substituted with glutamic acid (E).

[0039] In any of the above-mentioned examples, the variants provided by the present 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 embodiments, the mutant may be one in which the amino acid corresponding to position 42 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 embodiments, the mutant may be one in which the amino acid corresponding to position 42 from the N-terminus of SEQ ID NO: 1 is substituted with valine (V).

[0041] Meanwhile, a person skilled in the art can identify the amino acid in any amino acid sequence that corresponds to the 159th position and the 42nd position in the amino acid sequence of SEQ ID NO: 1 of the present application, by sequence alignment known in the art. In the present application, it goes without saying that "an amino acid at a specific position in a specific SEQ ID NO" includes "an amino acid at a corresponding position" in any amino acid sequence, even if not otherwise specified.

[0042] Furthermore, the variant of the present application may be 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 to the amino acid sequence represented by SEQ ID NO: 1, in which the amino acid at position 159 from the N-terminus of SEQ ID NO: 1 has been substituted with another amino acid, or in which the amino acid corresponding to position 159 from the N-terminus of SEQ ID NO: 1 has been substituted with another amino acid and further in which the amino acid corresponding to position 42 of the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid. Furthermore, it goes without saying 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 in the present application, as long as they have such homology or identity and exhibit efficacy equivalent to that of the variant of the present application.

[0043] For example, the variant of the present application may have, contain, or consist essentially of the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5. Alternatively, the variant may contain an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homologous or identical to the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5.

[0044] For example, the variants may have additions or deletions of sequences at the N-terminus, C-terminus and / or internally of the amino acid sequence that do not alter the function of the variants of the present application, naturally occurring mutations, silent mutations or conservative substitutions.

[0045] As used herein, the term "conservative substitution" refers to the substitution of an 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 aspartic acid; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Amino acids are further classified into those with electrically charged side chains and those with uncharged side chains, with charged amino acids including aspartic acid, glutamic acid, lysine, arginine, and histidine, and uncharged amino acids are further classified into nonpolar and polar amino acids, with nonpolar amino acids including glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, and polar amino acids including serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Typically, conservative substitutions have little or no effect on the activity of the resulting protein or polypeptide.

[0046] In one embodiment, the acetohydroxy acid synthase subunit (ilvN) mutant of the present application has enhanced acetohydroxy acid synthase subunit (ilvN) activity, but is not limited thereto. Furthermore, the mutant of the present application has an activity that improves L-valine production ability compared to a wild-type polypeptide having acetohydroxy acid synthase subunit (ilvN) activity, but is not limited thereto.

[0047] Another aspect of the present application provides polynucleotides encoding the variants of the present application.

[0048] 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 refers to a DNA or RNA chain longer than a predetermined length, and more specifically refers to a polynucleotide fragment encoding the above-mentioned variant.

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

[0050] The polynucleotides of the present application can be modified in various ways in the coding region, taking into account codon degeneracy or codons preferred in the organism in which the variant of the present application is to be expressed, as long as the amino acid sequence of the variant of the present application is not changed. Therefore, it goes without saying that polynucleotides that, due to codon degeneracy, are translated into a polypeptide consisting of the amino acid sequence of the variant of the present application, or a polypeptide having homology or identity thereto, are also included.

[0051] For example, the polynucleotide of the present application may 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% homologous or identical to the sequence of SEQ ID NO: 4 or SEQ ID NO: 6, or may contain said base sequence, or may 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% homologous or identical to the sequence of SEQ ID NO: 4 or SEQ ID NO: 6, or may be substantially composed of said base sequence, but is not limited thereto. 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 the present 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 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 Non-Patent Documents 3 and 4). For example, conditions include those 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; or conditions including washing once, specifically two to three times, at a salt concentration and temperature equivalent to those used in 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.

[0053] Hybridization requires that 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 to each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present 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 having homology or identity to the polynucleotides of the present application can be detected using the hybridization conditions described above, in which the hybridization step is performed at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and 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 of the polynucleotides and the degree of complementation, variables known in the art (eg, Non-Patent Document 3).

[0056] In this application, "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, expressed as a percentage. Homology and identity are often used interchangeably.

[0057] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard sequence algorithms, optionally with default gap penalties established by the program used. Substantially homologous or identical sequences will generally hybridize to all or part of the sequence under moderately or highly stringent conditions. Hybridization, of course, also includes hybridization to polynucleotides containing common codons or codons that take into account codon degeneracy in the polynucleotide.

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

[0059] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as that disclosed in Non-Patent Document 10, as described in Non-Patent Document 1. Briefly, the GAP program defines homology as the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include: (1) a binary comparison matrix (identity takes a value of 1, non-identity a value of 0) and a weighted comparison matrix (or EDNAFULL (the EMBOSS version of NCBINUC4.4) substitution matrix) as described in Non-Patent Document 11; (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, gap extension penalty of 0.5); and (3) no penalty for terminal gaps. Thus, "homology" or "identity" in this application indicates the relevance between sequences.

[0060] Yet another aspect of the present application provides 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.

[0061] In this application, the term "vector" refers to a DNA product comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable the expression of the target polypeptide in a suitable host. The expression control region includes a promoter that initiates transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence that regulates 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 vector used in the present application is not particularly limited, and any vector known in the art may 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 may be used as phage or cosmid vectors. Examples of plasmid vectors that may be used include pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors may be used.

[0063] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using a vector for intracellular chromosomal introduction. 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 determining whether or not the polynucleotide has been inserted into the chromosome may be further included. The selection marker is used to select cells transformed with the vector, i.e., to determine whether or not the target nucleic acid molecule has been inserted. Markers that confer selectable phenotypes, such as drug resistance, auxotrophy, 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 survive or exhibit a different phenotype, allowing the selection of transformed cells.

[0064] In the present application, "transformation" refers to introducing a vector containing a polynucleotide encoding a target polypeptide into a microorganism or microorganisms, thereby expressing the polypeptide encoded by the polynucleotide in the microorganism. The transformed polynucleotide may be any polynucleotide that can be expressed in the microorganism, regardless of whether it is located intrachromosomally or extrachromosomally. Furthermore, the polynucleotide may comprise DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form that can be 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. Typically, the expression cassette contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may 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 expression in the microorganism, but is not limited thereto.

[0065] Furthermore, 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.

[0066] Yet another aspect of the present application provides a microorganism comprising an acetohydroxyacid synthase subunit (ilvN) mutant of the present application, a polynucleotide encoding an acetohydroxyacid synthase subunit (ilvN) mutant of the present application, or a vector comprising a polynucleotide of the present application.

[0067] In one embodiment, the microorganism of the present 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 has the chemical formula (CH3)2CHCH(NH2)COOH.

[0069] In this application, "microorganism (or strain)" includes all wild-type microorganisms and naturally or artificially genetically modified microorganisms, and is a microorganism in which a specific mechanism has been weakened or strengthened by inserting an exogenous gene or by enhancing or inactivating the activity of an endogenous gene, and is a microorganism that has been genetically modified for the production of a desired polypeptide, protein, or product. In this application, the terms "microorganism" and "strain" are used interchangeably and are used interchangeably.

[0070] In the present application, the term "L-valine-producing microorganism" refers to a prokaryotic or eukaryotic microbial strain that produces L-valine in its organism, and includes microorganisms in which the ability to produce L-valine has been imparted to a parent strain that does not have the ability to produce L-valine, and microorganisms that have the ability to produce L-valine endogenously. The ability to produce L-valine can be imparted or improved by breeding.

[0071] In one embodiment, the microorganism of the present application may be a microorganism that naturally has an acetohydroxyacid synthase subunit (ilvN) mutant or the ability to produce L-valine, or may be a microorganism to 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 does not have the ability to produce acetohydroxyacid synthase subunit (ilvN) mutant or L-valine, and / or to which the ability to produce L-valine has been imparted, but is not limited to these.

[0072] In one embodiment, the microorganism of the present application includes, but is not limited to, a microorganism comprising an 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 a microorganism comprising an acetohydroxy acid synthase subunit (ilvN) variant of the present application due to the introduction of a vector comprising a polynucleotide encoding the acetohydroxy acid synthase subunit (ilvN) variant of the present application.

[0073] In the present application, the term "unmodified microorganism" does not exclude strains containing naturally occurring mutations in microorganisms, but refers to a wild-type strain or a naturally occurring strain itself, or a strain before its traits are changed due to genetic mutation caused by natural or artificial factors. For example, the unmodified microorganism refers to a strain into which the acetohydroxyacid synthase subunit (ilvN) mutant described herein has not been introduced or before it has been introduced. The term "unmodified microorganism" is also used interchangeably with "strain before modification," "microorganism before modification," "non-mutated strain," "unmodified strain," "non-mutated microorganism," or "reference microorganism."

[0074] Microorganisms capable of producing L-valine of the present application include, but are not limited to, microorganisms comprising at least one of the variants of the present application, the polynucleotides of the present application, and vectors comprising the polynucleotides of the present application, microorganisms modified to express the variants of the present application or the polynucleotides of the present application, microorganisms (e.g., recombinant strains) that express the variants of the present application or the polynucleotides of the present application, or microorganisms (e.g., recombinant strains) that have the activity of the variants of the present application.

[0075] For example, the strain of the present application is a cell or microorganism transformed with a vector containing the polynucleotide of the present application or a polynucleotide encoding the mutant of the present application and expressing the mutant of the present application. The strain of the present application may be any microorganism that contains the mutant of the present application and produces L-valine. For example, the microorganism of the present application may be a recombinant strain in which an acetohydroxy acid synthase subunit (ilvN) mutant is expressed and L-valine production ability is improved by introducing a polynucleotide encoding the mutant of the present application into a naturally occurring wild-type microorganism or a microorganism capable of producing L-valine. The recombinant strain with improved L-valine production ability is a microorganism that has improved L-valine production ability compared to a naturally occurring wild-type microorganism or a microorganism without an altered acetohydroxy acid synthase subunit (ilvN) (e.g., a microorganism expressing a wild-type acetohydroxy acid synthase subunit (ilvN) or a microorganism not expressing the mutant of the present application), but is not limited thereto. For example, the microorganisms of the present application with improved L-valine production ability are microorganisms with improved L-valine production ability compared to microorganisms containing the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding the same, but are not limited thereto.

[0076] The microorganisms of the present application include all microorganisms that express the acetohydroxyacid synthase subunit (ilvN) mutants of the present application by various known methods other than the introduction of the nucleic acid or vector.

[0077] For example, the L-valine-producing ability of the microorganism with improved L-valine-producing ability is 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, or about 14.5% or more, compared to the L-valine-producing ability of the parent strain or unmodified microorganism before mutation. , about 14.6% or more, about 14.7% or more, or about 14.8% or more (there is no particular upper limit, 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 any increase in the + value compared to the productivity of the parent strain or unmodified microorganism before mutation is acceptable. In other examples, the recombinant strain with improved L-valine production ability has an L-valine production ability that is about 1.1-fold or more, about 1.12-fold or more, about 1.13-fold or more, or about 1.14-fold or more (the upper limit is not particularly limited, 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, but is not limited thereto. The term "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and may be any number that is equal to or in a similar range to the number following the term "about," but is not limited thereto.

[0078] The microorganisms of the present application include Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens. Specifically, the microorganism of the present application is a microorganism of the genus Corynebacterium, more specifically, Corynebacterium glutamicum, but is not limited thereto.

[0079] On the other hand, although it is already known that microorganisms of the genus Corynebacterium produce L-valine, their production ability is very low, and the genes and mechanistic principles acting on the production mechanism have not been elucidated. Therefore, the L-valine-producing microorganisms of the genus Corynebacterium of the present application include all of the following: natural wild-type microorganisms themselves; Corynebacterium microorganisms whose L-valine-producing ability has been improved by strengthening or weakening the activity of genes involved in the L-valine production mechanism; and Corynebacterium microorganisms whose L-valine-producing ability has been improved by introducing or strengthening the activity of an exogenous gene.

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

[0081] In the present application, "enhancing" a polypeptide activity means improving the activity of the polypeptide compared to its endogenous activity. The term "enhancing" is used interchangeably with "activation," "up-regulation," "overexpression," and "increase." Here, activation, enhancement, up-regulation, overexpression, and enhancement all encompass the development of an activity not originally present, as well as an improvement in activity compared to endogenous activity or activity prior to modification. The term "endogenous activity" refers to the activity of a specific polypeptide inherently possessed by a parent strain or unmodified microorganism prior to phenotypic change, when the trait is altered through genetic mutation due to natural or artificial factors. This term is also used interchangeably with "activity prior to modification." "Enhancing," "up-regulating," "overexpressing," or "improving" a polypeptide activity compared to its endogenous activity means an improvement in the activity and / or concentration (expression level) of a specific polypeptide inherently possessed by a parent strain or unmodified microorganism prior to phenotypic change.

[0082] Various methods well known in the art can be applied to enhance the activity of the polypeptide, including, but not limited to, increasing the intracellular copy number of the gene encoding the mutant, introducing a mutation into the expression regulatory sequence of the gene on the chromosome encoding the mutant, replacing the expression regulatory sequence of the gene on the chromosome encoding the mutant with a sequence with stronger activity, replacing the gene encoding the protein on the chromosome with a gene mutated to improve the activity of the mutant, and introducing a mutation into the gene on the chromosome encoding the mutant protein so as to enhance the activity of the mutant.

[0083] In the present application, "transfection" refers to a method of delivering a polynucleotide encoding the acetohydroxyacid synthase variant or a vector containing the same into a host cell. Such introduction can be easily achieved by methods commonly used in the art. Commonly used methods include the CaCl precipitation method, the Hanahan method, which uses DMSO (dimethyl sulfoxide) as a reducing agent to improve the efficiency of the CaCl method, electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicon carbide fibers, PEG-based transformation, dextran sulfate, lipofectamine, and desiccation / repression-mediated transformation. The method for transforming the vector is not limited to these examples and may be any transformation or transfection method commonly used in the art. Furthermore, the delivered polynucleotide may be any polynucleotide that is expressed in the host cell, regardless of whether it is inserted into the host cell's chromosome or located extrachromosomally. Furthermore, the polynucleotide may be introduced in any form that is capable of being introduced and expressed in the host cell. For example, the polynucleotide may be introduced into a host cell 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. Typically, the expression cassette 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 host cell in its own form and operably linked to sequences necessary for expression in the host cell, but is not limited thereto.

[0084] Yet another aspect of the present application provides a method for producing L-valine, comprising culturing the microorganism of the present application in a medium.

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

[0086] The term "culturing" as used herein 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 a suitable medium and culture conditions known in the art. Those skilled in the art can easily adjust such a culturing process depending on the selected strain. Specifically, the culturing may be batch, continuous, and / or fed-batch culture, but is not limited thereto.

[0087] The term "culture medium" as used herein refers to a mixture of nutrients necessary for culturing the microorganism of the present application as its main components, and supplies nutrients such as water essential for survival and growth, growth factors, etc. Specifically, the medium and other culture conditions used to culture the microorganism of the present application may be any medium used for culturing ordinary microorganisms, and the microorganism of the present application can be cultured in an ordinary medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins under aerobic conditions by adjusting the temperature, pH, etc.

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

[0089] In the present application, examples of carbon sources that can be used include carbohydrates such as glucose, saccharose, 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. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid can also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) can be used. Any other carbon source can also be used as long as it is present in an appropriate amount. These carbon sources can be used alone or in combination of two or more, but are not limited to these.

[0090] Examples of the nitrogen source that can be used 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 degradation products, and defatted soybean cake or its degradation products. These nitrogen sources can be used alone or in combination of two or more, but are not limited to these.

[0091] Examples of the phosphorus source that can be used include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and sodium-containing salts corresponding thereto. Examples of inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate. Other examples include amino acids, vitamins, and / or suitable precursors. These components or precursors can be added to the medium in a batch or continuous manner. However, the present invention is not limited to these.

[0092] Furthermore, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the medium in a suitable manner during cultivation of the microorganism of the present application. Furthermore, during cultivation, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, oxygen or an oxygen-containing gas may be injected into the medium to maintain an aerobic state, and nitrogen, hydrogen, or carbon dioxide gas may be injected, or no gas may be injected, to maintain anaerobic and microaerobic states, but these are not limiting.

[0093] In the culture of the present application, the culture temperature is maintained at 20 to 45°C, specifically 25 to 40°C, and the culture is carried out for about 10 to 160 hours, but is not limited thereto.

[0094] The L-valine produced by the culture of the present invention is either secreted into the medium or remains intracellularly.

[0095] The method for producing L-valine of the present application may further include a step of preparing a 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.

[0096] 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 further included after the culturing step.

[0097] The recovery may involve collecting L-valine using a suitable method known in the art depending on the culture method of the microorganism of the present application, such as batch, continuous, or fed-batch culture. For example, centrifugation, filtration, crystallization, treatment with a 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 thereof can be used. L-valine can be recovered from the medium or the microorganism using a suitable method known in the art.

[0098] The L-valine production method of the present application may further include a purification step. The purification can be performed by any suitable method known in the art. For example, when the L-valine production method of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the order, simultaneously, or integrated into a single step, but is not limited thereto.

[0099] Yet another aspect of the present application provides a composition for producing L-valine, comprising an acetohydroxyacid synthase subunit (ilvN) mutant of the present application, a polynucleotide encoding the mutant of the present application, a vector comprising the polynucleotide of the present application, or a microorganism comprising the polynucleotide of the present application, a medium in which they have been cultured, or a combination of at least two of them.

[0100] The composition of the present application may further contain any suitable excipient commonly used in compositions for producing amino acids, including, but not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, isotonicity agents, etc. [Example]

[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 present application is not limited thereto. Note that technical matters not described in this specification are well understood and easily implemented by skilled artisans in the technical field of the present application or a similar technical field. [Example]

[0102] Selection of mutants with improved valine production ability by artificial mutagenesis Example 1-1. Induction of artificial mutations by UV irradiation To select mutants with improved valine production, the valine-producing strain Corynebacterium glutamicum KCCM11201P (Patent Document 3) was spread on a nutrient medium containing agar and cultured for 36 hours at 30°C. Several hundred colonies thus obtained were irradiated with UV light at room temperature to induce random mutations in the genome of the strain.

[0103] Example 1-2. Fermentation titer evaluation of mutagenized strains and selection of strains To select mutants with improved 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 then each strain was inoculated into a 250 ml corner-baffled flask containing 25 ml of production medium and cultured at 30°C and 200 rpm for 72 hours with shaking. The L-valine concentration was then 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 (in 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 hydrogen phosphate 1g, magnesium sulfate heptahydrate 0.5g, biotin 100μg, thiamine HCl 1mg, calcium pantothenate 2mg, nicotinamide 3mg, calcium carbonate 30g (in 1 liter of distilled water)

[0104] [Table 1]

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

[0106] Confirmation of mutations by gene sequencing The major genes of the strain were sequenced and compared with those of the KCCM11201P strain, and it was confirmed that the C6 strain, which had improved valine production, contained a mutation in the nucleotide sequence 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, contained the A42V mutation found in the parent strain KCCM11201P, and also contained a mutation 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 was confirmed to have a form in which the 159th amino acid, alanine, was replaced with glutamic acid (SEQ ID NO: 3).

[0108] Analysis of the A159E mutation region confirmed that it affects the effector binding domain of the valine biosynthetic enzyme, and predicted that the activity of the protein would be enhanced. In the following example, we examine the effect of the A159E mutation inserted into a specific ORF position in the ilvN gene on the ability to produce valine, a branched-chain amino acid, in Corynebacterium microorganisms. [Example]

[0109] Construction of the KCCM11201P strain carrying the ilvN mutation and confirmation of its valine-producing ability Example 3-1. Construction of a strain by introducing the ilvN mutation into Corynebacterium glutamicum KCCM11201P strain and evaluation of its L-valine production ability - 1 A vector containing the target mutation was constructed to insert the ilvN (A159E) mutant into Corynebacterium glutamicum KCCM11201P, which contains the A42V mutation. Specifically, genomic DNA from the C6 strain was extracted using a 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 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.

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

[0111] [Table 2]

[0112] The pDCM2-ilvN(A42V, A159E) was transformed into Corynebacterium glutamicum KCCM11201P by homologous recombination on the chromosome (Non-Patent Document 14). Strains in which the vector had been integrated into the chromosome by recombination of the homologous sequences were selected from medium containing 25 mg / L of kanamycin. Subsequently, PCR using SEQ ID NO: 7 and SEQ ID NO: 8 was performed on the Corynebacterium glutamicum transformants in which the secondary recombination had been completed. The strains were confirmed to contain the A42V mutation contained in the parent strain KCCM11201P, which has an alanine-to-valine substitution at amino acid position 42 of SEQ ID NO: 1 in the ORF of the ilvN gene on the chromosome, as well as a glutamic acid-to-alanine substitution at amino acid position 159 of SEQ ID NO: 1 in the ORF of the ilvN gene. The recombinant strain was named Corynebacterium glutamicum KCCM11201P::ilvN(A159E).

[0113] A flask assay was performed to compare the valine-producing abilities of the valine-producing strains Corynebacterium glutamicum KCCM11201P and KCCM11201P::ilvN(A159E). Each strain was subcultured in nutrient medium and then inoculated into a 250-ml corner-baffled flask containing 25 ml of production medium. The strains were then cultured at 30°C and 200 rpm for 72 hours with shaking. The L-valine concentrations were then 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 (in 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 hydrogen phosphate 1g, magnesium sulfate heptahydrate 0.5g, biotin 100μg, thiamine HCl 1mg, calcium pantothenate 2mg, nicotinamide 3mg, calcium carbonate 30g (in 1 liter of distilled water)

[0114] [Table 3]

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

[0116] Example 3-2. Construction of a Corynebacterium glutamicum KCCM11201P strain with an ilvN mutation and evaluation of its L-valine production ability - 2 To confirm the effect of the ilvN(A159E) mutation alone on wild-type ilvN enzyme, we constructed a strain in which alanine was substituted with glutamic acid at amino acid position 159 of SEQ ID NO: 1 in the ORF of the ilvN gene. Specifically, a vector containing the target mutation was constructed to insert ilvN(V42A,A159E) into C. glutamicum KCCM11201P. Genomic DNA from the C6 strain was extracted using a 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 as follows: 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. Using sequence numbers 9 and 10, a 515 bp PCR product (hereinafter referred to as "mutation-introduced fragment 2") was obtained, and using sequence numbers 11 and 12, a 518 bp PCR product (hereinafter referred to as "mutation-introduced fragment 3") was obtained.

[0117] The pDCM2 vector (Patent Documents 4 and 5) was treated with the restriction enzyme XbaI (New England Biolabs, Beverly, MA) and ligated with an Infusion Cloning Kit (Takara Bio Inc., Otsu, Japan) to the mutated fragments 2 and 3 obtained as described above. The gene prepared as described above was transformed into Escherichia coli DH5α, which was then selected on kanamycin-containing LB medium. DNA was then isolated using a DNA-spin Plasmid DNA Purification Kit (iNtRON), and the vector pDCM2-ilvN (V42A, A159E) containing the mutated fragments 2 and 3 was prepared.

[0118] [Table 4]

[0119] The pDCM2-ilvN(V42A, A159E) was transformed into Corynebacterium glutamicum KCCM11201P by homologous recombination on the chromosome (Non-Patent Document 14). The A42V mutation was restored and the A159E mutation was introduced by the homologous recombination. Strains in which the vector had been integrated into the chromosome by recombination of the homologous sequences were selected from medium containing 25 mg / L of kanamycin. Subsequently, for the Corynebacterium glutamicum transformants in which secondary recombination had been completed, PCR using SEQ ID NO: 9 and SEQ ID NO: 12 was used to confirm strains in which valine had been restored to alanine at amino acid position 42 of SEQ ID NO: 1 in the ORF of the ilvN gene on the chromosome and alanine had been replaced with glutamic acid at amino acid position 159. The recombinant strain was named Corynebacterium glutamicum KCCM11201P::ilvN(V42A,A159E).

[0120] To compare the L-valine-producing abilities of the prepared strains, they were cultured in the same manner as in Example 3-1 and the L-valine concentrations were analyzed. The analyzed L-valine concentrations are shown in Table 5.

[0121] [Table 5]

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

[0123] From the above description, those skilled in the art to which the present application pertains will understand that the present application can be implemented in other specific forms without changing the technical idea or essential features thereof. It should be understood that the above examples are merely illustrative and not limiting. The present application should be construed as including all modifications and variations derived from the meaning and scope of the claims, rather than the specification, and their equivalent concepts.

Claims

1. An acetohydroxyacid synthase subunit (ilvN) mutant in which the amino acid corresponding to position 159 in the amino acid sequence of SEQ ID NO: 1 has been 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 according to claim 1 , wherein the amino acid corresponding to position 159 of SEQ ID NO: 1 is substituted with glutamic acid.

4. The mutant of claim 2, wherein the amino acid corresponding to position 159 of SEQ ID NO: 1 is substituted with glutamic acid and the amino acid corresponding to position 42 is substituted with valine.

5. The mutant according to claim 1, wherein the mutant consists of the amino acid sequence represented by SEQ ID NO:

3.

6. The mutant according to claim 2, wherein the mutant consists of the amino acid sequence represented by SEQ ID NO:

5.

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

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

9. 9. The microorganism according to claim 8, wherein the microorganism has improved L-valine-producing ability compared to a microorganism containing the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding the polypeptide.

10. The microorganism according to claim 8 , wherein the microorganism is a Corynebacterium microorganism.

11. The microorganism according to claim 10, wherein the Corynebacterium microorganism is Corynebacterium glutamicum.

12. A method for producing L-valine, comprising culturing the microorganism according to claim 8 in a medium.

13. The method of claim 12 , further comprising the step of recovering the target substance from the culture medium.

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