Novel acetohydroxy acid synthase subunit variants and method for producing l-valine using same

Abstract

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

Description

Technical Field

[0001] The present disclosure relates to novel acetohydroxyacid synthase subunit (ilvN) variants, polynucleotides encoding the variants of the present disclosure, L-valine-producing microorganisms comprising the acetohydroxyacid synthase subunit (ilvN) variants of the present disclosure, and methods for producing L-valine using the microorganisms of the present disclosure. Background Art

[0002] L-amino acids are the basic structural units of proteins and are used as important materials for pharmaceuticals, food additives, animal feeds, nutrients, pesticides, fungicides, etc. In particular, branched-chain amino acids (BCAAs) are a general term for L-valine, L-leucine, and L-isoleucine, which are essential amino acids, and it is known that branched-chain amino acids have antioxidant effects and the effect of promoting protein synthesis in muscle cells.

[0003] Meanwhile, the production of branched-chain amino acids using microorganisms is mainly achieved by microorganisms of the genus Corynebacterium, and it is known that it is biosynthesized from pyruvate through several steps with 2-ketoisocaproic acid as a precursor [Korean Patent Nos. 10-0220018 and 10-0438146]. However, the production of L-branched-chain amino acids by these microorganisms has presented challenges for large-scale industrial production.

[0004] Under these circumstances, the present inventors have confirmed that, in order to improve the ability of microorganisms to produce L-valine, introducing an activity-enhanced variant of the gene ilvN (acetohydroxyacid synthase subunit), which encodes an enzyme involved in the biosynthesis of L-valine in microorganisms, significantly enhances the L-valine production ability of the microorganisms. Summary of the Invention

[0005] [Technical Problem]

[0006] An object of the present disclosure is to provide novel acetohydroxyacid synthase subunit (ilvN) variants, polynucleotides encoding the variants of the present disclosure, L-valine-producing microorganisms comprising the acetohydroxyacid synthase subunit (ilvN) variants of the present disclosure, and methods for producing L-valine using the microorganisms of the present disclosure.

[0007] [Technical Solution]

[0008] An object of the present disclosure is to provide an acetohydroxyacid synthase subunit (ilvN) variant in which the amino acid at position 159 in the amino acid sequence corresponding to SEQ ID NO: 1 is replaced with another amino acid.

[0009] Another object of the present disclosure is to provide a polynucleotide encoding the variant of the present disclosure.

[0010] Yet another object of the present disclosure is to provide a microorganism comprising a variant of the present disclosure or a polynucleotide encoding a variant of the present disclosure.

[0011] Another object of the present disclosure is to provide a method for producing L-valine, comprising culturing the microorganism in a culture medium.

[0012] Yet another object of the present disclosure is to provide a composition for producing L-valine, comprising: the microorganism of the present disclosure; a culture medium obtained by culturing the microorganism of the present disclosure; or a combination thereof.

[0013] [Beneficial effects]

[0014] Compared with a microorganism having a conventional, unmodified polypeptide, culturing a microorganism comprising a variant of the acetohydroxyacid synthase subunit of the present disclosure can produce L-valine in high yield. Detailed description of the specific embodiments

[0015] The present disclosure will be described in detail below. At the same time, the various descriptions and embodiments disclosed in the present disclosure can also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in the present disclosure fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the specific descriptions described below. In addition, many papers and patent documents are referenced and cited throughout the specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to further clarify the level of the prior art and the description of the present disclosure.

[0016] One aspect of the present disclosure provides a variant of the acetohydroxyacid synthase subunit (ilvN), wherein the amino acid at position 159 in the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by another amino acid.

[0017] As used herein, the term "variant of the acetohydroxyacid synthase subunit (ilvN)" refers to a variant of the acetohydroxyacid synthase subunit (ilvN) that includes one or more amino acid substitutions in the amino acid sequence of a polypeptide having acetohydroxyacid synthase subunit (ilvN) activity.

[0018] As used herein, the term "acetohydroxyacid synthase", which is the first enzyme involved in the biosynthesis of L-valine, is also referred to as acetolactate synthase. Acetohydroxyacid synthase can produce acetolactate (i.e., a precursor of valine) by catalyzing the decarboxylation of pyruvate and the condensation reaction with another pyruvate molecule, or produce aceto-hydroxybutyrate (i.e., a precursor of isoleucine) by catalyzing the decarboxylation of pyruvate and the condensation reaction with 2-ketobutyrate.

[0019] 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 acetohydroxyacid synthase. Among these two subunits, the small subunit encoded by the ilvN gene is considered to play a key role in feedback inhibition.

[0020] As used herein, the term "acetohydroxyacid synthase subunit (ilvN)" may be the acetohydroxyacid synthase subunit (ilvN) encoded by the ilvN gene, but is not limited thereto. In addition, the acetohydroxyacid synthase subunit (ilvN) may be the acetohydroxyacid synthase subunit (ilvN) protein derived from a microorganism of the genus Corynebacterium, or particularly Corynebacterium glutamicum, or a variant thereof, but is not limited thereto. Specifically, the acetohydroxyacid synthase subunit (ilvN) protein may include, for example, the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having 70% or higher homology or identity thereto, but is not limited thereto, as long as it retains the activity of the acetohydroxyacid synthase subunit (ilvN). Specifically, the amino acid sequence may include the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher homology or identity to the amino acid sequence of SEQ ID NO:1. The sequence of SEQ ID NO:1 can be obtained from publicly available databases such as Genbank of NCBI or the Kyoto Encyclopedia of Genes and Genomes (KEGG). For example, the amino acid sequence may be derived from a microorganism of the genus Corynebacterium, or particularly Corynebacterium glutamicum, and more particularly, it may be a polypeptide or protein containing the amino acid sequence of SEQ ID NO:1, but is not limited thereto. In addition, it is obvious that any auxiliary protein with a partially deleted, modified, substituted or added amino acid sequence may also be included within the scope of the present invention as long as the amino acid sequence has such homology or identity and exhibits the same efficacy as the protein.

[0021] In addition, the acetohydroxyacid synthase subunit (ilvN) protein having the amino acid sequence of SEQ ID NO:1 may have or contain the sequence of SEQ ID NO:2, or a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more and less than 100% homology or identity with the sequence of SEQ ID NO:2, or may be encoded by a polynucleotide consisting of the sequence of SEQ ID NO:2 or a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more and less than 100% homology or identity with the sequence of SEQ ID NO:2, or consisting essentially of the sequence of SEQ ID NO:2 or a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more and less than 100% homology or identity with the sequence of SEQ ID NO:2, but not limited thereto.

[0022] As used herein, the term "variant" refers to a polypeptide in which, due to conservative substitution and / or modification of one or more amino acids, the amino acid sequence is different from that of the variant prior to mutation, but retains its function or properties prior to mutation. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and assessing the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged or decreased compared to the polypeptide prior to mutation. In addition, some variants may include variants in which at least a portion (such as an N-terminal leader sequence or transmembrane domain) is removed. Other variants may be those in which portions of the N-terminus and / or C-terminus of the mature protein are removed. The term "variant" may be used interchangeably with "modification", "modified polypeptide", "modified protein", "mutant", "mutant protein", "variant", etc., and any term used in a mutational sense may be used, without limitation.

[0023] In addition, variants may include deletions or additions of amino acids that have a minimal impact on the properties and secondary structure of the polypeptide. For example, the polypeptide may be coupled to a signal (or leader) sequence at the N-terminus of a protein involved in co-translational or post-translational transfer. In addition, the polypeptide may also be coupled to another sequence or linker for identification, purification or synthesis of the polypeptide.

[0024] The acetohydroxyacid synthase subunit (ilvN) variant of the present disclosure can be an acetohydroxyacid synthase subunit (ilvN) variant, wherein the amino acid at position 159 in the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by another amino acid, but is not limited thereto.

[0025] In one embodiment, the acetohydroxyacid synthase subunit (ilvN) variant can, in addition to replacing the amino acid at position 159 in the amino acid sequence corresponding to SEQ ID NO: 1 with another amino acid, also replace the amino acid at position 42 in the amino acid sequence corresponding to SEQ ID NO: 1 with another amino acid.

[0026] "Another amino acid" or "other amino acid" is not restricted as long as the amino acid is different from the amino acid before replacement. Also, it is obvious that the statement "a specific amino acid is replaced" means that the amino acid is replaced by an amino acid different from the amino acid before replacement, even if it is not explicitly described that the amino acid is replaced by a different amino acid.

[0027] Amino acids can generally be classified based on the similarity of the residues in terms of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity.

[0028] For example, positively charged (basic) amino acids can 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) can include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; amino acids with polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In another example, amino acids with charged side chains (charged amino acids) include arginine, lysine, histidine, glutamic acid, and aspartic acid, and amino acids with uncharged side chains (also referred to as uncharged amino acids or neutral amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In another example, phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. In another example, valine, leucine, and isoleucine can be classified as branched-chain amino acids. In another example, the 20 amino acids can be divided into five groups according to size, starting from the group of amino acids with relatively small volume: glycine, alanine, and serine; cysteine, proline, threonine, aspartic acid, and asparagine; valine, histidine, glutamic acid, and glutamine; isoleucine, leucine, methionine, lysine, and arginine; and phenylalanine, tryptophan, and tyrosine. However, the classification of amino acids is not limited thereto.

[0029] For example, the phrase "the amino acid corresponding to position 159 in SEQ ID NO:1 is replaced by another amino acid" may mean that the amino acid is replaced by glutamic acid, phenylalanine, glycine, arginine, aspartic acid, cysteine, asparagine, glutamine, histidine, proline, serine, tyrosine, isoleucine, lysine, tryptophan, valine, methionine, threonine, or leucine (excluding alanine), and the phrase "the amino acid corresponding to position 42 in SEQ ID NO:1 is replaced by another amino acid" may mean that the amino acid is replaced by valine, asparagine, glycine, arginine, aspartic acid, cysteine, glutamic acid, histidine, proline, serine, tyrosine, isoleucine, leucine, lysine, phenylalanine, tryptophan, glutamine, methionine, or threonine (excluding alanine), but is not limited thereto.

[0030] When the expression "a protein having an amino acid sequence of a specific SEQ ID NO" is used in the present disclosure, it is obvious that a protein having an amino acid sequence with a partial deletion, modification, replacement, conservative replacement, or addition can also be used in the present disclosure, as long as the protein exhibits the same or equivalent activity as the protein composed of the amino acid sequence of the corresponding SEQ ID NO. For example, when exhibiting the same or equivalent activity as the variant protein, it is obvious that a protein having a sequence addition, a naturally occurring mutation or a silent mutation, or a conservative replacement thereof that does not change the protein function is not excluded, and such a protein having a sequence addition or mutation falls within the scope of the present disclosure.

[0031] As used herein, the term "position N" may include position N and the amino acid position corresponding to position N. Specifically, the term may include the amino acid position corresponding to any amino acid residue in the mature polypeptide of a specific amino acid sequence. The specific amino acid sequence may be the amino acid sequence of SEQ ID NO:1.

[0032] As used herein, the term "corresponding to" refers to the amino acid residue at the position listed in the polypeptide, or an amino acid residue that is similar to, identical to, or homologous to the amino acid residue listed in the polypeptide. Identifying the amino acid at the corresponding position may be determining a specific amino acid in a sequence, and the sequence refers to a specific sequence. As used herein, the term "corresponding region" generally refers to a similar or corresponding position in a related protein or a reference protein.

[0033] For example, any amino acid sequence can be aligned with SEQ ID NO:1, and based on this, each amino acid residue of the any amino acid sequence can be numbered with reference to the numerical position of the amino acid residue corresponding to the amino acid residue of SEQ ID NO:1. For example, the sequence alignment algorithm described in the present disclosure can determine the position of an amino acid or the position where a modification such as replacement, insertion, or deletion occurs by comparing with a query sequence (also referred to as a "reference sequence").

[0034] In such an alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), the Needleman program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), etc. can be used, but not limited thereto, as well as sequence alignment programs, pairwise sequence comparison algorithms, etc., which are known in the art and can be appropriately used.

[0035] In one embodiment, the acetohydroxyacid synthase subunit (ilvN) variant of the present disclosure may include an amino acid sequence in which one or two amino acids corresponding to positions 42 and 159 from the N-terminus in SEQ ID NO: 1 are replaced with different amino acids.

[0036] In one embodiment of the foregoing embodiment, the acetohydroxyacid synthase subunit (ilvN) variant of the present disclosure may be a polypeptide in which the amino acid (alanine) corresponding to position 159 in SEQ ID NO: 1 is replaced with glutamic acid, but not limited thereto.

[0037] In one embodiment of the foregoing embodiment, the acetohydroxyacid synthase subunit (ilvN) variant of the present disclosure may be a polypeptide in which the amino acid (alanine) corresponding to position 159 in SEQ ID NO: 1 is replaced with glutamic acid and the amino acid (alanine) corresponding to position 42 in SEQ ID NO: 1 is replaced with valine, but not limited thereto.

[0038] In one embodiment of the foregoing embodiment, the variant provided in the present disclosure may include replacing the amino acid corresponding to position 159 from the N-terminus in SEQ ID NO: 1 with another amino acid.

[0039] In one embodiment of the foregoing embodiment, the variant provided by the present invention may include replacing the amino acid corresponding to position 159 from the N-terminus in SEQ ID NO: 1 with an amino acid selected from arginine, lysine, histidine, glutamic acid, and aspartic acid (all of these amino acids are amino acids with charged side chains). For example, the amino acid may be selected from glutamic acid and aspartic acid, which are acidic amino acids. In one embodiment of the foregoing embodiment, the variant may be a variant in which the amino acid corresponding to position 159 from the N-terminus in SEQ ID NO: 1 is replaced with glutamic acid (E).

[0040] In one embodiment of the foregoing embodiments, the variant provided in the present disclosure may include replacing the amino acid corresponding to the 42nd position from the N-terminus in SEQ ID NO:1 with another amino acid.

[0041] In one embodiment of the foregoing embodiments, the variant may replace the amino acid corresponding to the 42nd position from the N-terminus in SEQ ID NO:1 with a non-polar amino acid. For example, the non-polar amino acid may be selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. In one embodiment of the foregoing embodiments, the variant may be a variant in which the amino acid corresponding to the 42nd position from the N-terminus in SEQ ID NO:1 is replaced by valine (V).

[0042] Meanwhile, those of ordinary skill in the art can identify the amino acids at positions 159 and 42 in the amino acid sequence corresponding to SEQ ID NO:1 of the present disclosure through sequence alignment known in the art. In addition, it is obvious that the expression "the amino acid at a specific position in a specific SEQ ID NO" also includes "the amino acid corresponding to a specific position" in any amino acid sequence, even if the present disclosure does not explicitly describe it.

[0043] In addition, the variants of the present disclosure may include amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or higher homology or identity with the amino acid sequence of SEQ ID NO:1, wherein the amino acid corresponding to the 159th position from the N-terminus in SEQ ID NO:1 is replaced by another amino acid, or include the amino acid sequence of SEQ ID NO:1, wherein in addition to the amino acid corresponding to the 159th position being replaced by another amino acid, the amino acid corresponding to the 42nd position is also replaced by another amino acid. In addition, it is obvious that variants with amino acid sequences having partial deletions, modifications, substitutions, conservative substitutions, or additions fall within the scope of the present disclosure as long as the amino acid sequence has the same homology or identity as the variant of the present disclosure and exhibits equivalent efficacy.

[0044] For example, the variant of the present disclosure may have or include the amino acid sequence of SEQ ID NO:3 or 5, or consist essentially of the amino acid sequence of SEQ ID NO:3 or 5. Alternatively, the variant may include 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 higher homology or identity with the amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOS:3 and 5.

[0045] For example, this includes adding or deleting sequences, naturally occurring mutations, silent mutations, or conservative substitutions that do not alter the function of the variants of the present disclosure at the N-terminus, C-terminus, and / or internal regions of the amino acid sequence.

[0046] As used herein, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structure and / or chemical properties. Such amino acid substitutions generally occur based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity 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. In addition, amino acids can be divided into amino acids with charged side chains and amino acids with uncharged side chains. Amino acids with charged side chains include aspartic acid, glutamic acid, lysine, arginine, and histidine. Amino acids with uncharged side chains can be further divided into non-polar amino acids and polar amino acids; non-polar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, and polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Generally, conservative substitutions have little or no effect on the activity of the resulting polypeptide. Generally, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0047] In one embodiment, the variants of the acetohydroxyacid synthase subunit (ilvN) of the present disclosure can have increased acetohydroxyacid synthase subunit (ilvN) activity, but are not limited thereto. In addition, the variants of the present disclosure can have an activity of increasing the ability to produce L-valine that is higher than that of the wild-type polypeptide having acetohydroxyacid synthase subunit (ilvN) activity, but are not limited thereto.

[0048] Another aspect of the present disclosure provides a polynucleotide encoding the variant of the present disclosure.

[0049] As used herein, the term "polynucleotide" refers to a polymer of nucleotides, in which nucleotide monomers are longitudinally chain-extended by covalent bonds, and it is a DNA or RNA chain having at least a certain length, and more specifically, a polynucleotide fragment encoding the protein.

[0050] The polynucleotide encoding the acetohydroxyacid synthase subunit (ilvN) variant disclosed herein can include any sequence without limitation, as long as the sequence is a polynucleotide sequence encoding the acetohydroxyacid synthase subunit (ilvN) variant disclosed herein. For example, the polynucleotide encoding the acetohydroxyacid synthase subunit (ilvN) variant disclosed herein can be a polynucleotide sequence encoding the amino acid sequence of the acetohydroxyacid synthase subunit (ilvN) variant disclosed herein, but is not limited thereto.

[0051] Considering codon degeneracy or the preferred codons of the organism in which the variant disclosed herein is to be expressed, the polynucleotides of the present invention can have various modifications in the coding region within the range of not changing the amino acid sequence of the variant disclosed herein. Thus, it is obvious that polynucleotides that can be translated into a polypeptide consisting of the amino acid sequence of the variant disclosed herein or a polypeptide homologous or identical thereto by codon degeneracy can also be included.

[0052] For example, the polynucleotide of the present disclosure can have or include a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more and less than 100% homology or identity with the sequence of SEQ ID NO: 4 or 6, or can consist of or consist essentially of a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more and less than 100% homology or identity with the sequence of SEQ ID NO: 4 or 6, but is not limited thereto. Alternatively, among the sequences having such homology or identity, the codon encoding the amino acid corresponding to position 159 in SEQ ID NO: 3 or SEQ ID NO: 5 can be one of the codons encoding glutamic acid, and the codon encoding the amino acid corresponding to position 42 in SEQ ID NO: 3 can be one of the codons encoding valine, but the codons are not limited thereto.

[0053] In addition, the polynucleotides of the present disclosure may include any probe that can be prepared from known gene sequences. For example, sequences that can hybridize with all or part of the complementary sequence of the polynucleotide sequences of the present disclosure under stringent conditions are not limited. The term "stringent conditions" refers to conditions that enable specific hybridization between polynucleotides. These 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; and F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50 - 9.51, 11.7 - 11.8). Examples of stringent conditions may include conditions under which polynucleotides with higher homology or identity hybridize to each other. For example, polynucleotides with 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology or identity hybridize to each other, while polynucleotides with lower homology or identity do not hybridize to each other; or typical washing conditions for Southern hybridization, i.e., washing once or twice or three times under salt concentrations and temperatures corresponding to 1×SSC, 0.1% SDS at 60°C, specifically 0.1×SSC, 0.1% SDS at 60°C, or more specifically 0.1×SSC, 0.1% SDS at 68°C.

[0054] Hybridization requires that two nucleic acids have complementary sequences, although mismatches between bases are also possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleobases that can hybridize to each other. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present disclosure may further include not only nucleic acid sequences that are substantially similar, but also isolated nucleic acid fragments that are complementary to the complete sequence.

[0055] Specifically, polynucleotides having homology or identity with the polynucleotides of the present disclosure can be detected by using hybridization conditions that include T at 55°C m and a hybridization step under the above conditions. In addition, the T m value can be 60°C, 63°C, or 65°C, but is not limited thereto. Those of ordinary skill in the art can appropriately adjust the T m value according to the purpose.

[0056] The appropriate stringency for polynucleotide hybridization depends on the length and degree of complementarity of the polynucleotide, and the variables are well known in the art (e.g., J. Sambrook et al., supra).

[0057] As used herein, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid sequences or nucleotide sequences, which can be expressed as a percentage. The terms "homology" and "identity" are often used interchangeably.

[0058] The sequence homology or identity of a conserved polynucleotide or polypeptide can be determined by standard alignment algorithms, and the default gap penalties established by the program used can be applied together. Basically, homologous or identical sequences can generally hybridize with each other, in whole or in part, under moderately or highly stringent conditions. Obviously, the hybridization also includes hybridization with polynucleotides containing normal codons or codons taking into account the codon degeneracy in the polynucleotide.

[0059] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined by using, for example, known computer algorithms such as the "FASTA" program, using default parameters as in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, they can be determined by using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as implemented in the Needleman program (version 5.0.0 or higher) of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (including the GCG program package (Devereux, J., et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S.F., et al., J Molec Biol 215: 403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and CARILLO et al. (1988) SIAM J Applied Math 48: 1073). For example, the homology, similarity or identity can be determined by using BLAST or ClustalW of the National Center for Biotechnology Information.

[0060] Homology, similarity or identity between polynucleotides or polypeptides can be determined by using the GAP computer program, as described in Needleman et al., (1970), J Mol Biol. 48:443), and as disclosed in Smith and Waterman, Adv Appl Math (1981) 2:482. Briefly, the GAP program defines similarity as the number of symbols (i.e., nucleotides or amino acids) in the similar alignment divided by the total number of symbols in the shorter of the two sequences. The default parameters of the GAP program can include: (1) a binary comparison matrix (including a value of 1 for identity and a value of 0 for non-identity) and a weighted comparison matrix as disclosed in Gribskov et al. (1986) Nucl. Acids Res. 14:6745, edited by Schwartz and Dayhoff, Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353–358 (1979) (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in the gap (or a gap opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for terminal gaps. Thus, the terms "homology" or "identity" as used herein refer to the relatedness between sequences.

[0061] Another aspect of the present disclosure provides a vector comprising the polynucleotide of the present disclosure. The vector can be an expression vector for expressing the polynucleotide in a microorganism, but is not limited thereto.

[0062] As used herein, the term "vector" can include a DNA construct comprising a nucleotide sequence of a polynucleotide encoding a target polypeptide, which is operably linked to a suitable expression control region (or expression control sequence) for expressing the target polypeptide in a suitable host. The expression control region can include a promoter capable of initiating transcription, any operator sequences for controlling such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences for controlling transcription and translation termination. After transformation into a suitable microorganism, the vector can replicate or function independently of the host genome or can integrate into the genome itself.

[0063] The vectors used in the present disclosure are not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses, and phages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and vectors based on 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.

[0064] In one embodiment, the polynucleotide encoding the target polypeptide can be inserted into a chromosome by a vector for chromosomal insertion in a cell. The polynucleotide can be inserted into the chromosome by using any method known in the art, such as homologous recombination, but is not limited thereto. The vector can further include a selection marker for confirming chromosomal insertion. The selection marker is used to select cells transformed with the vector, that is, to confirm the insertion of the target nucleic acid molecule, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, cytotoxic drug resistance, or surface polypeptide expression can be used. In an environment treated with a selection agent, only cells expressing the selection marker survive or exhibit different phenotypic expressions, thereby enabling the selection of transformed cells.

[0065] As used herein, the term "transformation" refers to introducing a vector containing a polynucleotide encoding a target polypeptide into a microorganism or host cell so that the polypeptide encoded by the polynucleotide can be expressed in the microorganism. The transformed polynucleotide can include any polypeptide as long as it can be expressed in the microorganism, regardless of whether it is inserted and located in the chromosome of the microorganism or outside the chromosome. In addition, the polynucleotide includes DNA and / or RNA encoding the target polypeptide. The polynucleotide can be introduced in any form as long as the polynucleotide can be introduced into the microorganism and expressed. For example, the polynucleotide can be introduced into the microorganism in the form of an expression cassette, which is a gene construct containing all the factors required for self-expression. The expression cassette generally may include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette can be in the form of an expression vector capable of self-replication. In addition, the polynucleotide can be introduced into the microorganism as it is and operably linked to the sequences required for expression in the microorganism, but is not limited thereto.

[0066] In addition, as used herein, the term "operably linked" refers to a functional linkage between a promoter sequence and a polynucleotide sequence that initiates and mediates the transcription of a polynucleotide encoding a target protein of the present disclosure.

[0067] Another aspect of the present disclosure provides a microorganism comprising a variant of the acetohydroxyacid synthase subunit (ilvN) of the present disclosure, a polynucleotide encoding the variant of the acetohydroxyacid synthase subunit (ilvN) of the present disclosure, or a vector comprising the polynucleotide of the present disclosure.

[0068] In one embodiment, the microorganism of the present disclosure may be a microorganism having the ability to produce L-valine.

[0069] As used herein, the term "L-valine" refers to the L-amino acid having the chemical formula (CH 3 ) 2 CHCH(NH 2 )COOH, which is an essential amino acid classified together with L-leucine and L-isoleucine as branched-chain amino acids.

[0070] As used herein, the term "microorganism (or strain)" includes all wild-type microorganisms and microorganisms having natural or artificial genetic modifications, and refers to microorganisms in which a specific mechanism is weakened or enhanced due to the insertion of foreign genes, the enhancement or inactivation of endogenous gene activities, etc. The microorganism may be a genetically modified microorganism used for producing a target polypeptide, protein, or product. As used herein, the terms "microorganism", "strain", and "microbe" have the same meaning and can be used interchangeably without limitation.

[0071] As used herein, the term "L-valine-producing microorganism" refers to a prokaryotic or eukaryotic microbial strain capable of producing L-valine within its cell system, and may include any microorganism in which the ability to produce L-valine has been conferred on a parental strain lacking such ability, or any microorganism inherently possessing the ability to produce L-valine. The ability to produce L-valine can be conferred or enhanced by strain improvement.

[0072] In one embodiment, the microorganism of the present disclosure may be a microorganism that naturally has a variant of the acetohydroxyacid synthase subunit (ilvN) or has the ability to produce L-valine; or a microorganism in which the variant of the present disclosure or a polynucleotide encoding the variant (or a vector comprising the polynucleotide) has been introduced and / or a parental strain lacking the variant of the acetohydroxyacid synthase subunit (ilvN) or the ability to produce L-valine has been conferred with the ability to produce L-valine, but is not limited thereto.

[0073] In one embodiment, the microorganisms of the present disclosure include any microorganism that contains the variant sequence of the acetohydroxyacid synthase subunit (ilvN) of the present disclosure by mutating the chromosomal gene encoding the acetohydroxyacid synthase subunit (ilvN) variant, and / or any microorganism that contains the variant sequence of the acetohydroxyacid synthase subunit (ilvN) of the present disclosure by introducing a vector containing a polynucleotide encoding the acetohydroxyacid synthase subunit (ilvN) variant of the present disclosure, but not limited thereto.

[0074] As used herein, the term "unmodified microorganism" does not exclude strains that may contain mutations that occur naturally in the microorganism, and may refer to the initial wild-type strain or natural strain, or the strain before its traits are changed due to genetic mutations caused by natural or artificial factors. For example, an unmodified microorganism may refer to a strain in which the variant of the acetohydroxyacid synthase subunit (ilvN) of the present disclosure has not been introduced or the strain before introduction. "Unmodified microorganism" may be used interchangeably with "strain before modification", "microorganism before modification", "unmutated strain", "unmodified strain", "unmutated microorganism", or "reference microorganism".

[0075] The microorganisms with the ability to produce L-valine of the present disclosure may be microorganisms that contain one or more selected from the variants of the present disclosure, the polynucleotides of the present disclosure, and vectors containing the polynucleotides of the present disclosure; microorganisms modified to express the variants or polynucleotides of the present disclosure; microorganisms expressing the variants or polynucleotides of the present disclosure (e.g., recombinant strains); or microorganisms having the activity of the variants of the present disclosure (e.g., recombinant strains), but not limited thereto.

[0076] For example, a strain of the present disclosure refers to a cell or microorganism that expresses a variant of the present disclosure by being transformed into a vector containing the polynucleotide of the present disclosure or a polynucleotide encoding a variant of the present disclosure, and the strain of the present disclosure may include any microorganism capable of producing L-valine by containing the variant of the present disclosure. For example, the microorganism of the present disclosure may be a recombinant strain with increased L-valine production ability, wherein the increased L-valine production ability is achieved by introducing a polynucleotide encoding a variant of the present disclosure into a natural wild-type microorganism or a microorganism with L-valine production ability, so as to be able to express a variant of acetohydroxyacid synthase subunit (ilvN). The recombinant strain with increased L-valine production ability may be a microorganism with increased L-valine production ability compared to a natural wild-type microorganism or a microorganism with an unmodified acetohydroxyacid synthase subunit (ilvN) (for example, a microorganism expressing a wild-type acetohydroxyacid synthase subunit (ilvN) or a microorganism not expressing the variant of the present disclosure), but is not limited thereto. For example, the microorganism of the present disclosure with increased L-valine production ability may be a microorganism with increased L-valine production ability compared to a microorganism containing the polypeptide of SEQ ID NO:1 or a polynucleotide encoding the polypeptide, but is not limited thereto.

[0077] In addition to introducing nucleic acids or vectors, the microorganism of the present disclosure may include any microorganism capable of expressing a variant of the acetohydroxyacid synthase subunit (ilvN) of the present disclosure by various known methods.

[0078] For example, compared with the L-valine production ability of the parental strain before modification or an unmodified microorganism, the L-valine production ability of a microorganism with enhanced L-valine production ability can be increased by 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 14.6% or more, about 14.7% or more, or about 14.8% or more (the upper limit is not particularly limited and can 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). However, the valine production ability is not limited thereto, as long as the microorganism shows a positive increase in L-valine production ability compared with the parental strain or the unmodified microorganism. In another example, compared with the L-valine production ability of the parental strain before modification or an unmodified microorganism, a recombinant strain with increased L-valine production ability can have an L-valine production ability increased by 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 and can 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), but the L-valine production ability is not limited thereto. As used herein, the term "about" refers to the entire range including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., which includes all values equivalent or similar to those within the range described after the term "about", but is not limited thereto.

[0079] The microorganisms of the present disclosure may be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens. Specifically, the microorganisms of the present disclosure may be microorganisms of the genus Corynebacterium, or more specifically Corynebacterium glutamicum, but are not limited thereto.

[0080] Meanwhile, it is already known that microorganisms of the genus Corynebacterium are capable of producing L-valine; however, their production ability is significantly lower, and the genes involved in the production mechanism and the basic principle of the mechanism have not been fully elucidated. Therefore, the Corynebacterium genus microorganisms having the ability to produce L-valine of the present disclosure include any Corynebacterium genus microorganisms having an increased L-valine production ability by enhancing or weakening the activity of genes related to the L-valine production mechanism, or Corynebacterium genus microorganisms having an increased L-valine production ability by introducing or enhancing the activity of exogenous genes.

[0081] In addition, compared with the parental strain, the microorganisms of the present disclosure may have enhanced activity of acetohydroxyacid synthase subunit (ilvN).

[0082] As used herein, the term "enhancement" of polypeptide activity refers to an increase in polypeptide activity as compared to the inherent activity. Such enhancement may be used interchangeably with terms such as "activation", "upregulation", "overexpression", and "increase". As used herein, the terms "activation", "enhancement", "upregulation", "overexpression", and "increase" may include the display of an activity that was not initially present, as well as the display of an increased activity as compared to the inherent activity or the activity prior to modification. The term "inherent activity" refers to the activity of a specific polypeptide that was originally possessed by the parental strain or unmodified microorganism prior to the change in trait when the trait is changed due to genetic variation caused by natural or artificial factors. This term may be used interchangeably with "activity prior to modification". An "enhancement", "upregulation", "overexpression", or "increase" in polypeptide activity as compared to its inherent activity refers to an increase in the activity and / or concentration (expression level) of a specific polypeptide that is naturally possessed by the parental strain or unmodified microorganism prior to transformation.

[0083] Enhancement of polypeptide activity can be achieved by applying various methods well-known in the art. These methods include, for example, increasing the intracellular copy number of the gene encoding the variant, introducing a mutation in the gene expression control sequence on the chromosome encoding the variant, replacing the gene expression control sequence on the chromosome encoding the variant with a sequence having enhanced activity, replacing the chromosomal gene encoding the protein with a gene mutated to have enhanced variant activity, and introducing a mutation in the chromosomal gene encoding the variant protein to enhance the activity of the variant, but are not limited thereto.

[0084] As used herein, the term "introducing" refers to delivering a polynucleotide encoding an acetohydroxyacid synthase variant or a vector containing the same into a host cell. Such introduction can be readily achieved using conventional methods in the art. Commonly used methods include CaCl 2 precipitation, the Hanahan method using the reducing agent dimethyl sulfoxide (DMSO) to increase the efficiency of CaCl 2Efficiency of the method, electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicon carbide fibers, transformation using PEG, and transformation mediated by dextran sulfate, Lipofectamine, or drying / inhibition. The method for the transformation vector is not limited to these examples, and any transformation or transfection method commonly used in the art can be used without limitation. In addition, the polynucleotide to be delivered can be introduced and located within the chromosome of the host cell or extrachromosomally, as long as it can be expressed within the host cell. Furthermore, the polynucleotide can be introduced into the host cell in any form as long as it can be expressed within the host cell. For example, the nucleotide can be introduced into the host cell in the form of an expression cassette, which is a polynucleotide construct containing all the necessary elements for self-expression, but the form is not limited thereto. The expression cassette generally includes a promoter operably linked to the open reading frame (hereinafter referred to as "ORF") of the gene, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette can be in the form of an expression vector capable of self-replication. In addition, the polynucleotide can be introduced into the host cell as it is and operably linked to the sequences necessary for expression in the host cell, but it is not particularly limited thereto.

[0085] Another aspect of the present disclosure provides a method for producing L-valine, including culturing the microorganism of the present disclosure in a medium.

[0086] Specifically, the method for producing L-valine of the present disclosure may include culturing a microorganism containing the variant, polynucleotide, or vector of the present disclosure in a medium, but is not limited thereto.

[0087] As used herein, the term "culturing" refers to culturing the microorganism of the present disclosure under appropriately controlled environmental conditions. The culturing process of the present disclosure can be carried out according to suitable media and culturing conditions known in the art. Those skilled in the art can easily adjust and use such a culturing method according to the selected strain. Specifically, the culturing can be of the batch type, continuous type, and / or fed-batch type, but is not limited thereto.

[0088] As used herein, the term "medium" refers to a mixture containing the nutrients required for culturing the microorganism of the present disclosure as the main components, and the medium provides nutrients, growth factors, etc., including water, which is indispensable for survival and development. Specifically, any medium and culturing conditions can be used for culturing the microorganism of the present disclosure without particular limitation as long as the medium is commonly used for culturing microorganisms. The microorganism described in the present disclosure can be cultured in a common medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, etc., while controlling conditions such as temperature and pH under aerobic conditions.

[0089] For example, the culture medium for Corynebacterium strains can be found in the document ["Manual of Methods for General Bacteriology", American Society for Bacteriology (Washington, D.C., USA, 1981)].

[0090] In the present disclosure, the carbon source includes carbohydrates such as glucose, sucrose (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. In addition, natural organic nutrients such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, sugarcane residues, and corn steep liquor can be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugars) can be used, and appropriate amounts of other carbon sources can be used in various ways without limitation. These carbon sources can be used alone or in combination of two or more, but are not limited thereto.

[0091] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition products, and defatted soybean cake or its decomposition products can be used. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0092] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or their corresponding sodium salts. As inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. may be used. In addition, amino acids, vitamins, and / or suitable precursors, etc. may be included. These components or precursors can be added to the culture medium in a batch or continuous manner, but the method is not limited thereto.

[0093] In addition, during the process of culturing the microorganisms of the present disclosure, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the culture medium in a suitable manner. In addition, during the culturing process, foaming can be inhibited by using an antifoaming agent such as fatty acid polyethylene glycol ester. In addition, oxygen or oxygen-containing gas can be injected into the culture medium to maintain an aerobic state of the culture medium, or nitrogen, hydrogen, or carbon dioxide gas can be injected, or no gas can be injected to maintain an anaerobic and micro-aerobic state, but the method is not limited thereto.

[0094] In the cultivation of the present disclosure, the cultivation temperature can be maintained at 20°C to 45°C, specifically, 25°C to 40°C, and the strain can be cultivated for about 10 to 160 hours, but the cultivation conditions are not limited thereto.

[0095] The L-valine produced by the cultivation according to the present disclosure can be secreted into the culture medium or retained in the cells.

[0096] The method for producing L-valine of the present disclosure may further include, for example, preparing the microorganism of the present disclosure before cultivation, preparing a culture medium for culturing the strain, or a combination of any order thereof.

[0097] The method for producing L-valine of the present disclosure may further include recovering L-valine from the cultivated culture medium (the culture medium in which cultivation has been carried out) or from the microorganism of the present disclosure. The method may further include recovery after cultivation.

[0098] According to the cultivation method of the microorganism of the present disclosure (such as batch, continuous or fed-batch cultivation methods), the recovery can be collecting L-valine using suitable methods known in the art. For example, L-valine can be recovered from the culture medium or microorganism using suitable methods known in the art, such as centrifugation, filtration, crystallization, treatment with a protein precipitant (salting-out method), extraction, sonication, 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.

[0099] In addition, the method for producing L-valine of the present disclosure may further include purification. Purification can be carried out using suitable methods known in the art. For example, when the method for producing L-valine of the present disclosure includes recovery and purification, the recovery and purification can be carried out sequentially or non-sequentially in any order, or they can be carried out simultaneously or integrated into a single step; however, the method is not limited thereto.

[0100] Another aspect of the present disclosure provides a composition for producing L-valine, which comprises a variant of the acetohydroxyacid synthase subunit (ilvN) of the present disclosure, a polynucleotide encoding the variant of the present disclosure, a vector comprising the polynucleotide of the present disclosure, or a microorganism comprising the polynucleotide of the present disclosure; a culture medium obtained by culturing the microorganism; or a combination of two or more thereof.

[0101] The composition of the present disclosure may further comprise any suitable excipient commonly used in compositions for amino acid production. Such excipients include, for example, preservatives, wetting agents, dispersants, suspending agents, buffering agents, stabilizers or isotonic agents, but are not limited thereto.

[0102] [Embodiments of the present invention]

[0103] The present disclosure will be described in detail by way of examples. However, these examples are for illustrative purposes only, and the scope of the present disclosure is not limited to these examples. At the same time, the technical descriptions missing in the present disclosure can be fully understood and easily practiced by those skilled in the art of the present disclosure or related fields

[0104] Example 1. Screening for mutant strains with increased valine production capacity by artificial mutagenesis

[0105] Example 1-1. Artificial mutagenesis by UV irradiation

[0106] To screen for mutant strains with increased valine production capacity, the valine-producing strain Corynebacterium glutamicum KCCM11201P (Korean Patent No. 10-1117022) was inoculated onto a nutrient medium containing agar and cultured at 30 °C for 36 hours. Hundreds of colonies thus obtained were irradiated with UV at room temperature to induce random mutagenesis in the genome of the strain

[0107] Example 1-2. Evaluation of the fermentation potential of mutagenized strains and strain screening

[0108] To select mutant strains with increased L-valine production capacity compared to the parental strain Corynebacterium glutamicum KCCM11201P, a fermentation potential test was performed on the randomly mutagenized strains. After subculturing in a nutrient medium, each strain was inoculated into a 250 mL Erlenmeyer flask with baffles containing 25 mL of production medium and incubated with shaking at 200 rpm at 30 °C for 72 hours. The concentration of L-valine was analyzed using HPLC, and the analyzed concentrations of L-valine are listed in Table 1

[0109] [Nutrient medium (pH 7.2)]

[0110] Glucose 10 g, meat extract 5 g, polypeptone 10 g, sodium chloride 2.5 g, yeast extract 5 g, agar 20 g, and urea 2 g (per 1 L of distilled water)

[0111] [Production medium (pH 7.0)]

[0112] Glucose 100 g, ammonium sulfate 40 g, soy protein 2.5 g, corn steep liquor solids 5 g, urea 3 g, dipotassium hydrogen phosphate 1 g, magnesium sulfate heptahydrate 0.5 g, biotin 100 μg, thiamine hydrochloride 1 mg, calcium pantothenate 2 mg, nicotinamide 3 mg, calcium carbonate 30 g (per 1 L of distilled water)

[0113] [Table 1]

[0114]

[0115] Based on the results shown in Table 1, select the C6 strain with the most increased valine production compared to the KCCM11201P strain (i.e., the control).

[0116] Example 2. Identification of mutations by gene sequencing

[0117] Sequence the main genes of this strain and compare them with the genes of the KCCM11201P strain. As a result, it was found that the C6 strain with enhanced valine production ability contains nucleotide sequence mutations at specific positions in the ORF region of the ilvN gene.

[0118] Specifically, it was found that the C6 strain with the highest increase in valine production also includes the A42V mutation in the parental strain KCCM11201P, as well as a mutation introduced 476 bp upstream of the start codon of the ilvN gene. This mutation changes the original codon GCA (SEQ ID NO: 2) to GAA (SEQ ID NO: 4), resulting in the substitution of the amino acid (alanine) at position 159 with glutamic acid (SEQ ID NO: 3).

[0119] When analyzing the A159E mutation region, it was found that this mutation affects the effector-binding domain of the valine biosynthetic enzyme, so it is expected that the activity of the corresponding protein will be enhanced. In the following, the following examples identify whether introducing the A159E mutation inserted at a specific position in the ilvN gene affects the ability of Corynebacterium microorganisms to produce the branched-chain amino acid valine.

[0120] Example 3. Preparation of the KCCM11201P strain with the ilvN mutation introduced and identification of its valine production ability

[0121] Example 3-1. Preparation of the Corynebacterium glutamicum KCCM11201P strain with the ilvN mutation introduced and evaluation of its L-valine production ability - 1

[0122] To insert the ilvN(A159E) mutation into the Corynebacterium glutamicum KCCM11201P strain with the A42V mutation, a vector containing the target mutation was constructed. Specifically, the genomic DNA of the C6 strain was extracted using the G-spin Total DNA Extraction Mini Kit (Intron, catalog number 17045) according to the protocol provided in the kit, and the genomic DNA was used as a template for PCR. The reaction conditions for PCR were as follows: First, denature at 94°C for 5 minutes; then perform 25 cycles, each cycle including denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 150 seconds; finally, extend at 72°C for 7 minutes. A 1010 bp PCR product (hereinafter referred to as "fragment 1 with the mutation introduced") was obtained using SEQ ID NO: 7 and 8.

[0123] The obtained fragment 1 with introduced mutations was ligated to the pDCM2 vector (Korean Patent Publication No. 10-2020-136813; International Patent Publication No. 2008-033001), treated with the restriction enzyme XbaI (New England Biolabs, Beverly, MA), used with an injection cloning kit (Takara Bio Inc., Otsu, Japan), and then transformed into Escherichia coli DH5α. After the prepared gene was transformed into Escherichia coli DH5α, the transformed strains were selected in LB medium containing kanamycin. DNA was obtained therefrom using a DNA-spin plasmid DNA purification kit (INTRON), which was used to construct the pDCM2-ilvN(A42V, A159E) vector containing the fragment 1 with introduced mutations.

[0124] [Table 2]

[0125]

[0126] The pDCM2-ilvN(A42V, A159E) vector 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 was inserted into the chromosome by homologous sequence recombination were selected in medium containing kanamycin (25 mg / L). Except that alanine at position 159 in the amino acid sequence of SEQ ID NO:1 within the ORF of the ilvN gene on the chromosome was replaced with glutamic acid, PCR was performed on the Corynebacterium glutamicum transformants that had undergone secondary recombination using SEQ ID NO:7 and 8 to identify strains that also included the A42V mutation (i.e., alanine at position 42 in the amino acid sequence of SEQ ID NO:1 within the ORF of the ilvN gene on the chromosome was replaced with valine), and the A42V mutation was included in the parental strain KCCM11201P. The recombinant strain was named Corynebacterium glutamicum KCCM11201P::ilvN(A159E).

[0127] To compare the valine-producing abilities of the valine-producing strains Corynebacterium glutamicum KCCM11201P and KCCM11201P::ilvN(A159E), a shake flask evaluation was conducted. Each strain was subcultured in a nutrient medium, inoculated into a 250 mL Erlenmeyer flask with baffles containing 25 mL of production medium, and incubated with shaking at 200 rpm at 30 °C for 72 hours. The concentration of L-valine was analyzed using HPLC, and the analyzed concentrations of L-valine are shown in Table 3.

[0128] [Nutrient medium (pH 7.2)]

[0129] Glucose 10 g, gravy 5 g, polypeptone 10 g, sodium chloride 2.5 g, yeast extract 5 g, agar 20 g, and urea 2 g (per 1 L of distilled water)

[0130] [Production medium (pH 7.0)]

[0131] Glucose 100 g, ammonium sulfate 40 g, soy protein 2.5 g, corn steep liquor solids 5 g, urea 3 g, dipotassium hydrogen phosphate 1 g, magnesium sulfate heptahydrate 0.5 g, biotin 100 μg, thiamine hydrochloride 1 mg, calcium pantothenate 2 mg, nicotinamide 3 mg, calcium carbonate 30 g (per 1 L of distilled water)

[0132] [Table 3]

[0133] KCCM11201P and KCCM11201P::ilvN(A42V+A159E),

[0134]

[0135] It was found that, compared with the KCCM11201P strain, the KCCM11201P::ilvN(A159E) strain showed a 14.8% increase in L-valine production ability.

[0136] Example 3-2. Preparation of Corynebacterium glutamicum KCCM11201P strain introduced with ilvN mutation and evaluation of L-valine production ability - 2

[0137] To identify the effect of the individual ilvN(A159E) mutation in the wild-type ilvN enzyme, a strain was constructed in which the 159th amino acid (alanine) of SEQ ID NO:1 in the ORF region of the ilvN gene was replaced with glutamic acid. Specifically, a vector containing the target mutation was constructed to insert ilvN(V42A, A159E) into Corynebacterium glutamicum strain KCCM11201P. Specifically, the genomic DNA of strain C6 was extracted using the G-spin Total DNA Extraction Mini Kit (Intron, catalog number 17045) according to the protocol provided in the kit, and the genomic DNA was used as a template for PCR. The conditions for PCR were as follows: First, denaturation at 94 °C for 5 minutes; then 25 cycles, each cycle including denaturation at 94 °C for 30 seconds, annealing at 55 °C for 30 seconds, and extension at 72 °C for 150 seconds; finally, extension at 72 °C for 7 minutes. PCR products of 515 bp (hereinafter referred to as "fragment 2 with introduced mutation") and 518 bp (hereinafter referred to as "fragment 3 with introduced mutation") were obtained using SEQ ID NO:9 and 10 and SEQ ID NO:11 and 12, respectively.

[0138] The obtained fragments 2 and 3 with introduced mutation were ligated to the pDCM2 vector (Korean Patent Application Publication No. 10-2020-136813; International Patent Publication No. 2008-033001), treated with the restriction enzyme XbaI (New England Biolabs, Beverly, MA), and then transformed into Escherichia coli DH5α using the In-Fusion Cloning Kit (Takara Bio Inc., Otsu, Japan). After the prepared gene was transformed into Escherichia coli DH5α, the transformed strains were selected in LB medium containing kanamycin. DNA was obtained therefrom using the DNA-spin Plasmid DNA Purification Kit (INTRON), which was used to construct the pDCM2-ilvN(V42A, A159E) vector containing fragments 2 and 3 with introduced mutation.

[0139] [Table 4]

[0140]

[0141] The pDCM2-ilvN(V42A, A159E) vector was transformed into Corynebacterium glutamicum KCCM11201P by homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). By homologous recombination, the A42V mutation was reversed and the A159E mutation was introduced. Strains in which the vector was inserted into the chromosome by homologous sequence recombination were selected in a medium containing kanamycin (25 mg / L). PCR using SEQ ID NO:9 and 12 was performed on Corynebacterium glutamicum transformants that had undergone secondary recombination to identify strains in which valine at position 42 in the amino acid sequence of SEQ ID NO:1 within the ORF of the ilvN gene on the chromosome was restored to alanine and alanine at position 159 in the amino acid sequence was replaced by glutamic acid. The recombinant strain was named Corynebacterium glutamicum KCCM11201P::ilvN(V42A, A159E).

[0142] To compare the L-valine production ability of the prepared strains, the strains were cultured in the same manner as in Example 3-1, and the concentration of L-valine was analyzed. The analyzed concentrations of L-valine are shown in Table 5 below.

[0143] [Table 5]

[0144] KCCM11201P, KCCM11201P::ilvN(V42A,A159E) and KCCM11201P::ilvN(A159E),

[0145]

[0146] As a result, compared with the KCCM11201P strain, the strains KCCM11201P::ilvN(V42A, A159E) and KCCM11201P::ilvN(A159E) showed 3.7% and 14.8% increases in L-valine production ability, respectively.

[0147] As described above, those skilled in the art will be able to understand that the present disclosure can be implemented in other specific forms without departing from the technical essence or basic features of the present disclosure. Therefore, the above embodiments should be construed as exemplary and not limiting the present disclosure. It should be understood that all changes or modifications derived from the definition and scope of the claims and their equivalents fall within the scope of the present disclosure.

Claims

1. A variant of the acetohydroxyacid synthase subunit (ilvN), wherein the amino acid at position 159 in the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by another amino acid.

2. The variant according to claim 1, wherein the amino acid at position 42 in the amino acid sequence corresponding to SEQ ID NO: 1 is further replaced by another amino acid.

3. The variant according to claim 1, wherein the amino acid at position 159 in the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by glutamic acid.

4. The variant according to claim 2, wherein the amino acid at position 159 in the amino acid sequence corresponding to SEQ ID NO: 1 is replaced by glutamic acid, and wherein the amino acid at position 42 in the amino acid sequence is replaced by valine.

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

3.

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

5.

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

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

9. The microorganism according to claim 8, wherein the microorganism has an increased L-valine production capacity compared to a microorganism comprising a polypeptide of SEQ ID NO: 1 or a polynucleotide encoding said polypeptide.

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

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

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

13. The method according to claim 12, wherein the method further comprises recovering the target substance from the culture medium.

Images