NOVEL BRANCHED CHAIN ​​AMINO ACID AMINOTRANSFERASE MUTANT AND METHOD FOR PRODUCING ISOLEUCINE USING THE SAME

Abstract

The present application relates to novel branched-chain amino acid aminotransferase mutants that reduce the amount of by-products generated during L-isoleucine production, polynucleotides encoding the mutants, vectors containing the polynucleotides, microorganisms containing the mutants, polynucleotides, or vectors, and methods for producing L-isoleucine using the microorganisms.

Description

[Technical field]

[0001] The present application relates to novel branched-chain amino acid aminotransferase mutants that reduce by-products generated during L-isoleucine production, polynucleotides encoding the mutants, vectors containing the polynucleotides, microorganisms containing the mutants, polynucleotides, or vectors, and methods for producing L-isoleucine using the microorganisms. [Background technology]

[0002] L-isoleucine is one of the branched-chain amino acids out of a total of 20 amino acids, and is classified as an essential amino acid and is used in animal feed, food additives, and pharmaceutical fields. L-isoleucine is increasingly used not only in infusions, nutritional supplements, and sports nutritional supplements, but also in animal feed, as it has functions such as energy production after metabolism, hemoglobin production, blood sugar regulation, and muscle production and repair.

[0003] Based on this trend, various microorganisms and their mutants are used to produce L-amino acids (US Patent No. 10113190), but even in such cases, a large number of by-products other than L-isoleucine are produced, which have a significant effect on the purity of L-isoleucine during the purification step, so a method for removing the by-products is required. In this regard, the L-isoleucine purification method developed to increase the purity of L-isoleucine has the disadvantage of requiring an additional purification process (US Patent No. 6072083), and there is currently a need to develop a method for increasing the purity of L-isoleucine. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 10113190 [Patent Document 2] U.S. Patent No. 6072083 [Patent Document 3] Korean Patent Publication No. 10-2020-0136813 [Patent Document 4] U.S. Patent No. 7662943 [Patent Document 5] U.S. Patent No. 10584338 [Patent Document 6] U.S. Patent No. 10273491 [Patent Document 7] Korea Registration Patent No. 10-1996769 [Patent Document 8] Korea Patent Application No. 10-2020-0078669 [Patent Document 9] U.S. Patent No. 10662450 [Patent Document 10] Korean Patent Registration No. 10-1335789

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[0006] As a result of our earnest efforts to increase the purity of L-isoleucine produced using microorganisms, we identified the gene ilvE, which encodes branched-chain amino acid aminotransferase that reduces by-products such as alpha-aminobutyric acid and L-valine and contributes to L-isoleucine production, and secured a mutation that further increases the purity of L-isoleucine. [Means for solving the problem]

[0007] One object of the present application is to provide branched-chain amino acid aminotransferase variants.

[0008] Another object of the present application is to provide polynucleotides encoding said variants. Another object of the present application is to provide a vector containing the polynucleotide.

[0009] Another object of the present application is to provide a microorganism comprising at least one of the mutant; a polynucleotide encoding the mutant; and a vector comprising the polynucleotide.

[0010] Another object of the present application is to provide a method for producing L-isoleucine. Another object of the present application is to provide a composition for producing L-isoleucine. Another object of the present application is to provide use of the mutant, a polynucleotide encoding the mutant, a vector comprising the polynucleotide, or the microorganism for producing L-isoleucine. Effect of the Invention

[0011] Microorganisms expressing the branched-chain amino acid aminotransferase mutants of the present invention can produce L-isoleucine with significantly improved purity compared to strains that do not express the mutants, and thus L-isoleucine can be effectively produced using the mutants. Therefore, the mutants can be expected to be used in a wide range of industrial applications, such as food, feed, and medicine, which utilize L-isoleucine. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The contents of the present application will be described in detail below. Meanwhile, the description and embodiment of one aspect disclosed in the present application may be applied to the description and embodiment of a different aspect for common matters. Furthermore, all combinations of the various elements disclosed in the present application belong to the scope of the present application. In addition, the following specific description is not considered to limit the scope of the present application.

[0013] One aspect of the present application provides a branched-chain amino acid aminotransferase mutant in which the amino acid corresponding to position 31 in the amino acid sequence of SEQ ID NO:1 is substituted with leucine, the amino acid corresponding to position 98 is substituted with isoleucine, the amino acid corresponding to position 256 is substituted with valine, or a combination thereof.

[0014] The mutant may be one in which the 31st amino acid in the amino acid sequence of SEQ ID NO:1 is substituted with leucine, the 98th amino acid is substituted with isoleucine, and the 256th amino acid is substituted with valine, or a combination thereof, but is not limited thereto.

[0015] Specifically, the variant may be, but is not limited to, an amino acid at a position corresponding to the 31st position in the amino acid sequence of SEQ ID NO:1 replaced with leucine; an amino acid at a position corresponding to the 98th position in the amino acid sequence of SEQ ID NO:1 replaced with isoleucine; an amino acid at a position corresponding to the 256th position in the amino acid sequence of SEQ ID NO:1 replaced with valine; an amino acid at a position corresponding to the 31st position in the amino acid sequence of SEQ ID NO:1 replaced with leucine and an amino acid at a position corresponding to the 98th position in the amino acid sequence of SEQ ID NO:1 replaced with isoleucine; an amino acid at a position corresponding to the 31st position in the amino acid sequence of SEQ ID NO:1 replaced with leucine and an amino acid at a position corresponding to the 256th position in the amino acid sequence of SEQ ID NO:1 replaced with valine; an amino acid at a position corresponding to the 98th position in the amino acid sequence of SEQ ID NO:1 replaced with isoleucine and an amino acid at a position corresponding to the 256 ... valine; or an amino acid at a position corresponding to the 31st position in the amino acid sequence of SEQ ID NO:1 replaced with leucine, an amino acid at a position corresponding to the 98th position in the amino acid sequence of SEQ ID NO:1 replaced with isoleucine, and an amino acid at a position corresponding to the 256th position in the amino acid sequence of SEQ ID NO:1 replaced with valine.

[0016] The mutant may be one in which the 31st amino acid, histidine, in the amino acid sequence of SEQ ID NO:1 is replaced with leucine, the 98th amino acid, arginine, is replaced with isoleucine, the 256th amino acid, serine, is replaced with valine, or a combination of these substitutions, but is not limited to these.

[0017] Specifically, the variant may be, but is not limited to, a variant in which the 31st amino acid in the amino acid sequence of SEQ ID NO:1 is substituted for histidine with leucine; a variant in which the 98th amino acid in the amino acid sequence of SEQ ID NO:1 is substituted for arginine with isoleucine; a variant in which the 256th amino acid in the amino acid sequence of SEQ ID NO:1 is substituted for serine with valine; a variant in which the 31st amino acid in the amino acid sequence of SEQ ID NO:1 is substituted for histidine with leucine and the 98th amino acid in the amino acid sequence of SEQ ID NO:1 is substituted for arginine with isoleucine; a variant in which the 31st amino acid in the amino acid sequence of SEQ ID NO:1 is substituted for histidine with leucine and the 256th amino acid in the amino acid sequence of SEQ ID NO:1 is substituted for serine with valine; a variant in which the 98th amino acid in the amino acid sequence of SEQ ID NO:1 is substituted for arginine with isoleucine and the 256th amino acid in the amino acid sequence of SEQ ID NO:1 is substituted for serine with valine; or a variant in which the amino acid at the position corresponding to the 31st amino acid in the amino acid sequence of SEQ ID NO:1 is substituted for leucine, the 98th amino acid in the amino acid sequence of SEQ ID NO:1 is substituted for serine with valine.

[0018] The mutant may be one or more sequences selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 7, but is not limited thereto. Specifically, the variant in which the 31st amino acid histidine in the amino acid sequence of SEQ ID NO:1 is replaced with leucine may be SEQ ID NO:2; the variant in which the 98th amino acid arginine in the amino acid sequence of SEQ ID NO:1 is replaced with isoleucine may be SEQ ID NO:3; the variant in which the 256th amino acid serine in the amino acid sequence of SEQ ID NO:1 is replaced with valine may be SEQ ID NO:4; the variant in which the 31st amino acid histidine in the amino acid sequence of SEQ ID NO:1 is replaced with leucine and the 98th amino acid arginine is replaced with isoleucine may be SEQ ID NO:5; the variant in which the 31st amino acid histidine in the amino acid sequence of SEQ ID NO:1 is replaced with leucine and the 256th amino acid serine is replaced with valine may be SEQ ID NO:6; and the variant in which the 98th amino acid arginine in the amino acid sequence of SEQ ID NO:1 is replaced with isoleucine and the 256th amino acid serine is replaced with valine may be SEQ ID NO:7, but is not limited thereto.

[0019] The mutant of the present application may be, but is not limited to, an amino acid corresponding to position 31 of the amino acid sequence of SEQ ID NO:1 substituted with leucine, an amino acid corresponding to position 98 with isoleucine, an amino acid corresponding to position 256 with valine, or a combination thereof, thereby reducing the by-product L-valine or AABA and increasing L-isoleucine productivity.

[0020] In this application, the term "L-isoleucine" refers to an L-amino acid with the chemical formula HO2CCH(NH2)CH(CH3)CH2CH3, which is one of the essential amino acids and structurally corresponds to a branched-chain amino acid along with L-valine and L-leucine.

[0021] In the present application, the term "branched-chain amino acid aminotransferase (IlvE)" refers to a polypeptide or protein having branched-chain amino acid aminotransferase activity, and is an enzyme in the L-isoleucine biosynthetic pathway. As an example, in the latter half of the L-isoleucine biosynthetic pathway, L-isoleucine is finally produced by the reaction of branched-chain amino acid aminotransferase (IlvE) via 2-keto-3-methylvalerate via dihydroxy acid dehydratase. The branched-chain amino acid aminotransferase may be used interchangeably with IlvE.

[0022] The branched-chain amino acid aminotransferase also produces α-aminobutyrate or L-valine from 2-ketobutyrate (2-keto butyric acid), which is a precursor of L-isoleucine, and may be one of the substances that significantly affect the purity of L-isoleucine, but is not limited thereto.

[0023] The branched-chain amino acid aminotransferase may be a protein comprising the amino acid sequence of SEQ ID NO: 1. The protein comprising the amino acid sequence of SEQ ID NO: 1 may be confused with a protein having the amino acid sequence of SEQ ID NO: 1 or a protein consisting of the amino acid sequence of SEQ ID NO: 1.

[0024] Specifically, the amino acid sequence of SEQ ID NO: 1 may be an amino acid sequence of a branched-chain amino acid aminotransferase encoded by the ilvE gene, and the amino acid sequence of SEQ ID NO: 1 may be obtained from various databases such as NCBI GenBank, which is a publicly known database, but is not limited thereto. For example, the amino acid sequence of SEQ ID NO: 1 may be derived from Escherichia sp. or Escherichia coli, but is not limited thereto, and may include sequences having the same activity as the amino acid sequence of SEQ ID NO: 1 without limitation. In addition, although the protein having branched-chain amino acid aminotransferase activity in the present application is described as a polypeptide or protein containing the amino acid sequence of SEQ ID NO: 1, this does not exclude meaningless sequence addition before or after the amino acid sequence of SEQ ID NO: 1, or naturally occurring mutations, or silent mutations thereof, and it is obvious to those skilled in the art that if it has the same or corresponding activity as the protein containing the amino acid sequence of SEQ ID NO: 1, it corresponds to the polypeptide or protein having branched-chain amino acid aminotransferase activity of the present application. Specifically, for example, a polypeptide having branched-chain amino acid aminotransferase activity of the present application may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto. It is also self-evident that a polypeptide having an amino acid sequence with such homology or identity and exhibiting efficacy corresponding to the above polypeptide, in which a part of the sequence has been deleted, modified, substituted, or added, is also included within the scope of the polypeptide of the present application.

[0025] In the present application, even if a "polypeptide or protein containing an amino acid sequence set forth in a specific SEQ ID NO," "a polypeptide or protein consisting of an amino acid sequence set forth in a specific SEQ ID NO," or "a polypeptide or protein having an amino acid sequence set forth in a specific SEQ ID NO" is described, it is self-evident that a protein having an amino acid sequence in which a part of the sequence has been deleted, modified, substituted, conservatively substituted, or added is also used in the present application, so long as it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of the SEQ ID NO. For example, a protein may have a sequence addition at the N-terminus and / or C-terminus of the amino acid sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation, or a conservative substitution thereof.

[0026] In the present application, the method for obtaining branched-chain amino acid aminotransferase may be various methods well known in the art, including, but not limited to, gene synthesis technology including codon optimization to obtain enzymes with high efficiency from microorganisms commonly used for enzyme expression, and screening of useful enzyme resources by bioinformatics based on large amounts of microbial genome information to obtain mutant strains.

[0027] In the present application, the term "mutation" or "variant" refers to a culture or individual that exhibits a stable phenotypic change, either genetically or non-genetically, and specifically, in the present application, the term may refer to a mutant in which an amino acid of a branched-chain amino acid aminotransferase having the amino acid sequence of SEQ ID NO: 1 has been mutated, and the activity of the mutant is efficiently increased compared to that of the wild-type, but is not limited thereto.

[0028] In the present application, the term "variant" or "modified polypeptide" refers to a protein that differs from the recited sequence by conservative substitutions and / or modifications of one or more amino acids, but maintains the functions or properties of the protein. The variant differs from the identified sequence by a few amino acid substitutions, deletions, or additions. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the protein and evaluating the properties of the modified protein. That is, the ability of the variant may be increased, unchanged, or decreased compared to the native protein. Some variants may also include variant polypeptides in which one or more portions, such as the N-terminal leader sequence or the transmembrane domain, have been removed. Other variants may include variants in which portions have been removed from the N- and / or C-terminus of the mature protein. The term "mutant" or "mutant polypeptide" may be used interchangeably with terms such as mutation, modification, mutated protein, mutation, etc., and is not limited thereto as long as it is a term used in the sense of a mutation.

[0029] For purposes of this application, the variant may be, but is not limited to, an increase in activity of the mutated protein compared to the native wild-type or non-mutated protein. As used herein, the term "conservative substitution" refers to the replacement of an amino acid with another amino acid having similar structural and / or chemical properties. Such variants still retain one or more biological activities and can, for example, have one or more conservative substitutions. Such amino acid substitutions can generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues.

[0030] For example, among amino acids with electrically charged side chains, the positively charged (basic) amino acids include arginine, lysine, and histidine, the negatively charged (acidic) amino acids include glutamic acid and aspartic acid; and the amino acids with uncharged side chains can be classified as including glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.

[0031] Variants can also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide can be conjugated to a protein N-terminal signal (or leader) sequence involved in co-translationally or post-translationally protein transfer. The polypeptide can also be conjugated to other sequences or linkers that allow the polypeptide to be identified, purified, or synthesized.

[0032] The variant of the present application may have branched-chain amino acid aminotransferase activity, or may have increased branched-chain amino acid aminotransferase activity, but is not limited thereto.

[0033] Although the variant having branched-chain amino acid aminotransferase activity of the present application is described as a polypeptide or protein in which the amino acid corresponding to the 31st position in the amino acid sequence of SEQ ID NO:1 is replaced with leucine, the amino acid corresponding to the 98th position in the amino acid sequence of SEQ ID NO:1 is replaced with isoleucine, the amino acid corresponding to the 256th position in the amino acid sequence of SEQ ID NO:1 is replaced with valine, or a combination thereof, it is not limited to the addition of meaningless sequences or naturally occurring mutations or silent mutations thereof before or after the amino acid sequence in which the amino acid corresponding to the 31st position in the amino acid sequence of SEQ ID NO:1 is replaced with leucine, the amino acid corresponding to the 98th position in the amino acid sequence of SEQ ID NO:1 is replaced with isoleucine, the amino acid corresponding to the 256th position in the amino acid sequence of SEQ ID NO:1 is replaced with valine, or a combination thereof. It will be obvious to those skilled in the art that a polypeptide, protein, or variant having branched-chain amino acid aminotransferase activity of the present application corresponds to a protein having an amino acid sequence in which the amino acid corresponding to the 31st position of SEQ ID NO: 1 is substituted with leucine, the amino acid corresponding to the 98th position of SEQ ID NO: 1 is substituted with isoleucine, the amino acid corresponding to the 256th position of SEQ ID NO: 1 is substituted with valine, or a combination thereof, if the polypeptide, protein, or variant has the same or corresponding activity as a protein having the amino acid sequence in which the amino acid corresponding to the 31st position of SEQ ID NO: 1 is substituted with leucine, the amino acid corresponding to the 98th position of SEQ ID NO: 1 is substituted with isoleucine, the amino acid corresponding to the 256th position of SEQ ID NO: 1 is substituted with valine, or a combination thereof, or a polypeptide having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto. It is also obvious that polypeptides having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted or added are also included within the scope of the polypeptides of the present application, so long as the amino acid sequence has such homology or identity and exhibits an efficacy corresponding to the above-mentioned polypeptide.

[0034] As used herein, the terms "corresponding" or "corresponding position" refer to the amino acid residue at the recited position in a protein or polypeptide, or to an amino acid residue that is similar or identical or homologous to the recited residue in a protein or polypeptide. As used herein, a "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0035] In this application, specific numbering is used for amino acid residue positions in the proteins used in this application. For example, it is possible to renumber positions corresponding to the amino acid residue positions of the proteins of this application by aligning the polypeptide sequences of the subject protein and the proteins of this application to be compared.

[0036] In this application, the terms "homology" or "identity" refer to the degree of relatedness between two given amino acid or nucleotide sequences and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0037] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences are generally capable of hybridizing under moderate or high stringent conditions over at least about 50%, 60%, 70%, 80% or 90% of the entire sequence or length. Hybridization is understood to include polynucleotides that contain common codons or codons that take into account codon degeneracy in the polynucleotide.

[0038] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms, such as, for example, the "FASTA" program using default parameters as in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444, or using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later versions) (GCG program package (Devereux, J., et al., Nucleic Acids Research 12:387 (1984), BLASTP, BLASTN, FASTA (including Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA / .] (1988) SIAM J Applied Math 48:1073). For example, BLAST from the National Center for Biotechnology Information, or ClustalW can be used to determine homology, similarity or identity.

[0039] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, e.g., as known in Smith and Waterman, Adv. Appl. Math (1981) 2:482, e.g., as described in Needleman et al. (1970), J Mol Biol. 48:443. In summary, the GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include (1) a binary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0040] Furthermore, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be confirmed by comparing the sequences in a Southern hybridization experiment under defined stringent conditions, and appropriate hybridization conditions as defined are within the skill of the art and can be determined by methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

[0041] Another aspect of the present application provides a polynucleotide encoding a branched-chain amino acid aminotransferase mutant of the present application, specifically, a polynucleotide encoding a branched-chain amino acid aminotransferase mutant in which the amino acid corresponding to position 31 in the amino acid sequence of SEQ ID NO:1 is substituted with leucine, the amino acid corresponding to position 98 is substituted with isoleucine, the amino acid corresponding to position 256 is substituted with valine, or a combination thereof.

[0042] The amino acid sequence of SEQ ID NO:1, the branched-chain amino acid aminotransferase, and the mutants are as described above. In the present application, the amino acid sequence of SEQ ID NO:1 can also be encoded by a polynucleotide comprising, for example, the nucleic acid sequence of SEQ ID NO:30.

[0043] In the present application, the term "polynucleotide" refers to a DNA or RNA chain of a certain length or more, which is a polymer of nucleotides in which nucleotide monomers are linked in a long chain by covalent bonds. In the present application, the polynucleotide may encode a polypeptide exhibiting branched-chain amino acid aminotransferase activity in which the amino acid corresponding to the 31st position of the amino acid sequence of SEQ ID NO: 1 is substituted with leucine, the amino acid corresponding to the 98th position is substituted with isoleucine, the amino acid corresponding to the 256th position is substituted with valine, or a combination thereof, but is not limited thereto.

[0044] The polynucleotide may include, without limitation, a polynucleotide encoding a polypeptide having the activity of the branched-chain amino acid aminotransferase according to the present application. In the present application, the gene encoding the amino acid sequence of the branched-chain amino acid aminotransferase is the ilvE gene, and the gene may be derived from a microorganism of the genus Escherichia (Escherichia sp.), specifically, but not limited to, Escherichia coli.

[0045] Specifically, the polynucleotide encoding the polypeptide exhibiting the activity of branched-chain amino acid aminotransferase may include a nucleotide sequence encoding an amino acid in which the amino acid corresponding to position 31 in the amino acid sequence of SEQ ID NO: 1 is replaced with leucine, the amino acid corresponding to position 98 is replaced with isoleucine, the amino acid corresponding to position 256 is replaced with valine, or a combination thereof. The polynucleotide may be modified in various ways in the coding region without changing the amino acid sequence of the polypeptide, taking into consideration the degeneracy of codons or the codons preferred in the organism in which the polypeptide is to be expressed. The polynucleotide may include, for example, the nucleotide sequence of SEQ ID NO: 30, and may be a nucleotide sequence having 80% homology or identity thereto, specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, or even more specifically 99% or more, but is not limited thereto.

[0046] Furthermore, the polynucleotide of the present application may include, without limitation, a sequence that hybridizes under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a complementary sequence to all or a part of the base sequence, and encodes an amino acid sequence in which the amino acid corresponding to position 31 in the amino acid sequence of SEQ ID NO:1 is substituted with leucine, the amino acid corresponding to position 98 is substituted with isoleucine, the amino acid corresponding to position 256 is substituted with valine, or a combination thereof.

[0047] Specifically, in the amino acid sequence of SEQ ID NO: 1, a sequence encoding a mutant in which the amino acid corresponding to position 31 is substituted with leucine may be SEQ ID NO: 31; a sequence encoding a mutant in which the amino acid corresponding to position 98 is substituted with isoleucine may be SEQ ID NO: 32; a sequence encoding a mutant in which the amino acid corresponding to position 256 is substituted with valine may be SEQ ID NO: 33; a sequence encoding a mutant in which the amino acid corresponding to position 31 is substituted with leucine and the amino acid corresponding to position 98 is substituted with isoleucine may be SEQ ID NO: 34; a sequence encoding a mutant in which the amino acid corresponding to position 31 is substituted with leucine and the amino acid corresponding to position 256 is substituted with valine may be SEQ ID NO: 35; and a sequence encoding a mutant in which the amino acid corresponding to position 98 is substituted with isoleucine and the amino acid corresponding to position 256 is substituted with valine may include, but are not limited to, SEQ ID NO: 36.

[0048] The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (e.g., J. Sambrook et al., supra). For example, conditions that allow hybridization between polynucleotides with high homology or identity, 40% or more, specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, and even more specifically 99% or more, to polynucleotides with a lower homology or identity than that, and conditions that allow washing once, specifically 2 to 3 times, at a salt concentration and temperature equivalent to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.×XSSC, 0.1% SDS, which are washing conditions for normal Southern hybridization, can be listed.

[0049] Hybridization requires that two nucleic acids have complementary sequences, even if the stringency of the hybridization allows for mismatches between bases. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Thus, the polynucleotides of the present application can also include isolated nucleic acid fragments that are complementary to an entire sequence, as well as substantially similar nucleic acid sequences.

[0050] Specifically, polynucleotides having homology or identity can be detected using the above-mentioned hybridization conditions including a hybridization step at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0051] The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0052] Another aspect of the present application provides a vector comprising a polynucleotide encoding a branched-chain amino acid aminotransferase mutant of the present application, specifically, a vector comprising a polynucleotide encoding a branched-chain amino acid aminotransferase mutant in which the amino acid corresponding to position 31 in the amino acid sequence of SEQ ID NO:1 is substituted with leucine, the amino acid corresponding to position 98 is substituted with isoleucine, the amino acid corresponding to position 256 is substituted with valine, or a combination thereof.

[0053] The amino acid sequence of SEQ ID NO:1, the branched-chain amino acid aminotransferase, the variant, and the polynucleotide are as described above. The term "vector" as used in this application refers to a DNA construct containing a polynucleotide sequence encoding a polypeptide of interest operably linked to an expression control region (or expression control sequence) suitable for expressing said polypeptide in a suitable host. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After being transformed into a suitable host cell, a vector can replicate and function independently of the host genome, or can be integrated into the genome itself.

[0054] The vector used in the present application is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pDCM2 (Korean Patent Publication No. 10-2020-0136813), pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0055] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome through a vector for intracellular chromosome insertion. The polynucleotide can be inserted into a chromosome by any method known in the art, for example, but not limited to, homologous recombination. A selection marker for checking the presence or absence of the chromosomal insertion can be further included. The selection marker is used to select cells transformed with the vector, i.e., to check the presence or absence of the insertion of the target nucleic acid molecule, and is a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide. In an environment treated with a selective agent, only cells expressing the selection marker survive or show other phenotypes, so that transformed cells can be selected.

[0056] In the present application, the term "expression cassette" refers to a unit cassette that includes a promoter and a gene of interest and can express the gene of interest operably linked to the promoter. Various factors that contribute to efficient expression of the gene of interest may be included inside or outside such a gene expression cassette. The gene expression cassette may typically include, but is not limited to, a transcription termination signal, a ribosome binding site, and a translation termination signal in addition to a promoter operably linked to the gene of interest.

[0057] In the present application, the term "transformation" means introducing a vector containing a polynucleotide encoding a target protein into a host cell or a microorganism so that the protein encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide can include all polynucleotides, regardless of whether they are inserted into the chromosome of the host cell or extrachromosomally located, as long as they can be expressed in the host cell. The polynucleotide also includes DNA and RNA encoding the target protein. The polynucleotide can be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a genetic structure containing all elements required for autonomous expression. The expression cassette usually includes a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replicating. The polynucleotide may also be introduced into the host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

[0058] In addition, the term "operably linked" means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a target polypeptide of the present application.

[0059] The method of transforming the vector of the present application includes any method of introducing nucleic acid into a cell, and can be performed by selecting a suitable standard technique as known in the art depending on the host cell, such as, but not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0060] Another aspect of the present application provides a microorganism comprising one or more of a branched-chain amino acid aminotransferase mutant in which the amino acid corresponding to position 31 in the amino acid sequence of SEQ ID NO:1 is substituted with leucine, the amino acid corresponding to position 98 is substituted with isoleucine, the amino acid corresponding to position 256 is substituted with valine, or a combination thereof; a polynucleotide encoding the mutant; and a vector comprising the polynucleotide.

[0061] In one embodiment, the microorganism of the present application may be a microorganism that produces L-isoleucine, but is not limited thereto. Specifically, the microorganism of the present application may be a microorganism that produces L-isoleucine as a target product, and may be a microorganism that includes any one or more of the mutant, polynucleotide, and vector of the present application and has increased L-isoleucine productivity, but is not limited thereto.

[0062] The amino acid sequence of SEQ ID NO:1, the branched-chain amino acid aminotransferase, the mutant, the polynucleotide, the vector, and L-isoleucine are as described above. In the present application, the term "microorganism" includes all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and is a concept that includes all microorganisms in which a specific mechanism has been weakened or strengthened due to the insertion of an exogenous gene, the strengthening or weakening of the activity of an endogenous gene, etc. In the present application, the term "microorganism" includes, without limitation, a branched-chain amino acid aminotransferase mutant in which the amino acid corresponding to position 31 in the amino acid sequence of SEQ ID NO: 1 is substituted with leucine, the amino acid corresponding to position 98 is substituted with isoleucine, the amino acid corresponding to position 256 is substituted with valine, or a combination thereof; a polynucleotide encoding the mutant; and a vector containing the polynucleotide, as long as the microorganism contains one or more of these, which have been introduced or contained therein.

[0063] The microorganism of the present application may be, but is not limited to, a microorganism that contains one or more of the above polypeptide, a polynucleotide encoding the same, and a vector containing the above polynucleotide, and has the ability to produce a target protein or a target product involved in the production of the target protein. The microorganism may be, but is not limited to, a microorganism that naturally has the ability to produce the target protein or the target product, or a microorganism in which the ability to produce the target protein or the target product has been imparted to a parent strain that does not have the ability to produce the target protein or the target product.

[0064] The microorganism is a cell or microorganism that has been transformed with a vector comprising the polynucleotide of the present application and a gene encoding a target protein, for example, expressing the target protein, and for the purposes of the present application, the host cell or microorganism can be any microorganism that contains the target protein and is capable of producing a target product.

[0065] The microorganism may be a recombinant microorganism, and the recombination may be achieved by genetic modification such as transformation. In the present application, the term "microorganism producing a target protein or product" includes all wild-type microorganisms and microorganisms that have been genetically modified, either naturally or artificially, and may be microorganisms in which a specific mechanism has been weakened or enhanced by, for example, inserting an exogenous gene or enhancing or inactivating the activity of an endogenous gene, and may also be a microorganism that contains a genetic modification for the production of the target protein or product.

[0066] The microorganism may be, but is not limited to, a microorganism genetically modified through one or more of the polypeptide, a polynucleotide encoding the polypeptide, and a vector containing the polynucleotide; a microorganism modified to express the polypeptide or a polynucleotide encoding the polypeptide; a recombinant microorganism expressing the polypeptide or a polynucleotide encoding the polypeptide; or a recombinant microorganism having activity of the polypeptide.

[0067] For the purposes of this application, the microorganism that produces the target protein or product may be a microorganism that contains the polynucleotide of this application and is characterized by having an increased ability to produce the target protein or product. Specifically, the microorganism that produces the target protein or product or has the ability to produce the target protein or product in this application may be a microorganism in which some of the genes in the biosynthetic pathway of the target protein or product are enhanced or weakened, or some of the genes in the degradation pathway of the target protein or product are enhanced or weakened.

[0068] The microorganism of the present application may be, but is not limited to, a branched-chain amino acid aminotransferase mutant in which the amino acid corresponding to position 31 in the amino acid sequence of SEQ ID NO: 1 is substituted with leucine, the amino acid corresponding to position 98 is substituted with isoleucine, the amino acid corresponding to position 256 is substituted with valine, or a combination thereof; a polynucleotide encoding the mutant; and a vector containing the polynucleotide. In addition, the microorganism of the present application may be, but is not limited to, a microorganism that produces L-isoleucine as a target product, and may have increased L-isoleucine productivity or reduced by-products generated during L-isoleucine production compared to non-modified or wild-type microorganisms.

[0069] In the present application, the term "expressing" a protein refers to a state in which a target protein is introduced into a microorganism or modified so as to be expressed in the microorganism. In the case where the target protein is a protein present in a microorganism, it refers to a state in which the activity of the target protein is enhanced compared to that of the endogenous protein or protein before modification.

[0070] A microorganism expressing a protein variant of the present application may be a microorganism that has been modified to express the protein variant, and therefore, another aspect of the present application provides a method for producing a microorganism expressing a protein variant of the present application.

[0071] In the present application, "introduction of a protein" means that a microorganism exhibits an activity of a specific protein that it did not originally have, or that the protein exhibits an improved activity compared to its intrinsic activity or activity before modification. For example, it may mean that a specific protein is introduced, a polynucleotide encoding a specific protein is introduced into a chromosome in a microorganism, or a vector containing a polynucleotide encoding a specific protein is introduced into a microorganism, and the activity is exhibited.

[0072] In the present application, the term "enhancement" of a polypeptide or protein activity means that the activity of the polypeptide or protein is increased compared to its intrinsic activity. The term "enhancement" may be used interchangeably with terms such as "up-regulation," "overexpression," and "increase." In this context, "increase" may include all of the following: exhibiting an activity that was not originally possessed, or exhibiting an activity that is improved compared to the intrinsic activity or activity before modification. The term "intrinsic activity" refers to the activity of a specific polypeptide or protein that was originally possessed by a parent strain or a non-modified microorganism before the modification, in the case where a trait is changed due to a genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before modification." The term "enhancement" or "increase" of a polypeptide or protein activity compared to its intrinsic activity means that the activity of a specific polypeptide or protein is improved compared to the activity of a parent strain or a non-modified microorganism before the modification. For example, the protein activity may be improved by at least 1%, 3%, 5%, 7%, 10%, 25%, 50%, 75%, 100%, 125%, 150%, 200%, 250%, 300%, 350%, 400%, or 500%, and up to 1000% or 2000% compared to the protein activity of the parent strain or unaltered microorganism before the transformation, 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 includes all numerical values ​​in a range that is equal to or similar to the numerical value following the term about, but is not limited thereto.

[0073] The "increase in activity" can be achieved by introducing an exogenous polypeptide or protein or by enhancing the activity of an endogenous polypeptide or protein, specifically, by enhancing the activity of an endogenous polypeptide or protein. The enhancement of the activity of the polypeptide or protein can be confirmed by an increase in the activity level, expression level, or amount of a product secreted from the polypeptide or protein.

[0074] The activity of the polypeptide or protein can be enhanced by various methods well known in the art, and may not be limited as long as the activity of the target polypeptide or protein can be enhanced compared to that of the microorganism before transformation. The method may be, but is not limited to, genetic engineering and / or protein engineering, which are routine methods in molecular biology and well known to those skilled in the art (Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2.1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0075] The method for enhancing polypeptide or protein activity using genetic engineering includes, for example, 1) increasing the intracellular copy number of a gene or polynucleotide encoding said polypeptide or protein; 2) a method of replacing the expression regulatory region of a gene on a chromosome encoding the polypeptide or protein with a sequence having a strong activity; 3) a method for modifying the nucleotide sequence of the initiation codon or 5'-UTR region of the polypeptide or protein; 4) a method for modifying a polynucleotide sequence on a chromosome so as to increase the activity of the polypeptide or protein; 5) introduction of an exogenous polynucleotide exhibiting the activity of said polypeptide or protein, or a codon-optimized mutant polynucleotide of said polynucleotide; or 6) Combinations of the above methods may be used, but are not limited to these.

[0076] The method for enhancing the activity of a polypeptide or protein using protein engineering can be carried out, for example, by analyzing the tertiary structure of a polypeptide or protein and selecting and deforming or chemically modifying exposed sites, but is not limited thereto.

[0077] The 1) increase in intracellular copy number of a gene or polynucleotide encoding a polypeptide or protein can be achieved by any method known in the art, for example, by introducing into a host cell a vector to which a gene or polynucleotide encoding the polypeptide or protein is operably linked, the vector being capable of replicating and functioning independently of a host. Alternatively, the vector to which the gene is operably linked is introduced into a host cell, the vector inserting the gene or polynucleotide into a chromosome in the host cell, but is not limited thereto. The vector is as described above.

[0078] The method of replacing the gene expression regulatory region (or expression regulatory sequence) on a chromosome encoding a polypeptide or protein with a sequence having a stronger activity may be any method known in the art, for example, by inducing a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so as to further enhance the activity of the expression regulatory region, or by replacing it with a nucleic acid sequence having a stronger activity. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating the termination of transcription and translation, etc. Specifically, the method may be, but is not limited to, linking a strong heterologous promoter in place of the original promoter.

[0079] Examples of known strong promoters include, but are not limited to, cj1 to cj7 promoters (US Patent US7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Patent US10584338 B2), O2 promoter (US Patent US10273491 B2), tkt promoter and yccA promoter.

[0080] The method for modifying the start codon or 5'-UTR region of a polypeptide or protein (3) may be any method known in the art, for example, replacing the endogenous start codon of the polypeptide or protein with another start codon that has a higher polypeptide or protein expression rate than the endogenous start codon, but is not limited thereto.

[0081] The method of modifying the polynucleotide sequence on the chromosome so as to increase the activity of the polypeptide or protein may be any method known in the art, for example, by inducing a mutation in the expression regulatory sequence by deleting, inserting, non-conservative or conservative substitution of the nucleic acid sequence or a combination thereof so as to further enhance the activity of the polynucleotide sequence, or by replacing the polynucleotide sequence with an improved polynucleotide sequence so as to have a stronger activity. The replacement may specifically be, but is not limited to, inserting the gene into the chromosome by homologous recombination.

[0082] The vector used in this case may further include a selection marker for confirming the presence or absence of chromosomal insertion. The selection marker is as described above.

[0083] The introduction of an exogenous polynucleotide exhibiting the activity of the polypeptide or protein in the above 5) can be carried out by any method known in the art, for example, by introducing an exogenous polynucleotide encoding a polypeptide or protein exhibiting the same / similar activity as the polypeptide or protein, or a codon-optimized mutant polynucleotide thereof, into a host cell. The exogenous polynucleotide can be used without any restriction on its origin or sequence, as long as it exhibits the same / similar activity as the polypeptide or protein. In addition, the introduced exogenous polynucleotide can be introduced into a host cell by optimizing its codons so that optimized transcription and translation can be performed in the host cell. The introduction can be carried out by a person skilled in the art by appropriately selecting a known transformation method, and the introduced polynucleotide can be expressed in the host cell to produce a polypeptide or protein and increase its activity.

[0084] Finally, 6) the combination of the methods can be carried out by simultaneously applying any one or more of the methods 1) to 5). Such enhanced polypeptide or protein activity may refer to, but is not limited to, an increase in the activity or concentration of the corresponding polypeptide or protein relative to the activity or concentration of the polypeptide or protein expressed in a wild-type or unaltered microbial strain, or an increase in the amount of a product produced from the polypeptide or protein.

[0085] In this application, the term "pre-transformed strain" or "pre-transformed microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, but refers to wild-type or naturally occurring strains themselves, or strains before their characteristics are changed due to genetic mutations caused by natural or artificial factors. The term "pre-transformed strain" or "pre-transformed microorganism" may be used interchangeably with "non-mutated strain," "non-mutated strain," "non-mutated microorganism," "non-mutated microorganism," or "reference microorganism."

[0086] In one embodiment, the microorganism of the present application may be, but is not limited to, a Corynebacterium sp. microorganism. In the present application, the term "microorganism of the genus Corynebacterium" can include all microorganisms of the genus Corynebacterium. Specifically, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens, and more specifically, Corynebacterium glutamicum, Corynebacterium callunae, Corynebacterium crenatum or Corynebacterium deserti.

[0087] In the present application, the parent strain of the microorganism may be, but is not limited to, a microorganism in which the biosynthetic pathway of L-isoleucine has been further enhanced in order to increase the amount of L-isoleucine produced.

[0088] Specifically, the microorganism may be a microorganism in which, for example, the ilvE promoter is replaced with the gdh promoter to enhance the function of the promoter in order to enhance the biosynthetic pathway of L-isoleucine, a genetic mutation (R407H) (Korea Patent Registration No. 10-1996769) is further introduced into the hom gene encoding homoserine dehydrogenase to eliminate feedback inhibition of threonine, a precursor of isoleucine, a genetic mutation (T381A, F383A) is further introduced into the ilvA gene encoding L-threonine dehydratase (Korea Patent Application No. 10-2020-0078669), or a genetic mutation (L377K) (US Patent Registration No. US10662450 B2) is further introduced into the lysC gene encoding aspartokinase. However, the present invention is not limited to the above, and the production amount of L-isoleucine can be increased by a gene expression regulation method known in the art.

[0089] As another example for increasing the production amount of L-isoleucine, the parent strain of the microorganism in the present application may be a microorganism in which a gene that weakens the biosynthetic pathway of L-isoleucine has been further inactivated in order to increase the production amount of L-isoleucine, but is not limited thereto.

[0090] In the present application, the term "inactivation" or "attenuation" of a polypeptide or protein includes all of the following: reduced activity or no activity compared to the intrinsic activity. The inactivation or attenuation may be used interchangeably with terms such as down-regulation, decrease, and reduction. The inactivation or attenuation may include a case where the activity of the protein itself is reduced or eliminated compared to the activity of the protein originally possessed by the microorganism due to a mutation in the gene encoding the protein, a case where the overall protein activity in the cell is lower than that of the wild-type strain due to inhibition of expression or translation of the gene encoding the protein, a case where the gene is not expressed at all, and a case where the gene is expressed but has no activity. The "intrinsic activity" refers to the activity of a specific polypeptide or protein originally possessed by a parent strain or a non-transformed microorganism before the transformation, when the trait is changed due to a genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before transformation." The term "decreased" activity of a polypeptide or protein compared to its intrinsic activity means that the activity of the particular polypeptide or protein is lower than that originally possessed by the parent strain or non-transformed microorganism before transformation. The term "about" includes, but is not limited to, ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, and other ranges that are equal to or similar to the number following the term "about."

[0091] Inactivation or attenuation of the activity of such proteins can be achieved by applying a variety of methods well known in the art, including but not limited to these (Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014; 15(2): 2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).

[0092] As an example of the method, 1) a method for deleting the whole or part of the gene encoding the protein; 2) modifying an expression control region (or an expression control sequence) so that expression of the gene encoding the protein is reduced; 3) modifying the gene sequence encoding the protein such that the activity of the protein is eliminated or attenuated; 4) introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to the transcript of the gene encoding the protein; 5) a method in which a sequence complementary to the Shine-Dalgarno sequence is added to the front end of the Shine-Dalgarno sequence of the gene encoding the protein to form a secondary structure, thereby disabling ribosome attachment; 6) A method of adding a promoter that transcribes in the opposite direction to the 3' end of the ORF (open reading frame) of the polynucleotide sequence of the gene encoding the protein (reverse transcription engineering, RTE), and the like, and a combination of these methods can also be used, but are not limited thereto.

[0093] Specifically, the method of deleting a part or the whole of the gene encoding the protein can be carried out by replacing a polynucleotide encoding an endogenous target protein in a chromosome with a polynucleotide having a deleted nucleotide sequence or a marker gene through a vector for insertion into a chromosome of a microorganism. As an example of a method of deleting a part or the whole of such a polynucleotide, a method of deleting a polynucleotide by homologous recombination can be used, but is not limited to this.

[0094] In addition, a method for deleting a part or the whole of the gene can be performed by inducing mutations using light such as ultraviolet light or chemicals, and selecting a strain in which the target gene is deleted from the obtained mutants. The gene deletion method includes a method using DNA recombination technology. The DNA recombination technology can be performed, for example, by injecting a nucleotide sequence or vector containing a nucleotide sequence that is homologous to the target gene into the microorganism to cause homologous recombination. The injected nucleotide sequence or vector can include, but is not limited to, a dominant selection marker.

[0095] In addition, the method of modifying the expression regulatory sequence can be achieved by applying various methods well known in the art. For example, the expression regulatory sequence can be modified by inducing a mutation in the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of a polynucleotide sequence to further weaken the activity of the expression regulatory region (or expression regulatory sequence), or by replacing the expression regulatory region (or expression regulatory sequence) with a polynucleotide sequence having a weaker activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0096] In addition, the method of modifying the gene sequence can be, but is not limited to, introducing mutations into the gene sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so as to further weaken the activity of the polypeptide, or by replacing the gene sequence with a gene sequence improved to have a weaker activity or a gene sequence improved to have no activity.

[0097] For example, expression of a gene can be prevented or attenuated by introducing a mutation into the gene sequence that results in the formation of a stop codon. Another embodiment of the present application provides a method for producing L-isoleucine, comprising the steps of culturing in a medium a microorganism comprising one or more of a branched-chain amino acid aminotransferase variant in which the amino acid corresponding to position 31 in the amino acid sequence of SEQ ID NO:1 is substituted with leucine, the amino acid corresponding to position 98 is substituted with isoleucine, the amino acid corresponding to position 256 is substituted with valine, or a combination thereof; a polynucleotide encoding the variant; and a vector comprising the polynucleotide.

[0098] Another embodiment of the present application provides a method for reducing by-products generated during L-isoleucine production, comprising the steps of culturing in a medium a microorganism comprising one or more of a branched-chain amino acid aminotransferase variant in which the amino acid corresponding to position 31 in the amino acid sequence of SEQ ID NO:1 is substituted with leucine, the amino acid corresponding to position 98 is substituted with isoleucine, the amino acid corresponding to position 256 is substituted with valine, or a combination thereof; a polynucleotide encoding the variant; and a vector containing the polynucleotide.

[0099] The L-isoleucine, the amino acid of SEQ ID NO: 1, the branched-chain amino acid aminotransferase, the mutant, the polynucleotide, the vector, and the microorganism are as described above. The microorganism may be of the genus Corynebacterium, specifically, but is not limited to, Corynebacterium glutamicum, as described above.

[0100] In the present application, the term "culture" means growing the microorganism under appropriately controlled environmental conditions. The culture process of the present application may be performed using an appropriate medium and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culture may be, but is not limited to, a batch, continuous, or fed-batch culture.

[0101] In the present application, the term "culture medium" refers to a substance containing a mixture of nutrients required for culturing the microorganism as the main components, and supplies nutrients and growth factors, including water essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the microorganism of the present application are not particularly limited and any medium used for culturing ordinary microorganisms may be used, and the microorganism of the present application may be cultured under aerobic conditions by adjusting the temperature, pH, etc. in an ordinary culture medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins.

[0102] In the present application, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid may be used, specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and other appropriate amounts of carbon sources may be used in a variety of ways without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited thereto.

[0103] Examples of the nitrogen source include 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 steeping liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more kinds, and are not limited thereto.

[0104] The phosphorus source may include potassium monophosphate, potassium diphosphate, or a corresponding sodium-containing salt. The inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and may also include amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the medium in a batch or continuous manner. However, they are not limited thereto.

[0105] During the cultivation of the microorganism, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the medium in an appropriate manner. During the cultivation, foam generation can be suppressed using an antifoaming agent such as fatty acid polyglycol ester. In addition, to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas can be injected into the medium, and to maintain anaerobic and microaerobic states, no gas can be injected or nitrogen, hydrogen, or carbon dioxide gas can be injected, but this is not limited thereto.

[0106] The temperature of the medium may be, but is not limited to, 20° C. to 50° C., specifically, 30° C. to 37° C. The culture period may be continued until the desired amount of useful substance is produced, specifically, 10 hours to 100 hours, but is not limited to this.

[0107] Specifically, culture media for Corynebacterium strains can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)], but are not limited thereto.

[0108] As a specific example, the method for producing L-isoleucine, the method for producing L-isoleucine, or the method for reducing by-products generated during L-isoleucine production may further include, but is not limited to, a step of recovering L-isoleucine from the medium or the microorganism.

[0109] The method for recovering the L-isoleucine may be to collect the target L-isoleucine using an appropriate method known in the art by the microorganism culture method of the present application, such as a batch, continuous or fed-batch culture method, etc. For example, the target L-isoleucine can be recovered from the medium or the microorganism using an appropriate method known in the art, such as centrifugation, filtration, treatment with a crystallized protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographies such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, and a combination of these methods.

[0110] The production method may further include a purification step. The purification step may purify the recovered L-isoleucine using any suitable method known in the art. The recovered L-isoleucine may be in a purified form or may be a microbial fermentation liquid containing the desired product. (Introduction to Biotechnology and Genetic Engineering, AJ Nair, 2008) In one embodiment, the method for producing or manufacturing L-isoleucine can reduce by-products generated during L-isoleucine production, but is not limited thereto.

[0111] In the present application, the term "by-product" includes any substance that may be generated during the production of L-isoleucine. The by-product may be any one or more selected from alpha-aminobutyrate (AABA) and amino acids other than L-isoleucine, such as arginine (Arg, R), histidine (His, H), glutamic acid (Glu, E), aspartic acid (Asp, D), glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe, F), tryptophan (Trp, W), proline (Pro, P), serine (Ser, S), cysteine ​​(Cys, C), tyrosine (Tyr, Y), asparagine (Asn, N), and glutamine (Gln, Q), but is not limited thereto. For purposes of this application, the by-product may be any one or more selected from the group consisting of alpha-aminobutyrate (AABA), valine, phenylalanine, and leucine, and specifically may be, but is not limited to, alpha-aminobutyric acid or valine.

[0112] Another aspect of the present application provides a composition for producing L-isoleucine, comprising a branched-chain amino acid aminotransferase variant in which the amino acid corresponding to position 31 in the amino acid sequence of SEQ ID NO:1 is substituted with leucine, the amino acid corresponding to position 98 is substituted with isoleucine, the amino acid corresponding to position 256 is substituted with valine, or a combination thereof, a microorganism or a culture of the microorganism, comprising one or more of a polynucleotide encoding the variant, and a vector containing the polynucleotide.

[0113] The amino acid of SEQ ID NO:1, the branched-chain amino acid aminotransferase, the mutant, the polynucleotide, the vector, the microorganism, and the culture are as described above. The composition for producing L-isoleucine may refer to a composition capable of producing L-isoleucine with high efficiency by the branched-chain amino acid aminotransferase mutant of the present application. The composition may include, without limitation, the branched-chain amino acid aminotransferase mutant or a structure for operating the branched-chain amino acid aminotransferase mutant. The branched-chain amino acid aminotransferase mutant may be in a form contained in a vector so as to express an operably linked gene in a host cell into which it is introduced.

[0114] The composition may further include a cryoprotectant or excipient. The cryoprotectant or excipient may be, but is not limited to, a non-naturally occurring or naturally occurring substance. In one embodiment, the cryoprotectant or excipient may be, but is not limited to, a substance that is not naturally in contact with the strain or is not naturally co-occurring with the strain.

[0115] Another aspect of the present application is to provide a branched-chain amino acid aminotransferase mutant in which the amino acid corresponding to position 31 in the amino acid sequence of SEQ ID NO:1 is substituted with leucine, the amino acid corresponding to position 98 is substituted with isoleucine, the amino acid corresponding to position 256 is substituted with valine, or a combination thereof; a polynucleotide encoding the mutant; and a vector comprising the polynucleotide or use of a microorganism for producing L-isoleucine, the microorganism comprising one or more of the mutant, the polynucleotide encoding the mutant, and the vector comprising the polynucleotide.

[0116] The mutant, polynucleotide, vector, microorganism, and isoleucine are as described above. The present application will be described in more detail with reference to the following examples. However, these examples and experimental examples are for illustrative purposes only and the scope of the present application is not limited to these examples and experimental examples.

[0117] Reference Example 1: Construction of a vector for overexpression of the ilvE gene In order to more clearly identify the phenotypic changes caused by the ilvE gene encoding branched-chain amino acid aminotransferase and its mutants according to the purpose of this application, the ilvE gene was overexpressed in the Corynebacterium glutamicum ATCC13032 strain.

[0118] To introduce the ilvE gene from E. coli while overexpressing the ilvE gene encoding branched-chain amino acid aminotransferase, a plasmid vector was constructed in which the ilvE gene promoter was replaced with the gdh promoter to enhance the ilvE promoter.

[0119] To prepare a strain in which the ilvE promoter was replaced with the gdh promoter, PCR was performed using the chromosome of ATCC13032 as a template and the primers SEQ ID NO:8 and SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, or SEQ ID NO:14 and SEQ ID NO:15. The primers used for each PCR are shown in Table 1 below.

[0120] [Table 1]

[0121] PfuUltraTM high fidelity DNA polymerase (Stratagene) was used as the polymerization enzyme for the PCR reaction, and the PCR conditions were denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute. The denaturation, annealing, and polymerization reactions under these conditions were repeated 28 times. As a result, a 5'-upper 737 bp DNA fragment centered on the ATCC13032 ilvE gene initiation codon, a 5'-upper 499 bp DNA fragment centered on the ATCC13032 gdh gene initiation codon, a 3'-lower 742 bp DNA fragment centered on the ATCC13032 ilvE gene termination codon, and a MG1655 ilvE gene 930 bp DNA fragment were obtained. PCR was performed again using the three amplified DNA fragments as templates and primers of SEQ ID NO:8 and SEQ ID NO:13. The PCR conditions were denaturation at 95°C for 5 minutes, followed by 6 cycles of denaturation at 95°C for 30 seconds, annealing, and polymerization at 72°C for 2 minutes, followed by 20 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 2 minutes, followed by polymerization at 72°C for 5 minutes. As a result, a 2868 bp DNA fragment encoding the gdh promoter and the E. coli ilvE gene was amplified. The amplified product was purified using QIAGEN's PCR Purification kit and used as an insert DNA fragment for vector construction.

[0122] Meanwhile, the vector was treated with the restriction enzyme smaI, and then the pDCM2 vector was heat-treated at 65°C for 20 minutes. The molar concentration (M) ratio of the pDCM2 vector and the insert DNA fragment amplified through the PCR was adjusted to 1:2. The vector was cloned according to the instructions provided with the Infusion Cloning Kit from Takara, where the ilvE gene and ilvE promoter of Corynebacterium were replaced with the gdh promoter of Corynebacterium glutamicum and ilvE of E. coli to construct the vector pDCM2-Pgdh-ilvE(eco) for introduction into the chromosome.

[0123] Reference Example 2: Construction of L-isoleucine producing strain Wild-type Corynebacterium glutamicum has the ability to produce L-isoleucine but does not overproduce it. Therefore, in order to confirm the genetic traits that increase L-isoleucine production ability according to the purpose of this application, a strain with increased L-isoleucine production ability was used.

[0124] First, an L-isoleucine producing strain was developed from wild-type Corynebacterium glutamicum ATCC13032. Specifically, to eliminate the feedback inhibition of threonine, the precursor of isoleucine in the L-isoleucine biosynthetic pathway, the gene hom encoding homoserine dehydrogenase was mutated and the 407th amino acid, arginine, of homoserine dehydrogenase was replaced with histidine (Korea Patent Registered No. 10-1996769). Specifically, the polynucleotide sequence encoding hom(R407H) is shown in SEQ ID NO:16.

[0125] Specifically, to prepare a strain having the hom(R407H) mutation, PCR was performed using the chromosome of Corynebacterium glutamicum ATCC13032 as a template and primers of SEQ ID NO: 17 and SEQ ID NO: 18, or SEQ ID NO: 19 and SEQ ID NO: 20. The primer sequences used for each PCR are shown in Table 2 below.

[0126] [Table 2]

[0127] The polymerase used for the PCR reaction was PfuUltraTM high fidelity DNA polymerase (Stratagene), and the PCR conditions were denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute. These denaturation, annealing, and polymerization reactions were repeated 28 times. As a result, a 1000 bp DNA fragment at the 5' upper end and a 1000 bp DNA fragment at the 3' lower end were obtained, each of which was centered on the mutation in the hom gene.

[0128] Using the two types of amplified DNA fragments as templates, PCR was performed with primers of SEQ ID NO: 17 and SEQ ID NO: 20. The PCR conditions were denaturation at 95°C for 5 minutes, followed by 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 2 minutes, followed by polymerization reaction at 72°C for 5 minutes.

[0129] As a result, a 2 kb DNA fragment containing a mutation in the hom gene encoding a homoserine dehydrogenase mutant in which arginine at position 407 was replaced with histidine was amplified. The amplified product was purified using a PCR Purification kit (QIAGEN) and used as an insert DNA fragment for vector construction. The purified amplified product was treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes. The molar concentration (M) ratio of the pDCM2 vector (Korean Patent Publication No. 10-2020-0136813) and the amplified product, the insert DNA fragment, was 1:2. The vector pDCM2-R407H for introducing the hom (R407H) mutation into the chromosome was constructed by cloning according to the manual provided using an Infusion Cloning Kit (TaKaRa).

[0130] The constructed vector was transformed into Corynebacterium glutamicum ATCC13032 by electroporation, and a strain containing the hom(R407H) mutation on the chromosome was obtained through a second crossover process, which was named Corynebacterium glutamicum ATCC13032 hom(R407H).

[0131] In order to increase the feedback release and activity of L-isoleucine in the constructed ATCC13032 hom (R407H) strain, ilvA, a gene encoding L-threonine dehydratase, was mutated to replace the 381st amino acid threonine with alanine and the 383rd amino acid phenylalanine with alanine in L-threonine dehydratase (SEQ ID NO: 35). In addition, a strain into which ilvA (T381A, F383A) was introduced was constructed, and the polynucleotide encoding ilvA (T381A, F383A) is shown in SEQ ID NO: 21.

[0132] Specifically, to prepare a strain having the ilvA (T381A, F383A) mutation, PCR was performed using the chromosome of Corynebacterium glutamicum ATCC13032 as a template and primers of SEQ ID NO: 22 and SEQ ID NO: 23, or SEQ ID NO: 24 and SEQ ID NO: 25. The primer sequences used for each PCR are shown in Table 3 below.

[0133] [Table 3]

[0134] PfuUltraTM high fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction, and the PCR conditions were denaturation at 95°C for 30 seconds, denaturation at 55°C for 30 seconds, and polymerization at 72°C for 1 minute, and these denaturation, annealing, and polymerization conditions were repeated 28 times. As a result, a 1126 bp DNA fragment at the 5' upper end and a 286 bp DNA fragment at the 3' lower end were obtained, each of which was centered on the mutation in the ilvA gene.

[0135] Using the two types of amplified DNA fragments as templates, PCR was performed with primers of SEQ ID NO: 22 and SEQ ID NO: 25. The PCR conditions were as follows: denaturation at 95°C for 5 minutes, followed by 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 2 minutes, followed by polymerization reaction at 72°C for 5 minutes.

[0136] As a result, a 1.4 kb DNA fragment containing a mutation in the ilvA gene encoding an L-threonine dehydratase mutant in which the 381st threonine was replaced with alanine and the 383rd phenylalanine was replaced with alanine was amplified. The amplified product was purified using a PCR Purification kit (QIAGEN) and used as an insert DNA fragment for vector construction. The purified amplified product was treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes to make the molar concentration (M) ratio of the pDCM2 vector (Korean Patent Publication No. 10-2020-0136813) and the amplified product, the insert DNA fragment, was adjusted to 1:2, and the vector pDCM2-ilvA (T381A, F383A) for introducing the hom (R407H) mutation into the chromosome was constructed by cloning according to the manual provided using an Infusion Cloning Kit (TaKaRa).

[0137] The constructed vector was transformed into Corynebacterium glutamicum ATCC13032 hom (R407H) by electroporation, and a strain containing the ilvA (T381A, F383A) mutation on the chromosome was obtained through a second crossover process and named Corynebacterium glutamicum CA10-3101.

[0138] The strain CA10-3101 was internationally deposited with the Korea Center for Microorganisms (KCCM), an international depository institution under the Budapest Treaty, on May 27, 2020 and assigned the deposit number KCCM12739P.

[0139] For reference, the following experiment was carried out to confirm whether the introduction of ilvA(T381A, F383A) into an L-isoleucine producing strain increases the efficiency of L-isoleucine production by releasing the feedback loop and increasing the activity of L-isoleucine. Specifically, the ilvA(T381A, F383A) mutation was introduced into KCJI-38 (KCCM11248P, Korean Patent No. 10-1335789), an L-isoleucine producing strain treated with NTG (N-Methyl-N'-nitro-N-nitrosoguanidine), by electric pulse method, and the L-isoleucine and L-threonine concentrations in the culture medium of KCCM11248P / pECCG117-ilvA(T381A, F383A) strain were measured. The results are shown in Table 4 below.

[0140] [Table 4]

[0141] As shown in Table 4, it was confirmed that the KCCM11248P / pECCG117-ilvA(T381A, F383A) strain into which the ilvA(T381A, F383A) mutation was introduced exhibited significantly increased L-isoleucine production and a higher L-threonine degradation rate than the KCCM11248P or KCCM11248P / pECCG117-ilvA(F383A) strains. In other words, for the purposes of this application, it was confirmed that the ilvA(T381A, F383A) mutation was introduced to release the feedback loop on L-isoleucine and increase activity.

[0142] Example 1: Construction of an L-isoleucine producing strain in which the ilvE gene is overexpressed and confirmation of the effect of ilvE on the fermentation purity of L-isoleucine In order to confirm whether overexpression or mutation of ilvE during L-isoleucine production reduces by-products and increases the purity of L-isoleucine, the ilvE gene was enhanced. For this purpose, the L-isoleucine producing strain CA10-3101 prepared in Reference Example 2 was used. Specifically, the vector prepared in Reference Example 1 was transformed into Corynebacterium glutamicum CA10-3101 by electroporation, and a secondary crossover process was carried out to obtain a strain in which the Corynebacterium glutamicum ilvE promoter and ilvE gene were replaced and enhanced with the Corynebacterium glutamicum gdh promoter and E. coli ilvE gene on the chromosome, and the strain was named Corynebacterium glutamicum CA10-3119. The parent strain (CA10-3101) and the ilvE-enhanced strain (CA10-3119) were inoculated into a 250-ml corner baffle flask containing 25 ml of isoleucine production medium, and then cultured at 32° C. for 60 hours with shaking at 200 rpm to produce L-isoleucine. The composition of the production medium used in this example is as follows.

[0143] <Production medium> Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, potassium phosphate monobasic 0.1%, magnesium sulfate heptahydrate 0.1%, ferrous sulfate heptahydrate 10mg / l, manganese sulfate monohydrate 10mg / l, biotin 200μg / l, pH 7.2 After the cultivation, the amount of L-isoleucine and by-products produced was measured using high performance liquid chromatography (HPLC), and the concentrations of L-isoleucine and by-products in the culture medium for each strain tested are shown in Table 5 below.

[0144] [Table 5]

[0145] As a result, as shown in Table 5, the parent strain Corynebacterium glutamicum CA10-3101 produced L-isoleucine at a concentration of 2.4 g / L, and the main by-products alpha-ketobutyric acid (AABA) and L-valine were detected at 0.9 g / L and 1.4 g / L, respectively, whereas the E. coli ilvE-enhanced strain Corynebacterium glutamicum CA10-3119 prepared in this embodiment produced L-isoleucine at a concentration of 2.7 g / L, confirming that the L-isoleucine productivity increased by 12% compared to the parent strain. In addition, the by-products alpha-ketobutyric acid and valine were detected at 0.8 g / L and 1.2 g / L, respectively, confirming that the by-products alpha-ketobutyric acid and valine were decreased by 12% and 15%, respectively, compared to the parent strain.

[0146] Based on the above results, it was confirmed that the ilvE gene can be a target for reducing L-isoleucine by-products, increasing L-isoleucine productivity, and increasing the fermentation purity of L-isoleucine. Therefore, random mutagenesis was used to further improve ilvE and develop a superior ilvE mutant.

[0147] Example 2: Construction of mutant ilvE library vector An ilvE mutation library was constructed based on the vector constructed in Reference Example 1. To this end, the library was constructed using an error-prone PCR kit (clontech Diversify™ PCR Random Mutagenesis Kit), and PCR was performed using SEQ ID NO: 14 and SEQ ID NO: 15 as primers under conditions that would allow mutations to occur.

[0148] Specifically, the reaction was pre-heated at 94°C for 30 seconds under conditions that would result in 0-3 mutations per 1000bp, followed by 25 cycles of 94°C for 30 seconds and 68°C for 1 minute and 30 seconds. The resulting product was then subjected to 25 cycles of 95°C for 50 seconds, 60°C for 50 seconds, and 68°C for 12 minutes using megaprimer (500-125ng), and then treated with DpnI, transformed into E. coli DH5α, and smeared on LB solid medium containing kanamycin (25mg / L). After selecting 20 transformed colonies, the plasmids were obtained and the polynucleotide sequences were analyzed, confirming that mutations were introduced at different positions with a frequency of 2 mutations / kb. Approximately 20,000 transformed E. coli colonies were taken and plasmids were extracted, which were named pTOPO-ilvE-library.

[0149] Example 3: Construction of an L-isoleucine strain carrying the ilvE library The pTOPO-ilvE-library prepared in Example 2 was transformed into Corynebacterium glutamicun CA10-3101 by electroporation, and then 5,000 colonies of the strain into which the mutant gene was inserted were obtained by plating on a nutrient medium containing 25 mg / L kanamycin. The colonies were named CA10-3101 / pTOPO-ilvEm1 to CA10-3101 / pTOPO-ilvEm5000.

[0150] Among the 5,000 colonies obtained, the fermentation titer of each colony was evaluated as follows to identify colonies with increased L-isoleucine productivity and reduced by-products. The parent strain and the mutant strain were inoculated into a 250 ml corner baffle flask containing 25 ml of isoleucine production medium, and then cultured at 32° C. for 60 hours with shaking at 200 rpm to produce L-isoleucine. The composition of the production medium used in this example is as follows.

[0151] <Production medium> Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, potassium phosphate monobasic 0.1%, magnesium sulfate heptahydrate 0.1%, ferrous sulfate heptahydrate 10mg / l, manganese sulfate monohydrate 10mg / l, biotin 200μg / l, pH 7.2 After the cultivation, the amounts of L-isoleucine and L-threonine produced were measured using high performance liquid chromatography (HPLC), and the concentrations of L-isoleucine, L-valine and AABA in the culture medium for each strain tested are shown in Table 6 below.

[0152] [Table 6]

[0153] - By-product ratio: By-product (AABA, Valine) concentration / (Isoleucine + by-product) concentration As a result, three mutant strains were identified that had similar L-isoleucine productivity and reduced by-products AABA and L-valine compared to the parent strain Corynebacterium glutamicum CA10-3101, as shown in Table 6. Sequencing of the three mutant strains was performed, and the ilvE gene was compared with that of the wild-type MG1655, confirming that the three mutant strains contained mutations in the ilvE gene.

[0154] Specifically, it was confirmed that the CA10-3101 / pTOPO-ilvEm1251 strain has the 98th amino acid in the amino acid sequence of ilvE, arginine, replaced with isoleucine (R98I), the CA10-3101 / pTOPO-ilvEm3528 strain has the 31st amino acid in the amino acid sequence of ilvE, histidine, replaced with leucine (H31L), and the CA10-3101 / pTOPO-ilvEm4401 strain has the 256th amino acid in the amino acid sequence of ilvE, serine, replaced with valine (S256V). Based on the above results, it was confirmed that the three mutant strains (R98I, H31L, S256V) can produce L-isoleucine more efficiently and in higher yields than the parent strain. More specifically, when comparing the strains into which ilvE(R98I), ilvE(H31L), and ilvE(S256V) had been introduced with the parent strain, L-isoleucine was produced at similar concentrations, but the AABA concentrations were reduced by approximately 67%, 56%, and 22%, respectively, and L-valine was reduced by approximately 58%, 50%, and 27%, respectively, and the ratios of by-products (AABA, L-valine) were reduced by 44%, 26%, and 11%, respectively, demonstrating that the three mutant strains were excellent in reducing by-products.

[0155] Example 4: Construction of an L-isoleucine strain carrying mutant ilvE The three mutant ilvE genes prepared in Example 3 were transformed into Corynebacterium glutamicum CA10-3101.

[0156] Specifically, as in Reference Example 1, PCR was performed using the chromosomes of ATCC13032 as a template and the primers of SEQ ID NO: 8 and SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, and the chromosomes of CA10-3101 / pTOPO-ilvEm1251(R98I), CA10-3101 / pTOPO-ilvEm3528(H31L), and CA10-3101 / pTOPO-ilvEm4401(S256V) as templates and the primers of SEQ ID NO: 14 and SEQ ID NO: 15. As the polymerization enzyme for the PCR reaction, PfuUltraTM high fidelity-DNA polymerase (Stratagene) was used, and the PCR conditions were denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization reaction at 72°C for 1 minute, and such denaturation, annealing, and polymerization reactions were repeated 28 times. As a result, a 5'-upper 737 bp DNA fragment centered on the ATCC13032 ilvE gene initiation codon, a 5'-upper 499 bp DNA fragment centered on the ATCC13032 gdh gene initiation codon, a 3'-lower 742 bp DNA fragment centered on the ATCC13032 ilvE gene termination codon, and 930 bp fragments of the ilvE genes of CA10-3101 / pTOPO-ilvEm1251, CA10-3101 / pTOPO-ilvEm3528, and CA10-3101 / pTOPO-ilvEm4401 were obtained. PCR was performed using the three amplified DNA fragments as templates and primers of SEQ ID NO:8 and SEQ ID NO:13. The PCR conditions were as follows: denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, and polymerization at 72°C for 2 minutes repeated 6 times, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 2 minutes repeated 20 times, followed by polymerization reaction at 72°C for 5 minutes. As a result, three types of 2868 bp DNA fragments encoding the ilvE gene including the gdh promoter were amplified. The amplified products were purified using QIAGEN's PCR Purification kit and used as insert DNA fragments for vector construction.Meanwhile, the pDCM2 vector, which had been treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes, and the insert DNA fragment amplified through the PCR were cloned according to the instructions provided using the Infusion Cloning Kit from Takara, with the molar concentration (M) ratio of 1:2 to prepare vectors pDCM2-Pgdh-ilvE(eco,H31L), pDCM2-Pgdh-ilvE(eco,R98I), and pDCM2-Pgdh-ilvE(eco,S256V) for replacing the ilvE gene and ilvE promoter of Corynebacterium glutamicum with the ilvE gene and gdh promoter of Corynebacterium glutamicum and introducing them into the chromosome.

[0157] The constructed vector was transformed into Corynebacterium glutamicum CA10-3101 by electroporation, and the mutant ilvE promoter was enhanced on the chromosome through a second crossover process to obtain strains in which mutant ilvE was replaced. The CA10-3101::Pgdh-ilvE(eco,H31L) strain was named CA10-3120, the CA10-3101::Pgdh-ilvE(eco,R98I) strain was named CA10-3121, and the CA10-3101::Pgdh-ilvE(eco,S256V) strain was named CA10-3122. In order to confirm the effect of increasing L-isoleucine productivity and reducing by-products for the L-isoleucine producing parent strain (CA10-3101) and the three mutant-introduced strains (CA10-3120, CA10-3121, CA10-3122) prepared in this example, the fermentation titer of each strain was evaluated by the following method. The parent strain and the three mutant strains were inoculated into a 250 ml corner baffle flask containing 25 ml of isoleucine production medium, and then cultured at 32°C for 60 hours with shaking at 200 rpm to produce L-isoleucine. The composition of the production medium used in this example is shown below.

[0158] <Production medium> Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, potassium phosphate monobasic 0.1%, magnesium sulfate heptahydrate 0.1%, ferrous sulfate heptahydrate 10mg / l, manganese sulfate monohydrate 10mg / l, biotin 200μg / l, pH 7.2 After the cultivation was completed, the concentrations of L-isoleucine, AABA, and L-valine were measured using high performance liquid chromatography (HPLC), and the results are shown in Table 7 below.

[0159] [Table 7]

[0160] - By-product ratio: By-product (AABA, L-valine) concentration / (Isoleucine + by-product) concentration As shown in Table 7, the amount of L-isoleucine produced by the ilvE(H31L)-introduced strain (CA10-3120), ilvE(R98I)-introduced strain (CA10-3121), and ilvE(S256V)-introduced strain (CA10-3122) was similar compared to the parent strain (CA10-3101), and the concentrations of the by-products AABA and L-valine were reduced. Furthermore, the ilvE(H31L), ilvE(R98I), and ilvE(S256V) mutant-introduced strains produced only about 56%, 44%, and 89% of the AABA produced by the parent strain, and only about 58%, 50%, and 83% of the L-valine produced by the parent strain. In addition, the ratios of by-products (AABA and L-valine) were reduced by approximately 27%, 32%, and 9% compared to the parent strain. We confirmed that the by-products were reduced in the strains carrying the ilvE(H31L), ilvE(R98I), and ilvE(S256V) mutants, while the productivity and purity of L-isoleucine were significantly increased.

[0161] The strain CA10-3125 was internationally deposited with the Korea Center for Microorganisms (KCCM), an international depository institution under the Budapest Treaty, on December 1, 2020 and was assigned the deposit number KCCM12859P.

[0162] Example 5: Construction of combined mutant ilvE plasmids In order to introduce two combinations of the E. coli ilvE mutants identified in Example 3 into the L-isoleucine strain, plasmids were constructed using the primers in Table 8 below.

[0163] [Table 8]

[0164] Specifically, PCR was performed using primers of SEQ ID NO: 8 and SEQ ID NO: 26 with pDCM2-Pgdh-ilvE (eco, H31L) as a template, and PCR was performed using primers of SEQ ID NO: 27 and SEQ ID NO: 13 with pDCM2-Pgdh-ilvE (eco, R98I) as a template. PfuUltraTM high fidelity-DNA polymerase (Stratagene) was used as the polymerization enzyme for the PCR reaction, and the PCR conditions were denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization reaction at 72°C for 1 minute, and these denaturation, annealing, and polymerization reactions were repeated 28 times. As a result, a 1370 bp DNA fragment of pDCM2-Pgdh-ilvE (eco, H31L) and a 1558 bp DNA fragment of pDCM2-Pgdh-ilvE (eco, R98I) were obtained. Using the two amplified DNA fragments as templates, PCR was performed with primers of SEQ ID NO:8 and SEQ ID NO:13. The PCR conditions were denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, and polymerization at 72°C for 2 minutes repeated 6 times, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 2 minutes repeated 20 times, followed by polymerization reaction at 72°C for 5 minutes. As a result, one DNA fragment of 2868 bp encoding the gdh promoter and the E. coli ilvE gene was amplified. The amplified product was purified using QIAGEN's PCR Purification kit and used as an insert DNA fragment for vector construction. Meanwhile, the pDCM2 vector, which had been treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes, and the insert DNA fragment amplified through the PCR were cloned according to the instructions provided using the Infusion Cloning Kit from Takara, with the molar concentration (M) ratio of 1:2 to prepare a vector pDCM2-Pgdh-ilvE(eco,H31L,R98I) for replacing the ilvE gene and ilvE promoter of Corynebacterium glutamicum with the gdh promoter of Corynebacterium glutamicum and the ilvE gene of E. coli for introduction into the chromosome.

[0165] Furthermore, PCR was performed using primers SEQ ID NO: 8 and SEQ ID NO: 26 with pDCM2-Pgdh-ilvE(eco,H31L) as a template, and primers SEQ ID NO: 27 and SEQ ID NO: 13 with pDCM2-Pgdh-ilvE(eco,S256V) as a template. PfuUltraTM high fidelity-DNA polymerase (Stratagene) was used as the polymerization enzyme for the PCR reaction, and the PCR conditions were denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization reaction at 72°C for 1 minute, and such denaturation, annealing, and polymerization reaction were repeated 28 times. As a result, a 1370 bp DNA fragment of pDCM2-Pgdh-ilvE(eco,H31L) and a 1558 bp DNA fragment of pDCM2-Pgdh-ilvE(eco,S256V) were obtained. Using the two amplified DNA fragments as templates, PCR was performed with primers of SEQ ID NO:8 and SEQ ID NO:13. The PCR conditions were denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, and polymerization at 72°C for 2 minutes repeated 6 times, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 2 minutes repeated 20 times, followed by polymerization at 72°C for 5 minutes. As a result, one DNA fragment of 2868 bp encoding the gdh promoter and the E. coli ilvE gene was amplified. The amplified product was purified using QIAGEN's PCR Purification kit and used as an insert DNA fragment for vector construction. Meanwhile, the pDCM2 vector, which had been treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes, and the insert DNA fragment amplified through the PCR were cloned according to the instructions provided using the Infusion Cloning Kit from Takara, with the molar concentration (M) ratio of 1:2 to prepare a vector pDCM2-Pgdh-ilvE(eco,H31L,S256V) for replacing the ilvE gene and ilvE promoter of Corynebacterium glutamicum with the gdh promoter of Corynebacterium glutamicum and ilvE of E. coli for introduction into the chromosome.

[0166] Finally, PCR was performed using primers of SEQ ID NO: 8 and SEQ ID NO: 28 with pDCM2-Pgdh-ilvE(eco,R98I) as a template, and primers of SEQ ID NO: 29 and SEQ ID NO: 13 with pDCM2-Pgdh-ilvE(eco,S256V) as a template. PfuUltraTM high fidelity-DNA polymerase (Stratagene) was used as the polymerization enzyme for the PCR reaction, and the PCR conditions were denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization reaction at 72°C for 1 minute. Such denaturation, annealing, and polymerization reactions were repeated 28 times. As a result, a 1581 bp DNA fragment of pDCM2-Pgdh-ilvE(eco,R98I) and a 1350 bp DNA fragment of pDCM2-Pgdh-ilvE(eco,S256V) were obtained. Using the two amplified DNA fragments as templates, PCR was performed with primers of SEQ ID NO:8 and SEQ ID NO:13. The PCR conditions were denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, and polymerization at 72°C for 2 minutes repeated 6 times, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 2 minutes repeated 20 times, followed by polymerization at 72°C for 5 minutes. As a result, one DNA fragment of 2868 bp encoding the gdh promoter and the E. coli ilvE gene was amplified. The amplified product was purified using QIAGEN's PCR Purification kit and used as an insert DNA fragment for vector construction. Meanwhile, the pDCM2 vector, which had been treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes, and the insert DNA fragment amplified through the PCR were cloned according to the instructions provided using the Infusion Cloning Kit from Takara, with the molar concentration (M) ratio of 1:2 to prepare a vector pDCM2-Pgdh-ilvE(eco, R98I, S256V) for replacing the ilvE gene and ilvE promoter of Corynebacterium glutamicum with the gdh promoter of Corynebacterium glutamicum and ilvE of E. coli for introduction into the chromosome.

[0167] Example 6: Construction of L-isoleucine strains carrying combined mutant ilvE The vector constructed in Example 5 was transformed into Corynebacterium glutamicum CA10-3101 by electroporation, and a secondary crossover process was performed to obtain strains in which mutant E. coli ilvE was replaced on the chromosome and the promoter was enhanced. The strains were named CA10-3124 (CA10-3124), CA10-3101::Pgdh-ilvE(eco, H31L, S256V) (CA10-3125), and CA10-3101::Pgdh-ilvE(eco, R98I, S256V) (CA10-3126).

[0168] To confirm the effect of increasing L-isoleucine productivity and reducing by-products of the three strains, the fermentation titer of each strain was evaluated. For this purpose, the parent strain and the mutant strain were inoculated into a 250 ml corner baffle flask containing 25 ml of isoleucine production medium, and then cultured at 32° C. for 60 hours with shaking at 200 rpm to produce L-isoleucine. The composition of the production medium used in this example is as follows.

[0169] <Production medium> Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, potassium phosphate monobasic 0.1%, magnesium sulfate heptahydrate 0.1%, ferrous sulfate heptahydrate 10mg / l, manganese sulfate monohydrate 10mg / l, biotin 200μg / l, pH 7.2 After the cultivation was completed, the amounts of L-isoleucine, alpha-aminobutyric acid (AABA) and L-valine produced were measured using high performance liquid chromatography (HPLC), and the concentrations are shown in Table 9 below.

[0170] [Table 9]

[0171] - By-product ratio: By-product (AABA, L-valine) concentration / (Isoleucine + by-product) concentration As a result, as shown in Table 9, it was confirmed that the L-isoleucine was similar but the by-products were reduced in the mutant strains into which the combined mutant E. coli ilvE was introduced compared to the parent strain Corynebacterium glutamicum CA10-3101. Specifically, the by-product AABA was reduced by 62% in CA10-3124, 37.5% in CA10-3125, and 25% in CA10-3126 compared to the parent strain, and L-valine was reduced by 60% in CA10-3124, 30% in CA10-3125, and 40% in CA10-3126 compared to the parent strain. In addition, it was confirmed that the ratio of by-products (AABA, L-valine) was reduced by 38% in CA10-3124, 14% in CA10-3125, and 14% in CA10-3126.

[0172] From the above results, it was confirmed that the combined mutant E. coli ilvE gene reduces by-products and increases the fermentation purity of L-isoleucine in L-isoleucine production. Example 6: Construction of mutant E. coli ilvE-enhanced strain and combination mutant E. coli ilvE-enhanced strain using L-isoleucine producing strain Corynebacterium glutamicum KCCM11248P In Examples 4 and 5, three types of E. coli ilvE mutants and three types of combination mutants effective in increasing L-isoleucine productivity and reducing by-products were introduced into KCJI-38 (KCCM11248P, Korean Patent No. 10-1335789) strain, which is an L-isoleucine producing strain treated with NTG (N-Methyl-N'-nitro-N-nitrosoguanidine), by electric pulse method, and then the strain was transformed by spreading on a selection medium containing 25 mgm / L kanamycin, and a promoter-enhanced mutant E. coli ilvE and combination mutant E. coli ilvE enhanced strains on the chromosome were obtained through a second crossover process. Thereafter, the L-isoleucine and by-product concentrations in the culture solution were measured in the same manner as in Example 3, and the results are shown in Table 10 below.

[0173] [Table 10]

[0174] - By-product ratio: By-product (AABA, L-valine) concentration / (Isoleucine + by-product) concentration As shown in Table 10, it was confirmed that the KCCM11248P strain into which the mutant and combined mutant E. coli ilvE were introduced produced less by-products AABA and L-valine than the parent strain. Specifically, it was confirmed that the KCCM11248P△Pn-ilvE::Pgdh-ilvE(Eco,H31L) and KCCM11248P△Pn-ilvE::Pgdh-ilvE(Eco,S256V) strains maintained L-isoleucine productivity and produced less by-products AABA and L-valine than the parent strain, and that the KCCM11248P△Pn-ilvE::Pgdh-ilvE(Eco,R98I) and the KCCM11248P strain into which the combined mutant E. coli ilvE was introduced produced less L-isoleucine but produced less by-products, thereby increasing the fermentation purity of L-isoleucine.

[0175] The above results suggest that the mutant E. coli ilvE and its combined enhanced strain of the present application increase L-isoleucine production. In addition, since the mutant E. coli ilvE and its combined enhanced strain are mutations that contribute to increasing the purity in the L-isoleucine production and purification process, it was confirmed that the mutations play a role in increasing L-isoleucine production.

[0176] From the above description, a person skilled in the art to which the present application pertains will understand that the present application may be implemented in other specific forms without changing its technical ideas or essential features. In this regard, it should be understood that the above described embodiments are merely illustrative and not limiting. The scope of the present application should be interpreted as including any modified or altered forms derived from the meaning and scope of the claims below, and their equivalent concepts, rather than the above detailed description.

[0177] [Table 11]

[0178] [Table 12]

Claims

1. A branched-chain amino acid aminotransferase mutant in which an amino acid corresponding to position 31 in the amino acid sequence of SEQ ID NO:1 is substituted with leucine, an amino acid corresponding to position 98 is substituted with isoleucine, an amino acid corresponding to position 256 is substituted with valine, or a combination thereof.

2. A polynucleotide encoding the variant of claim 1.

3. A vector comprising the polynucleotide of claim 2.

4. A microorganism comprising one or more of the following: a mutant according to claim 1; a polynucleotide encoding said mutant; and a vector comprising said polynucleotide.

5. The microorganism according to claim 4 , which produces L-isoleucine.

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

7. The microorganism according to claim 4, wherein the microorganism is Corynebacterium glutamicum.

8. A method for producing L-isoleucine, comprising: culturing in a medium a microorganism comprising one or more of a branched-chain amino acid aminotransferase mutant in which the amino acid corresponding to position 31 in the amino acid sequence of SEQ ID NO:1 is substituted with leucine, the amino acid corresponding to position 98 is substituted with isoleucine, the amino acid corresponding to position 256 is substituted with valine, or a combination thereof; a polynucleotide encoding the mutant; and a vector containing the polynucleotide.

9. 9. The method for producing L-isoleucine according to claim 8, further comprising the step of recovering L-isoleucine from the medium or the microorganism.

10. 9. The method for producing L-isoleucine according to claim 8, wherein the microorganism is a Corynebacterium sp.

11. 9. The method for producing L-isoleucine according to claim 8, wherein the method reduces by-products generated during L-isoleucine production.

12. 12. The method for producing L-isoleucine according to claim 11, wherein the by-product is at least one selected from the group consisting of alpha-aminobutyrate (AABA), valine, phenylalanine, and leucine.

13. A method for reducing by-products generated during L-isoleucine production, comprising: culturing in a medium a microorganism containing one or more of a branched-chain amino acid aminotransferase mutant in which the amino acid corresponding to position 31 in the amino acid sequence of SEQ ID NO:1 is substituted with leucine, the amino acid corresponding to position 98 is substituted with isoleucine, the amino acid corresponding to position 256 is substituted with valine, or a combination thereof; a polynucleotide encoding the mutant; and a vector containing the polynucleotide.

14. A composition for producing L-isoleucine, comprising: a branched-chain amino acid aminotransferase mutant in which the amino acid corresponding to position 31 in the amino acid sequence of SEQ ID NO:1 is substituted with leucine, the amino acid corresponding to position 98 is substituted with isoleucine, the amino acid corresponding to position 256 is substituted with valine, or a combination thereof; a polynucleotide encoding the mutant; and a microorganism containing one or more of vectors containing the polynucleotide, or a culture medium for the microorganism.

15. A branched-chain amino acid aminotransferase mutant, in which the amino acid corresponding to position 31 in the amino acid sequence of SEQ ID NO:1 is substituted with leucine, the amino acid corresponding to position 98 is substituted with isoleucine, the amino acid corresponding to position 256 is substituted with valine, or a combination thereof; or use of a microorganism comprising said mutant, a polynucleotide encoding said mutant, or a vector comprising said polynucleotide in producing L-isoleucine.

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