Novel acetate metabolism regulator A mutant and method for producing L-branched-chain amino acids using the same

Abstract

The present application relates to a novel acetate metabolism regulator A mutant, a polynucleotide encoding said mutant, a microorganism for producing L-branched-chain amino acids comprising said mutant or said polynucleotide, and a method for producing L-branched-chain amino acids using said microorganism.

Description

[Technical Field]

[0001] The present application relates to novel acetate metabolism regulator A mutants, polynucleotides encoding said mutants, microorganisms for producing branched-chain amino acids containing said mutants or said polynucleotides, and methods for producing branched-chain amino acids using said microorganisms. [Background technology]

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

[0003] Meanwhile, production of branched-chain amino acids using microorganisms is mainly carried out using Escherichia or Corynebacterium microorganisms, and it is known that they are biosynthesized from pyruvate through multiple steps to 2-ketoisocaproate, a precursor (Korean Patent Registration No. 10-0220018, Korean Patent Registration No. 10-0438146). However, the production of L-branched-chain amino acids using these microorganisms presents a problem in that it is difficult to mass-produce them industrially.

[0004] Against this background, the present inventors have demonstrated that introducing a mutant with enhanced activity of regulators of acetate metabolism A (hereinafter referred to as RamA, J Bacteriol. 2006 Apr;188(7):2554-67., Applied Microbiology and Biotechnology (2018) 102:5901-5910) into a microorganism for the purpose of improving L-branched-chain amino acid production using the microorganism significantly increases L-branched-chain amino acid production. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Republic of Korea Patent Registration No. 10-0220018 [Patent Document 2] Republic of Korea Patent Registration No. 10-0438146 [Patent Document 3] US Patent US 7662943 B2 [Patent Document 4] US Patent US 10584338 B2 [Patent Document 5] US Patent US 10273491 B2 [Patent Document 6] Republic of Korea Patent No. 10-1117022 [Patent Document 7] Republic of Korea Patent No. 10-0924065 [Patent Document 8] International Patent Publication No. 2008-033001 [Patent Document 9] Korean Patent No. 10-1335789 [Non-patent literature]

[0006] [Non-Patent Document 1] J Bacteriol. 2006 Apr;188(7):2554-67.,Applied Microbiology and Biotechnology (2018) 102:5901-5910 [Non-patent document 2] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444 [Non-patent document 3] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-patent document 4] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453

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[0007] The present inventors have developed novel acetate metabolism regulator A mutants that enhance L-branched-chain amino acid production, polynucleotides encoding said mutants, microorganisms for producing L-branched-chain amino acids that contain said mutants or said polynucleotides, and methods for producing L-branched-chain amino acids using said microorganisms, and have completed the present application. [Means for solving the problem]

[0008] One object of the present application is to provide a mutant acetate metabolism regulator A in which the amino acid corresponding to position 56 in the amino acid sequence of SEQ ID NO: 3 is substituted with another amino acid.

[0009] Another object of the present application is to provide polynucleotides encoding the variants of the present application.

[0010] Another object of the present application is to provide a microorganism for producing branched-chain L-amino acids, which comprises the mutant of the present application or a polynucleotide encoding the mutant.

[0011] Another object of the present application is to provide a method for producing branched-chain L-amino acids, which comprises culturing the above-mentioned microorganism in a medium.

[0012] Another object of the present application is to provide a composition for producing L-branched-chain amino acids, comprising a variant of the present application, a polynucleotide encoding the variant, a vector containing the polynucleotide, or a microorganism containing the polynucleotide of the present application; a medium in which these are cultured; or a combination of two or more of these.

[0013] Another object of the present application is to provide a use of a mutant acetate metabolism regulator A in which the amino acid corresponding to position 56 in the amino acid sequence of SEQ ID NO: 3 is substituted with another amino acid for the production of L-branched-chain amino acids. [Effects of the Invention]

[0014] When a microorganism containing a mutant of the acetate metabolism regulator A of the present application is cultured, it is possible to produce branched-chain L-amino acids in higher yields than in a microorganism containing an existing unmodified polypeptide. DETAILED DESCRIPTION OF THE INVENTION

[0015] This will be explained in more detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. In other words, any combination of various elements disclosed in the present invention belongs to the category of this application. Furthermore, the specific descriptions described below are not considered to limit the category of this application. Furthermore, numerous papers and patent documents are referenced throughout this specification, and citations thereof are provided. The disclosure contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention.

[0016] One aspect of the present application provides a mutant acetate metabolism regulator A in which the amino acid corresponding to position 56 in the amino acid sequence of SEQ ID NO: 3 is substituted with another amino acid.

[0017] The above-mentioned mutants of the present application may be those in which amino acids at specific positions in the amino acid sequence of an existing acetate metabolism regulator A are substituted to enhance activity, but are not limited thereto.

[0018] In one embodiment, the acetate metabolism regulator A mutant may be, but is not limited to, an acetate metabolism regulator A mutant containing one or more amino acid substitutions in the amino acid sequence of SEQ ID NO: 3.

[0019] Specifically, the variant of the present application may be, but is not limited to, a variant in which the amino acid corresponding to position 56 and / or position 52 of SEQ ID NO: 3 is substituted with another amino acid. More specifically, the variant may be, but is not limited to, a variant in which one or more or both of the above positions or the corresponding positions are substituted with another amino acid.

[0020] The "other amino acid" is not limited as long as it is different from the amino acid before substitution. For example, when describing "the amino acid corresponding to the 56th position in SEQ ID NO: 3 is substituted with another amino acid," the amino acid can be any of phenylalanine, glycine, alanine, arginine, aspartate, cysteine, glutamic acid, asparagine, glutamine, histidine, proline, serine, tyrosine, isoleucine, lysine, thiamin ... The phrase "the amino acid corresponding to position 52 in SEQ ID NO: 3 is substituted with another amino acid" may mean that the amino acid is substituted with asparagine, glycine, arginine, aspartate, cysteine, glutamic acid, glutamine, histidine, proline, serine, tyrosine, isoleucine, leucine, lysine, phenylalanine, tryptophan, valine, methionine, or threonine, excluding alanine, but is not limited thereto.

[0021] Meanwhile, a person skilled in the art can identify the amino acids corresponding to positions 56 and 52 of SEQ ID NO: 3 in any amino acid sequence through sequence alignment known in the art, and it is obvious that the term "amino acids at specific positions in a particular SEQ ID NO:" in this application also includes "amino acids at corresponding positions" in any amino acid sequence, even if not otherwise specified. Therefore, amino acid sequences in which one or more amino acids selected from the group consisting of amino acids corresponding to positions 56 and 52 of SEQ ID NO: 3 are substituted with other amino acids are also included within the scope of this application.

[0022] For example, if one or more amino acids corresponding to positions 56 and 52 of SEQ ID NO: 3 are replaced with other amino acids, a mutant can be provided that has even higher activity than the unsubstituted (unaltered) amino acid sequence.

[0023] Specifically, the mutant of the present application may be one in which the amino acids corresponding to positions 56 and 52 of SEQ ID NO: 3 are substituted with other amino acids, but is not limited thereto.

[0024] As a specific example, the variant of the present application may be one in which the leucine corresponding to the 56th amino acid in SEQ ID NO: 3 is replaced with alanine, and the alanine corresponding to the 52nd amino acid in SEQ ID NO: 3 is replaced with valine, but is not limited thereto.

[0025] As a more specific example, the variant of the present application may have the amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 5, or may essentially consist of the above amino acid sequence.

[0026] Furthermore, the variants of the present application can include amino acid sequences that have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 5, in which the amino acid at the position corresponding to amino acid 56 and / or amino acid 52 from the N-terminus of SEQ ID NO: 3 has been substituted with another amino acid. It is also clear that variants having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added are also included within the scope of the present application, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the variants of the present application.

[0027] For example, the above amino acid sequence may have additions or deletions of sequences at the N-terminus, C-terminus and / or internally that do not alter the function of the variant of the present application, naturally occurring mutations, silent mutations or conservative substitutions.

[0028] As used herein, the term "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartate; aromatic amino acids include phenylalanine, tryptophan, and tyrosine, and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Amino acids can also be classified as those with electrically charged side chains or those with uncharged side chains. Charged amino acids include aspartic acid, glutamic acid, lysine, arginine, and histidine. Uncharged amino acids can be further classified as nonpolar or polar amino acids. Nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Conservative substitutions typically have little or no effect on the activity of the resulting polypeptide. Conservative substitutions typically have little or no effect on the activity of the resulting protein or polypeptide.

[0029] Variants can also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide can be conjugated to an N-terminal signal (or leader) sequence of a protein involved in co-translational or post-translational protein transfer. The polypeptide can also be conjugated to other sequences or linkers that allow the polypeptide to be identified, purified, or synthesized.

[0030] As used herein, the term "variant" refers to a polypeptide in which one or more amino acids have been conservatively substituted and / or modified, resulting in a difference from the amino acid sequence of the variant but maintaining its functions or properties. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the polypeptide before the modification. Some variants may also include variants in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, have been deleted. Other variants may include variants in which portions have been deleted from the N- and / or C-termini of the mature protein. The term "mutant" may be interchangeably used with terms such as mutant, variant, mutant polypeptide, mutated protein, mutation, and variant (e.g., modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), and is not limited thereto as long as it is a term used to mean mutated. For purposes of this application, the mutant may be a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, in which the leucine corresponding to the 56th amino acid of SEQ ID NO: 3 is substituted with alanine; or the amino acid sequence set forth in SEQ ID NO: 5, in which the leucine corresponding to the 56th amino acid of SEQ ID NO: 3 is substituted with alanine and the alanine corresponding to the 52nd amino acid of SEQ ID NO: 3 is substituted with valine.

[0031] The variants may also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the N-terminus of the variant may be conjugated to a signal (or leader) sequence involved in co- or post-translational protein translocation. The variants may also be conjugated to other sequences or linkers to allow for identification, purification, or synthesis.

[0032] As used herein, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid or nucleotide sequences, which can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0033] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined using standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences can generally hybridize to the entire sequence or a portion thereof under moderately or highly stringent conditions. Hybridization obviously includes hybridization to polynucleotides containing common codons or codons that take codon degeneracy into account.

[0034] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using known computer algorithms such as the "FASTA" program using default parameters, for example, as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later), as implemented in the GCG program package (Devereux, J., et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.] [ET AL, J MOLEC BIOL 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [Ed.] Academic Press, San Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48:1073. For example, BLAST from the National Center for Biotechnology Information, or ClustalW can be used to determine homology, similarity, or identity.

[0035] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, e.g., as known in Smith and Waterman, Adv. Appl. Math (1981) 2:482, or as described in Needleman et al. (1970), J Mol Biol. 48:443. Briefly, 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.

[0036] As an example of the present application, the variant of the present application can have acetate metabolism regulator A activity. Furthermore, the variant of the present application can have an activity that increases L-branched amino acid producing ability compared to a wild-type polypeptide that has acetate metabolism regulator A activity.

[0037] In the present application, the term "regulators of acetate metabolism A" refers to regulatory proteins related to acetate metabolism as the target protein of the present application, and can be encoded by the ramA gene.

[0038] In the present application, the expression of the acetate metabolism regulator A may be enhanced, and the enhanced expression can result in an increase in the ability to produce branched-chain L-amino acids.

[0039] As used herein, the term "corresponding to" refers to the amino acid residue at the recited position in the polypeptide, or an amino acid residue that is similar, identical, or homologous to the recited residue in the polypeptide. Identifying the amino acid at the corresponding position may be determining the specific amino acid in a sequence that references a particular sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0040] For example, any amino acid sequence can be aligned with SEQ ID NO: 3, and based on this, each amino acid residue in the amino acid sequence can be numbered by reference to the numeric position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 3. For example, a sequence alignment algorithm such as that described in this application can identify the position of an amino acid relative to a query sequence (also referred to as a "reference sequence"), or the position where a variation such as a substitution, insertion, or deletion occurs.

[0041] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) or the Needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) can be used, but is not limited thereto. Sequence alignment programs, pairwise sequence comparison algorithms, and the like known in the art can also be used appropriately.

[0042] Another aspect of the present application is to provide polynucleotides encoding the variants of the present application.

[0043] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide units are linked in a long chain by covalent bonds, and a DNA or RNA chain of a certain length or more, and more specifically, refers to a polynucleotide fragment encoding the above-mentioned variant.

[0044] A polynucleotide encoding a variant of the present application may comprise a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 5. As an example of the present application, a polynucleotide of the present application may have or comprise the sequence of SEQ ID NO: 2 or SEQ ID NO: 6. Alternatively, a polynucleotide of the present application may consist of or consist essentially of the sequence of SEQ ID NO: 2 or SEQ ID NO: 6.

[0045] The polynucleotide of the present application may be modified in various ways in the coding region without changing the amino acid sequence of the variant of the present application, taking into consideration codon degeneracy or codons preferred in an organism in which the variant of the present application is to be expressed. Specifically, the polynucleotide of the present application may have or include a nucleotide sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to the sequence of SEQ ID NO: 2 or SEQ ID NO: 6, or may be composed of or essentially composed of a nucleotide sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to the sequence of SEQ ID NO: 2 or SEQ ID NO: 6, but is not limited thereto. In this case, in the above-mentioned homologous or identical sequences, the codon encoding the amino acid corresponding to the 56th position of SEQ ID NO: 1 or SEQ ID NO: 5 may be one of the codons encoding alanine, and the codon encoding the amino acid corresponding to the 52nd position of SEQ ID NO: 5 may be one of the codons encoding valine.

[0046] Furthermore, the polynucleotides of the present application may include, without limitation, probes prepared from known gene sequences, for example, sequences that can hybridize under stringent conditions to a complementary sequence to the entire or partial polynucleotide sequences of the present application. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, pp. 9.50-9.51, 11.7-11.8). For example, conditions include conditions under which polynucleotides with high homology or identity, such as polynucleotides with a homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, hybridize with each other, but polynucleotides with lower homology or identity do not hybridize with each other; or conditions under which washing is performed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions for standard Southern hybridization, such as 60°C, 1X SSC, and 0.1% SDS, specifically 60°C, 0.1X SSC, and 0.1% SDS, more specifically 68°C, 0.1X SSC, and 0.1% SDS.

[0047] Hybridization requires that two nucleic acids have complementary sequences, even though mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application can also include isolated nucleic acid fragments that are complementary to an entire sequence, as well as substantially similar nucleic acid sequences.

[0048] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the above-mentioned hybridization conditions, including a hybridization step at a Tm value of 55° C. The Tm value may 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.

[0049] The appropriate stringency for hybridizing such polynucleotides depends on the length and degree of complementation of the polynucleotides, variables well known in the art (eg, J. Sambrook et al., supra).

[0050] Another aspect of the present application is to provide a vector comprising the polynucleotide of the present application, which may be, but is not limited to, an expression vector for expressing the polynucleotide in a microorganism.

[0051] In this application, the term "vector" includes a DNA construct containing a base sequence of a polynucleotide encoding a polypeptide of interest operably linked to an expression control region (or expression control sequence) suitable for expressing the polypeptide of interest in a suitable host. The expression control region can include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating the termination of transcription and translation. After being transformed into a suitable microorganism, a vector can replicate and function independently of the host genome, or can be integrated into the genome itself.

[0052] The vectors used in the present application are not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0053] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a vector for chromosomal integration in a cell. The polynucleotide can be inserted into a chromosome by any method known in the art, including, but not limited to, homologous recombination. A selection marker for confirming the presence or absence of the chromosomal insertion can also be included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of the target nucleic acid molecule. A marker that confers a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide, is used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes, allowing the selection of transformed cells.

[0054] As used herein, the term "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a microorganism, thereby enabling the expression of the polypeptide encoded by the polynucleotide in the microorganism. A transformed polynucleotide can include any polynucleotide that can be expressed in the microorganism, regardless of whether it is located within the chromosome or extrachromosomally. The polynucleotide also includes DNA and / or RNA encoding the target polypeptide. The polynucleotide can be introduced in any form that can be introduced and expressed in the microorganism. For example, the polynucleotide can be introduced into the microorganism in the form of an expression cassette, which is a genetic construct containing all elements necessary for autonomous expression. The expression cassette typically contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of an autonomously replicating expression vector. The polynucleotide may also be introduced into the microorganism in its own form and operably linked to sequences necessary for expression in the microorganism, but is not limited thereto.

[0055] In addition, the term "operably linked" as used above means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target variant of the present application.

[0056] Another aspect of the present application is to provide a microorganism for producing L-branched-chain amino acids, comprising the variant of the present application or the polynucleotide of the present application.

[0057] Specifically, the microorganism may be a microorganism of the genus Corynebacterium, more specifically, Corynebacterium glutamicum, but is not limited thereto.

[0058] The microorganism of the present application can comprise a mutant polypeptide of the present application, a polynucleotide encoding said polypeptide, or a vector comprising a polynucleotide of the present application.

[0059] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and naturally or artificially genetically modified microorganisms, including those in which a specific mechanism has been weakened or enhanced by inserting an exogenous gene or by enhancing or inactivating the activity of an endogenous gene, and may also be a microorganism that contains a genetic modification for the production of a desired polypeptide, protein, or product.

[0060] The strain of the present application may be, but is not limited to, a strain containing any one or more of the variant of the present application, the polynucleotide of the present application, and the vector containing the polynucleotide of the present application; a strain modified to express the variant of the present application or the polynucleotide of the present application; a strain (e.g., a recombinant strain) that expresses the variant of the present application or the polynucleotide of the present application; or a strain (e.g., a recombinant strain) that has the activity of the variant of the present application.

[0061] The strain of the present application may be a strain capable of producing branched-chain L-amino acids.

[0062] The strain of the present application may be, but is not limited to, a microorganism that naturally has the ability to produce acetate metabolic regulator A or L-branched-chain amino acids, or a microorganism into which the mutant of the present application or a polynucleotide encoding the mutant (or a vector containing the polynucleotide) has been introduced into a parent strain that does not have the ability to produce acetate metabolic regulator A or L-branched-chain amino acids, and / or into which the ability to produce L-branched-chain amino acids has been imparted.

[0063] For example, the strain of the present application is a cell or microorganism that has been transformed with a vector containing a polynucleotide encoding the polynucleotide of the present application or a polynucleotide encoding the variant of the present application and expresses the variant of the present application. For purposes of this application, the strain of the present application can include all microorganisms capable of producing L-branched-chain amino acids, including the variant of the present application. For example, the strain of the present application may be a recombinant strain having increased L-branched-chain amino acid production ability by introducing a polynucleotide encoding the variant of the present application into a naturally occurring wild-type microorganism or a microorganism that produces L-branched-chain amino acids, thereby expressing an acetate metabolism regulator A variant. The recombinant strain with increased L-branched-chain amino acid production ability may be, but is not limited to, a naturally occurring wild-type microorganism or a microorganism that is not modified with an acetate metabolism regulator A (i.e., a microorganism that expresses the wild-type acetate metabolism regulator A (SEQ ID NO: 3) or a microorganism that does not express the mutant (SEQ ID NO: 1 or SEQ ID NO: 5) protein). For example, the strain of the present application having increased L-branched-chain amino acid production ability may be, but is not limited to, a microorganism having increased L-branched-chain amino acid production ability compared to a microorganism containing the polypeptide of SEQ ID NO: 3 or a polynucleotide encoding the same.

[0064] For example, the recombinant strain with the increased L-branched-chain amino acid production ability has an increased L-branched-chain amino acid production ability of about 1% or more, specifically about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 10.5% or more, about 11% or more, about 11.5% or more, about 12% or more, about 12.5% ​​or more, about 13% or more, about 13.5% or more, about 14% or more, about 14.1% or more, about 14.2% or more, about 14.3% or more, about 14.4% or more, about 14.5% or more, about 14.6% or more, about 14.7% or more, about 14.8% or more, about 14.9% or more, about 15.1% or more, about 15.2% or more, about 15.3% or more, about 15.4% or more, about 15.5% or more, about 15.6% or more, about 15.7% or more, about 15.8% or more, about 15.9% or more, about 15.9% or more, about 15.1% or more, about 15.1% or more, about 15.2% or more, about 15.3% or more, about 15.4% or more, about 15.5% or more, about 15.6% or more, about 15.7% or more, about 15.8% or more, about 15.9% or more, about 15.1% or more, about 15.1% or more, about 15.1% or more, about 15.2% or more, about 15.3% The increase may be 4.3% or more, about 14.4% or more, about 14.5% or more, or about 14.6% or more (the upper limit is not particularly limited, and may be, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, or about 15% or less), but is not limited thereto as long as there is an increase in the + value compared to the productivity of the parent strain or untransformed microorganism before mutation. In another example, the recombinant strain with increased L-branched-chain amino acid production ability may be, but is not limited to, at least about 1.1-fold, at least about 1.12-fold, at least about 1.13-fold, or at least 1.14-fold (the upper limit is not particularly limited, and may be, for example, at most about 10-fold, at most about 5-fold, at most about 3-fold, at most about 2-fold, at most about 1.5-fold, or at most about 1.2-fold) increased compared to the parent strain or unmodified microorganism before mutation. The term "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​in a range that is equal to or similar to the numerical value following the term "about," but is not limited thereto.

[0065] In the present application, the term "untransformed microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, and may refer to a wild-type or naturally occurring strain itself, or a strain before its traits are changed due to genetic mutations caused by natural or artificial factors. For example, the untransformed microorganism may refer to a strain into which the acetate metabolism regulator A mutant described herein has not been introduced or before it has been introduced. The term "untransformed microorganism" may be used interchangeably with "pre-transformed strain," "pre-transformed microorganism," "non-mutated strain," "non-transformed strain," "non-mutated microorganism," or "reference microorganism."

[0066] In yet another example of the present application, the microorganism of the present application is Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens.

[0067] As used herein, the term "enhancement" of polypeptide activity refers to an increase in polypeptide activity compared to its endogenous activity. The term "enhancement" may be used interchangeably with terms such as "activation," "up-regulation," "overexpression," and "increase." Here, activation, enhancement, up-regulation, overexpression, and increase can all encompass the display of an activity not inherently possessed, or the display of an activity that is improved compared to the endogenous activity or the activity prior to transformation. The term "endogenous activity" refers to the activity of a specific polypeptide inherently possessed by a parent strain or an unaltered microorganism prior to transformation, in cases where a trait is altered by genetic mutation due to natural or artificial factors. This term may be used interchangeably with "activity prior to transformation." "Enhancement," "up-regulation," "overexpression," or "increase" of a polypeptide activity compared to its endogenous activity refers to an improvement in the activity and / or concentration (expression level) of a specific polypeptide inherently possessed by a parent strain or an unaltered microorganism prior to transformation.

[0068] The enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. The enhancement of the activity of the polypeptide can be confirmed by an increase in the activity level, expression level, or amount of a product secreted from the polypeptide.

[0069] The activity of the polypeptide can be enhanced by various methods well known in the art, and is not limited thereto, as long as the activity of the target polypeptide can be enhanced compared to that of the microorganism before transformation. Specifically, the enhancement may be achieved by using genetic engineering and / or protein engineering, which are routine methods in molecular biology and well known to those skilled in the art, but is not limited thereto (e.g., Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2, pp. 1-16; Sambrook et al., Molecular Cloning 2012, etc.).

[0070] Specifically, the enhancement of the polypeptide activity of the present application is achieved by: 1) an increase in the intracellular copy number of the polynucleotide encoding the polypeptide; 2) replacing the expression regulatory region of a gene on a chromosome that encodes a polypeptide with a sequence with strong activity; 3) a modification of the nucleotide sequence encoding the initiation codon or 5'-UTR region of a gene transcript encoding a polypeptide; 4) modifying the amino acid sequence of the polypeptide so that the polypeptide activity is enhanced; 5) modifying a polynucleotide sequence encoding the polypeptide so as to enhance the polypeptide's activity (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the polypeptide's activity); 6) introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same; 7) codon optimization of the polynucleotide encoding the polypeptide; 8) analyzing the tertiary structure of a polypeptide and selectively modifying or chemically modifying exposed sites; or 9) It may be a combination of two or more selected from the above 1) to 8), but is not particularly limited to this.

[0071] More specifically, The above 1) increase in the intracellular copy number of a polynucleotide encoding a polypeptide may be achieved by introducing into a microorganism a vector operably linked to the polynucleotide encoding the polypeptide, which can replicate and function independently of a host. Alternatively, it may be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide into a chromosome of the microorganism. The introduction into a chromosome may be achieved by introducing into the microorganism a vector that inserts the polynucleotide into the chromosome of the microorganism, but is not limited to this. The vector is as described above.

[0072] The replacement of the expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide with a sequence having a stronger activity may involve, for example, mutation of the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further enhance the activity of the expression regulatory region, or replacement with a sequence 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, and a sequence regulating the termination of transcription and translation. For example, the original promoter may be replaced with a strong promoter, but this is not limiting.

[0073] Examples of known strong promoters include, but are not limited to, the CJ1 to CJ7 promoters (U.S. Patent No. 7,662,943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (U.S. Patent No. 10,584,338 B2), O2 promoter (U.S. Patent No. 10,273,491 B2), tkt promoter, and yccA promoter.

[0074] The modification of the base sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide (3) may be, for example, a substitution with a base sequence encoding another start codon that has a higher polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0075] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) above may be, but is not limited to, a mutation in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, such as deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to enhance the activity of the polypeptide, or a replacement with an improved amino acid sequence or polynucleotide sequence that has been modified to have stronger activity or to increase activity. Specifically, the replacement may be performed by, but is not limited to, inserting the polynucleotide into a chromosome via homologous recombination. The vector used in this case may further contain a selection marker to confirm the presence or absence of chromosomal insertion. The selection marker is as described above.

[0076] The introduction of an exogenous polynucleotide that exhibits the activity of the polypeptide (6) above may be the introduction of an exogenous polynucleotide that encodes a polypeptide that exhibits the same or similar activity as the polypeptide into a microorganism. The origin or sequence of the exogenous polynucleotide is not limited, as long as it exhibits the same or similar activity as the polypeptide. The method used for the introduction may be any publicly known transformation method appropriately selected by those skilled in the art. The introduced polynucleotide is expressed in the host cell to produce the polypeptide, and its activity is increased.

[0077] The codon optimization of the polynucleotide encoding the polypeptide (7) above may be codon optimization of an endogenous polynucleotide to increase transcription or translation within a microorganism, or codon optimization of an exogenous polynucleotide to optimize transcription and translation within a microorganism.

[0078] The above 8) analyzing the tertiary structure of a polypeptide and selecting and modifying or chemically modifying exposed sites may involve, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins to determine candidate template proteins according to the degree of sequence similarity, confirming the structure based on the candidate template proteins, and selecting and modifying exposed sites to be modified or chemically modified.

[0079] Such enhancement of polypeptide activity may mean, but is not limited to, an increase in the activity or expression level of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in a wild-type or untransformed microbial strain, or an increase in the amount of a product produced from the polypeptide.

[0080] Modification of a portion or the entire polynucleotide in the microorganism of the present application may be induced by, but is not limited to, (a) homologous recombination using a vector for chromosomal insertion into the microorganism or genome editing using engineered nucleases (e.g., CRISPR-Cas9) and / or (b) treatment with light and / or chemicals, such as ultraviolet light and radiation. Methods for modifying a portion or the entire gene may include DNA recombination techniques. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene is injected into the microorganism to cause homologous recombination, thereby deleting a portion or the entire gene. The injected nucleotide sequence or vector may contain, but is not limited to, a dominant selectable marker.

[0081] In the microorganism of the present application, the mutant, polynucleotide, L-branched-chain amino acid, etc. are as described above in other aspects.

[0082] Another aspect of the present application provides a method for producing L-branched-chain amino acids, comprising culturing the above-described microorganism in a medium.

[0083] Specifically, the method for producing L-branched-chain amino acids of the present application may include, but is not limited to, culturing a Corynebacterium glutamicum strain containing a mutant of the present application, a polynucleotide of the present application, or a vector of the present application in a medium.

[0084] In the present application, the term "culturing" refers to growing the microorganism of the present application under appropriately controlled environmental conditions. The culturing process of the present application is carried out using an appropriate medium and culture conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing may be, but is not limited to, a batch, continuous, and / or fed-batch culture.

[0085] In the present application, the term "culture medium" refers to a substance containing a mixture of nutrients required for culturing the microorganism of the present application as its 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 as long as they are media used for culturing conventional microorganisms. The microorganism of the present application can be cultured in a conventional culture medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins under aerobic conditions by adjusting the temperature, pH, etc.

[0086] For example, 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)].

[0087] In the present application, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid may also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted with reducing sugars) may also be used. A variety of other carbon sources may be used in appropriate amounts without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited thereto.

[0088] Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, 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, and are not limited thereto.

[0089] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and other compounds, including amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the culture medium in a batch or continuous manner. However, the present invention is not limited to these.

[0090] During the cultivation of the microorganisms of the present application, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the medium in an appropriate manner. During cultivation, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. To maintain an aerobic state in the medium, oxygen or an oxygen-containing gas can be injected into the medium, or to maintain an anaerobic or microaerobic state, no gas can be injected, or nitrogen, hydrogen, or carbon dioxide gas can be injected, but this is not limiting.

[0091] In the culture of the present application, the culture temperature may be maintained at 20 to 45°C, specifically 25 to 40°C, and the culture may be performed for about 10 to 160 hours, but is not limited thereto.

[0092] The L-branched-chain amino acids produced by the culture of the present invention can be secreted into the medium or remain intracellularly.

[0093] The method for producing L-branched-chain amino acids of the present application may further include a step of preparing the microorganism of the present application, a step of preparing a medium for culturing the strain, or a combination thereof (regardless of the procedure, in any order), for example, before the culturing step.

[0094] The method for producing L-branched-chain amino acids of the present application may further include a step of recovering L-branched-chain amino acids from the culture medium (the medium in which the culture was carried out) or the microorganism of the present application. The recovery step may be carried out after the culturing step.

[0095] The recovery may involve collecting the target L-branched-chain amino acid using a suitable method known in the art, such as a batch, continuous, or fed-batch culture method for culturing the microorganism of the present application. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination of these methods may be used. The target L-branched-chain amino acid can be recovered from the medium or the microorganism using a suitable method known in the art.

[0096] Furthermore, the method for producing L-branched-chain amino acids of the present application may additionally include a purification step. The purification can be carried out using an appropriate method known in the art. In one example, when the method for producing L-branched-chain amino acids of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be carried out continuously or discontinuously, simultaneously, or integrated into one step, regardless of the order, but are not limited thereto.

[0097] In the methods of the present application, the mutant, polynucleotide, vector, microorganism, etc. are as described in other aspects above.

[0098] Another aspect of the present application is to provide a composition for producing L-branched-chain amino acids, comprising a variant of the present application, a polynucleotide encoding the variant, a vector comprising the polynucleotide, or a microorganism comprising the polynucleotide of the present application; a medium in which these are cultured; or a combination of two or more of these.

[0099] The composition of the present application may further contain any suitable excipient commonly used in compositions for producing amino acids, and such excipients may be, for example, but are not limited to, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, or an isotonic agent.

[0100] In the compositions of the present application, the variants, polynucleotides, vectors, strains, media, L-branched-chain amino acids, etc. are as described above in other aspects.

[0101] Another object of the present application is to provide a use of a mutant acetate metabolism regulator A in which the amino acid corresponding to position 56 in the amino acid sequence of SEQ ID NO: 3 is substituted with another amino acid for the production of L-branched-chain amino acids.

[0102] For purposes of this application, variants and L-branched chain amino acids etc. are as described elsewhere above.

[0103] The present application will be described in more detail below with reference to examples. However, the following examples are merely preferred embodiments for illustrating the present application, and are not intended to limit the scope of the present application. Meanwhile, technical matters not described in this specification can be fully understood and easily performed by those of ordinary skill in the technical field of the present application or a similar technical field.

[0104] Example 1. Screening of mutants with increased valine production through artificial mutagenesis Example 1-1. Induction of artificial mutations through UV irradiation To select mutants with increased valine production, the valine-producing strain Corynebacterium glutamicum KCCM11201P (Korean Patent Registration No. 10-1117022) was spread onto a nutrient medium containing agar and cultured at 30°C for 36 hours. Several hundred colonies were then irradiated with UV light at room temperature to induce random mutations in the strain genome.

[0105] Example 1-2. Evaluation of fermentation titer of mutagenized strains and strain selection To select mutants with increased L-valine production compared to the parent strain, Corynebacterium glutamicum KCCM11201P, fermentation titer experiments were performed on randomly mutated strains. Each colony was subcultured in nutrient medium, and then each strain was inoculated into a 250 ml corner-baffled flask containing 25 ml of production medium and cultured at 30°C for 72 hours with shaking at 200 rpm. The compositions of the nutrient and production media are as follows:

[0106] [Nutrient medium (pH 7.2)] Glucose 10g, meat extract 5g, polypeptone 10g, sodium chloride 2.5g, yeast extract 5g, agar 20g, urea 2g (based on 1 liter of distilled water)

[0107] [Production medium (pH 7.0)] Glucose 100g, ammonium sulfate 40g, soy protein 2.5g, corn steep solids 5g, urea 3g, dipotassium phosphate 1g, magnesium sulfate heptahydrate 0.5g, biotin 100μg, thiamine-HCl 1mg, calcium pantothenate 2mg, nicotinamide 3mg, calcium carbonate 30g (based on 1 liter of distilled water)

[0108] After the cultivation was completed, the amount of L-valine produced was measured using HPLC, and the analyzed L-valine concentrations are shown in Table 1 below.

[0109] [Table 1]

[0110] As shown in Table 1 above, the C13 strain was selected, which showed the greatest increase in valine production compared to the control strain KCCM11201P.

[0111] Example 2. Mutation confirmation through gene sequencing The main genes of the C13 strain, which exhibited increased valine production, were sequenced and compared with those of the KCCM11201P strain and the wild-type Corynebacterium glutamicum ATCC14067 strain. The results confirmed that the C13 strain contained a mutation at a specific position in the ramA gene open reading frame (ORF). Specifically, two mutations were introduced into the ramA gene at 166-167 bp downstream from the initiation codon, changing the existing CTG to GCG and substituting alanine for leucine, the 56th amino acid from the N-terminus. This indicates that the C13 strain is an acetate metabolism regulator A (ramA) mutant.

[0112] Analysis of the above mutation region confirmed that it affects the effector binding domain of acetate metabolism regulator A protein, and predicted that the activity of the protein would be enhanced.

[0113] Example 3. Construction of KCCM11201P strain with ramA mutation and confirmation of valine production ability Example 3-1. Construction of a Corynebacterium glutamicum KCCM11201P strain with the ramA mutation introduced and evaluation of its L-valine production ability A vector containing the target mutation was constructed to insert the ramA(L56A) mutant shown in SEQ ID NO: 1 into C. glutamicum KCCM11201P. Specifically, genomic DNA from the C13 strain was extracted using a G-spin Total DNA Extraction Mini Kit (Intron, Cat. No. 17045) according to the protocol provided with the kit. PCR was then performed using the genomic DNA as a template. PCR conditions included denaturation at 94°C for 5 minutes, followed by 25 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 150 seconds, followed by polymerization at 72°C for 7 minutes. A 1000 bp PCR product (hereafter referred to as "mutagenesis fragment 1") was obtained using SEQ ID NO: 7 and SEQ ID NO: 8.

[0114] The resulting mutated fragment 1 was digested with the restriction enzyme XbaI (New England Biolabs, Beverly, MA) and then ligated with pDZ vector (Korean Patent Registration No. 10-0924065 and International Patent Publication No. 2008-033001) digested with the same restriction enzyme using T4 ligase (New England Biolabs, Beverly, MA). The resulting gene was transformed into E. coli DH5α, which was then selected in LB medium containing kanamycin. DNA was then isolated using a DNA-spin Plasmid DNA Purification Kit (iNtRON) to prepare vector pDZ-ramA(L56A) containing the mutated fragment 1.

[0115] [Table 2]

[0116] Next, we constructed a vector containing the targeted mutation to introduce the previously known ramA (A52V) mutant, in which the 52nd amino acid in the ramA gene, associated with lysine production, was mutated from alanine to valine (Metabolic Engineering, Volume 48, 2018, Pages 1-12, ISSN 1096-7176, https: / / doi.org / 10.1016 / j.ymben.2018.05.004.) into C. glutamicum KCCM11201P. Specifically, genomic DNA from the C13 strain was extracted using a G-spin Total DNA Extraction Mini Kit (Intron, Cat. No. 17045) according to the protocol provided with the kit, and PCR was performed using the genomic DNA as a template. The PCR conditions were as follows: denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 150 seconds, repeated 25 times; polymerization reaction at 72°C for 7 minutes. A 502 bp PCR result (hereinafter referred to as "mutagenesis fragment 2") was obtained using sequence numbers 9 and 10, and a 500 bp PCR result (hereinafter referred to as "mutagenesis fragment 3") was obtained using sequence numbers 11 and 12, respectively.

[0117] The resulting mutated fragments 2 and 3 were ligated to a pDZ vector (Korean Patent Registration No. 10-0924065 and International Patent Publication No. 2008-033001) treated with the restriction enzyme XbaI (New England Biolabs, Beverly, MA) using an Infusion Cloning Kit (Takara Bio Inc., Otsu, Japan), and then transformed into E. coli DH5α. The resulting gene was transformed into E. coli DH5α, which was then selected in kanamycin-containing LB medium. DNA was then isolated using a DNA-spin Plasmid DNA Purification Kit (iNtRON), yielding vector pDZ-ramA(A52V+L56A) containing the mutated fragments 2 and 3.

[0118] [Table 3]

[0119] Next, the pDZ-ramA(L56A) was transformed into Corynebacterium glutamicum KCCM11201P by homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which the vector had been integrated into the chromosome through recombination of the homologous sequences were selected on a medium containing 25 mg / L kanamycin. After the second recombination, the Corynebacterium glutamicum transformants were subjected to PCR using SEQ ID NO:7 and SEQ ID NO:8 to identify strains in which leucine was replaced with alanine at amino acid position 56 of SEQ ID NO:3 in the ramA gene ORF on the chromosome. The recombinant strain was designated Corynebacterium glutamicum KCCM11201P::ramA(L56A).

[0120] The pDZ-ramA(A52V+L56A) vector was transformed into Corynebacterium glutamicum KCCM11201P by homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which the vector had been integrated into the chromosome through recombination of the homologous sequences were selected on a medium containing 25 mg / L kanamycin. The Corynebacterium glutamicum transformants in which secondary recombination had been completed were then subjected to PCR using SEQ ID NO: 9 and SEQ ID NO: 12 to identify strains in which alanine was replaced with valine at amino acid position 52 and leucine was replaced with alanine at amino acid position 56 of SEQ ID NO: 3 in the ramA gene ORF on the chromosome. The recombinant strain was named Corynebacterium glutamicum KCCM11201P::ramA(A52V+L56A).

[0121] To compare the valine-producing strains Corynebacterium glutamicum KCCM11201P, KCCM11201P::ramA(L56A), and KCCM11201P::ramA(A52V+L56A), a flask assay was performed. After subculture in nutrient medium, each strain was inoculated into a 250-ml corner-baffled flask containing 25 ml of production medium and cultured at 30°C for 72 hours with shaking at 200 rpm.

[0122] [Nutrient medium (pH 7.2)] Glucose 10g, meat extract 5g, polypeptone 10g, sodium chloride 2.5g, yeast extract 5g, agar 20g, urea 2g (based on 1 liter of distilled water)

[0123] [Production medium (pH 7.0)] Glucose 100g, ammonium sulfate 40g, soy protein 2.5g, corn steep solids 5g, urea 3g, dipotassium phosphate 1g, magnesium sulfate heptahydrate 0.5g, biotin 100μg, thiamine-HCl 1mg, calcium pantothenate 2mg, nicotinamide 3mg, calcium carbonate 30g (based on 1 liter of distilled water)

[0124] After the cultivation was completed, the L-valine productivity was measured using HPLC. The analyzed L-valine concentrations are shown in Table 4 below.

[0125] [Table 4]

[0126] As shown in Table 4 above, the L-valine production ability of the mutant strains KCCM11201P::ramA(L56A) and KCCM11201P::ramA(A52V+L56A) was confirmed to be increased by 11% and 14.6%, respectively, compared to the parent wild-type strain KCCM11201P.

[0127] Example 3-2: Construction of a Corynebacterium glutamicum CJ7V strain with RamA mutation and evaluation of L-valine production To confirm whether the increased L-valine production effect also exists in other strains of Corynebacterium glutamicum that produce L-valine, a single mutation [ilvN(A42V); Biotechnology and Bioproducts Engineering, June 2014, Volume 19, Issue 3, pp. 456-467] was introduced into the wild-type Corynebacterium glutamicum ATCC14067 to create a strain with improved L-valine production ability.

[0128] Specifically, genomic DNA from the wild-type Corynebacterium glutamicum strain ATCC14067 was extracted using a G-spin Total DNA Extraction Mini Kit (Intron, Cat. No. 17045) according to the protocol provided with the kit. PCR was performed using the genomic DNA as a template. To construct a vector for introducing the A42V mutation into the ilvN gene, gene fragments (A and B) were obtained using the primer pair SEQ ID NOs: 13 and 14 and the primer pair SEQ ID NOs: 15 and 16, respectively. PCR conditions were as follows: denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 60 seconds, repeated 25 times; polymerization at 72°C for 7 minutes.

[0129] As a result, polynucleotides of 537 bp were obtained for both fragments A and B. Overlapping PCR was performed using SEQ ID NO: 13 and SEQ ID NO: 16 as templates to obtain a 1044 bp PCR product (hereinafter referred to as "mutagenesis fragment 2").

[0130] The resulting mutated fragment 2 was digested with the restriction enzyme XbaI (New England Biolabs, Beverly, MA) and then ligated with pDZ vector digested with the same restriction enzyme using T4 ligase (New England Biolabs, Beverly, MA). The resulting gene was transformed into Escherichia coli DH5α, which was then selected on LB medium containing kanamycin, and DNA was isolated using a DNA-spin plasmid DNA purification kit (iNtRON). The vector used to introduce the A42V mutation into the ilvN gene was designated pDZ-ilvN(A42V).

[0131] [Table 5]

[0132] The wild-type Corynebacterium glutamicum ATCC14067 was then transformed with pDZ-ilvN(A42V) via homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which the vector had been integrated into the chromosome via homologous recombination were selected on a medium containing 25 mg / L kanamycin. After the second recombination, the C. glutamicum transformants were subjected to PCR amplification of gene fragments using SEQ ID NOs: 13 and 16, and the mutant strains were identified through gene sequence analysis. The recombinant strain was designated Corynebacterium glutamicum CJ7V.

[0133] Next, the Corynebacterium glutamicum CJ7V was transformed with each vector as described in Example 3-1 to prepare strains named Corynebacterium glutamicum CJ7V::ramA(L56A) and CJ7V::ramA(A52V+L56A), respectively. To compare the L-valine production abilities of the prepared strains, they were cultured in the same manner as in Example 3-1 and analyzed for L-valine concentration. The analyzed L-valine concentrations are shown in Table 6 below.

[0134] [Table 6]

[0135] As shown in Table 6 above, the L-valine production ability of the CJ7V::ramA(L56A) and CJ7V::ramA(A52V+L56A) strains into which the mutations were introduced was confirmed to be increased by 8.5% and 12.3%, respectively, compared to the CJ7V strain.

[0136] Example 4. Construction of a strain containing the ramA mutation introduced into the Corynebacterium glutamicum KCCM11248P strain capable of producing L-isoleucine and evaluation of its L-isoleucine productivity To confirm whether an L-isoleucine-producing Corynebacterium glutamicum strain has an effect of increasing L-isoleucine productivity, a Corynebacterium glutamicum L-isoleucine-producing strain, KCCM11248P (Korea Patent Registered No. 10-1335789), was constructed by introducing a ramA mutation into the strain.

[0137] Specifically, the vector pDZ-ramA(L56A) constructed in Example 3-1 above was transformed into Corynebacterium glutamicum KCCM11248P by homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which the vector had been integrated into the chromosome through recombination of the homologous sequences were selected on a medium containing 25 mg / L kanamycin. After the secondary recombination, the Corynebacterium glutamicum transformants were subjected to PCR using SEQ ID NO: 7 and SEQ ID NO: 8 to identify strains in which leucine was replaced with alanine at amino acid position 56 of SEQ ID NO: 3 in the ramA gene ORF on the chromosome. The recombinant strain was designated Corynebacterium glutamicum KCCM11248P::ramA(L56A).

[0138] The vector pDZ-ramA(A52V+L56A) constructed in Example 3-1 above was transformed into Corynebacterium glutamicum KCCM11248P by homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which the vector had been integrated into the chromosome through recombination of the homologous sequences were selected on a medium containing 25 mg / L kanamycin. Subsequently, the Corynebacterium glutamicum transformants in which secondary recombination had been completed were subjected to PCR using SEQ ID NO: 9 and SEQ ID NO: 12 to identify strains in which alanine was replaced with valine at amino acid position 52 and leucine was replaced with alanine at amino acid position 56 of SEQ ID NO: 3 in the ramA gene ORF on the chromosome. The recombinant strain was named Corynebacterium glutamicum KCCM11248P::ramA(A52V+L56A).

[0139] A flask assay was performed to compare the isoleucine-producing abilities of the isoleucine-producing strains Corynebacterium glutamicum KCCM11248P, KCCM11248P::ramA(L56A), and KCCM11248P::ramA(A52V+L56A). Each strain was inoculated into a 250-ml corner-baffled flask containing 25 ml of isoleucine-producing medium and cultured at 32°C for 60 hours with shaking at 200 rpm.

[0140] [Production medium (pH 7.2)] Glucose 100g, yeast extract 2g, ammonium sulfate 16g, potassium monophosphate 1g, magnesium sulfate heptahydrate 1g, ferrous sulfate heptahydrate 10mg, manganese sulfate monohydrate 10mg, biotin 200μg (based on 1 liter of distilled water)

[0141] After the cultivation was completed, the L-isoleucine productivity was measured using HPLC. The analyzed L-isoleucine concentrations are shown in Table 7 below.

[0142] [Table 7]

[0143] As shown in Table 7 above, the L-isoleucine production ability of the mutant strains KCCM11248P::ramA(L56A) and KCCM11248P::ramA(A52V+L56A) was confirmed to be increased by 6.8% and 13.6%, respectively, compared to the parent strain KCCM11248P.

[0144] From the above description, those skilled in the art to which the present application pertains will understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present application should be interpreted as including all modifications and variations derived from the meaning and scope of the claims below, and equivalent concepts thereof, rather than the above detailed description.

Claims

1. An acetate metabolism regulator A mutant having acetate metabolism regulator A activity, the amino acid corresponding to position 56 in the amino acid sequence of SEQ ID NO: 3 is substituted with alanine; The acetate metabolism regulator A mutant has at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 5, and has an activity of increasing L-branched-chain amino acid producing ability compared to a wild-type acetate metabolism regulator A having the amino acid sequence set forth in SEQ ID NO:

3. Acetate metabolism regulator A mutant.

2. The mutant of claim 1 , wherein the amino acid corresponding to position 52 in the amino acid sequence of SEQ ID NO: 3 is further substituted with valine.

3. An acetate metabolism regulator A mutant having acetate metabolism regulator A activity, The acetate metabolism regulator A mutant comprises the amino acid sequence represented by SEQ ID NO:

1. Acetate metabolism regulator A mutant.

4. An acetate metabolism regulator A mutant having acetate metabolism regulator A activity, The acetate metabolism regulator A mutant comprises the amino acid sequence represented by SEQ ID NO:

5. Acetate metabolism regulator A mutant.

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

6. A microorganism for producing branched-chain L-amino acids, comprising the mutant according to any one of claims 1 to 4 or a polynucleotide encoding said mutant.

7. 7. The microorganism for producing L-branched-chain amino acids according to claim 6, wherein the microorganism has increased ability to produce L-branched-chain amino acids compared to a microorganism containing the polypeptide of SEQ ID NO: 3 or a polynucleotide encoding the polypeptide.

8. The microorganism for producing L-branched-chain amino acids according to claim 6, wherein the microorganism belongs to the genus Corynebacterium.

9. 9. The microorganism for producing L-branched-chain amino acids according to claim 8, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.

10. A method for producing branched-chain L-amino acids, comprising culturing the microorganism of claim 6 in a medium.

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

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