aroG aldolase variant and method for producing branched-chain amino acids using the same

Abstract

The present application relates to an aroG aldolase (phospho-2-dehydro-3-deoxyheptonate aldolase) mutant, a microorganism containing the same, and a method for producing amino acids using the same.

Description

Technical Field

[0001] This application relates to an aroG aldolase (Phospho-2-dehydro-3-deoxyheptonate aldolase) variant and a method for producing branched-chain amino acids using the same.

Background Art

[0002] L-amino acids are the basic building blocks of proteins and are used as important materials such as pharmaceutical raw materials, food additives, animal feeds, nutritional agents, insecticides, and fungicides. Therefore, the industrial production of amino acids has become an economically important industrial process.

[0003] Various studies have been made to efficiently produce amino acids, for example, efforts have been made to develop microorganisms with high amino acid production efficiency and fermentation process technologies. Specifically, approaches specific to target substances such as increasing the expression of genes encoding enzymes involved in amino acid biosynthesis in Corynebacterium strains or removing genes unnecessary for amino acid biosynthesis have been developed (Patent Document 1). In addition to these methods, methods for removing genes not involved in amino acid production and methods for removing genes whose specific functions are not known in amino acid production are also being utilized.

[0004] On the other hand, among amino acids, branched-chain amino acids refer to three types: valine, leucine, and isoleucine, and are mainly metabolized in muscles and are known to be used as an energy source during activity. As it is known that branched-chain amino acids play an important role in maintaining and increasing muscle mass during activity, their usage has been increasing. However, since a large amount of by-products are generated in the branched-chain amino acid biosynthesis pathway, it is important to reduce the amount of by-products generated in order to produce branched-chain amino acids in high yield and high purity.

Prior Art Documents

Patent Documents

[0005] [Patent Document 1] U.S. Patent No. 9,109,242 [Patent Document 2] U.S. Patent No. 7,662,943 [Patent Document 3] U.S. Patent No. 10,584,338 [Patent Document 4] U.S. Patent No. 10,273,491 [Patent Document 5] Korean Patent Publication No. 10-2020-0136813 [Patent Document 6] Korean Registered Patent No. 10-2143964 [Patent Document 7] U.S. Patent Application Publication No. 2020 / 0340022 [Patent Document 8] U.S. Patent No. 8,465,962 [Patent Document 9] Korean Registered Patent No. 10-0057684 [Patent Document 10] Korean Patent Publication No. 10-2018-0077008 [Non-Patent Document]

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

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

[0007] The applicants have confirmed that a microorganism introduced with a newly discovered aroG aldolase mutant can produce branched-chain amino acids in high yield and high purity, and have thus completed this application. [Means for Solving the Problems]

[0008] This application aims to provide an aroG aldolase (Phospho-2-dehydro-3-deoxyheptonate aldolase) mutant in which at least one amino acid corresponding to the 217th, 310th, 403rd, and 462nd positions from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.

[0009] Furthermore, this application aims to provide a polynucleotide encoding the mutant and a vector containing the same.

[0010] Furthermore, this application aims to provide a Corynebacterium microorganism containing at least one of the mutant, polynucleotide, and vector.

[0011] Furthermore, this application aims to provide a method for producing branched-chain amino acids, which includes the step of culturing the microorganism in a medium.

Advantages of the Invention

[0012] When the aroG aldolase mutant of this application is used, compared with the case where it is not used, the by-product production amount can be reduced, and branched-chain amino acids can be produced in high yields.

Modes for Carrying Out the Invention

[0013] Hereinafter, these will be specifically described. Note that each description and embodiment disclosed in this application is also applicable to other descriptions and embodiments. That is, all combinations of various elements disclosed in this application are included in this application. Also, this application is not limited to the following specific descriptions.

[0014] Moreover, those with ordinary knowledge in the relevant technical field will be able to recognize and confirm many equivalents of the specific aspects of this application described in this application using only ordinary experiments. Furthermore, such equivalents are also intended to be included in this application.

[0015] One aspect of the present application provides an aroG aldolase (Phospho-2-dehydro-3-deoxyheptonate aldolase) variant in which at least one amino acid among the amino acids corresponding to the 217th, 310th, 403rd, and 462nd positions from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.

[0016] The aroG aldolase variant means a polypeptide having aroG aldolase activity or a variant in which at least one amino acid among the amino acids corresponding to the 217th, 310th, 403rd, and 462nd positions from the N-terminus of the aroG aldolase of SEQ ID NO: 1 in the aroG aldolase is substituted with another amino acid.

[0017] The "aroG aldolase" in the present application means an enzyme that catalyzes the following reaction.

[0018]

Chemical formula

[0019] The aroG aldolase of the present application may be an aroG aldolase that has been modified to produce the aroG aldolase variant provided in the present application or a polypeptide having aroG aldolase activity. Specifically, it may be a naturally occurring polypeptide or a wild-type polypeptide, its mature polypeptide, may include its variant or functional fragment, and any polypeptide may be used as long as it can be a parent of the aroG aldolase variant of the present application.

[0020] The aroG aldolase in the present application is, but not limited to, the polypeptide of SEQ ID NO: 1. In one embodiment, it may be a polypeptide having about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity with the polypeptide of SEQ ID NO: 1, and any polypeptide having the same or equivalent activity as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is included in the aroG aldolase.

[0021] The aroG aldolase of the present application has its sequence obtained from GenBank of NCBI, a well-known database. Specifically, it is a polypeptide encoded by the aroG gene, but not limited thereto.

[0022] The "variant" in the present application means a polypeptide that is different from the amino acid sequence before mutation of the variant due to at least one conservative substitution and / or modification of an amino acid, but maintains its functions or properties. Such variants can generally be identified by modifying at least one amino acid of the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be improved, unchanged or decreased compared to the polypeptide before mutation. Some variants also include those in which at least one part such as an N-terminal leader sequence or a transmembrane domain is removed. Other variants also include those in which a part is removed from the N and / or C terminus of the mature protein. The "variant" is used interchangeably with terms such as modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc. in English, and any term that means mutation may be used.

[0023] Alternatively, the variant may include amino acid deletions or additions that have a minimal impact on the properties and secondary structure of the polypeptide. For example, at the N-terminus of the variant, a signal (or leader) sequence involved in protein translocation may be bound co-translationally or post-translationally. Also, the variant may be bound to other sequences or linkers so that it can be identified, purified, or synthesized.

[0024] The variant provided in the present application may be an aroG aldolase variant in which at least one amino acid corresponding to the 217th, 310th, 403rd, and 462nd positions from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid. For example, all of at least two, at least three, or four amino acids at the above positions may be substituted. However, it is not limited thereto.

[0025] The amino acid corresponding to the 217th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 may be arginine, the amino acid corresponding to the 310th amino acid may be lysine, the amino acid corresponding to the 403rd amino acid may be arginine, and / or the amino acid corresponding to the 462nd amino acid may be glutamic acid.

[0026] The variant provided in the present application includes at least one substitution of the amino acid corresponding to the 217th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 with an amino acid other than arginine, the amino acid corresponding to the 310th amino acid with an amino acid other than lysine, the amino acid corresponding to the 403rd amino acid with an amino acid other than arginine, and the amino acid corresponding to the 462nd amino acid with an amino acid other than glutamic acid, but is not limited thereto.

[0027] The "other amino acid" may be any amino acid as long as it is different from the amino acid before substitution. It goes without saying that in this application, "a specific amino acid is substituted" means that even if it is not stated as being substituted with another amino acid, it is substituted with an amino acid different from the amino acid before substitution.

[0028] In one embodiment, the variant of this application may be a variant in which at least one amino acid corresponding to the 217th, 310th, 403rd, and 462nd positions in the amino acid sequence of SEQ ID NO: 1, which is a reference protein, is substituted with an amino acid different from the amino acid before substitution among hydrophobic amino acids or aliphatic amino acids.

[0029] Specifically, the variant may be a variant in which at least one amino acid corresponding to the 217th, 310th, 403rd, and 462nd positions in the amino acid sequence of SEQ ID NO: 1 is substituted with either a hydrophobic (non-polar) amino acid or an aliphatic amino acid. The aliphatic amino acid is, for example, an amino acid selected from the group consisting of glycine, alanine, valine, leucine, and isoleucine, but is not limited thereto. The hydrophobic (non-polar) amino acid is, for example, an amino acid selected from the group consisting of glycine, methionine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, and tryptophan, but is not limited thereto.

[0030] In one embodiment, the variant of this application is a variant in which at least one amino acid corresponding to the 217th, 310th, 403rd, and 462nd positions in the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid different from the amino acid before substitution among amino acids with a small size, but is not limited thereto.

[0031] As used herein, the term "small amino acid" refers to glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, proline, and asparagine, which are relatively small amino acids among the 20 amino acids. Specifically, it means glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, and proline, but is not limited thereto. More specifically, it means glycine, alanine, valine, leucine, isoleucine, serine, and threonine, but is not limited thereto.

[0032] More specifically, substitution of other amino acids in the variant of the present application is substitution with alanine, but is not limited thereto.

[0033] As used herein, "corresponding to" means an amino acid residue at the position listed in a polypeptide, or an amino acid residue similar to, identical to, or corresponding to the residue listed in a polypeptide. Identifying the amino acid at the corresponding position will determine the specific amino acid of the sequence with reference to a specific sequence. As used herein, the term "corresponding region" generally means a similar or corresponding position in a related protein or a reference protein.

[0034] For example, when any amino acid sequence is aligned with SEQ ID NO: 1, based on this, each amino acid residue of the amino acid sequence can be numbered with reference to the number and position of the amino acid residue corresponding to the amino acid residue of SEQ ID NO: 1. For example, the sequence alignment algorithm used herein can identify the position of an amino acid or the position where modifications such as substitution, insertion, or deletion occur when compared with a query sequence (also referred to as a "reference sequence").

[0035] For such alignment, for example, the Needleman-Wunsch algorithm (Non-Patent Document 1), the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2), etc. can be used, but it is not limited thereto, and sequence alignment programs, pairwise sequence comparison algorithms, etc. known in the technical field can be appropriately used.

[0036] In one embodiment, the variant of the present application has at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the polypeptide of SEQ ID NO: 1, and at least one amino acid of the amino acids corresponding to positions 217, 310, 403 and 462 of SEQ ID NO: 1 may be substituted with other amino acids.

[0037] In one embodiment, the variant of the present application may include an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity with the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 23 or SEQ ID NO: 25.

[0038] Specifically, the variant of the present application may have the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 23 or SEQ ID NO: 25, may include the amino acid sequence, may consist of the amino acid sequence, or may essentially consist of the amino acid sequence.

[0039] In one embodiment, the variant of the present application has at least one amino acid of the amino acids corresponding to positions 217, 310, 403, and 462 in the amino acid sequence of SEQ ID NO: 1 being alanine, and having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity with the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 23 or SEQ ID NO: 25. Also, as long as it has such homology or identity and shows the efficacy corresponding to the variant of the present application, mutants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included in the present application, needless to say.

[0040] For example, those having an addition or deletion of a sequence that does not change the function of the variant of the present application at the N-terminus, C-terminus and / or inside of the amino acid sequence, naturally occurring mutations, silent mutations or conservative substitutions can be mentioned.

[0041] The "conservative substitution" means that one amino acid is substituted with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can generally occur based on the similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues. Usually, conservative substitutions have little or no effect on the activity of the protein or polypeptide.

[0042] The "homology" or "identity" in the present application means the degree to which two given amino acid sequences or base sequences are similar and can be expressed as a percentage. Homology and identity are often used interchangeably.

[0043] The sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard sequence algorithms and may use the default gap penalties established by the programs used. Substantially, homologous or identical sequences generally hybridize with all or part of the sequence under medium or high stringency conditions. Under stringent conditions, they generally can hybridize with all or part of the sequence. It goes without saying that hybridization also includes hybridization with a polynucleotide having codons that take into account general codons or codon degeneracy in a polynucleotide.

[0044] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as default parameters like those in Non-Patent Document 3 and the "FASTA" program. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 1) as performed by the needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later versions) (including the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, FASTA (Non-Patent Documents 5, 6 and 7)). For example, homology, similarity or identity can be determined using BLAST or Clustal W of the National Center for Biotechnology Information.

[0045] The homology, similarity or identity of a polynucleotide or polypeptide can be determined by comparing sequence information using a GAP computer program such as Non-Patent Document 1, as disclosed in Non-Patent Document 8, for example. Briefly, the GAP program defines it as the value obtained by dividing the number of similar sequence symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. The default parameters for the GAP program are: (1) a binary comparison matrix (with a value of 1 for identity and 0 for non-identity) and a weighted comparison matrix of Non-Patent Document 10 (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 9, and (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5), and (3) no penalty for terminal gaps.

[0046] In one embodiment, the variant of the present application has aroG aldolase activity. In one embodiment, the variant of the present application has an activity of improving the branched-chain amino acid production ability as compared with the wild-type or non-mutated aroG aldolase. In one embodiment, the variant of the present application has an activity of reducing the by-product production level of the branched-chain amino acid production pathway as compared with the wild-type or non-mutated aroG aldolase. In one embodiment, the variant of the present application has a weakened activity as compared with the wild-type or non-mutated aroG aldolase. However, it is not limited thereto.

[0047] Another aspect of the present application provides a polynucleotide encoding the variant of the present application.

[0048] The "polynucleotide" in the present application refers to a polymer of nucleotides in which nucleotide monomers are covalently linked in a long chain, meaning a DNA or RNA chain longer than a predetermined length, and more specifically refers to a polynucleotide fragment encoding the variant.

[0049] The polynucleotide encoding the variant of the present application may include a base sequence encoding the amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 23, or SEQ ID NO: 25. As an example of the present application, the polynucleotide of the present application may have the sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 24, or SEQ ID NO: 26, or may include the said sequence. Further, the polynucleotide of the present application may consist of the sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 24, or SEQ ID NO: 26, or may be essentially composed of the said sequence.

[0050] Due to the degeneracy of codons or considering the preferred codons in the organism in which the variant of the present application is to be expressed, various modifications can be made to the coding region within the range where the amino acid sequence of the variant of the present application does not change. Specifically, the polynucleotide of the present application has a base sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 24, or SEQ ID NO: 26, or includes the said base sequence, or consists of a base sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 24, or SEQ ID NO: 26, or is essentially composed of the said base sequence, but is not limited thereto.

[0051] Here, in the sequence having the said homology or identity, the codons encoding the amino acids corresponding to the 217th, 310th, 403rd, and 462nd positions of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 24, or SEQ ID NO: 26 may be one of the codons encoding alanine.

[0052] In addition, the polynucleotide of the present application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a complementary sequence to all or part of the polynucleotide sequence of the present application. The "stringent condition" means a condition that enables specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Documents 11 and 12). For example, conditions under which polynucleotides with high homology or identity, such as polynucleotides having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity hybridize with each other, and polynucleotides with lower homology or identity do not hybridize with each other, or washing conditions for ordinary Southern hybridization, such as 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS, and washing once, specifically 2 to 3 times, at the corresponding salt concentration and temperature can be mentioned.

[0053] Hybridization requires that two nucleic acids have complementary sequences, even if base mismatches are possible depending on the stringency of the hybridization. "Complementary" is used to represent the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotide of the present application may include not only nucleic acid sequences that are substantially similar but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0054] Specifically, a polynucleotide having homology or identity with the polynucleotide of the present application can be detected using hybridization conditions in which the hybridization step is performed at a Tm value of 55°C and the conditions described above. Further, the Tm value may be 60°C, 63°C or 65°C, but is not limited thereto, and can be appropriately adjusted by those skilled in the art according to the purpose.

[0055] The appropriate stringency for hybridizing the polynucleotide depends on the length and degree of complementarity of the polynucleotide, and the variables are known in the art (for example, Non-Patent Document 11).

[0056] Still another aspect of the present application provides a vector containing the polynucleotide of the present application. The vector is an expression vector for expressing the polynucleotide in a host cell, but is not limited thereto.

[0057] The "vector" in the present application means a DNA product containing the nucleotide sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable expression regulatory region (or expression regulatory sequence) so that the target polypeptide can be expressed in a suitable host. The expression regulatory region includes a promoter that initiates transcription, any operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating the termination of transcription and translation. When the vector is transformed into a suitable host cell, it can replicate and function regardless of the host genome and can be integrated into the genome itself.

[0058] The vector used in this 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 their natural or recombinant states. For example, as phage vectors or cosmid vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used, and as plasmid vectors, pDC series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, pET series, etc. can be used. Specifically, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector, etc. can be used.

[0059] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome by a vector for intracellular chromosome introduction. The insertion of the polynucleotide into the chromosome can be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. A selection marker for confirming whether it has been introduced into the chromosome may be further included. The selection marker is for selecting cells transformed with the vector, that is, for confirming whether the target nucleic acid molecule has been inserted, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, expression of surface polypeptides, etc. is used. In an environment treated with a selective agent, only cells expressing the selection marker survive or show different phenotypes, so transformed cells can be selected.

[0060] As used in this application, "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism to express the polypeptide encoded by the polynucleotide in the host cell. The transformed polynucleotide may be any polynucleotide as long as it is expressed in the host cell, regardless of whether it is inserted into the host cell chromosome or located extrachromosomally. Further, the polynucleotide includes DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced into the host cell in any form as long as it is introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a gene construct containing all the elements necessary for its own expression. Usually, the expression cassette contains a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of a self-replicable expression vector. Further, the polyn ucleotide may be introduced into the host cell in its own form and operably linked to the sequences necessary for expression in the host cell, but is not limited thereto.

[0061] Further, the "operably linked" means that the promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the target variant of this application is functionally linked to the polynucleotide sequence.

[0062] Another aspect of this application provides a Corynebacterium microorganism containing at least one of the variant of this application, the polynucleotide of this application, and the vector of this application.

[0063] The microorganism of this application may contain the variant polypeptide of this application, the polynucleotide encoding the polypeptide, or a vector containing the polynucleotide of this application.

[0064] As used in this application, the term "microorganism" or "strain" includes all wild-type microorganisms and microorganisms that have been genetically modified either naturally or artificially. These are microorganisms in which a specific mechanism has been weakened or strengthened due to reasons such as the insertion of foreign genes, or the enhancement or inactivation of the activity of endogenous genes, and which have been genetically modified for the production of a target polypeptide, protein, or product.

[0065] The strain of this application is a strain that includes at least one of the variant of this application, the polynucleotide of this application, and a vector containing the polynucleotide of this application; a strain modified to express the variant of this application or the polynucleotide of this application; a strain that expresses the variant of this application or the polynucleotide of this application (for example, a recombinant strain); or a strain having the activity of the variant of this application (for example, a recombinant strain), but is not limited thereto.

[0066] The strain of this application may be a strain having the ability to produce branched-chain amino acids.

[0067] The strain of this application is a microorganism in which the variant of this application or the polynucleotide encoding the same (or a vector containing the said polynucleotide) has been introduced into a microorganism that naturally has aroG aldolase or the ability to produce branched-chain amino acids, or into a parent strain that does not have aroG aldolase or the ability to produce branched-chain amino acids, and / or the ability to produce branched-chain amino acids has been imparted, but is not limited thereto.

[0068] For example, the strain of the present application is a cell or microorganism transformed with a vector containing the polynucleotide of the present application or a polynucleotide encoding a variant of the present application, and expressing the variant of the present application. For the purpose of the present application, the strain of the present application may be any microorganism that contains the variant of the present application and produces branched-chain amino acids. For example, the strain of the present application may be a recombinant strain in which an aroG aldolase variant is expressed and the ability to produce branched-chain amino acids is improved by introducing a polynucleotide encoding the variant of the present application into a natural wild-type microorganism or a microorganism that produces branched-chain amino acids. The recombinant strain with improved ability to produce branched-chain amino acids is a microorganism with improved ability to produce branched-chain amino acids compared to a natural wild-type microorganism or a microorganism without modified aroG aldolase (i.e., a microorganism expressing wild-type aroG aldolase), but is not limited thereto.

[0069] As an example, the aroG aldolase non-modified microorganism, which is the target strain for comparing whether the ability to produce branched-chain amino acids is improved, may be Corynebacterium glutamicum ATCC13032 strain. As another example, the aroG aldolase non-modified microorganisms, which are the target strains for comparing whether the ability to produce branched-chain amino acids is improved, are CJL-8109, KCCM12739P (CA10-3101), KCCM11201P, but are not limited thereto.

[0070] As an example, the recombinant strain is improved by about 1% or more, specifically about 3%, about 5% or more compared to the ability of the parent strain or non-modified microorganism before mutation to produce branched-chain amino acids. However, as long as it shows an increase in the + value compared to the production ability of the parent strain or non-modified microorganism before mutation, it may be any one.

[0071] As another example, the recombinant strain has a production amount of by-products generated in the branched-chain amino acid production pathway reduced to about 50% or less, specifically about 30% or less, about 10% or less, or no by-products are produced, compared to the parent strain or non-modified microorganism before mutation, but is not limited thereto.

[0072] The term "about" refers to a range that includes all of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and can be any numerical value within a range equivalent to or of the same order of magnitude as the numerical value following the term "about", but is not limited thereto.

[0073] In the present application, the "branched-chain amino acid" means an amino acid having a branched alkyl group in the side chain, and includes valine, leucine, and isoleucine. Specifically, the branched-chain amino acid in the present application is an L-branched-chain amino acid, and the L-branched-chain amino acid is at least one selected from L-valine, L-leucine, and L-isoleucine, but is not limited thereto.

[0074] The by-product generated in the branched-chain amino acid production pathway in the present application means a substance other than the branched-chain amino acid, and specifically is at least one selected from aromatic amino acids, more specifically L-tyrosine and L-phenylalanine. However, it is not limited thereto.

[0075] In the present application, the "unmodified microorganism" does not exclude strains containing mutations that can occur naturally in the microorganism, but means the wild-type strain or the natural strain itself, or the strain before being genetically mutated by natural or artificial factors and changing in traits. For example, the unmodified microorganism means a strain in which the aroG aldolase variant of the present application has not been introduced or the strain before being introduced. The "unmodified microorganism" is used interchangeably with the "strain before modification", "microorganism before modification", "non-mutant strain", "unmodified strain", "non-mutant microorganism", or "reference microorganism".

[0076] In one embodiment, the microorganism of the present application may be Corynebacterium stationis, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium glutamicum, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens.

[0077] The microorganism of the present application may further contain a mutation that improves the ability to produce branched-chain amino acids.

[0078] In one embodiment, the microorganism of the present application may include at least one activity change of isopropylmalate synthase, homoserine dehydrogenase, threonine dehydratase, branched-chain amino acid aminotransferase, and citrate synthase.

[0079] In one embodiment, the microorganism of the present application may be a microorganism in which the activity of at least one of isopropylmalate synthase, branched amino acid aminotransferase, homoserine dehydrogenase, and threonine dehydratase is further enhanced.

[0080] In one embodiment, the microorganism of the present application may be a microorganism in which the activity of citrate synthase is further weakened.

[0081] However, it is not limited to the above content. Depending on the branched-chain amino acid to be produced, those skilled in the art can appropriately select further modifications contained in the microorganism.

[0082] The "enhancement" of polypeptide activity in the present application means improving the activity of the polypeptide compared to the endogenous activity. The enhancement is used interchangeably with activation, up-regulation, overexpression, increase, etc. Here, activation, enhancement, up-regulation, overexpression, and increase all include the appearance of an activity that was not originally present and the improvement of the activity compared to the endogenous activity or the activity before modification. The "endogenous activity" means the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before the trait change when the trait changes due to genetic mutation by natural factors or artificial factors. This is used interchangeably with the "activity before modification". That the activity of a polypeptide is "enhanced", "up-regulated", "overexpressed" or "increased" compared to the endogenous activity means an improvement compared to the activity and / or concentration (expression level) of the specific polypeptide originally possessed by the parent strain or unmodified microorganism before the trait change.

[0083] The enhancement may be achieved by introducing a foreign polypeptide, or may also be achieved by enhancing the activity and / or increasing the concentration (expression level) of an endogenous polypeptide. Whether the activity of the polypeptide is enhanced can be confirmed by an increase in the degree of activity of the polypeptide, the expression level, or the amount of the product produced from the polypeptide.

[0084] For enhancing the activity of the polypeptide, various methods well-known in the art can be applied, and any method can be used as long as it can enhance the activity of the target polypeptide more than that of the microorganism before modification. Specifically, it is a usual method in molecular biology and uses genetic engineering and / or protein engineering well-known to those with ordinary knowledge in the technical field, but is not limited thereto (for example, Non-Patent Documents 13, 14, etc.).

[0085] Specifically, the enhancement of the polypeptide of the present application can be achieved by: 1) increasing the intracellular copy number of the polynucleotide encoding the polypeptide; 2) replacing the expression regulatory region of the gene on the chromosome encoding the polypeptide with a sequence having strong activity; 3) modifying the base sequence encoding the start codon or 5' UTR region of the gene transcript encoding the polypeptide; 4) modifying the amino acid sequence of the polypeptide so that the activity of the polypeptide is enhanced; 5) modifying the polynucleotide sequence encoding the polypeptide so that the activity of the polypeptide is enhanced (for example, modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so that the activity of the polypeptide is enhanced); 6) introducing a foreign polypeptide showing the activity of the polypeptide or a foreign polynucleotide encoding the same; 7) optimizing the codons of the polynucleotide encoding the polypeptide; 8) analyzing the tertiary structure of the polypeptide, selecting and modifying the exposed part, or chemically modifying it; or 9) carried out by a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0086] More specifically, increasing the intracellular copy number of the polynucleotide encoding the polypeptide 1) is achieved by introducing into the host cell a vector that replicates and functions regardless of the host, in which the polynucleotide encoding the polypeptide is operably linked. Alternatively, it may be achieved by introducing one copy or two or more copies of the polynucleotide encoding the polypeptide into the chromosome in the host cell. The introduction into the chromosome is carried out by introducing into the host cell a vector capable of inserting the polynucleotide into the chromosome in the host cell, but is not limited thereto. The vector is as described above. This is not the case. The vector is as described above.

[0087] Substituting the gene expression regulatory region (or expression regulatory sequence) on the chromosome encoding the polypeptide 2) with a sequence having strong activity is carried out, for example, by causing a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof so that the activity of the expression regulatory region is further enhanced, or by substituting with a sequence having higher activity. The expression regulatory region includes, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating the termination of transcription and translation, and the like. For example, it is carried out by substituting the original promoter with a strong promoter, but is not limited thereto.

[0088] Examples of known strong promoters include, but are not limited to, cj1 to cj7 promoters (Patent Document 2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (Patent Document 3), O2 promoter (Patent Document 4), tkt promoter, yccA promoter, and the like.

[0089] Modifying the base sequence encoding the start codon of the gene transcript encoding the polypeptide in 3) above or the 5'UTR region is performed, for example, by substituting it with a base sequence encoding another start codon with a higher polypeptide expression rate compared to the endogenous start codon, but is not limited thereto.

[0090] Modifying the amino acid sequence or polynucleotide sequence in 4) and 5) above is to cause sequence mutations in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide by deletion, insertion, non-conservative or conservative substitution, or a combination thereof so that the activity of the polypeptide is enhanced, or substituting it with an amino acid sequence or polynucleotide sequence improved to have higher activity, or an amino acid sequence or polynucleotide sequence improved to have improved activity, but is not limited thereto. Specifically, the substitution is performed by inserting a polynucleotide into the chromosome by homologous recombination, but is not limited thereto. Here, the vector used may further contain a selection marker for confirming whether it has been inserted into the chromosome. The selection marker is as described above.

[0091] Introducing a foreign polynucleotide showing the activity of the polypeptide in 6) above is performed by introducing a foreign polynucleotide encoding a polypeptide showing the same / similar activity as the polypeptide into the host cell. The origin and sequence of the foreign polynucleotide may be any as long as it shows the same / similar activity as the polypeptide. The introduction can be appropriately selected and performed by those skilled in the art using known transformation methods. When the polynucleotide introduced as described above is expressed in the host cell, a polypeptide is generated and its activity is improved.

[0092] Optimizing the codons of the polynucleotide encoding the polypeptide (7) is achieved by optimizing the codons of the endogenous polynucleotide so that transcription or translation increases in the host cell, or by optimizing the codons of the foreign polynucleotide so that optimized transcription and translation occur in the host cell.

[0093] Analyzing the tertiary structure of the polypeptide (8), selecting and modifying the exposed portions, or chemically modifying them is, for example, by comparing the sequence information of the polypeptide to be analyzed with a database in which the sequence information of known proteins is stored, determining candidates for the template protein according to the degree of sequence similarity, confirming the structure based on this, and selecting and modifying or chemically modifying the exposed portions to be modified.

[0094] Enhancement of such polypeptide activity is achieved by improving the activity or concentration, expression level of the corresponding polypeptide compared to the activity or concentration of the polypeptide expressed in the wild-type or unmodified microbial strain, or by increasing the amount of the product produced from the polypeptide, but is not limited thereto.

[0095] In the microorganism of the present application, partial or total modification of the polynucleotide can be induced by (a) homologous recombination using a chromosomal integration vector in the microorganism, or genome editing using an engineered nuclease (e.g., CRISPR-Cas9), and / or (b) treatment with light such as ultraviolet rays or radiation and / or chemical substances, but is not limited thereto. The methods for partial or total modification of the gene include methods by DNA recombination technology. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene is introduced into the microorganism to cause homologous recombination, resulting in partial or total deletion of the gene. The introduced nucleotide sequence or vector may contain a dominant selection marker, but is not limited thereto.

[0096] The "attenuation" of polypeptide activity in this application is a concept that includes all cases where the activity decreases or disappears compared to the intrinsic activity. The above attenuation is used interchangeably with inactivation, deficiency, down-regulation, decrease, reduce, attenuation, etc.

[0097] The above attenuation includes cases where the activity of the polypeptide itself is decreased or removed compared to the activity of the polypeptide originally possessed by the microorganism due to mutations in the polynucleotide encoding the polypeptide, etc., and cases where the overall degree and / or concentration (expression level) of polypeptide activity in the cell is decreased compared to the natural strain due to inhibition of gene expression of the polynucleotide encoding it or inhibition of translation into the polypeptide, etc., including cases where the expression of the polynucleotide is completely absent, and / or cases where the polypeptide has no activity even if the polynucleotide is expressed. The "intrinsic activity" means the activity of a specific polypeptide originally possessed by the parental strain, wild type or unmodified microorganism before the trait change when the trait changes genetically due to natural or artificial factors. This is used interchangeably with "activity before modification". That the activity of a polypeptide is "inactivated, deficient, decreased, down-regulated, reduced, attenuated" compared to the intrinsic activity means that it is decreased compared to the activity of a specific polypeptide originally possessed by the parental strain or unmodified microorganism before the trait change.

[0098] The attenuation of the activity of such a polypeptide is not limited to these, and can be achieved by applying various methods well-known in the art (for example, Non-Patent Documents 14, 15, etc.).

[0099] Specifically, the attenuation of the polypeptide of the present application can be achieved by: 1) deleting all or part of the gene encoding the polypeptide; 2) modifying the expression regulatory region (or expression regulatory sequence) so that the expression of the gene encoding the polypeptide is decreased; 3) modifying the amino acid sequence constituting the polypeptide so that the activity of the polypeptide is deleted or attenuated (for example, deleting / substituting / adding one or more amino acids in the amino acid sequence); 4) modifying the gene sequence encoding the polypeptide so that the activity of the polypeptide is deleted or attenuated (for example, deleting / substituting / adding one or more nucleobases in the nucleobase sequence of the polypeptide gene so as to encode a polypeptide modified so that the activity of the polypeptide is deleted or attenuated); 5) modifying the base sequence encoding the start codon or 5'UTR region of the gene transcript encoding the polypeptide; 6) introducing an antisense oligonucleotide (for example, antisense RNA) that binds complementarily to the gene transcript encoding the polypeptide; 7) adding a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide so that a secondary structure that makes it impossible for ribosomes to attach is formed; 8) adding a promoter so as to reverse transcribe at the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE); or 9) by a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0100] For example, the deletion of all or part of the gene encoding the polypeptide in 1) above may be carried out by deleting the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, substituting it with a polynucleotide with some nucleotides deleted or a marker gene.

[0101] In addition, modifying the 2) expression regulatory region (or expression regulatory sequence) may be performed by generating mutations on the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by substituting it with a sequence having lower 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.

[0102] In addition, modifying the base sequence encoding the start codon of the gene transcript encoding the 5) polypeptide or the 5'UTR region may be performed, for example, by substituting it with a base sequence encoding another start codon having a lower polypeptide expression rate compared to the endogenous start codon, but is not limited thereto.

[0103] Furthermore, modifying the amino acid sequence or polynucleotide sequence of 3) and 4) may be performed by deleting, inserting, non-conservatively or conservatively substituting, or a combination thereof to generate mutations in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide so that the activity of the polypeptide is weakened, or substituting it with an amino acid sequence or polynucleotide sequence improved to have lower activity, or an amino acid sequence or polynucleotide sequence improved to have no activity, but is not limited thereto. For example, gene expression can be inhibited or weakened by introducing a mutation into the polynucleotide sequence to form a stop codon, but is not limited thereto.

[0104] Introducing an antisense oligonucleotide (for example, antisense RNA) that binds complementarily to the gene transcript encoding the 6) polypeptide may be performed, for example, as described in Non-Patent Document 16.

[0105] Adding a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide so that a secondary structure that prevents the attachment of the 7) ribosome is formed may be carried out by making mRNA translation impossible or reducing the rate.

[0106] Adding a promoter so as to reverse transcribe at the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE) may be carried out by creating an antisense nucleotide complementary to the gene transcript encoding the polypeptide and weakening the activity.

[0107] The mutants, polynucleotides, vectors and branched-chain amino acids in the microorganisms of the present application are as described above.

[0108] Still another aspect of the present application provides a method for producing a branched-chain amino acid, which includes the step of culturing the Corynebacterium microorganism of the present application in a medium.

[0109] "Culturing" in the present application means growing the Corynebacterium microorganism of the present application under appropriately adjusted environmental conditions. The culturing process of the present application can be carried out in a known suitable medium and culturing conditions. Such a culturing process can be easily adjusted and used according to the selected strain by those skilled in the art. Specifically, the culturing is batch, continuous and / or fed-batch culturing, but is not limited thereto.

[0110] As used herein, the "medium" refers to a substance obtained by mixing nutrients necessary for culturing the Corynebacterium genus microorganisms of the present application as the main components, and supplies nutrients such as water essential for survival and growth, and growth factors. Specifically, the medium and other culture conditions used for culturing the Corynebacterium genus microorganisms of the present application may be any medium used for culturing ordinary microorganisms. The Corynebacterium genus microorganisms of the present application can be cultured by adjusting temperature, pH, etc. under aerobic conditions in an ordinary medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin.

[0111] Specifically, the culture medium for Corynebacterium genus microorganisms is disclosed in Non-Patent Document 17.

[0112] Examples of the carbon source in the present application include carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steep liquor can be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugar) can be used, and any other appropriate amount of carbon source can be used. These carbon sources can be used alone or in combination of two or more, but are not limited thereto.

[0113] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc., amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. can be used. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0114] As the phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate or their corresponding sodium-containing salts, etc. can be mentioned. As inorganic compounds, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. can be used, and in addition, amino acids, vitamins and / or suitable precursors, etc. can be used. These components or precursors can be added to the medium in a batch or continuous manner. However, it is not limited thereto.

[0115] Also, during the culture of the Corynebacterium genus microorganism of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. can be added to the medium in a suitable manner to adjust the pH of the medium. Furthermore, an antifoaming agent such as a fatty acid polyglycol ester can be used during the culture to suppress the generation of bubbles. Furthermore, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas can be injected into the medium, and in order to maintain the anaerobic and microaerobic states, it is not necessary to inject gas, and nitrogen, hydrogen or carbon dioxide gas can be injected, but it is not limited thereto.

[0116] In the culture of the present application, the culture temperature is maintained at 20 to 45 °C, specifically 25 to 40 °C, and cultured for about 10 to 160 hours, but is not limited thereto.

[0117] The branched-chain amino acids produced by culturing in the present application are either secreted into the culture medium or remain intracellular.

[0118] The method for producing branched-chain amino acids in the present application may further include, for example, in any order, the step of preparing the Corynebacterium microorganism of the present application, the step of preparing a culture medium for culturing the strain, or a combination thereof, even before the step of culturing.

[0119] The method for producing branched-chain amino acids in the present application may further include the step of recovering branched-chain amino acids from the culture medium (the medium in which the culture was performed) used in the culture or from the Corynebacterium microorganism of the present application. The step of recovering may further be included after the step of culturing.

[0120] The recovery may be to collect the target branched-chain amino acids using a suitable method known in the art according to the method of culturing the microorganism of the present application, such as batch, continuous, fed-batch culture methods, etc. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting-out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and other various chromatographies, HPLC, or a combination thereof may be used, and the target branched-chain amino acids can be recovered from the culture medium or the microorganism using a suitable method known in the art.

[0121] In addition, the method for producing branched-chain amino acids in the present application may further include a purification step. The purification can be performed by a suitable method known in the art. For example, when the method for producing branched-chain amino acids in the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed at different times (or continuously) regardless of the order, or may be performed simultaneously or integrated as one step, but are not limited thereto.

[0122] The mutants, polynucleotides, vectors, microorganisms, etc. in the method of the present application are as described above.

[0123] Still other aspects of the present application provide a composition for producing branched-chain amino acids, which comprises a mutant of the present application, a polynucleotide encoding the mutant, a vector containing the polynucleotide, or a Corynebacterium microorganism containing the polynucleotide of the present application, or comprises a medium in which the same is cultured, or comprises a combination of two or more of them.

[0124] The composition of the present application may further contain any suitable excipient commonly used in the composition for producing branched-chain amino acids. Such excipients include, for example, preservatives, wetting agents, dispersants, suspending agents, buffering agents, stabilizing agents, isotonic agents, etc., but are not limited thereto.

[0125] The mutants, polynucleotides, vectors, strains, media, branched-chain amino acids, etc. in the composition of the present application are as described above.

[0126] Still other aspects of the present application provide the use of the aroG aldolase mutant of the present application for producing branched-chain amino acids.

[0127] Still other aspects of the present application provide the use of a microorganism containing at least one of the aroG aldolase mutant of the present application, a polynucleotide encoding the aroG aldolase mutant, and a vector containing the polynucleotide for producing branched-chain amino acids.

[0128] The mutants, polynucleotides, vectors, microorganisms, etc. in the use of the present application are as described above.

Examples

[0129] Hereinafter, the present application will be described in more detail with reference to examples. However, these examples are merely illustrative of the present application, and the present application is not limited to these examples. This will be obvious to those having ordinary knowledge in the technical field to which the present application pertains.

Example

[0130] Discovery of aroG aldolase mutation Example 1-1. Preparation of a vector containing aroG aldolase To prepare a library of aroG aldolase mutants having the activity of Phospho-2-dehydro-3-deoxyheptonate aldolase, first, a recombinant vector containing aroG aldolase was prepared. To amplify the aroG gene (SEQ ID NO: 2) encoding aroG aldolase derived from wild-type Corynebacterium glutamicum (SEQ ID NO: 1, KEGG ID: NCgl2098), using the chromosome of Corynebacterium glutamicum ATCC13032 wild strain as a template, PCR was performed by conducting 25 cycles of denaturation at 94°C for 1 minute, annealing at 58°C for 30 seconds, and polymerization with Pfu DNA polymerase at 72°C for 1 minute using the primers of SEQ ID NOs: 11 and 12. The specific sequences of the primers used are shown in Table 1. The above amplification product was cloned into the Escherichia coli vector pCR2.1 using the TOPO Cloning Kit (Invitrogen) to obtain "pCR-aroG".

[0131]

Table 1

[0132] Example 1-2. Preparation of aroG aldolase mutant library Based on the vector prepared in Example 1-1, an aroG aldolase mutant library was prepared using an error-prone PCR kit (clontech Diversify® PCR Random Mutagenesis Kit). PCR reaction was performed using SEQ ID NO: 11 and SEQ ID NO: 12 as primers under conditions where 0 to 3 mutations occur per 1000 bp. Specifically, after pre-heating at 94°C for 30 seconds, the PCR reaction was carried out by performing 25 cycles of a process of 30 seconds at 94°C and 1 minute 30 seconds at 68°C. Here, the obtained PCR product was used as a megaprimer (50 - 125 ng), and a process of 50 seconds at 95°C, 50 seconds at 60°C, and 12 minutes at 68°C was performed for 25 cycles, followed by treatment with DpnI, transformation into Escherichia coli DH5α by the heat shock method, and plating on an LB solid medium containing kanamycin (25 mg / L). Twenty transformed colonies were selected to obtain plasmids, and nucleotide sequence analysis confirmed that mutations were introduced at different positions at a frequency of 2 mutations / kb. Approximately 20,000 transformed Escherichia coli colonies were collected and plasmids were extracted. This was named "pTOPO-aroG-library".

Example

[0133] Evaluation of the prepared library and selection of mutants Example 2-1. Selection of mutant strains with increased L-leucine production The pTOPO-aroG-library prepared in Example 1-2 was transformed into wild-type Corynebacterium glutamicum ATCC13032 by electroporation, and then plated on a nutrient medium (Table 2) containing 25 mg / L of kanamycin to select 10,000 colonies of strains into which the mutant gene was inserted. Each selected colony was named ATCC13032 / pTOPO_aroG(mt)1 - ATCC13032 / pTOPO_aroG(mt)10,000.

[0134] Of the 10,000 colonies obtained, in order to identify colonies in which L-leucine production increased and the aromatic amino acids L-tyrosine and L-phenylalanine decreased, the fermentation titer of each colony was evaluated by the following method.

[0135]

Table 2

[0136] Each colony was inoculated with a platinum loop into a 250-ml Erlenmeyer flask containing 25 ml of the autoclaved production medium (Table 2) and 25 μg / ml of kanamycin, and then cultured with shaking at 30 °C and 200 rpm for 60 hours. After the cultivation, L-leucine and the aromatic amino acids L-tyrosine and L-phenylalanine were measured by a method using high-performance liquid chromatography (HPLC, SHIMAZDU LC20A).

[0137] Among the 10,000 colonies obtained, four strains (ATCC13032 / pTOPO_aroG(mt)2256, ATCC13032 / pTOPO_aroG(mt)6531, ATCC13032 / pTOPO_aroG (mt)8316, ATCC13032 / pTOPO_aroG(mt)9426) with the highest L-leucine production ability compared to the wild-type Corynebacterium glutamicum strain (ATCC13032) were selected. The concentrations of L-leucine (Leu), L-tyrosine (Tyr), and L-phenylalanine (Phe) produced by the selected strains are shown in Table 3.

[0138]

Table 3

[0139] As shown in Table 3, it was confirmed that Corynebacterium glutamicum ATCC13032 / pTOPO_aroG(mt)2256 with a mutation in the aroG gene had about 1.4 times improved L-leucine production ability compared to the parent strain Corynebacterium glutamicum ATCC13032. It was confirmed that ATCC13032 / pTOPO_aroG(mt)6531, ATCC13032 / pTOPO_aroG(mt)8316, and ATCC13032 / pTOPO_aroG(mt)9426 each had about 1.4 times improved L-leucine production compared to the parent strain. It was confirmed that all four of ATCC13032 / pTOPO_aroG(mt)2256, ATCC13032 / pTOPO_aroG(mt)6531, ATCC13032 / pTOPO_aroG(mt)8316, and ATCC13032 / pTOPO_aroG(mt)9426 had a decrease in L-tyrosine to 1 / 2.4 to 1 / 8.5 and a decrease in L-phenylalanine to 1 / 3 to 1 / 10.

[0140] Example 2-2. Confirmation of mutations in mutant strains with increased L-leucine production and decreased aromatic amino acid production To confirm the aroG gene mutations in the four selected mutant strains, using the primers of SEQ ID NO: 11 and SEQ ID NO: 12 shown in Table 1, with the DNA of each mutant strain as a template, after denaturation at 94°C for 5 minutes, 30 cycles were performed at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute and 30 seconds, and then PCR was performed under the condition of 72°C for 5 minutes, followed by DNA sequencing.

[0141] As a result of the sequencing, it was confirmed that in the ATCC13032 / pTOPO_aroG(mt)2256 strain, the CGC at nucleotides 649, 650, and 651 of the aroG gene was replaced by GCG. This means that it encodes a mutant in which arginine, the 217th amino acid of aroG aldolase, is replaced by alanine (hereinafter referred to as R217A). The amino acid sequence of the aroG aldolase mutant (R217A) and the nucleotide sequence of the aroG aldolase mutant encoding the same are shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0142] In addition, it was confirmed that in the ATCC13032 / pTOPO_aroG(mt)6531 strain, the AA at the 928th and 929th nucleotides of the aroG gene was substituted with GC. This means that it encodes a mutant in which lysine, the 310th amino acid of aroG aldolase, is substituted with alanine (hereinafter referred to as K310A). The amino acid sequence of the aroG aldolase mutant (K310A) and the nucleotide sequence of the aroG aldolase mutant encoding the same are shown in SEQ ID NOs: 5 and 6.

[0143] In the ATCC13032 / pTOPO_aroG(mt)8316 strain, it was confirmed that the CGC at the 1207th to 1209th nucleotides of the aroG gene was substituted with GCG. This means that it encodes a mutant in which arginine, the 403rd amino acid of aroG aldolase, is substituted with alanine (hereinafter referred to as R403A). The amino acid sequence of the aroG aldolase mutant (R403A) and the nucleotide sequence of the aroG aldolase mutant encoding the same are shown in SEQ ID NOs: 7 and 8.

[0144] In the ATCC13032 / pTOPO_aroG(mt)9426 strain, it was confirmed that the AA at the 1385th and 1386th nucleotides of the DAHP gene was substituted with CG. This means that it encodes a mutant in which glutamic acid, the 462nd amino acid of aroG aldolase, is substituted with alanine (hereinafter referred to as E462A). The amino acid sequence of the aroG aldolase mutant (E462A) and the nucleotide sequence of the aroG aldolase mutant encoding the same are shown in SEQ ID NOs: 9 and 10.

[0145] In the following examples, it is confirmed whether the above mutations (R217A, K310A, R403A, E462A) affect the production of L-leucine and aromatic amino acids in Corynebacterium microorganisms.

Example

[0146] Confirmation of the ability of the selected mutant strain to produce L-leucine, L-tyrosine, and L-phenylalanine Example 3-1. Preparation of an insertion vector containing an aroG aldolase mutation An insertion vector was prepared to introduce the mutation selected in Example 2 into the strain. For the preparation of the vector for introducing the aroG(R217A,K310A,R403A,E462A) mutation, the site-directed mutagenesis method was used. Using the chromosome of Corynebacterium glutamicum ATCC13032 wild type as a template, to generate the R217A mutation, the primer pairs of SEQ ID NO: 13 and 14, and the primer pairs of SEQ ID NO: 15 and 16 were used, and to generate the K310A mutation, the primer pairs of SEQ ID NO: 13 and 17, and the primer pairs of SEQ ID NO: 15 and 18 were used for PCR. To generate the R403A mutation, the primer pairs of SEQ ID NO: 13 and 19, and the primer pairs of SEQ ID NO: 15 and 20 were used, and to generate the E462A mutation, the primer pairs of SEQ ID NO: 13 and 21, and the primer pairs of SEQ ID NO: 15 and 22 were used for PCR. Specifically, after denaturation at 94°C for 5 minutes, 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute 30 seconds were performed, and then PCR was performed under the condition of 72°C for 5 minutes. The specific sequences of the primers used are shown in Table 4.

[0147]

Table 4

[0148] The resulting PCR products were cloned by fusing the 15-base homologous sequences at the ends between DNA fragments using a linear pDCM2 vector (Patent Document 5) cleaved with SmaI restriction enzyme and In-Fusion enzyme, to prepare vectors "pDCM2-aroG(R217A)", "pDCM2-aroG(K310A)", "pDCM2-aroG(R403A)" and "pDCM2-aroG(E462A)" that substitute the amino acids of aroG. Further, by combining the mutants, vectors "pDCM2-aroG(R217A,K310A,E462A)" and "pDCM2-aroG(R217A,K310A,R403A,E462A)" that substitute the 217th, 310th, 403rd and 462nd amino acids of aroG were prepared. The amino acid sequence of the aroG aldolase mutant (R217A,K310A,E462A) and the nucleotide sequence of the aroG aldolase mutant encoding the same are shown in SEQ ID NOs: 23 and 24. Further, the amino acid sequence of the aroG aldolase mutant (R217A,K310A,R403A,E462A) and the nucleotide sequence of the aroG aldolase mutant encoding the same are shown in SEQ ID NOs: 25 and 26.

[0149] Example 3-2. Introduction and Evaluation of Mutants into ATCC 13032 Strain The pDCM2-aroG(R217A), pDCM2-aroG(K310A), pDCM2-aroG(R403A), pDCM2-aroG(E462A), pDCM2-aroG(R217A,K310A,E462A), and pDCM2-aroG(R217A,K310A,R403A,E462A) vectors prepared in Example 3-1 were transformed into ATCC 13032 by electroporation, and strains in which the vector was inserted into the chromosome by recombination of homologous sequences were selected from a medium containing 25 mg / L of kanamycin. The selected primary strains were further subjected to a secondary cross-over to select strains into which mutations in the target gene were introduced. Whether or not the aroG gene mutation was introduced into the finally transformed strains was determined by performing PCR using the primers of SEQ ID NO: 11 and SEQ ID NO: 12 shown in Table 1 and then analyzing the nucleotide sequences, and it was confirmed that mutations were introduced into the strains. A total of 5 types of strains were prepared and named ATCC13032_aroG_R217A, ATCC13032_aroG_K310A, ATCC13032_aroG_R403A, ATCC13032_aroG_E462A, ATCC13032_aroG_(R217A,K310A,E462A), and ATCC13032_aroG_(R217A,K310A,R403A,E462A).

[0150] To evaluate the L-leucine and aromatic amino acid production capabilities of all six strains prepared as described above, flask fermentation titer evaluations were conducted. In 250 ml corner baffle flasks each containing 25 ml of production medium, the parent strain Corynebacterium glutamicum ATCC13032, and ATCC13032_aroG_R217A, ATCC13032_aroG_K310A, ATCC13032_aroG_R403A, ATCC13032_aroG_E462A, ATCC13032_aroG_(R217A,K310A,E462A), ATCC13032_aroG_(R217A,K310A,R403A,E462A) prepared as described above were each inoculated with one platinum loop, and then cultured with shaking at 30 °C and 200 rpm for 60 hours to produce L-leucine. After the cultivation was completed, the production amounts of L-leucine, L-tyrosine and L-phenylalanine were measured by HPLC. The leucine concentrations in the culture solutions of each strain used in the experiment are shown in Table 5.

[0151]

Table 5

[0152] As shown in Table 5, it was confirmed that ATCC13032_aroG_R217A, ATCC13032_aroG_K310A, ATCC13032_aroG_R403A, ATCC13032_aroG_E462A, ATCC13032_aroG_(R217A,K310A,E462A), ATCC13032_aroG_(R217A,K310A,R403A,E462A) had a yield of L-leucine improved by about 1.35 to 1.55 times compared to the parent strain Corynebacterium glutamicum ATCC13032. Also, it was confirmed that the yield of L-tyrosine decreased to 1 / 2.5 to 1 / 8.5, and L-phenylalanine decreased to about 1 / 3.5 to 1 / 14.

Example

[0153] Confirmation of the ability of aroG selected mutants in leucine-producing strains to produce leucine, tyrosine, and phenylalanine Wild-type strains of the genus Corynebacterium produce only trace amounts of leucine, if any. Therefore, an experiment was conducted to prepare a leucine-producing strain derived from ATCC 13032, introduce the selected mutations, and confirm the ability to produce leucine, tyrosine, and phenylalanine. Specifically, it was as follows.

[0154] Example 4-1. Preparation of L-leucine-producing strain CJL-8109 strain As a strain for high-concentration L-leucine production, a strain derived from ATCC 13032 containing the following mutations was prepared: (1) a mutation (R558H) in which the G at nucleotide position 1673 of the leuA gene is replaced by A, and the arginine at amino acid position 558 of the LeuA protein is replaced by histidine; (2) a mutation (G561D) in which the GC at nucleotide positions 1682 and 1683 of the leuA gene is replaced by AT, and the glycine at amino acid position 561 is replaced by aspartic acid; and (3) a mutation (P247C) in which the CC at nucleotide positions 739 and 740 of the leuA gene is replaced by TG, and the proline at amino acid position 247 is replaced by cysteine.

[0155] Specifically, the pDCM2-leuA(P247C,R558H,G561D) vector containing the leuA gene mutation was transformed into Corynebacterium glutamicum ATCC13032 by electroporation, and a strain in which the vector was inserted into the chromosome by recombination of homologous sequences was selected from a medium containing 25 mg / L of kanamycin. The selected primary strain was further subjected to a secondary cross-over to select a strain into which the leuA gene mutation was introduced. Whether the mutation was introduced into the finally transformed strain was determined by performing PCR (5 minutes at 94°C, then 30 cycles of 30 seconds at 94°C / 30 seconds at 55°C / 90 seconds at 72°C, and then 5 minutes at 72°C) using the primers of SEQ ID NO: 27 and SEQ ID NO: 28 in Table 6 and analyzing the nucleotide sequence. It was confirmed that the P247C, R558H, and G561D mutations were introduced. The ATCC13032_leuA_(P247C,R558H,G561D) strain transformed with the pDCM2-leuA(P247C,R558H,G561D) vector was named "CJL-8105".

[0156]

Table 6

[0157] In order to improve the L-leucine-producing ability of the prepared CJL-8105 strain, a strain was prepared by introducing the ilvE mutant (V156A), which is a gene encoding branched-chain amino acid aminotransferase (Patent Document 6). Specifically, the pDCM2-ilvE(V156A) vector containing the ilvE gene mutation was transformed into Corynebacterium glutamicum CJL-8105 by electroporation, and a strain in which the vector was inserted into the chromosome by recombination of homologous sequences was selected from a medium containing 25 mg / L of kanamycin. The selected primary strain was further subjected to a secondary cross-over to select a strain into which the ilvE gene mutation was introduced. Whether or not the mutation was introduced into the finally transformed strain was determined by performing PCR (performed at 94°C for 5 minutes, then at 94°C for 30 seconds / 55°C for 30 seconds / 72°C for 90 seconds for 30 cycles, and then at 72°C for 5 minutes) using the primers of SEQ ID NO: 29 and SEQ ID NO: 30 in Table 7 and analyzing the nucleotide sequence. It was confirmed that the V156A mutation was introduced. The strain transformed with the pDCM2-ilvE(V156A) vector was named "CJL-8108".

[0158]

Table 7

[0159] In order to improve the L-leucine production ability in the prepared CJL-8108 strain, a strain was prepared by introducing a gltA mutant (M312I; SEQ ID NO: 41) with weakened citrate synthase activity. Specifically, the pDCM2-gltA(M312I) vector containing the gltA gene mutation was transformed into Corynebacterium glutamicum CJL-8108 by electroporation, and a strain in which the vector was inserted into the chromosome by recombination of homologous sequences was selected from a medium containing 25 mg / L of kanamycin. The selected primary strain was further subjected to a secondary cross-over to select a strain into which the gltA gene mutation was introduced. Whether or not the mutation was introduced into the finally transformed strain was determined by performing PCR (5 minutes at 94°C, then 30 cycles of 30 seconds at 94°C / 30 seconds at 55°C / 90 seconds at 72°C, and then 5 minutes at 72°C) using the primers of SEQ ID NO: 31 and SEQ ID NO: 32 in Table 8 and analyzing the nucleotide sequence. It was confirmed that the M312I mutation was introduced. The strain transformed with the pDCM2-gltA(M312I) vector was named "CJL-8109".

[0160]

Table 8

[0161] Example 4-2. Introduction and evaluation of aroG aldolase mutants into the CJL-8109 strain The leucine-producing strain CJL-8109 was transformed with the pDCM2-aroG(R217A), pDCM2-aroG(K310A), pDCM2-aroG(R403A), pDCM2-aroG(E462A), pDCM2-aroG(R217A,K310A,E462A), and pDCM2-aroG(R217A,K310A,R403A,E462A) vectors prepared in Example 3-1. Strains in which the vector was inserted into the chromosome by recombination of homologous sequences were selected from a medium containing 25 mg / L of kanamycin. The selected primary strains were further subjected to a secondary cross-over to select strains into which mutations in the target gene were introduced. Whether or not the aroG gene mutation was introduced into the finally transformed strain was determined by performing PCR using the primers of SEQ ID NO: 11 and SEQ ID NO: 12 and then analyzing the nucleotide sequence. It was confirmed that the aroG aldolase mutation was introduced into the strain. The prepared CJL-8109_aroG_R217A was named CJL-8110, CJL-8109_aroG_K310A was named CJL-8111, CJL-8109_aroG_R403A was named CJL-8112, CJL-8109_aroG_E462A was named CJL-8113, CJL-8109_aroG_(R217A,K310A,E462A) was named CA13-8114, and CJL-8109_aroG_(R217A,K310A,R403A,E462A) was named CJL-8115.

[0162] The leucine-producing abilities of the CJL-8110, CJL-8111, CJL-8112, CJL-8113, CA13-8114, CJL-8115, and ATCC13032, and CJL-8109 strains prepared as described above were evaluated. Flask culture was performed in the same manner as in Example 2. After the culture was completed, the production amounts of leucine and aromatic amino acids were measured by a method using HPLC. The culture results are shown in Table 9.

[0163]

Table 9

[0164] As shown in Table 9, Corynebacterium glutamicum CJL-8110, CJL-8111, CJL-8112, CJL-8113, CA13-8114, and CJL-8115, which are L-leucine-producing strains further containing R217A, K310A, R403A, and E462A mutations in the aroG gene, were confirmed to have about 4 to 5 times higher L-leucine production ability compared to the parent strain Corynebacterium glutamicum ATCC13032. In addition, Corynebacterium glutamicum CJL-8110, CJL-8111, CJL-8112, CJL-8113, CA13-8114, and CJL-8115, which are L-leucine-producing strains, were confirmed to have about 1.2 to 1.6 times higher L-leucine production ability and about 1 / 10 to 1 / 20 lower L-tyrosine and L-phenylalanine compared to the parent strain Corynebacterium glutamicum CJL-8109. From these results, it was confirmed that the amino acids at positions 217, 310, 403, and 462 in the amino acid sequence of aroG aldolase are important positions for L-leucine production and the production of aromatic amino acids L-tyrosine and L-phenylalanine.

[0165] The strain CA13-8114 prepared as described above was internationally deposited with the Korean Culture Center of Microorganisms (KCCM), an international depository authority under the Budapest Treaty, under the deposit number KCCM12931P on January 18, 2021.

Example

[0166] Confirmation of the ability to produce leucine, tyrosine, and phenylalanine of aroG selected mutations in isoleucine-producing strains To confirm whether the selected mutations also have an effect on isoleucine, which is a typical branched-chain amino acid like leucine, an experiment was conducted to introduce them into Corynebacterium genus isoleucine-producing strains and confirm the isoleucine production ability. Specifically, it is as follows.

[0167] Example 5-1. Preparation of L-isoleucine-producing strain CA10-3101 An L-isoleucine-producing strain was developed from the wild-type Corynebacterium glutamicum ATCC13032. Specifically, in order to relieve the feedback inhibition of threonine, which is a precursor of isoleucine in the biosynthetic pathway, arginine, the 407th amino acid of the hom gene encoding homoserine dehydrogenase, was replaced with histidine (Patent Document 7). More specifically, in order to prepare a strain into which the hom(R407H) mutation was introduced, the chromosome of Corynebacterium glutamicum ATCC13032 was used as a template, and PCR was performed using the primers of SEQ ID NO: 33 and SEQ ID NO: 34, or SEQ ID NO: 35 and SEQ ID NO: 36, respectively. The primer sequences used here are shown in Table 10.

[0168]

Table 10

[0169] As the polymerase for the PCR reaction, PfuUltraTM High-Fidelity DNA Polymerase (Stratagene) was used, and the PCR conditions were set to perform 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization reaction at 72°C for 1 minute. As a result, a 1000 bp DNA fragment upstream of the 5' end and a 1000 bp DNA fragment downstream of the 3' end were obtained, each centered on the mutation of the hom gene. Using the two amplified DNA fragments as templates, PCR was performed with the primers of SEQ ID NO: 34 and SEQ ID NO: 35. The PCR conditions were set to perform 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 after denaturation at 95°C for 5 minutes, and then a polymerization reaction at 72°C for 5 minutes.

[0170] As a result, a 2-kb DNA fragment containing a mutation in the hom gene encoding a homoserine dehydrogenase mutant in which the 407th arginine was replaced with histidine was amplified. The amplification product was purified using a PCR Purification kit (QUIAGEN) and used as an insert DNA fragment for vector construction. The purified amplification product was treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes. Using an Infusion Cloning Kit (TaKaRa), according to the provided manual, a vector pDCM2-R407H for introducing the hom(R407H) mutation onto the chromosome was constructed by cloning such that the molar concentration (M) ratio of the pDCM2 vector treated as above to the insert DNA fragment which was the amplification product was 1:2.

[0171] The constructed vector was transformed into Corynebacterium glutamicum ATCC13032 by electroporation, and through a secondary crossing process, a strain containing the hom(R407H) mutation on the chromosome was obtained. This was named Corynebacterium glutamicum ATCC13032 hom(R407H).

[0172] To relieve feedback inhibition and improve the activity against L-isoleucine, a strain was constructed by introducing the ilvA mutant (T381A, F383A), which is a gene encoding L-threonine dehydratase, into the constructed ATCC13032 hom(R407H) strain. More specifically, to construct a strain into which the ilvA(T381A, F383A) mutation was introduced, PCR was performed using the chromosome of Corynebacterium glutamicum ATCC13032 as a template and the primers of SEQ ID NO: 37 and SEQ ID NO: 38, or SEQ ID NO: 39 and SEQ ID NO: 40, respectively. The primer sequences used here are shown in Table 11.

[0173]

Table 11

[0174] As the polymerase for the PCR reaction, PfuUltraTM High Fidelity DNA Polymerase (Stratagene) was used, and the PCR conditions were set to perform 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization reaction at 72°C for 1 minute. As a result, a 1126 bp DNA fragment upstream of the 5' end and a 286 bp DNA fragment downstream of the 3' end were obtained, mainly centered on the mutation of the ilvA gene. Using the two amplified DNA fragments as templates, PCR was performed with the primers of SEQ ID NO: 37 and SEQ ID NO: 40. The PCR conditions were as follows: after denaturation at 95°C for 5 minutes, 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 2 minutes, and then a polymerization reaction at 72°C for 5 minutes.

[0175] As a result, a 1.4 kb DNA fragment containing a mutation in the ilvA gene encoding a threonine dehydratase mutant in which the 381st threonine was substituted with alanine and the 383rd phenylalanine was substituted with alanine was amplified. The amplification product was purified using a PCR purification kit (PCR Purification kit, QAIGEN) and used as an inserted DNA fragment for vector construction. The purified amplification product was treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes. Using an Infusion Cloning Kit (TaKaRa) according to the provided manual, a vector pDCM2-ilvA(T381A,F383A) for introducing the ilvA(T381A,F383A) mutation onto the chromosome was constructed by cloning such that the molar concentration (M) ratio of the pDCM2 vector after treatment and heat treatment to the inserted DNA fragment, which was the amplification product, was 1:2. The constructed vector was transformed into Corynebacterium glutamicum ATCC13032 hom(R407H) by electroporation, and through a secondary crossover process, a strain containing the ilvA(T381A,F383A) mutation on the chromosome was obtained. This was named Corynebacterium glutamicum CA10-3101.

[0176] Example 5-2. Introduction and evaluation of aroG aldolase mutants into strain CA10-3101 The L-isoleucine-producing strain CA10-3101 was transformed with the pDCM2-aroG(R217A), pDCM2-aroG(K310A), pDCM2-aroG(R403A), pDCM2-aroG(E462A), pDCM2-aroG(R217A,K310A,E462A), and pDCM2-aroG(R217A,K310A,R403A,E462A) vectors prepared in Example 3-1. Strains in which the vector was inserted into the chromosome by homologous sequence recombination were selected from a medium containing 25 mg / L of kanamycin. The selected primary strains were further subjected to a secondary cross-over to select strains into which mutations in the target gene were introduced. Whether or not the aroG gene mutation was introduced into the finally transformed strain was determined by performing PCR using the primers of SEQ ID NO: 11 and SEQ ID NO: 12 and then analyzing the nucleotide sequence. It was confirmed that the aroG aldolase mutation was introduced into the strain.

[0177] The prepared strains were named CA10-3101_aroG_R217A, CA10-3101_aroG_K310A, CA10-3101_aroG_R403A, CA10-3101_aroG_E462A, CA10-3101_aroG_(R217A,K310A,E462A), and CA10-3101_aroG_(R217A,K310A,R403A,E462A).

[0178] As described above, the L-isoleucine-producing abilities of CA10-3101_aroG_R217A, CA10-3101_aroG_K310A, CA10-3101_aroG_R403A, CA10-3101_aroG_E462A, CA10-3101_aroG_(R217A,K310A,E462A), CA10-3101_aroG_(R217A,K310A,R403A,E462A), and ATCC13032, and the CA10-3101 strain were evaluated. A 250-ml Erlenmeyer flask containing 25 ml of isoleucine production medium was inoculated with the parental strain and the above aroG aldolase mutant strains, and then cultured with shaking at 32 °C and 200 rpm for 60 hours to produce L-isoleucine.

[0179] The composition of the production medium used in this example is as follows. <Production Medium> Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, ferrous sulfate heptahydrate 10 mg / l, manganese sulfate monohydrate 10 mg / l, biotin 200 μg / l, pH 7.2

[0180] After the cultivation was completed, the production amounts of L-isoleucine and by-products were measured using high-performance liquid chromatography (HPLC). The concentrations of L-isoleucine and by-products in the culture broth of each strain tested are shown in Table 12.

[0181]

Table 12

[0182] As shown in Table 12, the L-isoleucine-producing strains further containing the R217A, K310A, R403A, and E462A mutations in the aroG gene had about 1.1 to 1.2 times improved L-isoleucine-producing ability compared to the parental strain Corynebacterium glutamicum CA10-3101, and it was confirmed that L-tyrosine and L-phenylalanine decreased.

[0183] From these results, it is confirmed that the amino acids at positions 217, 310, 403, and 462 in the amino acid sequence of aroG aldolase are important positions for the production of L-isoleucine and the production of the aromatic amino acids L-tyrosine and L-phenylalanine.

Example

[0184] Confirmation of the valine-producing ability of aroG selected mutations in a valine-producing strain To confirm whether the selected mutations also have an effect on L-valine, which is a typical branched-chain amino acid like leucine, an experiment was conducted to introduce the selected mutations into KCCM11201P, a valine-producing strain of the genus Corynebacterium, and confirm its valine-producing ability. Specifically, it was as follows.

[0185] Example 6-1. Introduction and evaluation of aroG aldolase mutants into Corynebacterium glutamicum KCCM11201P strain To confirm whether the mutation is effective in improving valine production ability, Corynebacterium glutamicum KCCM11201P (Patent Document 8) strain, which is an L-valine producing strain, was used. KCCM11201P, a valine producing strain, was transformed with the pDCM2-aroG(R217A), pDCM2-aroG(K310A), pDCM2-aroG(R403A), pDCM2-aroG(E462A), pDCM2-aroG(R217A,K310A,E462A), and pDCM2-aroG(R217A,K310A,R403A,E462A) vectors prepared in Example 3-1, and strains in which the vector was inserted into the chromosome by recombination of homologous sequences were selected from a medium containing 25 mg / L of kanamycin. The selected primary strains were further subjected to a secondary cross-over to select strains into which the mutation of the target gene was introduced. Finally, whether the aroG gene mutation was introduced into the transformed strain was determined by performing PCR using the primers of SEQ ID NO: 11 and SEQ ID NO: 12 and then analyzing the nucleotide sequence, and it was confirmed that the aroG aldolase mutation was introduced into the strain. The prepared strains were named KCCM11201P-aroG(R217A), KCCM11201P-aroG(K310A), KCCM11201P-aroG(R403A), KCCM11201P-aroG(E462A), KCCM11201P-aroG(R217A,K310A,E462A), and KCCM11201P-aroG(R217A,K310A,R403A,E462A), respectively.

[0186] The valine-producing abilities of the Corynebacterium glutamicum KCCM11201P-aroG(R217A), KCCM11201P-aroG(K310A), KCCM11201P-aroG(R403A), KCCM11201P-aroG(E462A), KCCM11201P-aroG(R217A,K310A,E462A), and KCCM11201P-aroG(R217A,K310A,R403A,E462A) strains prepared as described above were evaluated. Flask culture was performed in the same manner as in Example 2, and after completion of the culture, the valine production amount was measured by a method using HPLC. The culture results are shown in Table 13.

[0187] [Table 13]

[0188] As shown in Table 13, the Corynebacterium glutamicum KCCM11201P-aroG(R217A), KCCM11201P-aroG(K310A), KCCM11201P-aroG(R403A), KCCM11201P-aroG(E462A), KCCM11201P-aroG(R217A,K310A,E462A), and KCCM11201P-aroG(R217A,K310A,R403A,E462A) strains, which are L-valine-producing strains further containing the R217A, K310A, R403A, and E462A mutations in the aroG gene, had a valine-producing ability improved by up to 1.11-fold compared to the parent strain Corynebacterium glutamicum KCCM11201P, and it was confirmed that L-tyrosine and L-phenylalanine decreased to about 1 / 10 to 1 / 20.

[0189] From these results, it was confirmed that the amino acids at positions 217, 310, 403, and 462 in the amino acid sequence of the aroG aldolase are important positions for L-valine production and the production of the aromatic amino acids L-tyrosine and L-phenylalanine.

[0190] Reference Example 1: Confirmation of the effect of the gltA (M312I) mutation on leucine production Reference Example 1-1. Preparation of an insertion vector containing the gltA mutation For the preparation of a vector for introducing the gltA (M312I; SEQ ID NO: 41) mutation, the site-directed mutagenesis method was used.

[0191] Using the chromosome of wild-type Corynebacterium glutamicum as a template, PCR was performed using the primer pairs of SEQ ID NOs: 43 and 44, and the primer pairs of SEQ ID NOs: 45 and 46. PCR was carried out with denaturation at 94°C for 5 minutes, followed by 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute and 30 seconds, and then a polymerization reaction at 72°C for 5 minutes. The resulting gene fragment was cloned by ligating the 15-base homologous sequences at the ends between DNA fragments using a linear pDCM2 vector cleaved with SmaI restriction enzyme and In-Fusion enzyme, and a vector pDCM2-gltA (M312I) was prepared that substitutes isoleucine for methionine, which is the 312th amino acid.

[0192]

Table 14

[0193] Reference Example 1-2. Introduction and evaluation of the mutant into the ATCC13032 strain The pDCM2-gltA (M312I) vector prepared in Reference Example 1-1 was transformed into wild-type ATCC13032, and a strain in which the vector was inserted into the chromosome by homologous sequence recombination was selected from a medium containing 25 mg / L of kanamycin. The selected primary strain was further subjected to a secondary cross-over to select a strain into which the mutation of the target gene was introduced. Whether the gltA gene mutation was introduced into the finally transformed strain was determined by performing PCR using the primers of SEQ ID NOs: 31 and 32 (Example 4-1, Table 8) and then analyzing the nucleotide sequence. It was confirmed that the mutation (SEQ ID NO: 42) was introduced into the strain. The prepared strain was named ATCC13032_gltA_M312I.

[0194] To evaluate the leucine production ability of the ATCC13032_gltA_M312I strain prepared as described above, flask fermentation titer evaluation was performed. In 250 ml corner baffle flasks each containing 25 ml of the production medium, 1 platinum loop of the parent strain Corynebacterium glutamicum ATCC13032 and the ATCC13032_gltA_M312I prepared as described above were each inoculated, and then cultured with shaking at 30 °C and 200 rpm for 60 hours to produce leucine. After the culture was completed, the production amount of leucine was measured by HPLC. The leucine concentration in the culture broth of each strain in which the experiment was conducted is shown in Table 15. · Production medium: Glucose 100 g, (NH 4 ) 2 SO 4 40 g, Soy Protein 2.5 g, Corn Steep Solids 5 g, Urea 3 g, KH 2 PO 4 1 g, MgSO 4 ·7H 2 O 0.5 g, Biotin 100 μg, Thiamine hydrochloride 1000 μg, Calcium pantothenate 2000 μg, Nicotinamide 3000 μg, CaCO 3 30 g (in 1 liter of distilled water), pH 7.0

[0195]

Table 15

[0196] From these results, it was confirmed that the M312I substitution of gltA is a mutation effective for increasing leucine production.

[0197] Reference Example 2: Confirmation of the effect of ilvA (T381A, F383A) mutation on isoleucine production Reference Example 2-1: Preparation of pECCG117-ilvA(F383A) To amplify the ilvA (SEQ ID NO: 48) gene encoding threonine dehydratase (SEQ ID NO: 47), BamHI restriction enzyme sites were inserted at both ends of primers (SEQ ID NO: 49 and SEQ ID NO: 50) for amplifying from the promoter site (about 300 bp upstream of the start codon) to the terminator site (about 100 bp downstream of the stop codon) based on the ilvA sequence (Non-Patent Document 18) into which the known F383A mutation was introduced. Also, primers (SEQ ID NO: 51 and SEQ ID NO: 52) for introducing the F383A mutation into ilvA were used. The primer sequences used here are shown in Table 16.

[0198]

Table 16

[0199] Using the chromosome of wild type Corynebacterium glutamicum ATCC13032 as a template, PCR was performed using the primers of SEQ ID NO: 49 and SEQ ID NO: 52, and SEQ ID NO: 50 and SEQ ID NO: 51. The PCR conditions were as follows: after denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 90 seconds were performed, and then a polymerization reaction at 72°C for 5 minutes was carried out.

[0200] As a result, a 1460 bp DNA fragment upstream of the 5' end and a 276 bp DNA fragment downstream of the 3' end were obtained, mainly centered on the mutation of the ilvA gene.

[0201] Using the two amplified DNA fragments as templates, PCR was performed with the primers of SEQ ID NO: 49 and SEQ ID NO: 50.

[0202] As a result, a 1,531-bp DNA fragment containing an ilvA mutation in which the 383rd phenylalanine was replaced with alanine was amplified. The pECCG117 (Patent Document 9) vector and the ilvA DNA fragment were treated with the restriction enzyme BamHI, ligated using DNA ligase, and then cloned to obtain a plasmid. This was named pECCG117-ilvA(F383A).

[0203] Reference Example 2-2: Additional Introduction of Random Mutations into pECCG117-ilvA(F383A) To obtain a mutant of the gene encoding L-threonine dehydratase, a random mutagenesis kit (Agilent Technologies, USA) was used to prepare an ilvA mutant gene plasmid. Using the ilvA(F383A) chromosome of Reference Example 2-1 as a template, PCR was performed using the primers of SEQ ID NO: 49 and SEQ ID NO: 50. The PCR conditions were as follows: After denaturation at 95°C for 2 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 90 seconds were performed, followed by a polymerization reaction at 72°C for 10 minutes.

[0204] As a result, in addition to the mutation in which the 383rd phenylalanine was replaced with alanine, a 1,531-bp DNA fragment, which is an ilvA mutant encoding L-threonine dehydratase having additional random mutations, was amplified. The pECCG117 vector and the ilvA mutant DNA fragment were treated with the restriction enzyme BamHI, ligated using DNA ligase, and then cloned to obtain a group of plasmids.

[0205] Reference Example 2-3: Preparation of CJILE-301 Strain pECCG117-ilvA(F383A) was introduced into Corynebacterium glutamicum ATCC13032 hom(R407H) strain. The strain into which the prepared plasmid was introduced was named ATCC13032 hom(R407H) / pECCG117-ilvA(F383A). In addition, the group of mutant plasmids obtained in Reference Example 2-2 was introduced into Corynebacterium glutamicum ATCC13032 hom(R407H) strain, smeared on minimal medium, and the mortality rate was measured. As a result, the mortality rate was 70%. The surviving cells were inoculated into seed medium and cultured, and finally, a mutant strain showing excellent isoleucine-producing ability compared to the ATCC13032 hom(R407H) / pECCG117-ilvA(F383A) control group was selected and named Corynebacterium glutamicum CJILE-301.

[0206] When the plasmid was isolated from CJILE-301 strain and the ilvA gene was sequenced, it was confirmed that in addition to the substitution of the 1141st base sequence of the ilvA gene from A to G and the substitution of the 383rd F of the ilvA protein to A, it also encoded a mutant protein in which the 381st T was substituted with A. This is shown in SEQ ID NO: 54.

[0207] Reference Example 2-4: Introduction of ilvA Mutant (T381A,F383A) To introduce the ilvA mutant (T381A,F383A) into the wild-type strain, primers of SEQ ID NO: 49 and SEQ ID NO: 52 (Example 5-1, Table 16) were prepared.

[0208] To prepare a strain into which the ilvA mutant (T381A,F383A) was introduced, PCR was performed using the plasmid DNA extracted from CJILE-301 strain as a template and the primers of SEQ ID NO: 49 and SEQ ID NO: 52.

[0209] As the polymerase for the PCR reaction, PfuUltraTM High-Fidelity DNA Polymerase (Stratagene) was used. The PCR conditions were set to perform 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization reaction at 72°C for 2 minutes.

[0210] As a result, 1411-bp gene fragments each containing approximately 100-bp terminator sites of the 1311-bp ilvA gene were obtained.

[0211] The amplified products were purified using a PCR purification kit and used as inserted DNA fragments for vector construction. The purified amplified products were treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes. Using an Infusion cloning kit, according to the provided manual, cloning was performed such that the molar concentration (M) ratio of the pDCM2 vector to the inserted DNA fragment, which was the amplified product, was 1:2, thereby constructing a vector pDCM2-T381A_F383A for introducing the T381A and F383A mutations onto the chromosome.

[0212] The constructed vector was transformed into Corynebacterium glutamicum ATCC13032 hom(R407H) by electroporation. Through a secondary crossing process, a strain containing the ilvA(T381A,F383A; SEQ ID NO: 53) mutation on the chromosome was obtained. This was named CA10-3101.

[0213] The strain CA10-3101 was internationally deposited with the Korea Culture Center of Microorganisms (KCCM), an international depository authority under the Budapest Treaty, under the deposit number KCCM12739P on May 27, 2020.

[0214] The KCCM12739P strain was inoculated into a 250 ml Erlenmeyer flask containing 25 ml of the isoleucine production medium, and then cultured with shaking at 32 °C and 200 rpm for 60 hours to produce L-isoleucine. The composition of the production medium used was as follows. <Production Medium> Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, ferrous sulfate heptahydrate 10 mg / L, manganese sulfate monohydrate 10 mg / L, biotin 200 μg / L, pH 7.2

[0215] After the cultivation was completed, the concentrations of L-isoleucine and L-threonine in the culture broth were measured using high-performance liquid chromatography (HPLC). The results are shown in Table 17.

[0216]

Table 17

[0217] As shown in Table 17, Corynebacterium glutamicum ATCC13032 hom(R407H), the parental strain, does not produce L-isoleucine, but the ATCC13032 hom(R407H) ilvA(T381A,F383A) mutant produces L-isoleucine at a concentration of 3.9 g / L, and it was confirmed that the L-isoleucine productivity was significantly improved compared to the parental strain.

[0218] From these results, it was confirmed that the ilvA(T381A,F383A) mutation is an effective mutation for increasing isoleucine production.

[0219] Reference Example 3: Confirmation of the effect of leuA(P247C, R558H, G561D) on leucine production Reference Example 3-1. Preparation of CJL-8100 strain The pDCM2-leuA(R558H, G561D) vector containing the leuA gene mutation disclosed in Patent Document 10 was transformed into Corynebacterium glutamicum ATCC13032 by electroporation, and a strain in which the vector was inserted into the chromosome by recombination of homologous sequences was selected from a medium containing 25 mg / L of kanamycin. The selected primary strain was further subjected to a secondary cross-over to select a strain into which the leuA gene mutation was introduced. Whether or not the mutation was introduced into the finally transformed strain was determined by performing PCR (5 minutes at 94°C, then 30 cycles of 30 seconds at 94°C / 30 seconds at 55°C / 90 seconds at 72°C, and then 5 minutes at 72°C) using the primers of SEQ ID NO: 55 and SEQ ID NO: 56 and analyzing the nucleotide sequence. It was confirmed that the R558H and G561D mutations were introduced. The ATCC13032_leuA_(R558H, G561D) strain transformed with the pDCM2-leuA(R558H, G561D) vector was named "CJL-8100".

[0220] The primer sequences used in Reference Example 3 are shown in Table 18 below.

[0221]

Table 18

[0222] Reference Example 3-2. Preparation of an insertion vector containing the leuA mutation A vector for introducing the P247C mutation into CJL-8100, an L-leucine-producing strain in which two mutations (R558H, G561D) were introduced into LeuA, was prepared.

[0223] Using the chromosome of CJL-8100 strain as a template, PCR was performed using the primer pairs of SEQ ID NO: 57 and 58, and the primer pairs of SEQ ID NO: 59 and 60. The PCR was carried out with denaturation at 94°C for 5 minutes, followed by 30 cycles of 30 seconds at 94°C, 30 seconds at 55°C, and 1 minute and 30 seconds at 72°C, and then a polymerization reaction at 72°C for 5 minutes. The resulting PCR product was cloned by fusing the 15-base homologous sequences at the ends between DNA fragments using a linear pDCM2 vector cleaved with SmaI restriction enzyme and In-Fusion enzyme, and a leuA mutation encoding a LeuA variant in which arginine, the 558th amino acid in the wild-type strain LeuA amino acid sequence, was substituted with histidine, and glycine, the 561st amino acid, was substituted with aspartic acid, and proline (Pro), the 247th amino acid of LeuA, was substituted with cysteine (Cys), to generate vector pDCM2-leuA(P247C,R558H,G561D).

[0224] Reference Example 3-3. Introduction and Evaluation of LeuA Variant (P247C) into Strain CJL-8100 CJL-8100, a strain capable of producing L-leucine, was transformed with the pDCM2-leuA(P247C,R558H,G561D) vector prepared in Reference Example 3-2, and a strain in which the vector was inserted into the chromosome by recombination of homologous sequences was selected from a medium containing 25 mg / L of kanamycin. The selected primary strain was further subjected to a secondary cross-over to select a strain into which the mutation of the target gene was introduced. Finally, whether or not the leuA gene mutation of the transformed strain was introduced was determined by performing PCR (5 minutes at 94°C, then 30 cycles of 30 seconds at 94°C / 30 seconds at 55°C / 90 seconds at 72°C, and then 5 minutes at 72°C) using the primers of SEQ ID NO: 55 and SEQ ID NO: 61, and then analyzing the nucleotide sequence. As a result of analyzing the nucleotide sequence, G, the 1673rd nucleotide of the leuA gene in the chromosome of the strain, was replaced with A, GC, the 1682nd and 1683rd nucleotides, was replaced with AT, CC, the 739th and 740th nucleotides, was replaced with TG, arginine, the 558th amino acid of the LeuA protein, was replaced with histidine, glycine, the 561st amino acid, was replaced with aspartic acid, and proline (Pro), the 247th amino acid, was replaced with cysteine (Cys). It was confirmed that the leuA mutation encoding the LeuA variant (P247C,R558H,G561D) was introduced into the strain.

[0225] The prepared CJL8100_leuA_P247C was named "CA13-8105" and deposited with the Korean Culture Center of Microorganisms (KCCM), an international depositary authority under the Budapest Treaty, under the deposit number KCCM12709P on April 29, 2020.

[0226] The amino acid sequence of the LeuA variant (P247C,R558H,G561D) containing all three mutations described above and the nucleotide sequence of the leuA variant encoding the same are shown in SEQ ID NO: 62 and SEQ ID NO: 63, respectively.

[0227] The production capabilities of L-leucine of ATCC13032 and the prepared CJL-8100 and CA13-8105 strains were evaluated. Specifically, flask culture was performed in the same manner as in Example 2-1. After the completion of the culture, the L-leucine production amounts of the parent strain and the mutant strains were measured using HPLC. The results are shown in Table 19.

[0228]

Table 19

[0229] As shown in Table 19, Corynebacterium glutamicum CJL8100, which is an L-leucine-producing strain, had its L-leucine production ability improved by approximately 130% compared to the parent strain ATCC13032. The CA13-8105 strain, in which the leuA_P247C mutation was further introduced into the CJL8100 strain, had its L-leucine production ability improved by approximately 150% compared to the parent strain CJL8100.

[0230] From these results, it is confirmed that the leuA(R558H,G561D,P247C) mutation is an effective mutation for increasing leucine production.

[0231] From the above description, those skilled in the technical field to which the present application pertains will understand that the present application can be implemented in other specific forms without changing its technical idea and essential features. It should be understood that the above examples are merely illustrative and not restrictive. The present application should be construed as including all changes and modified forms derived from the meaning and scope of the claims rather than the specification and their equivalent concepts.

Claims

1. An aroG aldolase (Phospho-2-dehydro-3-deoxyheptonate aldolase) variant in which at least one amino acid corresponding to the 217th, 310th, 403rd, and 462nd positions from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine (Ala) and has 90% or more identity with SEQ ID NO:

1.

2. The aroG aldolase variant according to claim 1, which has 99% or more identity with SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 23, or SEQ ID NO:

25.

3. A polynucleotide encoding the aroG aldolase variant according to claim 1 or 2.

4. A vector containing the polynucleotide according to claim 3.

5. A Corynebacterium microorganism comprising at least one of the aroG aldolase variant according to claim 1 or 2, the polynucleotide encoding the aroG aldolase variant, and the vector containing the polynucleotide.

6. The microorganism according to claim 5, which is Corynebacterium glutamicum.

7. A method for producing branched-chain amino acids, comprising the step of culturing the microorganism according to claim 5 in a medium.

8. The method for producing branched-chain amino acids according to claim 7, further comprising the step of recovering the branched-chain amino acids from the microorganism or the medium.

9. The method for producing branched-chain amino acids according to claim 8, wherein the branched-chain amino acid is at least one selected from L-leucine, L-isoleucine, and L-valine.

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