Prephenate dehydratase mutant and method for producing branched-chain amino acids using the same

Abstract

The present application relates to prephenate dehydratase mutants.

Description

[Technical field]

[0001] The present application relates to a prephenate dehydratase mutant and a method for producing branched-chain amino acids using the same. [Background technology]

[0002] L-amino acids are the basic building blocks of proteins and are used as important raw materials for pharmaceuticals, food additives, animal feed, nutrients, insecticides, fungicides, etc. Therefore, industrial production of amino acids has become an economically important industrial process.

[0003] Various researches for efficient amino acid production, for example, efforts are being made to develop microorganisms and fermentation process technologies that can produce amino acids with high efficiency. Specifically, target substance-specific approaches have been developed, 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 (US 9109242 B2, US 8030036 B2), and in addition to these methods, methods for removing genes not involved in amino acid production or genes whose specific functions in amino acid production are unknown are also being used. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] US 9109242 B2 [Patent Document 2] US 8030036 B2 [Patent Document 3] US Patent US 7662943 B2 [Patent Document 4] US Patent US 10584338 B2 [Patent Document 5] US Patent US 10273491 B2 [Patent Document 6] Korean Patent Publication KR10-2020-0136813A [Patent Document 7] Republic of Korea Patent Registration KR10-2143964B1 [Patent Document 8] US2020-0340022A1 [Patent Document 9] US8465962B2 [Patent Document 10] Republic of Korea Patent Registration No. 10-0057684 [Patent Document 11] KR 10-2018-0077008 A [Non-Patent Document]

[0005] [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] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-Patent Document 5] Devereux, J., et al, Nucleic Acids Research 12: 387 (1984) [Non-Patent Document 6] Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990) [Non-Patent Document 7] Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego,1994

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Non-licensed literature 9

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[0006] Branched-chain amino acids (BCAs) are a group of three amino acids: valine, leucine, and isoleucine. They are known to be metabolized mainly in muscles and used as an energy source during activity. Although it is known that BCAs play an important role in maintaining and increasing muscle mass during activity, their use is increasing. [Means for solving the problem]

[0007] An object of the present application is to provide a prephenate dehydratase mutant in which the amino acid corresponding to position 182 from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.

[0008] Another object of the present application is to provide a polynucleotide encoding the above mutant and a vector containing the same.

[0009] Another object of the present application is to provide a Corynebacterium microorganism comprising one or more of the above mutants, polynucleotides and vectors.

[0010] Another object of the present application is to provide a method for producing branched-chain amino acids, comprising the step of culturing the above-mentioned microorganism in a medium. Effect of the Invention

[0011] When the priphenate dehydratase mutant of the present application is used, branched-chain amino acids can be produced in a higher yield than when it is not used. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in this application may be applied to each different description and embodiment. That is, all combinations of various elements disclosed in this application belong to the scope of this application. In addition, the specific description described below is not considered to limit the scope of this application.

[0013] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific aspects of the present application described herein, and such equivalents are intended to be encompassed by this application.

[0014] One aspect of the present application provides a prephenate dehydratase mutant in which the amino acid corresponding to position 182 from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.

[0015] The above-mentioned prephenate dehydratase mutant means a mutant of a polypeptide having prephenate dehydratase activity or prephenate dehydratase in which the amino acid corresponding to position 182 from the N-terminus of prephenate dehydratase in SEQ ID NO: 1 is replaced with another amino acid.

[0016] In the present application, "prephenate dehydratase" is an enzyme capable of catalyzing the following reaction:

[0017] prephenate ⇔ phenylpyruvate + H2O + CO2

[0018] The prephenate dehydratase of the present application may be a prephenate dehydratase or a polypeptide having prephenate dehydratase activity that is modified to produce the prephenate dehydratase variant provided in the present application. Specifically, it may be a naturally occurring polypeptide or wild-type polypeptide, may be a mature polypeptide thereof, or may include a mutant or functional fragment thereof, but is included without limitation as long as it can be the parent of the prephenate dehydratase variant of the present application.

[0019] In the present application, the prephenate dehydratase may be, but is not limited to, the polypeptide of SEQ ID NO: 1. In one embodiment, the prephenate dehydratase 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 is included in the scope of prephenate dehydratase without limitation as long as it has the same or corresponding activity as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0020] The sequence of the prephenate dehydratase of the present application can be obtained from the publicly known database, GenBank of NCBI. Specifically, it may be a polypeptide encoded by the pheA gene, but is not limited thereto.

[0021] In the present application, the term "mutant" refers to a polypeptide in which one or more amino acids are conservatively substituted and / or modified, and which differs from the amino acid sequence of the mutant before the mutation, but maintains its functions or properties. Such a mutant may generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the mutant may be increased, unchanged, or decreased compared to the polypeptide before the mutation. Some mutants may also include mutants in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, have been removed. Other mutants may include mutants in which a portion has been removed from the N- and / or C-terminus of a mature protein. The term "mutant" may be used interchangeably with terms such as mutant, modified, mutant polypeptide, mutant protein, mutation, and mutant (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), and is not limited to these terms as long as they are used in the sense of being mutated.

[0022] In addition, the variant may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the N-terminus of the variant may be conjugated to a signal (or leader) sequence involved in co-translationally or post-translationally protein translocation. The variant may also be conjugated to other sequences or linkers to allow identification, purification, or synthesis.

[0023] The mutant provided in the present application may be a prephenate dehydratase mutant in which the amino acid corresponding to the 182nd position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid, but is not limited thereto.

[0024] The amino acid corresponding to the 182nd position from the N-terminus in the amino acid sequence of SEQ ID NO:1 may be arginine.

[0025] The variants provided in the present application may include, but are not limited to, a substitution of an amino acid other than arginine for the amino acid corresponding to position 182 from the N-terminus in the amino acid sequence of SEQ ID NO:1.

[0026] The "other amino acid" is not limited as long as it is an amino acid different from the amino acid before substitution. On the other hand, when it is expressed in the present application that "a specific amino acid is substituted", it is self-evident that the amino acid before substitution is substituted with an amino acid different from the amino acid before substitution, even if it is not specifically stated that it is substituted with another amino acid.

[0027] In one embodiment, the variant of the present application may be a variant in which the amino acid corresponding to position 182 in the amino acid sequence of SEQ ID NO: 1, which is the reference protein, is replaced with another amino acid among hydrophobic amino acids or aliphatic amino acids.

[0028] Specifically, the mutant may be a mutant in which the amino acid corresponding to position 182 in the amino acid sequence of SEQ ID NO: 1 is replaced with one of hydrophobic (non-polar) amino acids or aliphatic amino acids. The aliphatic amino acid may be, 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 may be, 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.

[0029] In one embodiment, the variant of the present application may be a variant in which the amino acid corresponding to position 182 in the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid in an amino acid sequence having a small size, but is not limited thereto.

[0030] In the present application, the term "small amino acid" includes glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, proline, and asparagine, which are amino acids that are relatively small in size among the 20 amino acids, and specifically means, but is not limited to, glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, and proline, and more specifically may mean, glycine, alanine, valine, leucine, isoleucine, serine, and threonine, and may refer to, for example, alanine, serine, and glycine, but is not limited to these.

[0031] More specifically, the substitution with another amino acid in the variant of the present application may be, but is not limited to, alanine substitution.

[0032] In this application, the term "corresponding to" refers to an amino acid residue at a recited position in a polypeptide, or an amino acid residue that is similar or identical or homologous to a recited residue in a polypeptide. Identifying an amino acid at a corresponding position may be determining the particular amino acid of a sequence that references a particular sequence. As used in this application, a "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0033] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, and based on this, each amino acid residue in the amino acid sequence can be numbered with reference to the numeric position of the corresponding amino acid residue in SEQ ID NO: 1. For example, sequence alignment algorithms such as those described in this application can identify the position of an amino acid relative to a query sequence (also referred to as a "reference sequence"), or the position at which a modification such as a substitution, insertion or deletion occurs.

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

[0035] In one embodiment, the variant of the present application may have 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 the amino acid corresponding to position 182 of SEQ ID NO: 1 may be replaced with another amino acid.

[0036] In one embodiment, the variants 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 to the amino acid sequence set forth in SEQ ID NO:5.

[0037] Specifically, the variant of the present application may have, comprise, consist of, or essentially consist of the amino acid sequence set forth in SEQ ID NO:5.

[0038] As an embodiment, the variant of the present application may include an amino acid sequence in which the amino acid corresponding to position 182 in the amino acid sequence of SEQ ID NO: 1 is alanine, and which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity to the amino acid sequence set forth in SEQ ID NO: 5. It is also obvious that variants having an amino acid sequence in which a part of the sequence is deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application, so long as the amino acid sequence has such homology or identity and exhibits an efficacy corresponding to the variant of the present application.

[0039] For example, the above amino acid sequence may have additions or deletions of sequences at the N-terminus, C-terminus and / or internally that do not change the function of the variant of the present application, naturally occurring mutations, silent mutations or conservative substitutions.

[0040] The term "conservative substitution" refers to the replacement of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0041] In this application, the term "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, which can be expressed as a percentage. The terms homology and identity can often be used interchangeably.

[0042] Sequence homology or identity of conserved polynucleotides or polypeptides may be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences can generally hybridize to all or part of the sequence under moderate or high stringent conditions. Hybridization obviously includes hybridization to polynucleotides containing common codons in polynucleotides or codons that take into account codon degeneracy.

[0043] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity may be determined using known computer algorithms such as the "FASTA" program using default parameters, for example, as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, it may be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later versions) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ET AL.] (1988) SIAM J Applied Math 48: 1073. For example, BLAST or ClustalW from the National Database Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0044] Homology, similarity or identity of polynucleotides or polypeptides may be determined by comparing sequence information using the GAP computer program, e.g., as known in Smith and Waterman, Adv. Appl. Math (1981) 2:482, e.g., Needleman et al. (1970), J Mol Biol. 48:443. In summary, the GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include: (1) a binary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14: 6745 (or the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, gap extension penalty of 0.5); (3) no penalty for end gaps.

[0045] In one embodiment, the variant of the present application may have prephenate dehydratase activity. In one embodiment, the variant of the present application may have an activity of increasing branched-chain amino acid production ability compared to wild-type or non-mutated prephenate dehydratase. In one embodiment, the variant of the present application may have an activity of decreasing the by-product production level of the branched-chain amino acid production pathway compared to wild-type or non-mutated prephenate dehydratase. In one embodiment, the variant of the present application may have an activity that is weakened compared to wild-type or non-mutated prephenate dehydratase. However, the present application is not limited thereto.

[0046] Another aspect of the present application provides a polynucleotide encoding the variant of the present application.

[0047] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and is a DNA or RNA chain of a certain length or more, and more specifically, refers to a polynucleotide fragment encoding the above-mentioned mutant.

[0048] A polynucleotide encoding a variant of the present application may comprise a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 5. As an example of the present application, a polynucleotide of the present application may have or comprise the sequence of SEQ ID NO: 6. Also, a polynucleotide of the present application may consist of or consist essentially of the sequence of SEQ ID NO: 6.

[0049] The polynucleotide of the present application may be modified in various ways in the coding region without changing the amino acid sequence of the variant of the present application, taking into consideration the degeneracy of codons or the codons preferred in the organism in which the variant of the present application is to be expressed. Specifically, the polynucleotide of the present application may have or include a 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 to the sequence of SEQ ID NO: 6, or may consist of or essentially consist 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 to the sequence of SEQ ID NO: 6, but is not limited thereto.

[0050] In this case, in the above-mentioned sequence having homology or identity, the codon encoding the amino acid corresponding to position 182 of SEQ ID NO:6 may be one of the codons encoding alanine.

[0051] Furthermore, the polynucleotide of the present application includes, without limitation, a sequence that can hybridize under stringent conditions with a probe prepared from a known gene sequence, for example, a sequence complementary to the entire or partial polynucleotide sequence of the present application. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, conditions include conditions under which polynucleotides having high homology or identity, such as polynucleotides having a homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, hybridize with each other, and polynucleotides having lower homology or identity than that do not hybridize with each other, or conditions under which washing is performed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions for normal Southern hybridization, which are 60°C, 1 x SSC, 0.1% SDS, specifically 60°C, 0.1 x SSC, 0.1% SDS, more specifically 68°C, 1 x SSC, 0.1% SDS.

[0052] Hybridization requires that two nucleic acids have complementary sequences, even though mismatches between bases are possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application may also include isolated nucleic acid fragments that are complementary to the entire sequence, as well as substantially similar nucleic acid sequences.

[0053] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the above-mentioned hybridization conditions including a hybridization step at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0054] The appropriate stringency for hybridizing such polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables well known in the art (eg, J. Sambrook et al., supra).

[0055] Another aspect of the present application is to provide a vector comprising a polynucleotide of the present application, which may be, but is not limited to, an expression vector for expressing the polynucleotide in a host cell.

[0056] The "vector" of the present application may include a DNA construct comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the target polypeptide in a suitable host. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and decoding. After being transformed into a suitable host cell, the vector may be capable of replicating or functioning independently of the host genome, or may be integrated into the genome itself.

[0057] The vector used in the present application is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage or cosmid vectors, and pDC, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0058] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome through a vector for chromosomal insertion in a cell. The polynucleotide can be inserted into a chromosome by any method known in the art, for example, but not limited to, homologous recombination. A selection marker for checking the presence or absence of insertion into the chromosome may be further included. The selection marker is for selecting cells transformed with the vector, i.e., for checking the presence or absence of insertion of the target nucleic acid molecule, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide may be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or show other expression traits, so that transformed cells can be selected.

[0059] The term "transformation" in this application means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include both, regardless of whether it is inserted and located in the chromosome of the host cell or located extrachromosomally, as long as it can be expressed in the host cell. The polynucleotide may also include DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form as long as it can be introduced and expressed in the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic structure containing all elements necessary for its own expression. The expression cassette may usually include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replicating. The polynucleotide may also be introduced into the host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0060] In addition, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target mutant of the present application.

[0061] Another aspect of the present application provides a Corynebacterium microorganism comprising the mutant of the present application, the polynucleotide of the present application, or the vector of the present application.

[0062] The microorganism of the present application may comprise a mutant polypeptide of the present application, a polynucleotide encoding said polypeptide, or a vector comprising a polynucleotide of the present application.

[0063] In the present application, the term "microorganism" or "strain" includes all wild-type microorganisms and microorganisms that have been genetically modified, either naturally or artificially, and may be a microorganism in which a specific mechanism has been weakened or enhanced by inserting an exogenous gene or by enhancing or inactivating the activity of an endogenous gene, and may also be a microorganism that contains a genetic modification for the production of a desired polypeptide, protein, or product.

[0064] A strain of the present application may be, but is not limited to, a strain comprising one or more of a variant of the present application, a polynucleotide of the present application, and a vector comprising a polynucleotide of the present application; a strain modified to express a variant of the present application or a polynucleotide of the present application; a strain (e.g., a recombinant strain) that expresses a variant of the present application or a polynucleotide of the present application; or a strain (e.g., a recombinant strain) that has an activity of a variant of the present application.

[0065] The strain of the present application may be a strain capable of producing branched-chain amino acids.

[0066] The strain of the present application may be, but is not limited to, a microorganism that naturally has the ability to produce prephenate dehydratase or branched-chain amino acids, or a parent strain that does not have the ability to produce prephenate dehydratase or branched-chain amino acids into which the mutant of the present application or a polynucleotide encoding it (or a vector containing the above-mentioned polynucleotide) has been introduced and / or to which the ability to produce branched-chain amino acids has been imparted.

[0067] As an example, the strain of the present application is a cell or microorganism that is transformed with a vector containing a polynucleotide of the present application or a polynucleotide encoding the variant of the present application and expresses the variant of the present application, and for the purposes of this application, the strain of the present application may include all microorganisms capable of producing branched-chain amino acids, including the variant of the present application. For example, the strain of the present application may be a recombinant strain having increased branched-chain amino acid production ability by expressing a prephenate dehydratase variant by introducing a polynucleotide encoding the variant of the present application into a naturally occurring wild-type microorganism or a microorganism that produces branched-chain amino acids. The recombinant strain having increased branched-chain amino acid production ability may be, but is not limited to, a naturally occurring wild-type microorganism or a microorganism not modified with prephenate dehydratase (i.e., a microorganism expressing wild-type prephenate dehydratase).

[0068] For example, the prephenate dehydratase non-modified microorganism, which is a target strain for comparing the increase in the branched-chain amino acid producing ability, may be Corynebacterium glutamicum ATCC 13032. For other examples, the prephenate dehydratase non-modified microorganism, which is a target strain for comparing the increase in the branched-chain amino acid producing ability, may be, but is not limited to, CJL-8109, KCCM12739P (CA10-3101), or KCCM11201P.

[0069] As an example, the recombinant strain may have a branched-chain amino acid production ability that is at least about 1%, specifically at least about 3%, at least about 5%, higher than that of the parent strain or unmodified microorganism before mutation, but is not limited thereto as long as there is an increase in the + value compared to the production ability of the parent strain or unmodified microorganism before mutation.

[0070] In another example, the recombinant strain may produce less than about 50% of by-products in the branched chain amino acid production pathway, specifically less than about 30%, less than about 10%, compared to the parent strain or unmodified microorganism before mutation, or may not produce any by-products, but is not limited thereto.

[0071] The term "about" refers to a range including, but not limited to, ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​in a range equal to or similar to the numerical value following the term about.

[0072] In the present application, the term "branched chain amino acid" refers to an amino acid having a branched alkyl group in the side chain, including valine, leucine, and isoleucine. Specifically, in the present application, the branched chain amino acid may be an L-branched chain amino acid, and the L-branched chain amino acid may be one or more selected from L-valine, L-leucine, and L-isoleucine, but is not limited thereto.

[0073] In the present application, the by-products generated in the branched-chain amino acid production pathway refer to substances other than branched-chain amino acids, and specifically may be one or more selected from aromatic amino acids, more specifically, L-tyrosine and L-phenylalanine, but are not limited thereto.

[0074] In the present application, the term "non-mutated microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, but refers to a wild-type or naturally occurring strain itself, or a strain before its characteristics are changed by genetic mutation due to natural or artificial factors. For example, the non-mutated microorganism refers to a strain before the prephenate dehydratase mutant of the present application is introduced or before it is introduced. "Non-mutated microorganism" may be used interchangeably with "strain before mutation", "microorganism before mutation", "non-mutated strain", "non-mutated strain", "non-mutated microorganism", or "reference microorganism".

[0075] In one embodiment, the microorganism of the present application is Corynebacterium glutamicum, Corynebacterium stationis, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium The bacterium may be Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens.

[0076] The microorganism of the present application may further comprise additional mutations that increase the branched-chain amino acid producing ability.

[0077] In one embodiment, the microorganism of the present application may include altered activities of one or more of isopropylmalate synthase, homoserine dehydrogenase, threonine dehydratase, branched-chain amino acid aminotransferase, and citrate synthase.

[0078] In one embodiment, the microorganism of the present application may be a microorganism in which one or more activities of isopropylmalate synthase, homoserine dehydrogenase, branched amino acid aminotransferase, and threonine dehydratase are further enhanced.

[0079] However, the present invention is not limited to the above, and those skilled in the art can appropriately select additional modifications to be included in the microorganism depending on the branched-chain amino acid to be produced.

[0080] In the present application, the term "enhancement" of a polypeptide activity means that the activity of the polypeptide is increased compared to the endogenous activity. The term "enhancement" may be used interchangeably with terms such as "activation," "up-regulation," "overexpression," and "increase." Here, the terms "activation," "enhancement," "up-regulation," "overexpression," and "increase" may all include the display of an activity that was not originally possessed, or the display of an activity that is improved compared to the endogenous activity or the activity before transformation. The term "endogenous activity" refers to the activity of a specific polypeptide that was originally possessed by a parent strain or a non-transformed microorganism before transformation, when a trait has been changed by a genetic mutation due to natural or artificial factors. This term may be used interchangeably with "activity before transformation." The term "enhancement," "up-regulation," "overexpression," or "increase" of a polypeptide activity compared to the endogenous activity means that the activity and / or concentration (expression level) of a specific polypeptide is improved compared to the activity and / or concentration (expression level) of a specific polypeptide that was originally possessed by a parent strain or a non-transformed microorganism before transformation.

[0081] The enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. The enhancement of the activity of the polypeptide can be confirmed by an increase in the activity, expression level, or amount of a product secreted from the polypeptide.

[0082] The activity of the polypeptide can be enhanced by various methods well known in the art, and is not limited as long as the activity of the target polypeptide can be enhanced compared to that of the microorganism before modification. Specifically, the activity of the polypeptide can be enhanced by, but is not limited to, genetic engineering and / or protein engineering, which are routine methods in molecular biology and well known to those skilled in the art (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0083] Specifically, the enhancement of the polypeptide of the present application includes: 1) increasing the intracellular copy number of a polynucleotide encoding a polypeptide; 2) replacing the expression regulatory region of a gene on a chromosome that encodes a polypeptide with a sequence with a strong activity; 3) a modification of the nucleotide sequence encoding the initiation codon or the 5′-UTR region of a gene transcript encoding a polypeptide; 4) modifying the amino acid sequence of the polypeptide so that the activity of the polypeptide is enhanced; 5) modifying a polynucleotide sequence encoding a polypeptide so as to enhance the activity of the polypeptide (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the activity of the polypeptide); 6) introduction of a foreign polypeptide exhibiting a polypeptide activity or a foreign polynucleotide encoding the same; 7) codon optimization of a polynucleotide encoding a polypeptide; 8) analyzing the tertiary structure of a polypeptide and selectively altering or chemically modifying exposed sites; or 9) A combination of two or more selected from the above 1) to 8) may be used, but is not particularly limited thereto.

[0084] More specifically, The 1) increase in the intracellular copy number of the polynucleotide encoding the polypeptide may be achieved by introducing into the host cell a vector to which the polynucleotide encoding the polypeptide is operably linked and which can replicate and function independently of the host. Alternatively, the increase in the intracellular copy number of the polynucleotide encoding the polypeptide may be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide into a chromosome in the host cell. The introduction into the chromosome can be achieved by, but is not limited to, introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome in the host cell. The vector is as described above.

[0085] The replacement of the gene expression regulatory region (or expression regulatory sequence) on the chromosome encoding the polypeptide with a sequence having a stronger activity may be, for example, the occurrence of a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so as to further enhance the activity of the expression regulatory region, or the replacement with a sequence having a stronger activity. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and decoding. As an example, the original promoter may be replaced with a strong promoter, but is not limited to these.

[0086] Examples of known strong promoters include, but are not limited to, cj1 to cj7 promoters (U.S. Patent US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (U.S. Registered Patent US 10584338 B2), O2 promoter (U.S. Registered Patent US 10273491 B2), tkt promoter, and yccA promoter.

[0087] The modification of the nucleotide sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide (3) may be, for example, but is not limited to, replacement with a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate than the endogenous start codon.

[0088] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) may be, but is not limited to, a mutation in the sequence of 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 as to enhance the activity of the polypeptide, or an amino acid sequence or polynucleotide sequence improved to have stronger activity or an amino acid sequence or polynucleotide sequence improved to increase activity. The replacement may be, but is not limited to, specifically, by inserting the polynucleotide into a chromosome by homologous recombination. The vector used in this case may further include a selection marker for checking whether or not the chromosome is inserted. The selection marker is as described above.

[0089] The introduction of a foreign polynucleotide exhibiting the activity of the polypeptide 6) may be the introduction of a foreign polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide into a host cell. The foreign polynucleotide is not limited in origin or sequence as long as it exhibits the same / similar activity as the polypeptide. The method used for the introduction can be a known transformation method appropriately selected by those skilled in the art, and the introduced polynucleotide may be expressed in a host cell to produce a polypeptide, and its activity may be increased.

[0090] The codon optimization of the polynucleotide encoding the polypeptide (7) may be that of an endogenous polynucleotide that has been codon-optimized to increase transcription or translation in a host cell, or that of an exogenous polynucleotide that has been codon-optimized to optimize transcription or translation in a host cell.

[0091] The above 8) analyzing the tertiary structure of a polypeptide and selecting and modifying or chemically modifying an exposed site may involve, for example, comparing sequence information of a polypeptide to be analyzed with a database storing sequence information of known proteins, determining template protein candidates according to the degree of sequence similarity, confirming the structure based on the sequence information, and selecting and modifying an exposed site to be modified or chemically modified.

[0092] Such enhanced polypeptide activity may refer to, but is not limited to, an increase in the activity or concentration of the corresponding polypeptide expressed relative to the activity or concentration of the polypeptide expressed in a wild-type or unaltered microbial strain, or an increase in the amount of a product produced from the polypeptide.

[0093] In the microorganism of the present application, modification of a part or the whole of a polynucleotide may be induced by, but is not limited to, (a) homologous recombination using a vector for chromosomal insertion in the microorganism or genome editing using engineered nuclease (e.g., CRISPR-Cas9) and / or (b) treatment with light and / or chemicals such as ultraviolet light and radiation. A method for modifying a part or the whole of the gene may include a method using DNA recombination technology. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene may be injected into the microorganism to cause homologous recombination, thereby causing a deletion of a part or the whole of the gene. The injected nucleotide sequence or vector may contain, but is not limited to, a dominant selection marker.

[0094] The term "attenuation" of a polypeptide activity in the present application is a concept that includes all of a decrease in activity or no activity compared to the endogenous activity. The attenuation may be mixed with terms such as inactivation, deficiency, down-regulation, decrease, reduction, and attenuation.

[0095] The attenuation may include cases where the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide originally possessed by the microorganism due to a mutation in the polynucleotide encoding the polypeptide, where the overall polypeptide activity and / or concentration (expression amount) in the cell is lower than that of a wild-type strain due to inhibition of gene expression of the encoding polynucleotide or inhibition of translation into the polypeptide, where the polynucleotide is not expressed at all, and / or where the polypeptide activity is absent despite the expression of the polynucleotide. The "intrinsic activity" refers to the activity of a specific polypeptide originally possessed by a parent strain, a wild-type or a non-transformed microorganism before transformation when the trait is changed due to a genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before transformation." The activity of a polypeptide being "inactivated, deficient, reduced, downregulated, decreased, attenuated" compared to the intrinsic activity means that the activity of a specific polypeptide is reduced compared to that originally possessed by a parent strain or a non-transformed microorganism before transformation.

[0096] Such attenuation of the activity of a polypeptide can be achieved by any method known in the art, but is not limited to these, and may be achieved by application of various methods well known in the art (e.g., Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).

[0097] Specifically, the attenuation of the polypeptide of the present application is 1) A deletion of all or part of a gene encoding a polypeptide; 2) modification of an expression control region (or expression control sequence) so that expression of a gene encoding a polypeptide is reduced; 3) modification of the amino acid sequence constituting the polypeptide (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence) so that the activity of the polypeptide is eliminated or weakened; 4) modification of a gene sequence encoding a polypeptide such that the activity of the polypeptide is eliminated or attenuated (e.g., deletion / substitution / addition of one or more nucleic acid bases on the nucleic acid base sequence of the polypeptide gene to encode a polypeptide that has been altered such that the activity of the polypeptide is eliminated or attenuated); 5) a modification of the nucleotide sequence encoding the initiation codon or the 5′-UTR region of a gene transcript encoding a polypeptide; 6) introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to the transcript of the gene encoding the polypeptide; 7) Addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide in order to form a secondary structure to which ribosomes cannot attach; 8) Reverse transcription engineering (RTE) of a promoter transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of a gene sequence encoding a polypeptide; or 9) A combination of two or more selected from the above 1) to 8) may be used, but is not particularly limited thereto.

[0098] for example, The 1) deletion of a part or the whole of the gene encoding the polypeptide may be removal of the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, replacement with a polynucleotide lacking some nucleotides, or replacement with a marker gene.

[0099] The modification of the expression regulatory region (or expression regulatory sequence) in 2) above may be a mutation in the expression regulatory region (or expression regulatory sequence) caused by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or may be replaced with a sequence having a weaker activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0100] In addition, the above 3) nucleotide sequence modification encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, a replacement with a nucleotide sequence encoding another start codon having a lower polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0101] Furthermore, the modification of the amino acid sequence or polynucleotide sequence in 4) and 5) above may be, but is not limited to, the occurrence of mutations in the sequence of the amino acid sequence of the polypeptide or the sequence of the polynucleotide encoding the polypeptide by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or replacement with an amino acid sequence or polynucleotide sequence improved to have a weaker activity or an amino acid sequence or polynucleotide sequence improved to have no activity, so as to weaken the activity of the polypeptide. For example, but not limited to, the expression of a gene can be inhibited or weakened by introducing a mutation in a polynucleotide sequence to form a stop codon.

[0102] The introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide 6) can be carried out by referring to, for example, the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

[0103] 7) Addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide in order to form a secondary structure to which ribosomes cannot attach may disable or slow down mRNA translation.

[0104] 8) The addition of a promoter transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE) may create an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide, thereby weakening the activity.

[0105] In the microorganisms of the present application, the mutants, polynucleotides, vectors and branched chain amino acids are as described in other embodiments.

[0106] Another aspect of the present application provides a method for producing branched-chain amino acids, comprising the step of culturing the Corynebacterium microorganism of the present application in a medium.

[0107] In the present application, the term "culture" means growing the Corynebacterium microorganism of the present application under appropriately controlled environmental conditions. The culture process of the present application can be carried out according to suitable media and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culture may be, but is not limited to, a batch, continuous and / or fed-batch culture.

[0108] In the present application, the term "culture medium" refers to a substance containing a mixture of nutrients required for culturing the microorganism of the genus Corynebacterium of the present application as the main components, and supplies nutrients such as water essential for survival and growth, growth factors, etc. Specifically, the medium and other culture conditions used for culturing the microorganism of the genus Corynebacterium of the present application can be any medium used for culturing ordinary microorganisms without any particular limitation, and the microorganism of the genus Corynebacterium of the present application can be cultured under aerobic conditions while controlling the temperature, pH, etc. in an ordinary medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins, etc.

[0109] Specifically, culture media for Corynebacterium microorganisms can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0110] In the present application, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, baca, and corn steeping liquid may be used, specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and other suitable carbon sources may be used in a variety of ways without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited thereto.

[0111] The nitrogen source may be an inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; an organic nitrogen source such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more kinds, and are not limited thereto.

[0112] The phosphorus source may include potassium monophosphate, potassium diphosphate, or the corresponding sodium-containing salts. The inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and may also include amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the medium in a batch or continuous manner. However, they are not limited thereto.

[0113] In addition, during the cultivation of the Corynebacterium microorganism of the present application, a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. may be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during the cultivation, foam generation may be suppressed using an antifoaming agent such as fatty acid polyglycol ester, etc. In addition, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, or in order to maintain the anaerobic and microaerobic states, no gas may be injected or nitrogen, hydrogen or carbon dioxide gas may be injected, but this is not limited thereto.

[0114] In the culture of the present application, the culture temperature can be maintained at 20 to 45° C., specifically 25 to 40° C., and the culture can be performed for about 10 to 160 hours, but is not limited thereto.

[0115] The branched-chain amino acids produced by the culture of the present application are either secreted into the medium or remain intracellularly.

[0116] The method for producing branched-chain amino acids of the present application may further include a step of preparing a Corynebacterium microorganism of the present application, a step of preparing a medium for culturing the strain, or a combination thereof (in any order), for example, prior to the culturing step.

[0117] The method for producing branched-chain amino acids of the present application may further include a step of recovering branched-chain amino acids from the culture medium (cultured medium) or the Corynebacterium microorganism of the present application. The recovery step may be further included after the culturing step.

[0118] The recovery may be a collection of the desired branched-chain amino acids using a suitable method known in the art by the culture method of the microorganism of the present application, such as a batch, continuous or fed-batch culture method, etc. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographies such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination of these methods can be used, and the desired branched-chain amino acids can be recovered from the medium or the microorganism using a suitable method known in the art.

[0119] In addition, the branched chain amino acid production method of the present application may further include a purification step. The purification can be performed using a suitable method known in the art. As an example, when the branched chain amino acid production method of the present application includes both a recovery step and a purification step, the recovery step and the purification step can be performed chronologically (or consecutively) regardless of the order, or can be performed simultaneously or integrated into one step, but is not limited thereto.

[0120] In the methods of the present application, the variants, polynucleotides, vectors and microorganisms etc. are as described in other aspects above.

[0121] Another aspect of the present application is to provide a composition for producing branched-chain amino acids, comprising 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; a medium in which the same is cultured; or a combination of two or more of them.

[0122] The composition of the present application may further contain any suitable excipient that is commonly used in compositions for producing branched chain amino acids, and such excipients may be, for example, but are not limited to, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, or an isotonicity agent.

[0123] In the compositions of the present application, the variants, polynucleotides, vectors, strains, media and branched chain amino acids, etc. are as described in other aspects above.

[0124] Another aspect of the present application provides a use of a microorganism comprising one or more of the prephenate dehydratase mutant of the present application; a polynucleotide encoding the prephenate dehydratase mutant; and a vector containing the polynucleotide, for producing branched-chain amino acids.

[0125] For the purposes of this application, the variants, polynucleotides, vectors and microorganisms etc. are as described in the other aspects above.

[0126] The present application will be described in more detail with reference to the following examples and experimental examples. However, these examples and experimental examples are provided to exemplify the present application, and the scope of the present application is not limited to these examples and experimental examples.

[0127] Example 1: Discovery of pheA mutations Example 1-1. Construction of a vector containing pheA To prepare a pheA mutant library with prephenate dehydratase activity, a recombinant vector containing pheA was first prepared. To amplify the pheA gene (SEQ ID NO: 2) encoding the pheA protein (SEQ ID NO: 1, Uniprot ID: P10341) derived from wild-type Corynebacterium glutamicum, a PCR method was performed using the chromosome of the wild-type Corynebacterium glutamicum ATCC13032 strain as a template and primers SEQ ID NO: 3 and 4, which were denatured at 94°C for 1 minute, bound at 58°C for 30 seconds, and polymerized with Pfu DNA polymerase at 72°C for 1 minute, repeated 25 times. The specific sequences of the primers used are listed in Table 1. The above amplification product was cloned into the E. coli vector pCR2.1 using the TOPO Cloning Kit (Invitrogen) to obtain "pCR-pheA".

[0128] [Table 1]

[0129] Example 1-2. Construction of a pheA mutation library A pheA mutation library was prepared using an error-prone PCR kit (Clontech Diversify® PCR Random Mutagenesis Kit) based on the vector prepared in Example 1-1. PCR was performed using SEQ ID NO: 3 and SEQ ID NO: 4 as primers under conditions that 0-3 mutations per 1000 bp could occur. Specifically, PCR was performed by pre-heating at 94°C for 30 seconds, followed by 25 cycles of 94°C for 30 seconds and 68°C for 1 minute and 30 seconds. The PCR product obtained was used as a megaprimer (50-125 ng) and the cycle of 95°C for 50 seconds, 60°C for 50 seconds, and 68°C for 12 minutes was repeated 25 times, followed by DpnI treatment, transformation into E. coli DH5α by heat shock method, and smearing on LB solid medium containing kanamycin (25 mg / L). After selecting 20 transformed colonies, the plasmids were obtained and the base sequences were analyzed, confirming that mutations were introduced at different positions with a frequency of 2 mutations / kb. Plasmids were extracted from approximately 20,000 transformed E. coli colonies and named "pTOPO-pheA-library."

[0130] Example 2: Evaluation of constructed libraries and selection of mutants Example 2-1. Screening of mutant strains with increased and decreased L-leucine and L-phenylalanine production The pTOPO-pheA-library constructed in Example 1-2 was transformed into wild-type Corynebacterium glutamicum ATCC13032 by electroporation, and then 10,000 colonies of the strain into which the mutant gene was inserted were selected by plating on a nutrient medium (Table 2) containing 25 mg / L kanamycin. The selected colonies were named ATCC13032 / pTOPO_pheA(mt)1 to ATCC13032 / pTOPO_pheA(mt)10,000.

[0131] Among the 10,000 colonies obtained, in order to identify colonies in which L-leucine production was increased and L-phenylalanine production among aromatic amino acids was increased or decreased, fermentation titer evaluation was performed for each colony as follows.

[0132] [Table 2]

[0133] Each colony was inoculated into a 250 ml corner baffle flask containing 25 ug / ml kanamycin in 25 ml of pressure-sterilized production medium (Table 2) using a platinum loop, and then cultured at 30°C for 60 hours with shaking at 200 rpm. After the culture was completed, L-leucine and L-phenylalanine in aromatic amino acids were measured using a method using high performance liquid chromatography (HPLC, SHIMAZDU LC20A).

[0134] Among the 10,000 colonies obtained, one strain (ATCC13032 / pTOPO_pheA(mt)3891) was selected that showed the highest L-leucine production and the lowest L-phenylalanine production compared to the wild-type Corynebacterium glutamicum strain (ATCC13032). The concentrations of L-leucine (Leu) and L-phenylalanine (Phe) produced by the selected strain are shown in Table 3 below.

[0135] [Table 3]

[0136] As shown in Table 3 above, it was confirmed that Corynebacterium glutamicum ATCC13032 / pTOPO_pheA(mt)3891, which has a mutation in the pheA gene, had approximately 1.4-fold higher L-leucine production and approximately 7-fold lower L-phenylalanine production than the parent strain.

[0137] Example 2-2. Confirmation of mutations in strains with increased and decreased L-leucine and L-phenylalanine production To confirm the pheA gene mutation of the selected mutant strain ATCC13032 / pTOPO_pheA(mt)3891, DNA of each mutant strain was used as a template using primers of sequence numbers 3 and 4 in Table 1, and PCR was performed under the following conditions: 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute and 30 seconds, 30 times, followed by DNA sequencing at 72°C for 5 minutes.

[0138] As a result of sequencing, it was confirmed that the 544-546 nucleotides of the pheA gene in the ATCC13032 / pTOPO_pheA(mt)3891 strain had been substituted with GCG for CGC. This means that the strain can encode a mutant in which the 182nd amino acid, arginine, of the pheA protein is substituted with alanine (hereinafter referred to as R182A). The amino acid sequence of the pheA mutant (R182A) and the base sequence of the pheA mutant encoding it are shown in SEQ ID NOs: 5 and 6.

[0139] Therefore, in the following Example, it was decided to confirm whether the above mutation (R182A) affects the production of L-leucine and aromatic amino acids in microorganisms of the genus Corynebacterium.

[0140] Example 3: Confirmation of L-leucine and L-phenylalanine production ability of the selected mutant strain Example 3-1. Construction of an insertion vector containing a pheA mutation An insertion vector was constructed to introduce the mutation selected in Example 2 into the strain. A site-directed mutagenesis method was used to construct a vector for introducing the pheA (R182A) mutation. To generate the R182A mutation using the wild-type chromosome of Corynebacterium glutamicum as a template, PCR was performed using primers of SEQ ID NOs: 7 and 8 and a primer pair of SEQ ID NOs: 9 and 10. Specifically, PCR was performed under the conditions of 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 72°C for 5 minutes. The specific sequences of the primers used are shown in Table 4.

[0141] [Table 4]

[0142] As a result, the obtained PCR product was cut with SmaI restriction enzyme into a linear pDCM2 vector (Korean published patent KR10-2020-0136813A), and the terminal 15 base homologous sequence between the DNA fragments was fused and cloned using In-Fusion enzyme to create the vector "pDCM2-pheA(R182A)" that replaces the 182nd amino acid of pheA with alanine.

[0143] Example 3-2. Introduction and evaluation of mutants in ATCC13032 strain The pDCM2-pheA(R182A) vector prepared in Example 3-1 was transformed into ATCC13032 by electroporation, and the strains in which the vector was inserted into the chromosome by recombination of the homologous sequence were selected in a medium containing 25 mg / L of kanamycin. The selected primary strains were then subjected to a second crossover to select strains in which the target gene mutation was introduced. Finally, the pheA gene mutation of the transformed strains was confirmed by analyzing the base sequence after performing PCR using primers of SEQ ID NO: 3 and SEQ ID NO: 4. A total of three strains were prepared, and named ATCC13032_pheA_R182A.

[0144] In order to evaluate the L-leucine and aromatic amino acid production ability of the three strains prepared above, flask fermentation titer evaluation was carried out. A loopful of the parent strain Corynebacterium glutamicum ATCC13032 and the above prepared ATCC13032_pheA_R182A were inoculated into a 250 ml corner baffle flask containing 25 ml of each production medium, and then cultured at 30°C for 60 hours with shaking at 200 rpm to produce L-leucine. After the culture was completed, the production amounts of L-leucine, L-tyrosine, and L-phenylalanine were measured by HPLC. The leucine concentration in the culture solution for each strain tested is shown in Table 5 below.

[0145] [Table 5]

[0146] As shown in Table 5 above, it was confirmed that ATCC13032_pheA_R182A had an approximately 1.5-fold increase in L-leucine yield compared to the parent strain Corynebacterium glutamicum ATCC13032. ATCC13032_pheA_R182A had an approximately 8-fold decrease in L-phenylalanine.

[0147] Example 4: Confirmation of leucine and phenylalanine production ability of pheA-selected mutants in leucine-producing strains Wild-type strains of the genus Corynebacterium produce only very small amounts of leucine. We created a leucine-producing strain derived from ATCC13032 and introduced selected mutations to confirm the ability to produce leucine and phenylalanine. The specific experiment is as follows.

[0148] Example 4-1. Construction of L-leucine producing strain CJL-8109 As strains for producing high concentrations of L-leucine, we prepared the following ATCC13032-derived strains: (1) a mutation in which the 1673rd nucleotide of the leuA gene, G, is replaced by A and the 558th amino acid, arginine, is replaced by histidine (R558H); (2) a mutation in which the 1682nd and 1683rd nucleotides of the leuA gene, GC, are replaced by AT and the 561st amino acid, glycine, is replaced by aspartic acid (G561D); and (3) a mutation in which the 739th and 740th nucleotides of the leuA gene, CC, are replaced by TG and the 247th amino acid, proline, is replaced by cysteine ​​(P247C).

[0149] Specifically, the pDCM2-leuA (P247C, R558H, G561D) vector containing the above leuA gene mutation was transformed into Corynebacterium glutamicum ATCC13032 by electroporation, and a strain in which the vector had been inserted into the chromosome by recombination of homologous sequences was selected in a medium containing 25 mg / L kanamycin. The selected primary strain was then subjected to a second crossover to select a strain in which a leuA gene mutation had been introduced. Finally, the introduction of mutations into the transformed strain was confirmed by performing PCR (94°C for 5 minutes, followed by 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 90 seconds, followed by 72°C for 5 minutes) using primers of SEQ ID NO: 11 and SEQ ID NO: 12 in Table 6, and analyzing the base sequence to confirm that the P247C, R558H, and G561D mutations had been introduced. The ATCC13032_leuA_(P247C, R558H, G561D) strain transformed with the pDCM2-leuA(P247C, R558H, G561D) vector was named “CJL-8105”.

[0150] [Table 6]

[0151] In order to increase the L-leucine production ability of the constructed CJL-8105 strain, a strain was constructed in which the ilvE mutant (V156A), a gene encoding branched-chain amino acid aminotransferase, was introduced (Korea Patent KR10-2143964B1). Specifically, the pDCM2-ilvE(V156A) vector containing the above 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 in a medium containing 25 mg / L kanamycin. The selected primary strain was then subjected to a second crossover to select a strain in which the ilvE gene mutation was introduced. Finally, the introduction of the mutation into the transformed strain was confirmed by PCR (94°C for 5 minutes, followed by 30 cycles of 94°C for 30 seconds / 55°C for 30 seconds / 72°C for 90 seconds, and then 72°C for 5 minutes) using the primers of SEQ ID NO: 13 and SEQ ID NO: 14 in Table 7, and analyzing the base sequence to confirm that the V156A mutation was introduced. The strain transformed with the pDCM2-ilvE(V156A) vector was named "CJL-8108".

[0152] [Table 7]

[0153] In order to increase the L-leucine production ability of the constructed CJL-8108 strain, a strain was constructed in which a gltA mutant (M312I; SEQ ID NO: 25) with weakened citrate synthase activity was introduced. Specifically, the pDCM2-gltA(M312I) vector containing the above 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 in a medium containing 25 mg / L kanamycin. The selected primary strain was then subjected to a second crossover to select a strain in which a gltA gene mutation was introduced. Finally, the introduction of the M312I mutation into the transformed strain was confirmed by PCR (94°C for 5 minutes, followed by 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 90 seconds, and then 72°C for 5 minutes) using primers of SEQ ID NO: 15 and SEQ ID NO: 16 in Table 8, and analyzing the base sequence. The strain transformed with the pDCM2-gltA(M312I) vector was named "CJL-8109".

[0154] [Table 8]

[0155] Example 4-2. Introduction and evaluation of pheA mutants in CJL-8109 strain The leucine-producing strain CJL-8109 was transformed with the pDCM2-pheA(R182A) vector prepared in Example 3-1 above, and the strain in which the vector was inserted into the chromosome by recombination of the homologous sequence was selected in a medium containing 25 mg / L kanamycin. The selected primary strain was subjected to a second crossover to select a strain in which a mutation of the target gene was introduced. Finally, the pheA gene mutation of the transformed strain was confirmed by performing PCR using primers of SEQ ID NO: 3 and SEQ ID NO: 4, and analyzing the base sequence to confirm that the pheA mutation was introduced into the strain. The prepared CJL8109_pheA_R182A was named CA13-8116 and was internationally deposited with the Korea Center for Microorganisms (KCCM), an international depository institution under the Budapest Treaty, on January 22, 2021, and was assigned the deposit number KCCM12943P.

[0156] The leucine producing ability of the CA13-8116, ATCC13032, and CJL-8109 strains prepared above was evaluated. Flask culture was carried out in the same manner as in Example 2, and the amount of leucine produced was measured by HPLC after the culture was completed. The culture results are shown in Table 9 below.

[0157] [Table 9]

[0158] As shown in Table 9, the L-leucine producing strain Corynebacterium glutamicum CA13-8116, which has the R182A mutation in the pheA gene, was confirmed to have about 4 times higher L-leucine productivity than the parent strain Corynebacterium glutamicum ATCC13032. In addition, the L-leucine producing strain Corynebacterium CA13-8116 was confirmed to have about 1.2 times higher L-leucine productivity than the parent strain Corynebacterium glutamicum CJL-8109. CA13-8116 showed about 5.4 times lower L-phenylalanine productivity than the parent strain Corynebacterium glutamicum CJL-8109.

[0159] From the above results, it can be confirmed that the amino acid at position 182 in the amino acid sequence of pheA protein is an important position for increasing L-leucine production.

[0160] Example 5: Confirmation of leucine and phenylalanine production ability of pheA selected mutation in isoleucine producing strain In order to confirm whether the selected mutations are effective for leucine and isoleucine, a representative branched-chain amino acid, an experiment was conducted to introduce the selected mutations into an isoleucine-producing strain of Corynebacterium sp. and confirm the isoleucine-producing ability. The specific experiment is as follows.

[0161] Example 5-1. Construction of L-isoleucine producing strain CA10-3101 An L-isoleucine producing strain was developed from wild-type Corynebacterium glutamicum ATCC13032. Specifically, the 407th amino acid, arginine, of the hom gene, which encodes homoserine dehydrogenase, was replaced with histidine to eliminate feedback inhibition of threonine, the precursor of isoleucine in the biosynthetic pathway (US2020-0340022A1). Specifically, to produce a strain with the hom(R407H) mutation introduced, PCR was performed using the chromosome of Corynebacterium glutamicum ATCC13032 as a template and primers of SEQ ID NO:17 and SEQ ID NO:18, or SEQ ID NO:19 and SEQ ID NO:20, respectively. The primer sequences used here are as shown in Table 10 below.

[0162] [Table 10]

[0163] Polymerase (Stratagene) was used, and PCR conditions were denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute, repeated 28 times. As a result, a 1000 bp DNA fragment at the 5' upper end and a 1000 bp DNA fragment at the 3' lower end were obtained, focusing on the mutation of the hom gene. PCR was performed using the two amplified DNA fragments as templates and primers of SEQ ID NO: 17 and SEQ ID NO: 20. PCR conditions were denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 2 minutes, repeated 28 times, and polymerization at 72°C for 5 minutes.

[0164] As a result, a 2 kb DNA fragment containing a mutation in the hom gene encoding a homoserine dehydrogenase mutant in which arginine at position 407 was replaced with histidine was amplified. The amplified product was purified using a PCR Purification kit (QIAGEN) and used as an insert DNA fragment for vector construction. The purified amplified product was treated with the restriction enzyme smaI, and then heat-treated at 65°C for 20 minutes to obtain a molar concentration (M) ratio of pDCM2 vector and the above amplified product, the insert DNA fragment, at 1:2. The vector pDCM2-R407H for introducing the hom(R407H) mutation into the chromosome was constructed by cloning using an Infusion Cloning Kit (TaKaRa) according to the manual provided.

[0165] The constructed vector was transformed into Corynebacterium glutamicum ATCC13032 by electroporation, and a strain containing the hom(R407H) mutation on the chromosome was obtained through a second crossover process, which was named Corynebacterium glutamicum ATCC13032hom(R407H).

[0166] In order to enhance the feedback release and activity of L-isoleucine, a strain was constructed by introducing the ilvA mutant (T381A, F383A), which is a gene encoding L-threonine dehydratase, into the ATCC13032hom(R407H) strain thus constructed. More specifically, to construct a strain into which the ilvA(T381A, F383A) mutation was introduced, PCR was performed using the primers SEQ ID NO:21 and SEQ ID NO:22, or SEQ ID NO:23 and SEQ ID NO:24, respectively, with the chromosome of Corynebacterium glutamicum ATCC13032 as a template. The primer sequences used here are shown in Table 11 below.

[0167] [Table 11]

[0168] Polymerase (Stratagene) was used, and PCR conditions were denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute, repeated 28 times. As a result, a 1126 bp DNA fragment at the 5' upper end and a 286 bp DNA fragment at the 3' lower end were obtained, focusing on the mutation of the ilvA gene. PCR was performed using the two types of amplified DNA fragments as templates and primers of SEQ ID NO: 21 and SEQ ID NO: 24. PCR conditions were denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 2 minutes, repeated 28 times, and polymerization at 72°C for 5 minutes.

[0169] 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 replaced by alanine and the 383rd phenylalanine was replaced by alanine was amplified. The amplified product was purified using a PCR Purification kit (QIAGEN) and used as an insert DNA fragment for vector construction. The purified amplified product was treated with the restriction enzyme smal, and then heat-treated at 65°C for 20 minutes to make the molar concentration (M) ratio of the pDCM2 vector and the above amplified product, the insert DNA fragment, 1:2. The vector pDCM2-ilvA(T381A, F383A) for introducing the ilvA(T381A, F383A) mutation into the chromosome was constructed by cloning using an Infusion Cloning Kit (TaKaRa) according to the manual provided.

[0170] The constructed vector was transformed into Corynebacterium glutamicum ATCC13032hom(R407H) by electroporation, and a strain containing the ilvA(T381A, F383A) mutation on the chromosome was obtained through a second crossover process and named Corynebacterium glutamicum CA10-3101.

[0171] Example 5-2. Introduction and evaluation of pheA mutants in CA10-3101 strain CA10-3101, an L-isoleucine producing strain, was transformed with the pDCM2-pheA(R182A) vector prepared in Example 3-1 above, and the strains in which the vector was inserted into the chromosome by recombination of the homologous sequence were selected in a medium containing 25 mg / L kanamycin. The selected primary strains were then subjected to a second cross-over to select strains in which the target gene mutation was introduced. Finally, the introduction of the pheA gene mutation into the transformed strain was confirmed by analyzing the base sequence after performing PCR using the primers of SEQ ID NO:3 and SEQ ID NO:4 in Table 1.

[0172] The L-isoleucine and L-phenylalanine producing abilities of the CA10-3101_pheA_R182A, ATCC13032, and CA10-3101 strains prepared above were evaluated. The parent strain and the pheA mutant strain were inoculated into a 250 ml corner baffle flask containing 25 ml of isoleucine production medium, and then cultured at 32°C for 60 hours with shaking at 200 rpm to produce L-isoleucine. The composition of the production medium used in this example is as follows.

[0173] <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 10mg / l, manganese sulfate monohydrate 10mg / l, biotin 200μg / l, pH 7.2

[0174] After the cultivation was completed, the amounts of L-isoleucine and L-phenylalanine produced were measured using high performance liquid chromatography (HPLC), and the concentrations of L-isoleucine and by-products in the culture medium for each strain tested are shown in Table 12 below.

[0175] [Table 12]

[0176] As shown in Table 12 above, the L-isoleucine producing strain with the R182A mutation added to the pheA gene had approximately 1.1-fold improved L-isoleucine productivity compared to the parent strain Coryneglutamicum CA10-3101, and it was confirmed that the CA10-3101_pheA_R182A strain reduced the by-product L-phenylalanine.

[0177] From the above results, it can be confirmed that the amino acid at position 182 in the amino acid sequence of pheA protein is an important position for increasing L-isoleucine production.

[0178] Example 6: Confirmation of valine and phenylalanine production ability of pheA selected mutation in valine-producing strain In order to confirm whether the selected mutations are effective for L-valine, a representative branched-chain amino acid like leucine, an experiment was conducted to introduce the selected mutations into the Corynebacterium sp. valine-producing strain KCCM11201P and confirm the ability to produce valine and phenylalanine. The specific experiment is as follows.

[0179] Example 6-1. Introduction and evaluation of pheA mutants in KCCM11201P strain To confirm whether the mutation is effective in increasing L-valine productivity, Corynebacterium glutamicum KCCM11201P (US8465962B2), an L-valine producing strain, was used. The valine producing strain KCCM11201P was transformed with the pDCM2-pheA(R182A) vector prepared in Example 3-1 above, and the strain in which the vector was inserted into the chromosome by recombination of the homologous sequence was selected in a medium containing 25 mg / L kanamycin. The selected primary strain was subjected to a second cross-over again to select a strain in which a mutation of the target gene was introduced. Finally, the introduction of the pheA gene mutation into the transformed strain was confirmed by analyzing the base sequence after performing PCR using the primers of SEQ ID NO: 3 and SEQ ID NO: 4 in Table 1. The constructed strain was named KCCM11201P-pheA(R182A), respectively.

[0180] The valine producing ability of the KCCM11201P-pheA(R182A) strain thus constructed was evaluated. The flask culture was carried out in the same manner as in Example 2, and the amount of valine produced was measured by HPLC after the culture was completed. The culture results are shown in Table 13 below.

[0181] [Table 13]

[0182] As shown in Table 13, it was confirmed that the L-valine producing strain Corynebacterium glutamicum KCCM11201P-pheA(R182A) with the R182A mutation in the pheA gene had an approximately 1.1-fold increase in L-valine production compared to the parent strain Corynebacterium glutamicum KCCM11201P. It was confirmed that KCCM11201P-pheA(R182A) had a 5.36-fold decrease in L-phenylalanine production compared to the parent strain Corynebacterium glutamicum KCCM11201P. From the above results, it can be confirmed that the amino acid at the 182nd position in the amino acid sequence of the pheA protein is an important position for increasing L-valine production.

[0183] Reference Example 1: Confirmation of the effect of gltA(M312I) mutation on leucine production Reference Example 1-1. Construction of an insertion vector containing the gltA mutation A vector for introducing the gltA (M312I; SEQ ID NO: 25) mutation was constructed using the site-directed mutagenesis method.

[0184] Using the wild-type Corynebacterium glutamicum chromosome as a template, PCR was performed with the primer pair of SEQ ID NOs: 27 and 28 and the primer pair of SEQ ID NOs: 29 and 30. PCR was performed by denaturing at 94°C for 5 minutes, repeating 30 times at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute and 30 seconds, followed by polymerization reaction at 72°C for 5 minutes. The resulting gene fragment was cut with SmaI restriction enzyme and linearized pDCM2 vector, and cloned by combining the terminal 15 bases of homologous sequences between the DNA fragments using In-Fusion enzyme to create the vector pDCM2-gltA(M312I), which replaces the 312th amino acid, methionine, with isoleucine.

[0185] [Table 14]

[0186] Reference Example 1-2. Introduction and evaluation of mutants in ATCC13032 strain The pDCM2-gltA(M312I) vector prepared in Reference Example 1-1 was transformed into wild-type ATCC13032, and the strain in which the vector was inserted into the chromosome by recombination of the homologous sequence was selected in a medium containing 25 mg / L kanamycin. The selected primary strain was subjected to a second crossover to select a strain in which a mutation of the target gene was introduced. The gltA gene mutation of the final transformed strain was confirmed by performing PCR using primers of SEQ ID NO: 15 and SEQ ID NO: 16 (Example 4-1, Table 8) and analyzing the base sequence to confirm that a mutation (SEQ ID NO: 26) was introduced into the strain. The prepared strain was named ATCC13032_gltA_M312I.

[0187] To evaluate the leucine producing ability of the ATCC13032_gltA_M312I strain, a flask fermentation titer evaluation was performed. A loopful of the parent strain Corynebacterium glutamicum ATCC13032 and the ATCC13032_gltA_M312I strain prepared above were inoculated into a 250 ml corner-baffle flask containing 25 ml of production medium, and then cultured at 30°C for 60 hours with shaking at 200 rpm to produce leucine. After the culture was completed, the amount of leucine produced was measured by HPLC. The leucine concentration in the culture solution for each strain tested is shown in Table 15 below.

[0188] -Production medium: Glucose 100g, (NH4)2SO4 40g, Soy Protein 2.5g, Corn Steep Solids 5g, Urea 3g, KH2PO4 1g, MgSO4·7H2O 0.5 g, Biotin 100μg, Thiamine Hydrochloride 1,000μg, Calcium Pantothenate 2000μg, Nicotinamide 3,000μg, CaCO3 30g (based on 1 liter of distilled water), pH 7.0

[0189] [Table 15]

[0190] This confirmed that the M312I substitution in gltA is an effective mutation for increasing leucine production.

[0191] Reference Example 2: Confirmation of the effect of ilvA (T381A, F383A) mutation on isoleucine production Reference Example 2-1: Construction of pECCG117-ilvA(F383A) To amplify ilvA (SEQ ID NO: 32), a gene encoding threonine dehydratase (SEQ ID NO: 31), a BamHI restriction enzyme site was inserted at the terminal end of primers (SEQ ID NO: 33 and SEQ ID NO: 34) for amplifying from the promoter site (about 300 bp above the initiation codon) to the terminator site (about 100 bp below the termination codon) based on the previously reported ilvA sequence with the F383A mutation (World J Microbiol Biotechnol (2015) 31:1369-1377). Primers (SEQ ID NO: 35 and SEQ ID NO: 36) for introducing the F383A mutation into ilvA were also used. The primer sequences used here are shown in Table 16 below.

[0192] [Table 16]

[0193] PCR was performed using the chromosome of wild-type Corynebacterium glutamicum ATCC13032 as a template and primers of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36. The PCR conditions were denaturation at 95°C for 5 minutes, followed by 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, followed by polymerization at 72°C for 5 minutes.

[0194] As a result, a 1460 bp DNA fragment at the 5'-top and a 276 bp DNA fragment at the 3'-bottom were obtained, which were mainly composed of the ilvA gene mutation.

[0195] Using the two types of amplified DNA fragments as templates, PCR was performed with primers of SEQ ID NO:35 and SEQ ID NO:36.

[0196] As a result, a 1531 bp DNA fragment containing the ilvA mutation in which phenylalanine at position 383 was replaced with alanine was amplified. The pECCG117 (Korea Patent Registration No. 10-0057684) vector and the ilvA DNA fragment were treated with the restriction enzyme BamHI, ligated using DNA joining enzyme, and then cloned to obtain a plasmid, which was named pECCG117-ilvA(F383A).

[0197] Reference Example 2-2: Additional random mutations introduced into PECCG117-ilvA(F383A) To obtain a mutant of the gene encoding L-threonine dehydratase, an ilvA mutant gene plasmid was prepared using a random mutagenesis kit (Agilent Technologies, USA). PCR was performed using the primers of SEQ ID NO: 35 and SEQ ID NO: 36 and the ilvA(F383A) chromosome of Reference Example 2-1 as a template. The PCR conditions were denaturation at 95°C for 2 minutes, followed by 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, followed by polymerization at 72°C for 10 minutes.

[0198] As a result, a 1531 bp DNA fragment was amplified, which is an ilvA mutant capable of encoding L-threonine dehydratase, which has an additional random mutation in addition to the mutation in which phenylalanine at position 383 was replaced with alanine. The pECCG117 vector and the ilvA mutant DNA fragment were treated with the restriction enzyme BamHI, ligated with DNA joining enzyme, and then cloned to obtain a plasmid group.

[0199] Reference example 2-3: CJILE-301 strain production pECCG117-ilvA(F383A) was introduced into the wild-type Corynebacterium glutamicum ATCC13032hom(R407H) strain, and the strain carrying the constructed plasmid was named ATCC13032hom(R407H) / pECCG117-ilvA(F383A). In addition, the mutant plasmid group obtained in Reference Example 2-2 was introduced into the Corynebacterium glutamicum ATCC13032hom (R407H) strain, and the death rate was determined by smearing it on a minimal medium. The death rate was 70%. The surviving cells were inoculated and cultured on a seed medium, and finally, a mutant strain showing superior isoleucine production ability to the ATCC13032hom (R407H) / pECCG117-ilvA (F383A) control group was selected and named Corynebacterium glutamicum CJILE-301 (Corynebacterium glutamicum, CJILE-301).

[0200] A plasmid was isolated from the CJILE-301 strain and the ilvA gene was sequenced. As a result, it was confirmed that the 1141st base sequence of the ilvA gene was substituted from A to G, and that in addition to the mutation in which F at position 383 of the ilvA protein was substituted with A, the 381st base sequence of the ilvA gene could encode a mutant protein in which T at position 381 was substituted with A, which is shown in SEQ ID NO: 38.

[0201] Reference Example 2-4: Introduction of ilvA mutants (T381A, F383A) To introduce the ilvA mutant (T381A, F383A) into the wild-type strain, primers of SEQ ID NO: 21 and SEQ ID NO: 24 (Example 5-1, Table 11) were constructed.

[0202] To prepare a strain into which ilvA mutants (T381A, F383A) were introduced, PCR was performed using the plasmid DNA extracted from the CJILE-301 strain as a template and the primers of SEQ ID NO:21 and SEQ ID NO:24.

[0203] The polymerase used for the PCR reaction was PfuUltra™ high fidelity DNA polymerase (Stratagene). The PCR conditions were denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and polymerization at 72° C. for 2 minutes, repeated 28 times.

[0204] As a result, a 1411 bp gene fragment containing an approximately 100 bp terminator region of the ilvA gene, which is 1311 bp, was obtained.

[0205] The amplified product was purified using a PCR purification kit and used as an insert DNA fragment for vector construction. The purified amplified product was treated with the restriction enzyme smaI, and then heat-treated at 65°C for 20 minutes to make the molar concentration (M) ratio of the amplified product (inserted DNA fragment) to the pDCM2 vector was adjusted to 1:2. The vector pDCM2-T381A_F383A for introducing the T381A and F383A mutations into the chromosome was constructed by cloning according to the manual provided with the infusion cloning kit.

[0206] The constructed vector was transformed into Corynebacterium glutamicum ATCC 13032hom (R407H) by electroporation, and a strain containing the ilvA (T381A, F383A; SEQ ID NO: 37) mutation on the chromosome was obtained through a second crossover process and named CA10-3101.

[0207] The above strain CA10-3101 was internationally deposited with the Korea Center for Microorganisms (KCCM), an international depository institution under the Budapest Treaty, on May 27, 2020 and was assigned the deposit number KCCM12739P.

[0208] The above-mentioned KCCM12739P strain was inoculated into a 250 ml corner baffle flask containing 25 ml of isoleucine production medium, and then cultured at 32° C. for 60 hours with shaking at 200 rpm to produce L-isoleucine. The composition of the production medium used is as follows.

[0209] <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 10mg / L, manganese sulfate monohydrate 10mg / L, biotin 200μg / L, pH 7.2

[0210] After the cultivation was completed, the concentrations of L-isoleucine and L-threonine in the culture medium were measured using high-performance liquid chromatography (HPLC). The results are shown in Table 17 below.

[0211] [Table 17]

[0212] As shown in Table 17 above, the parent strain Corynebacterium glutamicum ATCC13032hom(R407H) did not produce L-isoleucine, but the ATCC13032hom(R407H)ilvA(T381A, F383A) mutant produced L-isoleucine at a concentration of 3.9 g / L, demonstrating a significant increase in L-isoleucine productivity compared to the parent strain.

[0213] This confirmed that the ilvA (T381A, F383A) mutation is an effective mutation for increasing isoleucine production.

[0214] Reference Example 3: Confirmation of the effect of leuA (P247C, R558H, G561D) on leucine production Reference example 3-1.CJL-8100 strain production The pDCM2-leuA(R558H,G561D) vector containing the leuA gene mutation disclosed in KR 10-2018-0077008 A 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 in a medium containing 25 mg / L kanamycin. The selected primary strain was then subjected to a second crossover to select a strain in which a mutation in the leuA gene was introduced. Finally, the mutation introduction of the transformed strain was confirmed by performing PCR (94℃ 5 minutes, 94℃ 30 seconds / 55℃ 30 seconds / 72℃ 90 seconds 30 times, 72℃ 5 minutes) using primers of SEQ ID NO: 39 and SEQ ID NO: 43, and analyzing the base sequence to confirm that the R558H,G561D mutation was introduced. The ATCC13032_leuA_(R558H,G561D) strain transformed with the pDCM2-1euA(R558H,G561D) vector was named “CJL-8100”.

[0215] The primer sequences used in Reference Example 3 are shown in Table 18 below.

[0216] [Table 18]

[0217] Reference Example 3-2. Construction of an insertion vector containing a leuA mutation A vector was constructed to introduce the P247C mutation into CJL-8100, an L-leucine producing strain that has two mutations (R558H, G561D) introduced into LeuA.

[0218] Using the chromosome of the CJL-8100 strain as a template, PCR was carried out using the primers of SEQ ID NOs: 39 and 40 and the primer pair of SEQ ID NOs: 41 and 42. After denaturation at 94°C for 5 minutes, PCR was repeated 30 times at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute and 30 seconds, followed by polymerization reaction at 72°C for 5 minutes. The resulting PCR product was cloned by fusing the terminal 15-base homologous sequence between the DNA fragments using In-Fusion enzyme with a linear pDCM2 vector cut with SmaI restriction enzyme, to create the vector pDCM2-leuA (P247C, R558H, G561D) containing a leuA mutation encoding a LeuA mutant in which the 558th amino acid, arginine, is replaced with histidine and the 561st amino acid, glycine, is replaced with aspartic acid in the LeuA amino acid sequence of the wild-type strain, and in which the 247th amino acid, proline (Pro), of LeuA is replaced with cysteine ​​(Cys).

[0219] Reference Example 3-3. Introduction and evaluation of LeuA mutant (P247C) into CJL-8100 strain The L-leucine producing strain CJL-8100 was transformed with the pDCM2-leuA (P247C, R558H, G561D) vector prepared in Reference Example 3-2 above, and the strains in which the vector was inserted into the chromosome by recombination of the homologous sequence were selected in a medium containing 25 mg / L of kanamycin. The selected primary strains were then subjected to a second cross-over to select strains in which the mutation of the target gene was introduced. Finally, the leuA gene mutation of the transformed strains was analyzed by performing PCR (94°C for 5 minutes, followed by 30 cycles of 94°C for 30 seconds / 55°C for 30 seconds / 72°C for 90 seconds, and 72°C for 5 minutes) using primers of SEQ ID NO: 39 and SEQ ID NO: 45, followed by analysis of the base sequence. As a result of the base sequence analysis, it was confirmed that the leuA mutations encoding LeuA mutants (P247C, R558H, G561D) in which the 1673th nucleotide of the leuA gene in the chromosome of the strain, G, was replaced by A, the 1682nd and 1683rd nucleotides, GC, were replaced by AT, the 739th and 740th nucleotides, CC, were replaced by TG, the 558th amino acid, arginine, was replaced by histidine, the 561st amino acid, glycine, was replaced by aspartic acid, and the 247th amino acid, proline (Pro), was replaced by cysteine ​​(Cys), were introduced into the strain. The resulting strain, CJL8100_leuA_P247C, was named "CA13-8105" and deposited with the Korean Culture Center of Microorganisms (KCCM), an international depository institution under the Budapest Treaty, on April 29, 2020 and was assigned the deposit number KCCM12709P.

[0220] The amino acid sequence of the LeuA mutant containing the above three mutations (P247C, R558H, G561D) and the nucleotide sequence of the leuA mutant encoding it are as set forth in SEQ ID NO: 46 and SEQ ID NO: 47, respectively.

[0221] The L-leucine productivity of ATCC13032, the constructed CJL-8100, and CA13-8105 strains was evaluated. Specifically, flask culture was carried out in the same manner as in Example 2-1, and after the culture was completed, the L-leucine production amount of the parent strain and the mutant strain was measured using HPLC, and the results are shown in Table 19.

[0222] [Table 19]

[0223] As shown in Table 19 above, the L-leucine producing strain Corynebacterium glutamicum CJL8100 had an L-leucine productivity that was improved by about 130% compared to the parent strain ATCC 13032. The CA13-8105 strain, which was further introduced with the leuA_P247C mutation in the CJL8100 strain, had an L-leucine productivity that was improved by about 150% compared to the parent strain CJL8100.

[0224] This confirmed that the leuA (R558H, G561D, P247C) mutation is an effective mutation for increasing leucine production.

[0225] From the above description, a person skilled in the art to which the present invention pertains will understand that the present application may be implemented in other specific forms without changing the technical idea or essential features of the present application. In this regard, it should be understood that the above described embodiments are merely illustrative and not limiting. The scope of the present application should be interpreted as including any modified or altered forms derived from the meaning and scope of the claims below, and their equivalent concepts, rather than the above detailed description. The present application provides the following aspects of the invention. (Aspect 1) A prephenate dehydratase mutant in which the amino acid corresponding to the 182nd position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid. (Aspect 2) The prephenate dehydratase mutant according to embodiment 1, wherein the mutant comprises a substitution of an amino acid corresponding to position 182 from the N-terminus in the amino acid sequence of SEQ ID NO:1 with an amino acid other than arginine. (Aspect 3) A prephenate dehydratase mutant according to embodiment 1, wherein the substitution with another amino acid is a substitution with a nonpolar amino acid or an amino acid of small size. (Aspect 4) The prephenate dehydratase variant according to embodiment 1, wherein the other amino acid is alanine (Ala). (Aspect 5) The prephenate dehydratase variant of embodiment 1, wherein the prephenate dehydratase has 99% or more homology or identity to SEQ ID NO:5. (Aspect 6) The prephenate dehydratase variant of embodiment 1, wherein the prephenate dehydratase variant has an attenuated activity compared to the prephenate dehydratase of SEQ ID NO:1. (Aspect 7) A polynucleotide encoding the prephenate dehydratase mutant according to any one of aspects 1 to 6. (Aspect 8) A vector comprising the polynucleotide according to embodiment 7. (Aspect 9) A Corynebacterium microorganism comprising one or more of the prephenate dehydratase mutant according to any one of aspects 1 to 6; a polynucleotide encoding the prephenate dehydratase mutant; and a vector comprising the polynucleotide. (Aspect 10) 10. The microorganism of claim 9, wherein the microorganism is Corynebacterium glutamicum. (Aspect 11) Aspect 10. The Corynebacterium microorganism according to aspect 9, wherein the microorganism is for producing branched-chain amino acids. (Aspect 12) 12. The Corynebacterium microorganism according to aspect 11, wherein the branched-chain amino acids are one or more selected from the group consisting of L-leucine, L-isoleucine, and L-valine. (Aspect 13) A method for producing branched chain amino acids, comprising culturing the microorganism of embodiment 9 in a medium. (Aspect 14) 14. The method of producing branched chain amino acids according to embodiment 13, further comprising recovering the branched chain amino acids from the microorganism or the medium. (Aspect 15) Aspect 14. The method for producing a branched-chain amino acid according to aspect 13, wherein the branched-chain amino acid is one or more selected from the group consisting of L-leucine, L-isoleucine, and L-valine.

[0226]

Table 20

[0227]

Table 21

[0228]

Table 22

Claims

1. A prephenate dehydratase mutant, in which the amino acid of prephenate dehydratase corresponding to position 182 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is arginine and is replaced with alanine, and the prephenate dehydratase has a sequence identity of 90% or more with the amino acid sequence of SEQ ID NO:

1.

2. The prephenate dehydratase mutant of claim 1 , wherein the prephenate dehydratase mutant has 99% or more identity to SEQ ID NO:

5.

3. The prephenate dehydratase variant of claim 1 , wherein the prephenate dehydratase variant has reduced activity compared to the prephenate dehydratase of SEQ ID NO:

1.

4. A polynucleotide encoding the prephenate dehydratase mutant according to any one of claims 1 to 3.

5. A vector comprising the polynucleotide of claim 4.

6. A Corynebacterium microorganism comprising at least one of the prephenate dehydratase mutant according to any one of claims 1 to 3, a polynucleotide encoding the prephenate dehydratase mutant, and a vector comprising the polynucleotide.

7. The microorganism according to claim 6 , wherein the microorganism is Corynebacterium glutamicum.

8. The microorganism according to claim 6 , which is for producing branched-chain amino acids.

9. The microorganism according to claim 8, wherein the branched-chain amino acid is one or more selected from the group consisting of L-leucine, L-isoleucine, and L-valine.

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

11. The method for producing branched-chain amino acids described in claim 10, further comprising a step of recovering the branched-chain amino acids from the microorganism or the culture medium.

12. The method for producing a branched-chain amino acid according to claim 10, wherein the branched-chain amino acid is one or more selected from the group consisting of L-leucine, L-isoleucine and L-valine.

13. Use of a prephenate dehydratase mutant described in any one of claims 1 to 3 for producing branched-chain amino acids.

14. Use of a Corynebacterium microorganism described in any one of claims 6 to 9 for producing branched-chain amino acids.

15. The use described in claim 14, wherein the microorganism is Corynebacterium glutamicum.

16. The use according to claim 14, wherein the branched chain amino acid is one or more selected from L-leucine, L-isoleucine and L-valine.

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