Novel gamma-aminobutyrate permease variant and method for producing isoleucine using the same

Abstract

The present application relates to a novel γ-aminobutyrate permease variant that decreases the amount of by-products generated during the production of Lisoleucine, a polynucleotide encoding the variant, and a vector comprising the polynucleotide, a microorganism comprising the variant, polynucleotide or vector, and a method for producing L-isoleucine using the microorganism.

Description

Novel gamma-aminobutyric acid permease mutant and method for producing isoleucine using the same

[0001] The present application relates to a novel gamma-aminobutyrate permease variant that reduces byproducts generated during L-isoleucine production, a polynucleotide encoding the variant, a vector comprising the polynucleotide, a microorganism comprising the variant, polynucleotide, or vector, and a method for producing L-isoleucine using the microorganism.

[0002]

[0003] L-isoleucine is a branched-chain amino acid among the 20 amino acids. Classified as an essential amino acid, it is used in animal feed, food additives, and pharmaceuticals. Because it plays a role in post-metabolism energy production, hemoglobin production, blood sugar regulation, muscle growth, and repair, its use is increasing not only in fluids, nutritional supplements, and sports nutrition, but also in animal feed.

[0004] Based on this trend, various microorganisms and their variants are used for the production of L-amino acids (US Patent No. 1,011,3190). Even in these cases, many by-products other than L-isoleucine are generated, and since these are substances that greatly affect the purity of L-isoleucine during the purification step, a method for removing the by-products is necessary. In this regard, L-isoleucine purification methods developed to increase the purity of L-isoleucine have the disadvantage of requiring a separate additional purification process (US Patent No. 6,072,083), and therefore, there is a need for the development of a method for increasing the purity of L-isoleucine.

[0005]

[0006] As a result of our efforts to increase the purity of L-isoleucine production using microorganisms, we identified the gene aroP encoding γ-aminobutyrate permease, which can contribute to the production of L-isoleucine by reducing byproducts such as alpha-aminobutyric acid and L-valine, and secured a mutation that can further increase the purity.

[0007]

[0008] One object of the present application is to provide a γ-aminobutyrate permease mutant.

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

[0010] Another object of the present application is to provide a vector comprising the polynucleotide.

[0011] Another object of the present application is to provide a microorganism comprising at least one of the variant; a polynucleotide encoding the variant; and a vector comprising the polynucleotide.

[0012] Another object of the present application is to provide a method for producing L-isoleucine.

[0013] Another object of the present application is to provide a composition for producing L-isoleucine.

[0014] Another object of the present application is to provide a variant, a polynucleotide encoding the variant, a vector comprising the polynucleotide, or a use of the microorganism for producing L-isoleucine.

[0015]

[0016] Microorganisms expressing the gamma-aminobutyric acid permease variant of the present invention can significantly improve the purity of L-isoleucine compared to strains that do not express it, and thus can be used to effectively produce L-isoleucine. Therefore, L-isoleucine is expected to find wide-ranging industrial applications, including in foods, feeds, and pharmaceuticals.

[0017]

[0018] Hereinafter, the contents of this application will be described in detail. The description and embodiments of one aspect disclosed in this application may also be applied to other aspects regarding common elements. Furthermore, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0019]

[0020] One aspect of the present application provides a γ-aminobutyrate permease mutant, wherein the amino acid corresponding to position 212 in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid corresponding to position 114 is substituted with phenylalanine, or the amino acid corresponding to position 28 is substituted with valine.

[0021] The above variant may be one in which the 212th amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the 114th amino acid is substituted with phenylalanine, or the 28th amino acid is substituted with valine. Specifically, the variant may be one in which glycine, the amino acid corresponding to the 212th position in the amino acid sequence of SEQ ID NO: 1, is substituted with threonine, isoleucine, the amino acid corresponding to the 114th position, is substituted with phenylalanine, or alanine, the amino acid corresponding to the 28th position, is substituted with valine, but is not limited thereto.

[0022] The above variant may be composed of one or more sequences selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 20, and SEQ ID NO: 21, but is not limited thereto.

[0023] Specifically, a variant in which glycine, the 212th amino acid in the amino acid sequence of SEQ ID NO: 1, is substituted with threonine may be represented by SEQ ID NO: 3, a variant in which isoleucine, the 114th amino acid in the amino acid sequence of SEQ ID NO: 1, is substituted with phenylalanine may be represented by SEQ ID NO: 20, and a variant in which alanine, the 28th amino acid in the amino acid sequence of SEQ ID NO: 1, is substituted with valine may be represented by SEQ ID NO: 21, but is not limited thereto.

[0024] The variant of the present application may be one in which the amino acid at the position corresponding to position 212, 114, or 28 in the gamma-aminobutyric acid permease having the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid, thereby increasing the absorption of by-products, thereby enhancing the activity of the gamma-aminobutyric acid permease, but is not limited thereto.

[0025]

[0026] In this application, the term "L-isoleucine" means an L-amino acid with the chemical formula HO2CCH(NH2)CH(CH3)CH2CH3, which is one of the essential amino acids and structurally corresponds to a branched-chain amino acid along with L-valine and L-leucine.

[0027] The term "γ-aminobutyrate permease" in the present application refers to a polypeptide or protein having γ-aminobutyrate permease activity, and a protein or polypeptide having an activity to uptake α-aminobutyrate or L-valine. The γ-aminobutyrate permease may be a protein or polypeptide having an activity to uptake not only α-aminobutyrate or L-valine, which are byproducts generated during the production of L-isoleucine, but also L-phenylalanine, L-tyrosine, L-tryptophan, or L-histidine, but is not limited thereto. The above gamma-aminobutyric acid permease can be mixed with a cyclic amino acid permease (aromatic amino-acid permease), and the gene encoding the gamma-aminobutyric acid permease can be mixed with the aroP gene, and the gamma-aminobutyric acid permease can be mixed with AroP.

[0028]

[0029] The above gamma-aminobutyric acid permease may be a protein comprising an amino acid sequence of SEQ ID NO: 1. The protein comprising the amino acid sequence of SEQ ID NO: 1 may be used interchangeably with a protein having an amino acid sequence of SEQ ID NO: 1 or a protein composed of an amino acid sequence of SEQ ID NO: 1.

[0030] Specifically, the above sequence number 1 may be an amino acid sequence of gamma-aminobutyric acid permease encoded by the aroP gene, and the amino acid sequence of the above sequence number 1 may be obtained from various databases such as the known database NCBI GenBank, but is not limited thereto. For example, the amino acid sequence of the above sequence number 1 may be derived from Corynebacterium sp. and may be derived from Corynebacterium glutamicum, but is not limited thereto, and a sequence having the same activity as the amino acid sequence of the above sequence number 1 may be included without limitation. In addition, although the protein having gamma-aminobutyric acid permease activity in the present application is described as a polypeptide or protein including an amino acid of SEQ ID NO: 1, it does not exclude meaningless sequence additions before and after the amino acid sequence of SEQ ID NO: 1, mutations that may occur naturally, or silent mutations thereof, and it will be apparent to those skilled in the art that if it has the same or corresponding activity as the protein including the amino acid sequence of SEQ ID NO: 1, it corresponds to the polypeptide or protein having gamma-aminobutyric acid permease activity of the present application. For a specific example, the polypeptide having gamma-aminobutyric acid permease activity of the present application may be a polypeptide composed of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity therewith. In addition, it may be obvious that a polypeptide having an amino acid sequence in which some sequences are deleted, modified, substituted or added is also included within the scope of the polypeptide of the present application, provided that the amino acid sequence has such homology or identity and exhibits an effect corresponding to the polypeptide.

[0031]

[0032] Even if the present application describes a "polypeptide or protein comprising an amino acid sequence described by a specific sequence number," a "polypeptide or protein consisting of an amino acid sequence described by a specific sequence number," or a "polypeptide or protein having an amino acid sequence described by a specific sequence number," it is obvious that a protein having an amino acid sequence in which a portion of the sequence is deleted, modified, substituted, conservatively substituted, or added can also be used in the present application, as long as it has the same or corresponding activity as the polypeptide consisting of the amino acid sequence of the corresponding sequence number. For example, this includes cases in which the protein has an addition of a sequence that does not alter the function of the protein at the N-terminus and / or C-terminus, a mutation that may occur naturally, a silent mutation thereof, or a conservative substitution.

[0033] In the present application, various methods well known in the art can be applied to secure gamma-aminobutyrate permease. Examples of such methods include, but are not limited to, gene synthesis techniques including codon optimization to efficiently secure enzymes from microorganisms commonly used for enzyme expression, and screening of useful enzyme resources using bioinformatics methods based on large-scale microbial genome information to secure mutant strains.

[0034]

[0035] In this application, the term "mutant" or "variant" means a culture or individual that exhibits a stable phenotypic change, either genetically or non-genetically, and specifically, in this application, it may be a mutant in which the amino acid of gamma-aminobutyrate permease having the amino acid sequence of SEQ ID NO: 1 is mutated so that its activity is efficiently increased compared to the wild type, but is not limited thereto.

[0036] As used herein, the term "variant" or "modified polypeptide" refers to a protein in which one or more amino acids differ from the recited sequence by conservative substitution and / or modification, but the functions or properties of the protein are maintained. The variant differs from the identified sequence by several amino acid substitutions, deletions, or additions. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the protein and evaluating the properties of the modified protein. That is, the ability of the variant may be increased, unchanged, or decreased compared to the native protein. Additionally, some variants may include variant polypeptides in which one or more portions, such as the N-terminal leader sequence or the transmembrane domain, are deleted. Other variants may include variants in which portions are removed from the N- and / or C-terminus of the mature protein. The term "variant" or "variant polypeptide" may be used interchangeably with terms such as variant, modified protein, mutant, mutein, divergent, variant, etc., and is not limited thereto as long as the term is used in the meaning of variant.

[0037] For the purposes of this application, the mutant may be, but is not limited to, a mutant protein having increased activity compared to the native wild-type or unmodified protein.

[0038] The term "conservative substitution" in this application refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. Such variants may, for example, have one or more conservative substitutions while still retaining one or more biological activities. Such amino acid substitutions may generally be based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues.

[0039] For example, among amino acids having electrically charged side chains, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; and amino acids having uncharged side chains include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.

[0040] Additionally, variants may include deletions or additions of amino acids that have minimal impact on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated to a signal (or leader) sequence at the N-terminus of a protein that is involved in co-translational or post-translational protein transfer. Furthermore, the polypeptide may be conjugated to other sequences or linkers to facilitate identification, purification, or synthesis of the polypeptide.

[0041]

[0042] The variant of the present application may have γ-aminobutyrate permease activity, and may have increased γ-aminobutyrate permease activity, but is not limited thereto.

[0043] Although the variant having gamma-aminobutyric acid permease activity of the present application is described as a polypeptide or protein in which the amino acid corresponding to the 212th position in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid corresponding to the 114th position is substituted with phenylalanine, or the amino acid corresponding to the 28th position is substituted with valine, it does not exclude meaningless sequence additions or mutations that may occur naturally before or after the amino acid sequence in which the amino acid corresponding to the 212th position in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid corresponding to the 114th position is substituted with phenylalanine, or the amino acid corresponding to the 28th position is substituted with valine, or a silent mutation thereof, and has the same or corresponding activity as a protein including an amino acid sequence in which the amino acid corresponding to the 212th position in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid corresponding to the 114th position is substituted with phenylalanine, or the amino acid corresponding to the 28th position is substituted with valine. In such a case, it may be apparent to those skilled in the art that the polypeptide, protein, or variant having the gamma-aminobutyric acid permease activity of the present application corresponds to the polypeptide, protein, or variant of the present application. For example, the polypeptide or variant having the gamma-aminobutyric acid permease activity of the present application may be a polypeptide consisting of an amino acid sequence in which the amino acid corresponding to position 212 in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid corresponding to position 114 is substituted with phenylalanine, or the amino acid corresponding to position 28 is substituted with valine, or an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto.In addition, it may be obvious that a polypeptide having an amino acid sequence in which some sequences are deleted, modified, substituted or added is also included within the scope of the polypeptide of the present application, provided that the amino acid sequence has such homology or identity and exhibits an effect corresponding to the polypeptide.

[0044]

[0045] As used herein, the term "corresponding" or "corresponding position" refers to an amino acid residue at a position listed in a protein or polypeptide, or an amino acid residue that is similar, identical, or homologous to the listed residue in the protein or polypeptide. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related protein or reference protein.

[0046] In the present application, specific numbering may be used for amino acid residue positions within the protein used in the present application. For example, by aligning the polypeptide sequences of the target protein to be compared with the protein of the present application, it is possible to renumber positions corresponding to amino acid residue positions in the protein of the present application.

[0047]

[0048] In this application, the terms "homology" or "identity" refer to the degree to which two given amino acid sequences or base sequences are related, which may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0049] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and may be combined with default gap penalties established by the program being used. In practice, homologous or identical sequences are generally capable of hybridizing under moderate or high stringency conditions, typically along at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or its entire length. It should be appreciated that hybridization also encompasses polynucleotides containing common codons or codons considered codon degeneracy.

[0050] Whether any two polynucleotide or polypeptide sequences are homologous, similar or identical can be determined using known computer algorithms such as the "FASTA" program using default parameters, for example as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, 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) can be determined using the GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW of the National Center for Biotechnology Information Database.

[0051] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information, for example, using a GAP computer program such as that of Needleman et al. (1970), J Mol Biol. 48:443, as disclosed, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In brief, the GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and (2) a comparison matrix as disclosed by Gribskov et al. (1986) Nucl. Acids Res. 48:443, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979). 14: 6745 weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) permutation matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0052] Additionally, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined by comparing the sequences by Southern hybridization experiments under defined stringent conditions, and appropriate hybridization conditions are within the skill of the art and can be determined by methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).

[0053]

[0054] Another aspect of the present application provides a polynucleotide encoding a gamma-aminobutyrate permease variant of the present application. Specifically, the present application provides a polynucleotide encoding a gamma-aminobutyrate permease variant, wherein the amino acid corresponding to position 212 in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid corresponding to position 114 is substituted with phenylalanine, or the amino acid corresponding to position 28 is substituted with valine.

[0055] The amino acid sequence of the above sequence number 1, gamma-aminobutyric acid permease, and variants are as described above.

[0056] In the present application, the amino acid sequence of SEQ ID NO: 1 may be encoded by a polynucleotide including, for example, the nucleotide sequence of SEQ ID NO: 2, and a gamma-aminobutyric acid permease variant in which the amino acid corresponding to position 212 in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid corresponding to position 114 is substituted with phenylalanine, or the amino acid corresponding to position 28 is substituted with valine may be encoded by a polynucleotide including any one of the nucleotide sequences of SEQ ID NO: 22 to SEQ ID NO: 24, but is not limited thereto.

[0057] The polynucleotide encoding the gamma-aminobutyric acid permease variant may include any one of the base sequences of SEQ ID NO: 22 to SEQ ID NO: 24, but is not limited thereto.

[0058] In this application, the term "polynucleotide" means a polymer of nucleotides in which nucleotide units are covalently bonded to form a long chain, and refers to a DNA or RNA strand of a certain length or longer. In this application, the polynucleotide may encode a polypeptide exhibiting the activity of gamma-aminobutyric acid permease, in which the amino acid corresponding to position 212 in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid corresponding to position 114 is substituted with phenylalanine, or the amino acid corresponding to position 28 is substituted with valine, but is not limited thereto.

[0059] The above polynucleotide may be included without limitation as long as it is a polynucleotide encoding a polypeptide having the activity of gamma-aminobutyric acid permease according to the present application. In the present application, the gene encoding the amino acid sequence of gamma-aminobutyric acid permease is the aroP gene, and the gene may be derived from a microorganism of the genus Corynebacterium sp., and specifically, may be derived from Corynebacterium glutamicum, but is not limited thereto.

[0060] Specifically, the polynucleotide encoding the polypeptide exhibiting the activity of the gamma-aminobutyric acid permease may include a base sequence encoding an amino acid in which the amino acid corresponding to the 212th position in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 1 is substituted with threonine, the amino acid corresponding to the 114th position is substituted with phenylalanine, or the amino acid corresponding to the 28th position is substituted with valine. The polynucleotide may be subjected to various modifications in the coding region within a range that does not change the amino acid sequence of the polypeptide due to the degeneracy of the codon or in consideration of the codon preferred in an organism that is to express the polypeptide. The polynucleotide may include, for example, the base sequence of SEQ ID NO: 2, and may be composed of a base sequence having a homology or identity of 80% or more, specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, or even more specifically 99% or more, but is not limited thereto.

[0061] In addition, the polynucleotide of the present application may include, without limitation, a probe that can be prepared from a known gene sequence, for example, a sequence that encodes an amino acid sequence in which the amino acid corresponding to position 212 in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid corresponding to position 114 is substituted with phenylalanine, or the amino acid corresponding to position 28 is substituted with valine by hybridizing under stringent conditions with a complementary sequence for all or part of the base sequence.

[0062] Specifically, in the amino acid sequence of SEQ ID NO: 1, the sequence encoding a variant in which the amino acid corresponding to position 212 is substituted with threonine may include SEQ ID NO: 22, the sequence encoding a variant in which the amino acid corresponding to position 114 is substituted with phenylalanine may include SEQ ID NO: 23, and the sequence encoding a variant in which the amino acid corresponding to position 28 is substituted with valine may include SEQ ID NO: 24, but is not limited thereto.

[0063] The above "stringent conditions" refer to conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (e.g., J. Sambrook et al., supra). For example, conditions under which polynucleotides having a high degree of homology or identity hybridize with each other, specifically at least 40%, specifically at least 90%, more specifically at least 95%, at least 96%, at least 97%, at least 98%, and even more specifically at least 99%, and polynucleotides having a lower degree of homology or identity than that hybridize, or conditions under which washing is performed once, specifically twice or three times, at a salt concentration and temperature equivalent to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, and more specifically 68°C, 0.1×SSC, 0.1% SDS, which are washing conditions of typical southern hybridization, can be listed.

[0064] Hybridization requires that two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of the present application may also include isolated nucleic acid fragments that are complementary in their entirety, as well as substantially similar nucleic acid sequences.

[0065] Specifically, polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the conditions described above. In addition, 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.

[0066] The appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, variables well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0067]

[0068] Another aspect of the present application provides a vector comprising a polynucleotide encoding a gamma-aminobutyrate permease variant of the present application. Specifically, the vector provides a vector comprising a polynucleotide encoding a gamma-aminobutyrate permease variant, wherein the amino acid corresponding to position 212 in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid corresponding to position 114 is substituted with phenylalanine, or the amino acid corresponding to position 28 is substituted with valine.

[0069] The amino acid sequence of the above sequence number 1, gamma-aminobutyric acid permease, mutant, and polynucleotide are as described above.

[0070] The term "vector" as used herein refers to a DNA construct containing 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 sequences regulating the termination of transcription and translation. After being transformed into a suitable host cell, the vector may replicate or function independently of the host genome, or may be integrated into the genome itself.

[0071] The vector used in the present application is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used as plasmid vectors. Specifically, pDCM2 (Korean Patent Publication No. 10-2020-0136813), pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

[0072] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using a vector for intracellular chromosomal insertion. The insertion of the polynucleotide into the chromosome can be achieved by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker for confirming the chromosomal insertion can be additionally included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the insertion of the target nucleic acid molecule. Markers that confer a selectable phenotype, such as drug resistance, nutrient requirement, cytotoxic agent resistance, or expression of a surface polypeptide, can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypic traits, so that transformed cells can be selected.

[0073] In this application, the term "expression cassette" refers to a unit cassette that includes a promoter and a target gene, and is capable of expressing the target gene operably linked to the promoter. Various factors that can facilitate efficient expression of the target gene may be included inside or outside of such a gene expression cassette. The gene expression cassette may typically include, but is not limited to, a transcription termination signal, a ribosome binding site, and a translation termination signal in addition to a promoter operably linked to the target gene.

[0074] The term "transformation" in this application refers to introducing a vector containing a polynucleotide encoding a target protein into a host cell or microorganism, thereby enabling expression of the protein encoded by the polynucleotide within the host cell. The transformed polynucleotide may be located within the chromosome of the host cell or located extrachromosomally, as long as it can be expressed within the host cell. Furthermore, the polynucleotide includes DNA and RNA encoding the target protein. The polynucleotide may be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for autonomous expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, all of which are operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. Additionally, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0075] Additionally, the term "operably linked" as used herein means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding the target polypeptide of the present application.

[0076] The method for transforming the vector of the present application includes any method for introducing nucleic acids into cells, and can be performed by selecting an appropriate standard technique known in the art depending on the host cell. Examples include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0077]

[0078] Another aspect of the present application provides a microorganism comprising at least one of: a γ-aminobutyrate permease variant, wherein the amino acid corresponding to position 212 in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid corresponding to position 114 is substituted with phenylalanine, or the amino acid corresponding to position 28 is substituted with valine; a polynucleotide encoding the variant; and a vector comprising the polynucleotide.

[0079] As a specific example, the microorganism of the present application may be a microorganism that produces L-isoleucine, but is not limited thereto. Specifically, the microorganism of the present application may be a microorganism that produces L-isoleucine as a target product, and may be a microorganism with increased L-isoleucine production ability, including at least one of the mutant, polynucleotide, and vector of the present application, but is not limited thereto.

[0080] The amino acid sequence of the above sequence number 1, gamma-aminobutyric acid permease, variant, polynucleotide, vector, and L-isoleucine are as described above.

[0081]

[0082] In this application, the term "microorganism" includes both wild-type microorganisms and microorganisms that have undergone genetic modification, either naturally or artificially, and is a concept that includes all microorganisms whose specific mechanisms are weakened or strengthened due to causes such as the insertion of an external gene or the strengthening or weakening of the activity of an endogenous gene. In this application, the microorganism may be included without limitation as long as it is a microorganism into which one or more of a gamma-aminobutyrate permease mutant in which the amino acid corresponding to position 212 in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid corresponding to position 114 is substituted with phenylalanine, or the amino acid corresponding to position 28 is substituted with valine; a polynucleotide encoding the mutant; and a vector comprising the polynucleotide.

[0083] In the present application, the microorganism may comprise one or more of the polypeptide; a polynucleotide encoding the polypeptide; and a vector comprising the polynucleotide.

[0084] The microorganism of the present application may be a microorganism having the ability to produce a target protein or a target product involved in the production of the target protein, including at least one of the polypeptide; a polynucleotide encoding the polypeptide; and a vector comprising the polynucleotide, but is not limited thereto. The microorganism may be a microorganism naturally having the ability to produce the target protein or the target product, or a microorganism in which the ability to produce the target protein or the target product has been conferred on a parent strain that does not have the ability to produce the target protein or the target product, but is not limited thereto.

[0085] The above microorganism is a cell or microorganism that is transformed with a vector containing the polynucleotide of the present application and a gene encoding the target protein, and expresses the target protein. For the purpose of the present application, the host cell or microorganism may be any microorganism capable of producing a target product including the target protein.

[0086] The above microorganism may be a recombinant microorganism, and the recombination may be achieved by genetic modification such as transformation.

[0087]

[0088] In this application, the term "microorganism producing a target protein or target product" includes both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and may be a microorganism that has a specific mechanism weakened or strengthened due to causes such as the insertion of an external gene or the enhancement or inactivation of the activity of an endogenous gene, and may be a microorganism that includes genetic modification for the production of a target protein or product.

[0089] The microorganism may be, but is not limited to, a microorganism genetically modified through one or more of the polypeptide, a polynucleotide encoding the polypeptide, and a vector comprising the polynucleotide; a microorganism modified to express the polypeptide or the polynucleotide encoding the polypeptide; a recombinant microorganism expressing the polypeptide or the polynucleotide encoding the polypeptide; or a recombinant microorganism having the polypeptide activity.

[0090] For the purposes of this application, the microorganism producing the target protein or target product may be a microorganism characterized by an increased ability to produce the target protein or target product, including the polynucleotide of this application. Specifically, the microorganism producing the target protein or target product in this application, or the microorganism having the ability to produce the target protein or target product, may be a microorganism in which some of the genes in the biosynthetic pathway of the target protein or target product are strengthened or weakened, or some of the genes in the degradation pathway of the target protein or target product are strengthened or weakened.

[0091] The microorganism of the present application may be a microorganism comprising at least one of a gamma-aminobutyrate permease mutant in which the amino acid corresponding to position 212 in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid corresponding to position 114 is substituted with phenylalanine, or the amino acid corresponding to position 28 is substituted with valine; a polynucleotide encoding the mutant; and a vector including the polynucleotide, but is not limited thereto. In addition, the microorganism of the present application may be a microorganism that produces L-isoleucine as a target product, and may have increased L-isoleucine productivity or reduced byproducts generated during L-isoleucine production compared to an unmodified or wild-type microorganism, but is not limited thereto.

[0092] In this application, the term "protein to be / is expressed" refers to a state in which a target protein is introduced into a microorganism or modified to be expressed within the microorganism. If the target protein is a protein existing within the microorganism, this refers to a state in which its activity is enhanced compared to its inherent state or prior to modification.

[0093] The microorganism expressing the protein variant of the present application may be a microorganism modified to express the protein variant, and therefore another aspect of the present application provides a method for producing a microorganism expressing the protein variant of the present application.

[0094] In this application, "introduction of a protein" means that a microorganism exhibits the activity of a specific protein that it did not originally possess, or exhibits enhanced activity compared to the protein's inherent activity or activity prior to modification. For example, a specific protein may be introduced, a polynucleotide encoding a specific protein may be introduced into the chromosome of a microorganism, or a vector containing a polynucleotide encoding a specific protein may be introduced into the microorganism, resulting in the expression of its activity.

[0095] As used herein, the term "enhancement" of polypeptide or protein activity means that the activity of the polypeptide or protein is increased compared to the intrinsic activity. The term "enhancement" may be used interchangeably with terms such as up-regulation, overexpression, and increase. Here, the term "increase" may include both exhibiting an activity that was not originally present, and exhibiting an enhanced activity compared to the intrinsic activity or activity before modification. The term "intrinsic activity" refers to the activity of a specific polypeptide or protein that was originally present in a parent strain or unmodified microorganism before the transformation, when the trait is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before modification." The term "enhancement" or "increase" of the activity of a polypeptide or protein compared to the intrinsic activity means that the activity of a specific polypeptide or protein is enhanced compared to the activity that was originally present in the parent strain or unmodified microorganism before the transformation. For example, the protein activity may be improved by at least 1%, 3%, 5%, 10%, 25%, 50%, 75%, 100%, 125%, 150%, 200%, 250%, 300%, 350%, 400%, or 500%, or up to 1000% or 2000%, but is not limited thereto, compared to the protein activity of the parent strain or unmodified microorganism before the transformation. The term "about" includes, but is not limited to, a range including all of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values ​​in a range equal to or similar to the value following the term "about."

[0096] The above "increased activity" may be achieved by introducing an exogenous polypeptide or protein, or by enhancing the activity of an endogenous polypeptide or protein. Specifically, it may be achieved by enhancing the activity of an endogenous polypeptide or protein. Whether the activity of the polypeptide or protein is enhanced can be confirmed by an increase in the level of activity of the polypeptide or protein, the amount of expression, or the amount of a product secreted from the protein.

[0097] Enhancement of the activity of the polypeptide or protein may be achieved by applying various methods well known in the art, and may not be limited as long as the activity of the target polypeptide or protein can be enhanced compared to the microorganism before modification. The method may utilize, but is not limited to, genetic engineering and / or protein engineering, which are routine methods of molecular biology and are well known to those skilled in the art (Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0098]

[0099] Methods for enhancing polypeptide or protein activity using the above genetic engineering include, for example,

[0100] 1) Increase in the intracellular copy number of a gene or polynucleotide encoding the above polypeptide or protein;

[0101] 2) A method of replacing the gene expression control region on a chromosome encoding the above polypeptide or protein with a highly active sequence;

[0102] 3) A method for modifying the base sequence of the initiation codon or 5'-UTR region of the above polypeptide or protein;

[0103] 4) A method for modifying a polynucleotide sequence on a chromosome so as to increase the activity of the polypeptide or protein;

[0104] 5) Introduction of a foreign polynucleotide exhibiting the activity of the above polypeptide or protein or a codon-optimized variant polynucleotide of the above polynucleotide, or

[0105] 6) It can be performed by a combination of the above methods, but is not limited thereto.

[0106]

[0107] The method of enhancing polypeptide or protein activity using the above protein engineering can be performed, for example, by analyzing the tertiary structure of a polypeptide or protein, selecting an exposed portion, and modifying or chemically modifying the exposed portion, but is not limited thereto.

[0108] The above 1) increase in the intracellular copy number of a gene or polynucleotide encoding a polypeptide or protein can be performed by any method known in the art, for example, by introducing a vector capable of replicating and functioning independently of a host, to which the gene or polynucleotide encoding the polypeptide or protein is operably linked, into a host cell. Alternatively, the method can be performed by introducing a vector capable of inserting the gene or polynucleotide into a chromosome within the host cell, to which the gene is operably linked, into the host cell, but is not limited thereto. The vector is as described above.

[0109] The method of replacing the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide or protein in the above 2) with a sequence having strong activity can be performed by any method known in the art, for example, by inducing a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof of the nucleic acid sequence to further enhance the activity of the expression control region, or by replacing it with a nucleic acid sequence having stronger activity. The expression control region may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating the termination of transcription and translation, etc. The method may specifically be, but is not limited to, linking a strong heterologous promoter instead of the original promoter.

[0110] Examples of known strong promoters include, but are not limited to, the cj1 to cj7 promoters (US Patent No. US 7662943 B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (US Patent No. US 10584338 B2), the O2 promoter (US Patent No. US 10273491 B2), the tkt promoter, and the yccA promoter.

[0111] The method of modifying the base sequence of the initiation codon or 5'-UTR region of the polypeptide or protein, as described above, may be any method known in the art, for example, replacing the endogenous initiation codon of the polypeptide or protein with another initiation codon having a higher polypeptide or protein expression rate than the endogenous initiation codon, but is not limited thereto.

[0112] The method of modifying a polynucleotide sequence on a chromosome to increase the activity of the polypeptide or protein described above 4) can be performed by any method known in the art, for example, by inducing a mutation in an expression regulatory sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof of the nucleic acid sequence to further enhance the activity of the polynucleotide sequence, or by replacing it with a polynucleotide sequence that has been improved to have stronger activity. Specifically, the replacement may be, but is not limited to, inserting the gene into the chromosome by homologous recombination.

[0113] The vector used at this time may additionally include a selection marker to confirm whether chromosomal insertion has occurred. The selection marker is as described above.

[0114] 5) The introduction of a foreign polynucleotide exhibiting the activity of the polypeptide or protein may be performed by any method known in the art, for example, by introducing a foreign polynucleotide encoding a polypeptide or protein exhibiting the same / similar activity as the polypeptide or protein, or a codon-optimized mutant polynucleotide thereof, into a host cell. The foreign polynucleotide may be used without limitation in its origin or sequence as long as it exhibits the same / similar activity as the polypeptide or protein. In addition, the introduced foreign polynucleotide may be introduced into a host cell by optimizing its codons so that optimized transcription and translation can occur within the host cell. The introduction may be performed by a person skilled in the art appropriately selecting a known transformation method, and the introduced polynucleotide may be expressed within the host cell, thereby producing a polypeptide or protein and increasing its activity.

[0115] Finally, 6) a combination of the above methods can be performed by applying one or more of the above methods 1) to 5) together.

[0116] Such enhancement of polypeptide or protein activity may be, but is not limited to, an increase in the activity or concentration of the corresponding polypeptide or protein relative to the activity or concentration of the polypeptide or protein expressed in the wild-type or unmodified microbial strain, or an increase in the amount of a product produced from the polypeptide or protein.

[0117] In this application, the term "pre-transformation strain" or "pre-transformation microorganism" does not exclude a strain that contains a mutation that can occur naturally in a microorganism, and may refer to a wild-type strain or a natural strain itself, or a strain before its characteristics are changed by genetic mutation due to natural or artificial factors. The "pre-transformation strain" or "pre-transformation microorganism" may be used interchangeably with "non-mutated strain", "non-transformed strain", "non-mutated microorganism", "non-transformed microorganism", or "reference microorganism".

[0118]

[0119] In one specific example, the microorganism of the present application may be, but is not limited to, Corynebacterium sp.

[0120] The “genus Corynebacterium” in the present application may include all microorganisms of the genus Corynebacterium. Specifically, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli pollutisoli), Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens, and more specifically, Corynebacterium glutamicum, Corynebacterium callunae, Corynebacterium crenatum or Corynebacterium deserti.

[0121] In the present application, the parent strain of the microorganism may be a microorganism whose biosynthetic pathway of L-isoleucine has been additionally strengthened to increase the production of L-isoleucine, but is not limited thereto.

[0122] Specifically, the microorganism may be a microorganism that has, for example, replaced the aroP promoter with the gapA promoter to strengthen the function of the promoter in order to strengthen the biosynthetic pathway of L-isoleucine, or has additionally introduced a genetic mutation (R407H) (Korean Patent No. 10-1996769) into the hom gene encoding homoserine dehydrogenase to eliminate feedback inhibition of threonine, a precursor of isoleucine, or has introduced a genetic mutation (T381A, F383A) into the ilvA gene encoding L-threonine dehydratase (Korean Patent Application No. 10-2020-0078669), or has additionally introduced a genetic mutation (L377K) (U.S. Registration Publication No. US 10662450 B2) into the lysC gene encoding aspartokinase to eliminate feedback inhibition of threonine, a precursor of isoleucine. However, the production of L-isoleucine can be increased by any method of controlling gene expression known in the art without being limited to the above.

[0123]

[0124] As another example for increasing the production of L-isoleucine, the parent strain of the microorganism in the present application may be a microorganism in which a gene that weakens the biosynthetic pathway of L-isoleucine is additionally inactivated to increase the production of L-isoleucine, but is not limited thereto.

[0125] In this application, the term "inactivation" or "weakening" of a polypeptide or protein includes both a decrease in activity or absence of activity compared to the intrinsic activity. The term "inactivation" or "weakening" may be used interchangeably with terms such as down-regulation, decrease, and reduce. The term "inactivation" or "weakening" may also include cases where the activity of the protein itself is reduced or eliminated compared to the activity of the protein originally possessed by the microorganism due to mutation of the gene encoding the protein, etc., cases where the overall protein activity level in the cell is lower than that of the natural strain due to inhibition of expression or translation of the gene encoding the protein, cases where the gene is not expressed at all, and cases where the gene is expressed but is inactive. The term "intrinsic activity" refers to the activity of a specific polypeptide or protein originally possessed by a parent strain or unmodified microorganism before the phenotype change, when the phenotype changes due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before modification." A "reduction" in the activity of a polypeptide or protein relative to its intrinsic activity means that the activity of the polypeptide or protein is reduced compared to the activity of the parent strain or unmodified microorganism prior to the transformation. For example, the activity may be reduced compared to the protein activity of the parent strain or unmodified microorganism prior to the transformation, but is not limited thereto.

[0126]

[0127] Inactivation or attenuation of the activity of these proteins can be achieved by the application of various methods well known in the art, including but not limited to (Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Sambrooket al. Molecular Cloning 2012, etc.).

[0128] As an example of the above method,

[0129] 1) A method of deleting all or part of the gene encoding the protein;

[0130] 2) Modification of the expression control region (or expression control sequence) so that the expression of the gene encoding the protein is reduced;

[0131] 3) Modification of the gene sequence encoding the protein such that the activity of the protein is eliminated or weakened;

[0132] 4) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that complementarily binds to the transcript of the gene encoding the protein;

[0133] 5) A method of forming a secondary structure by adding a sequence complementary to the Shine-Dalgarno sequence to the front of the Shine-Dalgarno sequence of the gene encoding the protein, thereby making attachment of the ribosome impossible;

[0134] 6) There is a method of adding a promoter transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the polynucleotide sequence of the gene encoding the protein (Reverse transcription engineering, RTE), and a combination of these can also be achieved, but there is no particular limitation thereto.

[0135] Specifically, the method for deleting part or all of the gene encoding the protein can be performed by replacing a polynucleotide encoding the endogenous target protein within the chromosome with a polynucleotide or marker gene having a partial nucleotide sequence deleted through a vector for chromosomal insertion into a microorganism. As an example of the method for deleting part or all of the polynucleotide, a method of deleting the polynucleotide by homologous recombination can be used, but is not limited thereto.

[0136] In addition, the method of deleting part or all of the above gene can be performed by inducing mutations using light such as ultraviolet rays or chemicals, and selecting a strain with a deletion of the target gene from the resulting mutants. The gene deletion method includes a method using DNA recombination technology. The DNA recombination technology can be performed, for example, by injecting a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism to cause homologous recombination. In addition, the injected nucleotide sequence or vector may include, but is not limited to, a dominant selection marker.

[0137] In addition, the method of modifying the above expression control sequence can be achieved by applying various methods well known in the art. As an example of the above method, it can be performed by inducing a mutation in the expression control region (or expression control sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so as to further weaken the activity of the expression control region (or expression control sequence), or by replacing it with a polynucleotide sequence having weaker activity. The expression control 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.

[0138]

[0139] In addition, the method of modifying the gene sequence may be performed by inducing a sequence mutation in the gene sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof to further weaken the activity of the polypeptide, or by replacing the gene sequence with a gene sequence modified to have weaker activity or a gene sequence modified to have no activity, but is not limited thereto.

[0140] For example, the expression of a gene can be inhibited or weakened by introducing a mutation in the gene sequence to form a stop codon.

[0141]

[0142] Another aspect of the present application provides a method for producing L-isoleucine. Specifically, the method comprises the step of culturing a microorganism comprising at least one of a gamma-aminobutyrate permease variant, wherein the amino acid corresponding to position 212 in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid corresponding to position 114 is substituted with phenylalanine, or the amino acid corresponding to position 28 is substituted with valine; a polynucleotide encoding the variant; and a vector comprising the polynucleotide.

[0143] Another aspect of the present application provides a method for reducing byproducts generated during the production of L-isoleucine. Specifically, the method comprises the step of culturing a microorganism comprising at least one of a gamma-aminobutyrate permease variant, wherein the amino acid corresponding to position 212 in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid corresponding to position 114 is substituted with phenylalanine, or the amino acid corresponding to position 28 is substituted with valine; a polynucleotide encoding the variant; and a vector comprising the polynucleotide.

[0144] The above L-isoleucine, amino acid of sequence number 1, gamma-aminobutyric acid permease, mutant, polynucleotide, vector, and microorganism are as described above.

[0145] The above microorganism may be of the genus Corynebacterium, and specifically may be Corynebacterium glutamicum, but is not limited thereto. This is as described above.

[0146]

[0147] In this application, the term "cultivation" refers to growing the microorganism under appropriately controlled environmental conditions. The cultivation process of this application can be performed using appropriate media and culture conditions known in the art. This cultivation process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the cultivation may be batch, continuous, or fed-batch, but is not limited thereto.

[0148] In this application, the term "medium" refers to a material containing nutrients necessary for culturing the microorganism as a main component, and supplies nutrients and growth factors, including water, which is essential for survival and growth. Specifically, the medium and other culture conditions used for culturing the microorganism of this application may be any medium used for culturing general microorganisms without particular limitation, but the microorganism of this application may be cultured under aerobic conditions while controlling temperature, pH, etc. in a general medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin.

[0149] 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 nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane bagasse, and corn steep liquor may be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugar) may be used, and other appropriate amounts of carbon sources may be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0150] The nitrogen source may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc.; peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0151] The above-mentioned components may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added to the medium in batch or continuous manner, but are not limited thereto.

[0152] In addition, during the cultivation of the above microorganism, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. can be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during the cultivation, foaming can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. In addition, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas can be injected into the medium, or in order to maintain the anaerobic and microaerobic state, nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection, but is not limited thereto.

[0153] The temperature of the medium may be, but is not limited to, 20°C to 50°C, specifically 30°C to 37°C. The incubation period may continue until the desired amount of useful material is produced, and specifically may be, but is not limited to, 10 to 100 hours.

[0154] Specifically, culture media for strains of the genus Corynebacterium can be found in, but are not limited to, the "Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981).

[0155]

[0156] As a specific example, the method for producing L-isoleucine, the method for manufacturing L-isoleucine, or the method for reducing byproducts generated during L-isoleucine production may additionally include a step of recovering L-isoleucine from the medium or microorganism, but is not limited thereto.

[0157] The method for recovering the above L-isoleucine may be to collect the desired L-isoleucine using a suitable method known in the art according to the culture method of the microorganism of the present application, for example, a batch, continuous or fed-batch culture method. For example, various chromatographies such as centrifugation, filtration, treatment with a crystallized protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC and a combination of these methods may be used, and the desired L-isoleucine may be recovered from the medium or microorganism using a suitable method known in the art.

[0158] The above production method may include an additional purification process. The purification process may purify the recovered L-isoleucine using any suitable method known in the art. The recovered L-isoleucine may be in purified form or as a microbial fermentation broth containing the desired product (Introduction to Biotechnology and Genetic Engineering, AJ Nair, 2008).

[0159]

[0160] As a specific example, the method for producing or manufacturing L-isoleucine may reduce byproducts generated during L-isoleucine production, but is not limited thereto.

[0161] In the present application, the term "by-product" includes all substances that may be generated during the production of L-isoleucine. The by-product may be any one or more selected from, but is not limited to, alpha-aminobutyrate (AABA) and amino acids other than L-isoleucine, such as arginine (Arg, R), histidine (His, H), glutamic acid (Glu, E), aspartic acid (Asp, D), glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe, F), tryptophan (Trp, W), proline (Pro, P), serine (Ser, S), cysteine ​​(Cys, C), tyrosine (Tyr, Y), asparagine (Asn, N), and glutamine (Gln, Q). For the purpose of the present application, the by-product may be at least one selected from the group consisting of alpha-aminobutyric acid (α-aminobutyrate; AABA), valine, phenylalanine, and leucine, and specifically, may be alpha-aminobutyric acid or valine, but is not limited thereto.

[0162]

[0163] Another aspect of the present application provides a composition for producing L-isoleucine. Specifically, the composition comprises a microorganism or a culture of the microorganism, which comprises at least one of a gamma-aminobutyrate permease variant, wherein the amino acid corresponding to position 212 in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid corresponding to position 114 is substituted with phenylalanine, or the amino acid corresponding to position 28 is substituted with valine; a polynucleotide encoding the variant; and a vector comprising the polynucleotide.

[0164] The amino acid, gamma-aminobutyric acid permease, variant, polynucleotide, vector, microorganism, and culture of the above sequence number 1 are as described above.

[0165] The composition for producing L-isoleucine may refer to a composition capable of producing L-isoleucine with high efficiency using the gamma-aminobutyric acid permease variant of the present application. The composition may include, without limitation, the variant of gamma-aminobutyric acid permease or a component capable of operating the gamma-aminobutyric acid permease variant. The variant of gamma-aminobutyric acid permease may be in a form included in a vector so as to be capable of expressing an operably linked gene in an introduced host cell.

[0166] The composition may further comprise a cryoprotectant or excipient. The cryoprotectant or excipient may be, but is not limited to, a non-naturally occurring substance or a naturally occurring substance. In another specific example, the cryoprotectant or excipient may be, but is not limited to, a substance with which the strain does not naturally come into contact or a substance that is not naturally co-incorporated with the strain.

[0167]

[0168] Another aspect of the present application provides a use of a microorganism for producing L-isoleucine, comprising a γ-aminobutyrate permease variant, wherein the amino acid corresponding to position 212 in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid corresponding to position 114 is substituted with phenylalanine, or the amino acid corresponding to position 28 is substituted with valine; a polynucleotide encoding the variant; and a vector comprising the polynucleotide, or at least one of the variant, the polynucleotide encoding the variant, and the vector comprising the polynucleotide.

[0169] The above mutant, polynucleotide, vector, microorganism, and isoleucine are as described above.

[0170]

[0171] Hereinafter, this application will be described in more detail through examples. However, these examples and experimental examples are intended to exemplify this application and the scope of this application is not limited to these examples and experimental examples.

[0172]

[0173] Reference Example 1: Construction of a vector for overexpression of the aroP gene

[0174]

[0175] In order to more clearly identify the phenotypic changes caused by the aroP gene encoding gamma-aminobutyrate permease and its mutants according to the purpose of the present application, the aroP gene was overexpressed in the Corynebacterium glutamicum ATCC13032 strain.

[0176] To overexpress the aroP gene encoding gamma-aminobutyric acid permease, a plasmid vector with an enhanced aroP promoter was constructed by replacing the aroP gene promoter with the gapA promoter.

[0177] To create a strain in which the aroP promoter was replaced with the gapA promoter, PCR was performed using the chromosome of ATCC13032 as a template and the primers of SEQ ID NO: 4 and SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, or SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The primers used to perform each of the PCRs are as shown in Table 1 below.

[0178]

[0179] SEQ ID NO NAME Sequence 4primerAATTCGAGCTCGGTACCCTTCCCCCTGGAGTCCGCA5primerATAGGCTCGGTCGTTTTTAGGCTTGTATCAACCGTAAACCCA6primerTGGGTTTACGGTTGATACAAGCCTAAAAACGACCGAGCCT AT7primerCCTTCATTAGATTTAGCCATGTGTCTCCTCTAAAGATTGT8primerACAATCTTTAGAGGAGACACATGGCTAAATCTAATGAAGG9primerGTCGACTCTAGAGGATCCCCTCAGTTCAAGTCGGAAGGGGTGCGA

[0180] PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the above PCR reaction, and the PCR conditions were denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute, and these denaturation, annealing, and polymerization conditions were repeated 28 times. As a result, a 1000 bp DNA fragment at the 5' upstream region and a 1392 bp DNA fragment at the 3' downstream region centered on the gapA promoter region and the aroP gene start codon were obtained, respectively. Using the three amplified DNA fragments as templates, PCR was performed again with the primers of SEQ ID NO: 4 and SEQ ID NO: 9. The PCR conditions were denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds and polymerization at 72°C for 2 minutes, repeated six times, and then denaturation at 95°C for 30 seconds; After 20 cycles of 30-second annealing at 55°C and 2-minute polymerization at 72°C, the polymerization reaction was performed at 72°C for 5 minutes. As a result, a 2.4-kb DNA fragment encoding the aroP gene containing the gapA promoter was amplified. The amplified product was purified using a PCR Purification kit from QUIAGEN and used as an insert DNA fragment for vector construction.

[0181] Meanwhile, after treating the vector with the restriction enzyme smaI, the molar concentration (M) ratio of the pDCM2 vector heat-treated at 65°C for 20 minutes and the inserted DNA fragment amplified through the PCR was made 1:2, and the vector pDCM2-PgapA-aroP was constructed by cloning according to the provided manual using TaKaRa's Infusion Cloning Kit, thereby replacing the aroP promoter with the gapA promoter and introducing it into the chromosome.

[0182]

[0183] Reference Example 2: Production of L-isoleucine producing strain

[0184]

[0185] Wild-type Corynebacterium glutamicum has the ability to produce L-isoleucine, but does not produce excessive amounts. Therefore, to identify genetic traits that increase L-isoleucine production in accordance with the purpose of this application, a strain with increased L-isoleucine production was utilized.

[0186] First, an L-isoleucine-producing strain was developed from wild-type Corynebacterium glutamicum ATCC13032. Specifically, to eliminate feedback inhibition of threonine, a precursor of isoleucine in the L-isoleucine biosynthetic pathway, the hom gene encoding homoserine dehydrogenase was mutated to substitute arginine, the 407th amino acid of homoserine dehydrogenase, with histidine (Korean Patent No. 10-1996769). Specifically, the polynucleotide sequence encoding hom(R407H) is shown in SEQ ID NO: 10.

[0187] Specifically, to produce strains in which the hom(R407H) mutation was introduced, PCR was performed using the chromosome of Corynebacterium glutamicum ATCC13032 as a template and primers of SEQ ID NO: 11 and SEQ ID NO: 12 or SEQ ID NO: 13 and SEQ ID NO: 14, respectively. The primer sequences used to perform each of the PCRs are as shown in Table 2 below.

[0188]

[0189] SEQ ID NO NAME Sequence 11primerTCGAGCTCGGTACCCCGCTTTTGCACTCATCGAGC12primerCACGATCAGATGTGCATCATCAT13primerATGATGATGCACATCTGATCGTG14primerCTCTAGAGGATCCCCGAGCATCTTCCAAAACCTTG

[0190] PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction. The PCR conditions were denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute. These denaturation, annealing, and polymerization cycles were repeated 28 times. As a result, a 1,000-bp DNA fragment from the 5' upper region and a 1,000-bp DNA fragment from the 3' lower region were obtained, respectively, centered on the mutation in the hom gene.

[0191] Using the two amplified DNA fragments as templates, PCR was performed with primers of SEQ ID NO: 11 and SEQ ID NO: 14. The PCR conditions were denaturation at 95°C for 5 minutes, followed by 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 2 minutes, followed by polymerization at 72°C for 5 minutes.

[0192] As a result, a 2 kb DNA fragment containing a mutation in the hom gene encoding a homoserine dehydrogenase mutant in which the 407th arginine is substituted with histidine was amplified. The amplified product was purified using a PCR purification kit (PCR Purification kit, QUIAGEN) and used as an insert DNA fragment for vector construction. After treating the purified amplified product with the restriction enzyme smaI, the molar concentration (M) ratio of the pDCM2 vector (Korean Patent Publication No. 10-2020-0136813) heat-treated at 65°C for 20 minutes and the amplified product, the insert DNA fragment, was made 1:2, and the vector pDCM2-R407H was constructed to introduce the hom (R407H) mutation into the chromosome using an Infusion Cloning Kit (TaKaRa) according to the provided manual.

[0193] 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, and this was named Corynebacterium glutamicum ATCC13032 hom(R407H).

[0194] In order to increase the feedback release and activity for L-isoleucine in the ATCC13032 hom(R407H) strain produced, ilvA, a gene encoding L-threonine dehydratase, was mutated so that the 381st amino acid, threonine, of L-threonine dehydratase (SEQ ID NO: 25) was substituted with alanine, and the 383rd amino acid, phenylalanine, was substituted with alanine. Specifically, a strain into which ilvA(T381A, F383A) was introduced was produced, and the polynucleotide encoding ilvA(T381A, F383A) is shown in SEQ ID NO: 15.

[0195]

[0196] Specifically, to produce strains in which the above ilvA (T381A, F383A) mutation was introduced, PCR was performed using the chromosome of Corynebacterium glutamicum ATCC13032 as a template and primers of SEQ ID NO: 16 and SEQ ID NO: 17 or SEQ ID NO: 18 and SEQ ID NO: 19, respectively. The primer sequences used to perform each of the above PCRs are as shown in Table 3 below.

[0197]

[0198] SEQ ID NO NAME Sequence 16primerTCGAGCTCGGTACCCATGAGTGAAACATACGTGTC17primerGCGCTTGAGGTACTCtgcCAGCGcGATGTCATCATCCGG18primerCCGGATGATGACATCgCGCTGgcaGAGTACCTCAAGCGC19primerCTCTAGAGGATCCCCCGTCACCGACACCTCCACA

[0199] PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction. The PCR conditions were denaturation at 95°C for 30 seconds; denaturation at 55°C for 30 seconds; and polymerization at 72°C for 1 minute. These denaturation, annealing, and polymerization cycles were repeated 28 times. As a result, a 1,126 bp DNA fragment from the 5' upper region and a 286 bp DNA fragment from the 3' lower region were obtained, respectively, centered on the mutation in the ilvA gene.

[0200] Using the two amplified DNA fragments as templates, PCR was performed with primers of SEQ ID NO: 16 and SEQ ID NO: 19. The PCR conditions were denaturation at 95°C for 5 minutes, followed by 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 2 minutes, followed by polymerization at 72°C for 5 minutes.

[0201] As a result, a 1.4 kb DNA fragment containing a mutation in the ilvA gene encoding an L-threonine dehydratase mutant in which the 381st threonine was replaced with alanine and the 383rd phenylalanine was replaced with alanine was amplified. The amplified product was purified using a PCR purification kit (PCR Purification kit, QUIAGEN) and used as an insert DNA fragment for vector construction. The purified amplification product was treated with the restriction enzyme smaI, and then heat-treated at 65°C for 20 minutes. The molar concentration (M) ratio of the pDCM2 vector (Korean Patent Publication No. 10-2020-0136813) and the amplification product, the inserted DNA fragment, was set to 1:2, and the vector pDCM2- ilvA (T381A, F383A) was constructed to introduce the hom (R407H) mutation into the chromosome by cloning according to the provided manual using the Infusion Cloning Kit (TaKaRa).

[0202] The constructed vector was transformed into Corynebacterium glutamicum ATCC13032 hom (R407H) by electroporation, and through a second crossover process, a strain containing the ilvA (T381A, F383A) mutation on the chromosome was obtained, which was named Corynebacterium glutamicum CA10-3101.

[0203] The above strain CA10-3101 was deposited internationally with the Korea Center for Microbiological Cultures (KCCM), an international depository under the Budapest Treaty, on May 27, 2020 and assigned the accession number KCCM12739P.

[0204]

[0205] For reference, in order to confirm whether introducing the above ilvA (T381A, F383A) into the L-isoleucine producing strain increases the efficiency of L-isoleucine production by increasing the feedback release and activity for L-isoleucine, the following experiment was performed. Specifically, the concentrations of L-isoleucine and L-threonine in the culture solution of the KCCM11248P / pECCG117-ilvA (T381A, F383A) strain, which was an L-isoleucine producing strain treated with NTG (N-Methyl-N'-nitro-N-nitrosoguanidine) and in which the above ilvA (T381A, F383A) mutation was introduced by the electric pulse method, are shown in Table 4 below.

[0206]

[0207] Strain name L-isoleucine (g / L) L-threonine (g / L) KCCM11248P1.5 0.5 KCCM11248P / pECCG117-ilvA (F383A) 2.8 0.6 KCCM11248P / pECCG117-ilvA (T381A, F383A) 4.0 0.0

[0208] As shown in Table 4 above, the KCCM11248P / pECCG117-ilvA(T381A, F383A) strain, which introduced the ilvA(T381A, F383A) mutation, was confirmed to have a significantly increased L-isoleucine production and a higher L-threonine degradation rate compared to the KCCM11248P or KCCM11248P / pECCG117-ilvA(F383A) strains. In other words, for the purpose of the present application, it was confirmed that the ilvA(T381A, F383A) mutation was introduced to increase the feedback release and activity for L-isoleucine.

[0209]

[0210] Example 1: Production of an L-isoleucine-producing strain with the aroP gene overexpressed and confirmation of the effect of aroP on the fermentation purity of L-isoleucine.

[0211]

[0212] To determine whether aroP overexpression or mutation reduces byproducts and increases the purity of L-isoleucine during L-isoleucine production, the aroP gene was strengthened. For this purpose, the L-isoleucine-producing strain CA10-3101 constructed in Reference Example 2 was used as a target. Specifically, the vector constructed in Reference Example 1 was transformed into Corynebacterium glutamicum CA10-3101 by electroporation, and a strain with an aroP promoter strengthened on the chromosome was obtained through a secondary crossing process, which was named Corynebacterium glutamicum CA10-3117. The parent strain (CA10-3101) and the aroP-enhanced strain (CA10-3117) were inoculated into a 250 ml corner-bottom flask containing 25 ml of isoleucine production medium, and then cultured with shaking at 200 rpm for 60 hours at 32°C to produce L-isoleucine. The composition of the production medium used in this example is as follows.

[0213]

[0214] <Production medium>

[0215] Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, potassium phosphate monobasic 0.1%, magnesium sulfate heptahydrate 0.1%, iron sulfate heptahydrate 10 mg / ℓ, manganese sulfate monohydrate 10 mg / ℓ, biotin 200 μg / ℓ, pH 7.2

[0216]

[0217] After the culture was completed, the production amount of L-isoleucine and by-products was measured using high-performance liquid chromatography (HPLC), and the concentrations of L-isoleucine and by-products in the culture medium for each strain tested are shown in Table 5 below.

[0218]

[0219] Strain name L-isoleucine concentration (g / L) AABA concentration (g / L) L-valine concentration (g / L) CA10-3101 (parent strain) 2.5 0.9 1.4 CA10-3117 (aroP-enhanced strain) 2.7 0.4 1.1

[0220] As a result, as shown in Table 5 above, the parent strain Corynebacterium glutamicum CA10-3101 produced L-isoleucine at a concentration of 2.5 g / ℓ, and the major by-products, alpha-ketobutyric acid (AABA) and L-valine, were detected at 0.9 g / L and 1.4 g / L, respectively, whereas the aroP-enhanced strain Corynebacterium glutamicum CA10-3117 produced L-isoleucine at a concentration of 2.7 g / ℓ, confirming that L-isoleucine productivity increased by 108% compared to the parent strain. In addition, the by-products, alpha-ketobutyric acid and valine, were detected at 0.4 g / L and 1.1 g / L, confirming that the by-products, alpha-ketobutyric acid and valine, were decreased by 55% and 21%, respectively, compared to the parent strain.

[0221] Based on the above results, we confirmed that the aroP gene can be a target for increasing L-isoleucine fermentation purity by reducing L-isoleucine byproducts and increasing L-isoleucine productivity. Therefore, random mutagenesis was used to further improve aroP and develop superior aroP variants.

[0222]

[0223] Example 2: Construction of a mutant aroP library vector

[0224]

[0225] An aroP mutant library was constructed based on the vector constructed in Reference Example 1 above. To this end, the library was constructed using an error-prone PCR kit (clontech Diversify® PCR Random Mutagenesis Kit), and a PCR reaction was performed using SEQ ID NO: 8 and SEQ ID NO: 9 as primers under conditions where mutations may occur.

[0226] Specifically, under the condition that 0 to 3 mutations occur per 1000 bp, preheating 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, was performed. At this time, the obtained product was treated with DpnI and transformed into E. coli DH5α using megaprimer (500-125 ng) at 95°C for 50 seconds, 60°C for 50 seconds, and 68°C for 12 minutes, and then plated on LB solid medium containing kanamycin (25 mg / L). After selecting 20 transformed colonies, the plasmid was obtained and the polynucleotide sequence was analyzed, confirming that mutations were introduced at different positions at a frequency of 2 mutations / kb. Approximately 20,000 transformed E. coli colonies were taken, the plasmid was extracted, and it was named pTOPO-aroP-library.

[0227]

[0228] Example 3: Production of L-isoleucine strains with the aroP library introduced.

[0229]

[0230] The pTOPO-aroP-library produced in Example 2 was transformed into Corynebacterium glutamicum CA10-3101 by electroporation, and then spread on a nutrient medium containing 25 mg / L of kanamycin to obtain 5,000 colonies of strains with inserted mutant genes, and each colony was named CA10-3101 / pTOPO-aroPm1 to CA10-3101 / pTOPO-aroPm5000.

[0231] Among the 5,000 colonies secured, the fermentation activity of each colony was evaluated using the following method to identify colonies with increased L-isoleucine productivity and reduced byproducts. The parent strain and the mutant strain were inoculated into a 250 ml corner-bottom flask containing 25 ml of isoleucine production medium, and then cultured with shaking at 200 rpm for 60 hours at 32°C to produce L-isoleucine. The composition of the production medium used in this example is as follows.

[0232]

[0233] <Production medium>

[0234] Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, potassium phosphate monobasic 0.1%, magnesium sulfate heptahydrate 0.1%, iron sulfate heptahydrate 10 mg / ℓ, manganese sulfate monohydrate 10 mg / ℓ, biotin 200 μg / ℓ, pH 7.2

[0235]

[0236] After the culture was completed, the production amounts of L-isoleucine and L-threonine were measured using liquid high-performance chromatography (HPLC), and the concentrations of L-isoleucine, L-valine, and AABA in the culture medium for each strain tested are shown in Table 6 below.

[0237]

[0238] Strain name L-isoleucine concentration (g / L) AABA concentration (g / L) L-valine concentration (g / L) By-product ratio to isoleucine + by-product (AABA, L-valine) CA10-3101 (parent strain) 2.6 0.9 1.2 0.45 CA10-3101 / pTOPO-aroPm 138 3.0 0.6 1.2 0.38 CA10-3101 / pTOPO-aroPm 254 22.7 0.20 70.25 CA10-3101 / pTOPO-aroPm 379 82.8 0.4 1.10.35

[0239]

[0240] - By-product ratio: By-product (AABA, L-valine) concentration / (isoleucine + by-product) concentration

[0241]

[0242] As a result, as shown in Table 6 above, three mutant strains were identified that had increased L-isoleucine productivity and decreased byproducts AABA and L-valine compared to the parent strain, Corynebacterium glutamicum CA10-3101. Sequencing of the three mutant strains was performed, and the aroP gene was compared with that of the wild-type ATCC13032, confirming that the three mutant strains contained a mutation in the aroP gene.

[0243] Specifically, it was confirmed that the CA10-3101 / pTOPO-aroPm138 strain had a substitution of glycine, the 212th amino acid in the amino acid sequence of aroP, with threonine (G212T), the CA10-3101 / pTOPO-aroPm2542 strain had a substitution of isoleucine, the 114th amino acid in the amino acid sequence of aroP, with phenylalanine (I114F), and the CA10-3101 / pTOPO-aroPm3798 strain had a substitution of alanine, the 28th amino acid in the amino acid sequence of aroP, with valine (A28V). Based on the above results, it was confirmed that the three mutant strains could produce L-isoleucine with higher efficiency and yield than the parent strain. More specifically, when comparing each strain into which aroP(G212T), aroP(I114F), and aroP(A28V) were introduced with the parent strain, L-isoleucine was produced at a similar concentration, but the AABA concentration was reduced by 33%, 78%, and 56%, L-valine was reduced by 0%, 42%, and 8%, and the by-product (AABA, Valine) ratio was reduced by 16%, 44%, and 22%, confirming that the strain into which aroP(G212T), aroP(I114F), or aroP(A28V) was introduced had an excellent by-product reduction effect.

[0244]

[0245] Example 4: Production of L-isoleucine strains with introduced mutant aroP

[0246]

[0247] Among the aroP mutant strains produced in Example 3, the aroP (I114F) mutant with the highest L-isoleucine productivity and by-product reduction rate was transformed into Corynebacterium glutamicum CA10-3101.

[0248] Specifically, as in Reference Example 1, PCR was performed using the primers of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 using the chromosome of ATCC13032 as a template, or using the primers of SEQ ID NO: 8 and SEQ ID NO: 9 using the chromosome of pTOPO-aroPm2542 as a template. PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as a polymerase for the PCR reaction, and the PCR conditions were denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute, and these denaturation, annealing, and polymerization reactions were repeated 28 times. As a result, a 1000 bp DNA fragment of the 5' upper region and a 1392 bp DNA fragment of the 3' lower region centered on the gapA promoter region and the aroP gene start codon were obtained, respectively. Using the three amplified DNA fragments as templates, PCR was performed with primers of SEQ ID NO: 4 and SEQ ID NO: 9. The PCR conditions were denaturation at 95°C for 5 minutes, followed by six cycles of denaturation at 95°C for 30 seconds and polymerization at 72°C for 2 minutes, followed by 20 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 2 minutes, followed by polymerization at 72°C for 5 minutes. As a result, a 2.4 kb DNA fragment encoding the aroP gene containing the gapA promoter was amplified. The amplified product was purified using a PCR Purification kit from QUIAGEN and used as an insert DNA fragment for vector construction. Meanwhile, after treating with restriction enzyme smaI, the molar concentration (M) ratio of the pDCM2 vector and the inserted DNA fragment amplified through the PCR was made 1:2 by heat-treating at 65°C for 20 minutes, and cloning was performed using TaKaRa's Infusion Cloning Kit according to the provided manual, thereby constructing vector pDCM2-PgapA-aroP(I114F) for introduction into the chromosome by replacing the mutant aroP promoter with the gapA promoter.

[0249] The vector constructed above was transformed into Corynebacterium glutamicum CA10-3101 by electroporation, and a strain with a strengthened mutant aroP promoter on the chromosome was obtained through a second crossing process, and the CA10-3101::PgapA-aroP(I114F) strain was named CA10-3118. In order to confirm the effect of increasing L-isoleucine productivity and reducing by-products for the L-isoleucine-producing parent strain (CA10-3101), the aroP-enhanced strain constructed in Example 1 (CA10-3117), and the aroP(I114F) mutant-introduced strain constructed in this Example (CA10-3118), each strain was subjected to a fermentation activity evaluation using the following method. After inoculating the parent strain and the above strains into a 250 ml corner-bottom flask containing 25 ml of isoleucine production medium, L-isoleucine was produced by shaking and culturing at 200 rpm for 60 hours at 32°C. The composition of the production medium used in this example is shown below.

[0250]

[0251] <Production medium>

[0252] Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, monobasic potassium phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, iron sulfate heptahydrate 10 mg / ℓ, manganese sulfate monohydrate 10 mg / ℓ, biotin 200 μg / ℓ, pH 7.2

[0253] The results of measuring the concentrations of L-isoleucine, AABA, and L-valine using liquid high-performance chromatography (HPLC) after the completion of culture are shown in Table 7 below.

[0254]

[0255] Strain name L-isoleucine concentration (g / L) AABA concentration (g / L) L-valine concentration (g / L) By-product ratio to isoleucine + by-product (AABA, L-valine) CA10-3101 (parent strain) 2.6 0.9 1.2 0.45 CA10-3117 (aroP-enhanced strain) 2.8 0.4 1.0 0.33 aroP-enhanced and aroP (G212T) introduced strain 3.10.5 1.00.32 CA10-3118 (aroP-enhanced and aroP (I114F) introduced strain) 2.8 0.20.7 0.24 aroP-enhanced and aroP (A28V) introduced strain 3.0 0.4 0.9 0.30

[0256]

[0257] - By-product ratio: By-product (AABA, L-valine) concentration / (isoleucine + by-product) concentration

[0258]

[0259] As shown in Table 7 above, compared to the parent strain (CA10-3101), the L-isoleucine produced in the aroP-enhanced strain (CA10-3117), the aroP(G212T) mutant-introduced strain, the aroP(I114F) mutant-introduced strain (CA10-3118), and the aroP(A28V) mutant-introduced strain increased, and the concentrations of the byproducts AABA and L-valine decreased.

[0260] Specifically, L-isoleucine produced by the aroP(G212T) mutant introduced strain increased by 15% and 7%, respectively, compared to the parent strain and the aroP-enhanced strain, while AABA decreased by 44% compared to the parent strain and L-valine decreased by 20% compared to the parent strain.

[0261] It was confirmed that AABA produced by the aroP(I114F) mutant introduced strain was reduced by 88% and 50% compared to the parent strain and the aroP-enhanced strain, respectively, and L-valine was reduced by 42% and 30% compared to the parent strain and the aroP-enhanced strain, respectively.

[0262] L-isoleucine produced by the aroP(A28V) mutant introduced strain increased by 19% and 11%, respectively, compared to the parent strain and the aroP-enhanced strain, while AABA decreased by 56% compared to the parent strain, and L-valine decreased by 25% and 10%, respectively, compared to the parent strain and the aroP-enhanced strain.

[0263] In addition, the ratio of by-products (AABA, L-valine) to the production of L-isoleucine and by-products (AABA, L-valine) in each mutant strain was reduced by 29%, 47%, and 33%, respectively, compared to the parent strain in the aroP(G212T) mutant-introduced strain, the aroP(I114F) mutant-introduced strain, and the aroP(A28V) mutant-introduced strain, and was reduced by 3%, 27%, and 9%, respectively, compared to the aroP-enhanced strain, confirming that by-products were reduced in the aroP(G212T), aroP(I114F), or aroP(A28V) mutant-introduced strains, and L-isoleucine productivity and purity were significantly increased.

[0264]

[0265] The above strain CA10-3118 was deposited internationally with the Korea Center for Microbiological Cultures (KCCM), an international depository under the Budapest Treaty, on December 1, 2020, and was assigned the accession number KCCM12857P.

[0266]

[0267] Example 5: Production of aroP-enhanced strains and mutant aroP-introduced strains in L-isoleucine-producing strains.

[0268]

[0269] In Example 4, each of the aroP mutants (aroP (I114F) mutants) that showed the best aroP enhancement (gapA promoter introduction) and increased L-isoleucine production and reduced by-products were introduced into the KCJI-38 (KCCM11248P, Republic of Korea Patent No. 10-1335789) strain, an L-isoleucine-producing strain treated with NTG (N-Methyl-N'-nitro-N-nitrosoguanidine), by electric pulse method, and then transformed by spreading on a selection medium containing 25 mg / L of kanamycin. After a second crossing process, aroP promoter-enhanced and aroP mutant-enhanced strains were obtained on the chromosome. Thereafter, the concentrations of L-isoleucine and by-products in the culture medium were measured in the same manner as in Example 1, and the results are shown in Table 8 below.

[0270]

[0271] Strain name L-isoleucine concentration (g / L) AABAS concentration (g / L) L-valine concentration (g / L) Isoleucine + by-product (AABA, L-valine) to by-product (AABA, L-valine) ratio KCCM11248P (Parent strain)1.50.71.20.56KCCM11248P△Pn-aroP::PgapA-aroP1.80.41.00.44KCCM11248P△Pn-aroP::PgapA-aroP(G212T)2.00.41 .10.43KCCM11248P△Pn-aroP::PgapA-aroP(I114F)1.90.30.80.37KCCM11248P△Pn-aroP::PgapA-aroP(A28V)1.90.41.00.43

[0272]

[0273] - By-product ratio: By-product (AABA, L-valine) concentration / (isoleucine + by-product) concentration

[0274]

[0275] As shown in Table 8 above, the KCCM11248P△Pn-aroP::PgapA-aroP strain, which introduced aroP, was confirmed to have increased L-isoleucine production and decreased byproducts compared to the parent strain KCCM11248P, and the KCCM11248P△Pn-aroP::PgapA-aroP(G212T), KCCM11248P△Pn-aroP::PgapA-aroP(I114F), and KCCM11248P△Pn-aroP::PgapA-aroP(A28V) strains, which introduced aroP mutants, were confirmed to have increased L-isoleucine production compared to the KCCM11248P and KCCM11248P△Pn-aroP::PgapA-aroP strains.

[0276] The KCCM11248P△Pn-aroP::PgapA-aroP(I114F) strain showed a decrease in the by-product AABA by about 57% and 25%, and a decrease in L-valine by about 33% and 20%, respectively, compared to the KCCM11248P and KCCM11248P△Pn-aroP::PgapA-aroP strains.

[0277] Compared to the KCCM11248P strain, the KCCM11248P△Pn-aroP::PgapA-aroP(G212T) and KCCM11248P△Pn-aroP::PgapA-aroP(A28V) strains showed a decrease in AABA, a by-product, by about 43% and 43%, respectively, and a decrease in L-valine by about 8% and 17%, respectively, confirming that the strains introducing the aroP mutant had a significantly higher by-product reduction rate.

[0278] In addition, the ratio of by-products (AABA, L-valine) to the production of L-isoleucine and by-products (AABA, L-valine) of each mutant strain was decreased by 23%, 34%, and 2%, respectively, in the KCCM11248P△Pn-aroP::PgapA-aroP(G212T), KCCM11248P△Pn-aroP::PgapA-aroP(I114F), and KCCM11248P△Pn-aroP::PgapA-aroP(A28V) strains compared to the parent strain, and decreased by 23%, 16%, and 2%, respectively, compared to the aroP-enhanced strain, confirming that by-products were significantly reduced in the aroP(G212T), aroP(I114F), or aroP(A28V) mutant-introduced strains, and L-isoleucine productivity and purity were significantly increased.

[0279]

[0280] From the above results, it was confirmed that the aroP-enhanced strain and its variants of the present application can increase the production of L-isoleucine. In addition, since it was confirmed that the aroP-enhanced strain and its variants are mutations that help increase the purity in the production and purification process of L-isoleucine, it was confirmed that the mutations play a role in increasing the production of L-isoleucine.

[0281]

[0282] From the above description, those skilled in the art will understand that the present application can be implemented in other specific forms without altering its technical concept or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of this application should be interpreted to include all changes or modifications derived from the meaning and scope of the following claims and their equivalents, rather than the detailed description above.

[0283]

[0284]

[0285]

Claims

1. A gamma-aminobutyrate permease mutant in which the amino acid corresponding to position 212 in the amino acid sequence of sequence number 1 is substituted with threonine, the amino acid corresponding to position 114 is substituted with phenylalanine, or the amino acid corresponding to position 28 is substituted with valine.

2. A polynucleotide encoding the mutant of paragraph 1.

3. A vector comprising the polynucleotide of paragraph 2.

4. A microorganism comprising at least one of the following: a variant of the first paragraph; a polynucleotide encoding the variant; and a vector comprising the polynucleotide.

5. In paragraph 4, the microorganism is a microorganism that produces L-isoleucine.

6. In paragraph 4, the microorganism is a microorganism of the genus Corynebacterium sp.

7. In paragraph 4, the microorganism is Corynebacterium glutamicum.

8. A method for producing L-isoleucine, comprising the step of culturing a microorganism including at least one of a gamma-aminobutyrate permease mutant, wherein the amino acid corresponding to position 212 in the amino acid sequence of sequence number 1 is substituted with threonine, the amino acid corresponding to position 114 is substituted with phenylalanine, or the amino acid corresponding to position 28 is substituted with valine; a polynucleotide encoding the mutant; and a vector including the polynucleotide.

9. A method for producing L-isoleucine in claim 8, wherein the method further comprises a step of recovering L-isoleucine from the medium or microorganism.

10. A method for producing L-isoleucine in claim 8, wherein the microorganism is Corynebacterium sp.

11. A method for producing L-isoleucine in claim 8, wherein the method reduces by-products generated during the production of L-isoleucine.

12. A method for producing L-isoleucine in claim 11, wherein the byproduct is at least one selected from the group consisting of alpha-aminobutyrate (AABA), valine, phenylalanine, and leucine.

13. A method for reducing by-products generated during the production of L-isoleucine, comprising the step of culturing a microorganism including at least one of a gamma-aminobutyrate permease mutant, wherein the amino acid corresponding to position 212 in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid corresponding to position 114 is substituted with phenylalanine, or the amino acid corresponding to position 28 is substituted with valine; a polynucleotide encoding the mutant; and a vector including the polynucleotide.

14. A composition for producing L-isoleucine, comprising a microorganism or a culture of the microorganism, comprising at least one of a gamma-aminobutyrate permease mutant, wherein the amino acid corresponding to position 212 in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid corresponding to position 114 is substituted with phenylalanine, or the amino acid corresponding to position 28 is substituted with valine; a polynucleotide encoding the mutant; and a vector comprising the polynucleotide.

15. A mutant of gamma-aminobutyrate permease, wherein the amino acid corresponding to position 212 in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid corresponding to position 114 is substituted with phenylalanine, or the amino acid corresponding to position 28 is substituted with valine; or a use of a microorganism for producing L-isoleucine, the mutant, a polynucleotide encoding the mutant, or a vector comprising the polynucleotide.