Novel acetohydroxyacid synthase mutant and method for producing L-isoleucine using the same

Abstract

The present application relates to a novel acetohydroxy acid synthase (AHAS) mutant having improved L-isoleucine-producing ability, a microorganism containing the same, and a method for producing L-isoleucine using the microorganism.

Description

[Technical Field]

[0001] The present application relates to a novel acetohydroxy acid synthase (AHAS) mutant that improves L-isoleucine-producing ability, a microorganism containing the same, and a method for producing L-isoleucine using the microorganism. [Background technology]

[0002] L-isoleucine is a branched-chain amino acid among the 20 amino acids in total, and is classified as an essential amino acid and is used in animal feed, food additives, and pharmaceuticals. L-isoleucine is increasingly used in infusions, nutritional supplements, sports nutrition, and animal feed because it plays a role in metabolic energy production, hemoglobin production, blood sugar regulation, muscle growth and repair, etc.

[0003] Based on this trend, various microorganisms and their mutants are used to produce L-amino acids (U.S. Patent No. 10,113,190). However, even in such cases, a large number of by-products other than L-isoleucine are produced, which have a significant impact on the purity of L-isoleucine during the purification process, so a method for removing the by-products is needed. In this regard, L-isoleucine purification methods developed to increase the purity of L-isoleucine have the disadvantage of requiring an additional purification process (U.S. Patent No. 6,072,083). Therefore, there is currently a need to develop a method for increasing the purity of L-isoleucine. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 10113190 [Patent Document 2] US Patent No. 6072083 [Patent Document 3] Republic of Korea Registration License No. 10-1335789 [License 4] US Registry Permit US 7662943 B2 [Patent Document 5] US Registry Permit US 10584338 B2 [License 6] US Registry Permit US 10273491 B2 [License 7] Republic of Korea Registration License No. 10-1996769 [License 8] Korean Patent No. 10-2020-0136813 [Non-licensed literature]

[0005] [Non-licensed Document 1] Pearson et al (1988)[Proc. Natl. Acad. Sci. USA 85]:2444 [Non-licensed Document 2] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-licensed Document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-licensed Document 4] Devereux, J., et al, Nucleic Acids Research 12: 387 (1984) [Non-licensed Document 5] Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990) [Non-licensed Document 6] Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994 [Non-licensed Document 7] [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073

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Summary of the Invention

[0006] The present inventors have identified a mutant of acetohydroxy acid synthase (AHAS), one of the proteins in the L-isoleucine production pathway, and confirmed that the mutant improves the L-isoleucine production ability of a strain, thereby completing the present application. [Means for solving the problem]

[0007] One object of the present application is to provide an acetohydroxy acid synthase (AHAS) mutant in which the amino acid corresponding to position 17 in the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid.

[0008] Another object of the present application is to provide polynucleotides encoding the variants of the present application.

[0009] Another object of the present application is to provide a Corynebacterium strain comprising a variant of the present application or a polynucleotide encoding the variant.

[0010] Another object of the present application is to provide a method for producing L-isoleucine, which comprises culturing in a medium a Corynebacterium strain containing a variant of the present application or a polynucleotide encoding the variant. [Effects of the Invention]

[0011] In the present application, microorganisms expressing acetohydroxyacid synthase mutants exhibit significantly improved L-isoleucine production compared to strains that do not express the mutants, and can be used to effectively produce L-isoleucine. Therefore, a wide range of industrial applications utilizing L-isoleucine, such as food, feed, and pharmaceuticals, are expected. DETAILED DESCRIPTION OF THE INVENTION

[0012] This will be explained in more detail as follows. Meanwhile, each description and embodiment disclosed in this application may also be applied to different descriptions and embodiments. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the following specific descriptions are not intended to limit the scope of this application. Furthermore, numerous papers and patent documents are referenced throughout this specification, and citations thereof are provided. The disclosures of the cited papers and patent documents are hereby incorporated by reference in their entirety into this specification to more clearly explain the state of the art to which this application pertains and the contents of this application.

[0013] One aspect of the present application provides an acetohydroxy acid synthase (AHAS) mutant in which the amino acid corresponding to position 17 in the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid.

[0014] In one embodiment, the other amino acid may be alanine.

[0015] The variants of the present application may include an amino acid sequence in which the asparagine corresponding to the 17th position in the amino acid sequence set forth in SEQ ID NO: 1, the parent sequence, is substituted with alanine, and which has at least 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.7% or more, but less than 100%, homology or identity to the amino acid sequence set forth in SEQ ID NO: 1. It is clear that variants having amino acid sequences in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added are also included within the scope of the present application, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the variants of the present application.

[0016] For example, the above amino acid sequence may have additions or deletions of sequences at the N-terminus, C-terminus and / or internally that do not alter the function of the variant of the present application, naturally occurring mutations, silent mutations or conservative substitutions.

[0017] The term "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions are generally made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0018] As used herein, the term "variant" refers to a polypeptide that differs from the amino acid sequence of the variant by conservative substitution and / or modification of one or more amino acids, but maintains its functions or properties. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the performance of the variant may be increased, unchanged, or decreased compared to the polypeptide of the variant. Some variants may also include variants in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, have been deleted. Other variants may include variants in which portions have been deleted from the N- and / or C-termini of the mature protein. The term "mutant" may be used interchangeably with terms such as mutation, variant, mutant polypeptide, mutated protein, mutation, and variant (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), and is not limited to these terms as long as they are used in the sense of mutation.

[0019] The variants may also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the N-terminus of the variant may be conjugated to a signal (or leader) sequence involved in co- or post-translational protein translocation. The variants may also be conjugated to other sequences or linkers to allow for identification, purification, or synthesis.

[0020] In the present application, the term "parent sequence" refers to a reference sequence into which modifications are introduced to form a mutant polypeptide. That is, the parent sequence may be used as a starting sequence into which mutations such as substitutions, insertions, and / or deletions are introduced. The parent sequence may be a naturally occurring or wild-type sequence, or may be a variant of the naturally occurring or wild-type sequence in which one or more substitutions, insertions, or deletions have occurred, or may be an artificially synthesized sequence.

[0021] As used herein, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid or nucleotide sequences, which can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0022] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined using standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences can hybridize under moderately or highly stringent conditions, generally over at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or full length. Hybridization, of course, also includes hybridization with polynucleotides containing common codons or codons that take codon degeneracy into account.

[0023] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using known computer algorithms such as the "FASTA" program using default parameters, for example, as in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later), as implemented in the GCG program package (Devereux, J., et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.] [ET AL, J MOLEC BIOL 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [Ed.] Academic Press, San Diego, 1994, and [CARILLO ET AL / .] (1988) SIAM J Applied Math 48:1073. For example, BLAST from the National Center for Biotechnology Information, or ClustalW, can be used to determine homology, similarity, or identity.

[0024] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, e.g., as known in Smith and Waterman, Adv. Appl. Math (1981) 2:482, or as described in, e.g., Needleman et al. (1970), J Mol Biol. 48:443. Briefly, the GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include: (1) a binary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0025] The variants of the present application can have acetohydroxy acid synthase (AHAS) activity and can have an activity that increases L-isoleucine productivity compared to a wild-type polypeptide having acetohydroxy acid synthase activity.

[0026] In this application, the term "acetohydroxy acid synthase (AHAS)" refers to the first enzyme in L-valine biosynthesis, also known as acetolactate synthase. Acetohydroxy acid synthase can catalyze the decarboxylation of pyruvate and its condensation with another pyruvate molecule to produce acetolactate, a precursor of valine, and the decarboxylation of pyruvate and its condensation with 2-ketobutyrate to produce acetohydroxybutyrate, a precursor of isoleucine.

[0027] The acetohydroxy acid synthase is encoded by two genes, ilvB and ilvN. The ilvB gene encodes the large subunit of acetohydroxy acid synthase, and the ilvN gene encodes the small subunit. The small subunit encoded by the ilvN gene is believed to be important in feedback inhibition. The term "feedback inhibition" refers to the inhibition of an early reaction in an enzyme system by a product of the enzyme system. For purposes of this application, the acetohydroxy acid synthase may be the acetohydroxy acid synthase encoded by the ilvN gene.

[0028] The sequence of the acetohydroxy acid synthase encoded by the ilvN gene can be obtained from the publicly known database, NCBI GenBank, and specifically may have the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

[0029] As used herein, the term "corresponding to" refers to the amino acid residue at the recited position in a polypeptide, or an amino acid residue that is similar, identical, or homologous to the recited residue in a polypeptide. Identifying the amino acid at the corresponding position may be determining the specific amino acid in a sequence that references a particular sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0030] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, and based on this, each amino acid residue in the amino acid sequence can be numbered by reference to the numeric position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, a sequence alignment algorithm such as that described in this application can identify the position of an amino acid relative to a query sequence (also referred to as a "reference sequence"), or the position where a variation such as a substitution, insertion, or deletion occurs.

[0031] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), the Needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16:276-277), etc. can be used, but are not limited to these, and any sequence alignment program, pairwise sequence comparison algorithm, etc. known in the art can be appropriately used.

[0032] The variants of the present application may further include one or more substitutions selected from the following: substitution of the amino acid corresponding to position 42 with another amino acid, based on the amino acid sequence set forth in SEQ ID NO: 1; substitution of the amino acid corresponding to position 47 with another amino acid; or a combination thereof.

[0033] In one embodiment, the amino acid corresponding to position 42 may be substituted with valine.

[0034] In another embodiment, the amino acid corresponding to the 47th position may be substituted with leucine.

[0035] Specifically, the variants of the present application may have, comprise, consist of, or essentially consist of the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6.

[0036] Specifically, based on the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3 may be an amino acid sequence in which the asparagine corresponding to the 17th position is substituted with alanine; SEQ ID NO: 4 may be an amino acid sequence in which the asparagine corresponding to the 17th position is substituted with alanine and the amino acid corresponding to the 42nd position is substituted with valine; SEQ ID NO: 5 may be an amino acid sequence in which the asparagine corresponding to the 17th position is substituted with alanine and the amino acid corresponding to the 47th position is substituted with leucine; and SEQ ID NO: 6 may be an amino acid sequence in which the asparagine corresponding to the 17th position is substituted with alanine, the amino acid corresponding to the 42nd position is substituted with valine, and the amino acid corresponding to the 47th position is substituted with leucine.

[0037] Another aspect of the present application is to provide polynucleotides encoding the variants of the present application.

[0038] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide units are linked in a long chain by covalent bonds, and is a DNA or RNA chain of a certain length or more, and more specifically, refers to a polynucleotide fragment encoding the above-mentioned variant.

[0039] A polynucleotide encoding a variant of the present application may comprise a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6. As an example of the present application, a polynucleotide of the present application may have or comprise the sequence of SEQ ID NO: 23 or SEQ ID NO: 24 or SEQ ID NO: 25 or SEQ ID NO: 26. Alternatively, a polynucleotide of the present application may consist of or consist essentially of the sequence of SEQ ID NO: 23 or SEQ ID NO: 24 or SEQ ID NO: 25 or SEQ ID NO: 26.

[0040] The polynucleotide of the present application may be modified in various ways in the coding region without changing the amino acid sequence of the variant of the present application, taking into consideration codon degeneracy or codons preferred in an organism in which the variant of the present application is to be expressed. Specifically, the polynucleotide of the present application may have or include a nucleotide sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more and less than 100% homologous or identical to the sequence of SEQ ID NO: 2, or may be composed of or essentially composed of a nucleotide sequence that is 70% or more, 75% or more, 6% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more and less than 100% homologous or identical to the sequence of SEQ ID NO: 2, but is not limited thereto. In this case, in the above-mentioned homologous or identical sequences, the codon encoding the amino acid corresponding to the 17th position of SEQ ID NO: 1 may be one of the codons encoding alanine, the codon encoding the amino acid corresponding to the 42nd position may be one of the codons encoding valine, and the codon encoding the amino acid corresponding to the 47th position may be one of the codons encoding leucine.

[0041] Furthermore, the polynucleotides of the present application may include, without limitation, probes prepared from known gene sequences, for example, sequences that can hybridize under stringent conditions to a complementary sequence to the entire or partial polynucleotide sequences of the present application. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., "Molecular Cloning, A Laboratory Manual," 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., "Current Protocols in Molecular Biology," John Wiley & Sons, Inc., New York, pp. 9.50-9.51, 11.7-11.8). For example, conditions include conditions under which polynucleotides with high homology or identity, such as polynucleotides with a homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, hybridize with each other, but polynucleotides with lower homology or identity do not hybridize with each other; or conditions under which washing is performed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions for standard Southern hybridization, such as 60°C, 1X SSC, and 0.1% SDS, specifically 60°C, 0.1X SSC, and 0.1% SDS, more specifically 68°C, 0.1X SSC, and 0.1% SDS.

[0042] Hybridization requires that two nucleic acids have complementary sequences, even though mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application can also include isolated nucleic acid fragments that are complementary to an entire sequence, as well as substantially similar nucleic acid sequences.

[0043] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the above-mentioned hybridization conditions, including a hybridization step at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0044] The appropriate stringency for hybridizing such polynucleotides depends on the length and degree of complementation of the polynucleotides, variables well known in the art (eg, J. Sambrook et al., supra).

[0045] Another aspect of the present application is to provide a vector comprising the polynucleotide of the present application. The vector may be, but is not limited to, an expression vector for expressing the polynucleotide in a host cell.

[0046] The vector of the present application can comprise a DNA construct comprising a base sequence of a polynucleotide encoding a polypeptide of interest operably linked to an expression control region (or expression control sequence) suitable for expressing the polypeptide of interest in a suitable host. The expression control region can comprise a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating the termination of transcription and translation. After being transformed into an appropriate host cell, the vector can replicate and function independently of the host genome, or can be integrated into the genome itself.

[0047] The vectors used in the present application are not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0048] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using a vector for intracellular chromosomal insertion. The polynucleotide can be inserted into a chromosome by any method known in the art, including, but not limited to, homologous recombination. A selection marker for confirming the presence or absence of the chromosomal insertion can also be included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of the target nucleic acid molecule. A marker that confers a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide, is used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes, allowing the selection of transformed cells.

[0049] As used herein, the term "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism, thereby enabling the expression of the polypeptide encoded by the polynucleotide in the host cell. A transformed polynucleotide can include any polynucleotide that can be expressed in the host cell, regardless of whether it is located intrachromosomally or extrachromosomally. The polynucleotide also includes DNA and / or RNA encoding the target polypeptide. The polynucleotide can be introduced in any form that can be introduced and expressed in the host cell. For example, the polynucleotide can 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 typically contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of an autonomously replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form and operably linked to sequences necessary for expression in the host cell, but is not limited thereto.

[0050] In addition, the term "operably linked" as used above means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target mutant of the present application.

[0051] Another aspect of the present application is to provide a strain of the genus Corynebacterium comprising a variant of the present application or a polynucleotide of the present application.

[0052] The strains of the present application can comprise a mutant polypeptide of the present application, a polynucleotide encoding said polypeptide, or a vector comprising a polynucleotide of the present application.

[0053] In this application, the term "strain (or microorganism)" includes all wild-type microorganisms and naturally or artificially genetically modified microorganisms, including those in which specific mechanisms have been weakened or enhanced by inserting an exogenous gene or by enhancing or inactivating the activity of an endogenous gene, and may also be a microorganism that contains genetic modifications for the production of a desired polypeptide, protein, or product.

[0054] The strain of the present application may be, but is not limited to, a strain comprising one or more of the variants of the present application, the polynucleotides of the present application, and the vectors comprising the polynucleotides of the present application; a strain modified to express the variants of the present application or the polynucleotides of the present application; a strain (e.g., a recombinant strain) that expresses the variants of the present application or the polynucleotides of the present application; or a strain (e.g., a recombinant strain) that has the activity of the variants of the present application.

[0055] The strain of the present application may be a strain capable of producing L-isoleucine.

[0056] The strain of the present application may be, but is not limited to, a microorganism that naturally has the ability to produce acetohydroxy acid synthase or L-isoleucine, or a parent strain that does not have the ability to produce acetohydroxy acid synthase or L-isoleucine, to which the mutant of the present application or a polynucleotide encoding the mutant (or a vector containing the polynucleotide) has been introduced and / or to which the ability to produce L-isoleucine has been imparted.

[0057] For example, the strain of the present application is a cell or microorganism that has been transformed with a vector containing a polynucleotide encoding the polynucleotide of the present application or a polynucleotide encoding the variant of the present application and expresses the variant of the present application. For purposes of this application, the strain of the present application can include all microorganisms capable of producing L-isoleucine, including the variant of the present application. For example, the strain of the present application may be a recombinant strain having increased L-isoleucine production ability, as a result of introducing a polynucleotide encoding the variant of the present application into a naturally occurring wild-type microorganism or a microorganism that produces L-isoleucine, thereby expressing an acetohydroxy acid synthase variant. The recombinant strain having increased L-isoleucine production ability may be, but is not limited to, a naturally occurring wild-type microorganism or a microorganism not modified with acetohydroxy acid synthase (i.e., a microorganism that expresses wild-type acetohydroxy acid synthase (SEQ ID NO: 1) or a microorganism that does not express a mutant (SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6) protein). For example, the acetohydroxyacid synthase non-transformed microorganism, which is the subject strain for comparing whether or not the L-isoleucine productivity is increased, may be, but is not limited to, the Corynebacterium glutamicum ATCC13032 strain (CA10-3101, KCCM12739P) into which the hom (R407H) and ilvA (T381A, F383A) mutations have been introduced, or the KCJI-38 strain (KCCM11248P, Korean Patent Registered No. 10-1335789), an L-isoleucine producing strain treated with NTG (N-Methyl-N'-nitro-N-nitrosoguanidine).

[0058] For example, the recombinant strain with the increased production ability has an L-isoleucine production ability of about 1% or more, specifically about 2% or more, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 32% or more, about 34% or more, about 35% or more, about 39% or more, about 40% or more, about 45% or more, about 46% or more, about 47% or more, about 50% or more, about 53% or more, about 54% or more, about 55% or more, or The increase may be about 57% or more, about 59% or more, about 60% or more, about 62% or more, about 63% or more, about 64% or more, or about 73% or more (the upper limit is not particularly limited, and may be, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, or about 15% or less), but is not limited thereto as long as there is an increase in the + value compared to the productivity of the parent strain or untransformed microorganism before mutation. In another example, the recombinant strain having the increased L-isoleucine production ability is about 1.01-fold or more, about 1.02-fold or more, about 1.05-fold or more, about 1.10-fold or more, about 1.15-fold or more, about 1.20-fold or more, about 1.25-fold or more, about 1.30-fold or more, about 1.32-fold or more, about 1.34-fold or more, about 1.35-fold or more, about 1.39-fold or more, about 1.40-fold or more, about 1.45-fold or more, or about 1.46-fold or more, compared to the parent strain or non-transformed microorganism before mutation. The increase may be, but is not limited to, about 1.47 times or more, about 1.50 times or more, about 1.53 times or more, about 1.54 times or more, about 1.55 times or more, about 1.57 times or more, about 1.59 times or more, about 1.60 times or more, about 1.62 times or more, about 1.63 times or more, about 1.64 times or more, or about 1.73 times or more (there is no particular restriction on the upper limit, and it may be, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less).

[0059] As used herein, the term "unmodified microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, but refers to a wild-type or naturally occurring strain itself, or a strain before its traits are changed due to genetic mutations caused by natural or artificial factors. For example, the unmodified microorganism refers to a strain into which the acetohydroxyacid synthase mutant described herein has not been introduced or before it has been introduced. The term "unmodified microorganism" can be used interchangeably with "pre-modified strain," "pre-modified microorganism," "non-mutated strain," "non-modified strain," "non-mutated microorganism," or "reference microorganism."

[0060] In yet another example of the present application, the microorganism of the present application is Corynebacterium stationis, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens, and specifically, may be, but is not limited to, Corynebacterium glutamicum.

[0061] As used herein, the term "enhancement" of polypeptide activity refers to an increase in polypeptide activity compared to its endogenous activity. The term "enhancement" can be used interchangeably with terms such as "activation," "up-regulation," "overexpression," and "increase." Here, activation, enhancement, up-regulation, overexpression, and increase can all encompass the display of an activity not inherently possessed, or the display of an activity that is improved compared to the endogenous activity or the activity prior to transformation. The term "endogenous activity" refers to the activity of a specific polypeptide inherently possessed by a parent strain or an unaltered microorganism prior to transformation, in cases where a trait is altered by genetic mutation due to natural or artificial factors. This term can be used interchangeably with "activity prior to transformation." "Enhancement," "up-regulation," "overexpression," or "increase" of a polypeptide activity compared to its endogenous activity refers to an improvement in the activity and / or concentration (expression level) of a specific polypeptide relative to the activity of a parent strain or an unaltered microorganism prior to transformation.

[0062] The enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. The enhancement of the activity of the polypeptide can be confirmed by an increase in the activity level, expression level, or amount of a product secreted from the polypeptide.

[0063] The activity of the polypeptide can be enhanced by various methods well known in the art, and is not limited thereto, as long as the activity of the target polypeptide can be enhanced compared to that of the microorganism before transformation. Specifically, the enhancement may be achieved by using genetic engineering and / or protein engineering, which are routine methods in molecular biology and well known to those skilled in the art (e.g., Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2, pp. 1-16; Sambrook et al., Molecular Cloning 2012, etc.).

[0064] Specifically, the enhancement of the polypeptides of the present application 1) an increase in the intracellular copy number of the polynucleotide encoding the polypeptide; 2) replacing the expression regulatory region of a gene on a chromosome that encodes a polypeptide with a sequence with strong activity; 3) a modification of the nucleotide sequence encoding the initiation codon or 5'-UTR region of a gene transcript encoding a polypeptide; 4) modifying the amino acid sequence of the polypeptide so that the polypeptide activity is enhanced; 5) modifying a polynucleotide sequence encoding the polypeptide so as to enhance the polypeptide's activity (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the polypeptide's activity); 6) introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same; 7) codon optimization of the polynucleotide encoding the polypeptide; 8) analyzing the tertiary structure of a polypeptide and selectively modifying or chemically modifying exposed sites; or 9) It may be a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0065] More specifically, The above 1) increase in intracellular copy number of a polynucleotide encoding a polypeptide may be achieved by introducing into a host cell a vector operably linked to the polynucleotide encoding the polypeptide, which can replicate and function independently of the host. Alternatively, it may be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide into a chromosome in the host cell. The introduction into a chromosome can be achieved by, but is not limited to, introducing into the host cell a vector that inserts the polynucleotide into a chromosome of the host cell. The vector is as described above.

[0066] The replacement of the expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide with a sequence having a stronger activity may involve, for example, mutation of the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further enhance the activity of the expression regulatory region, or replacement with a sequence having a stronger activity. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. For example, the original promoter may be replaced with a strong promoter, but this is not limiting.

[0067] Examples of known strong promoters include, but are not limited to, the cj1 to cj7 promoters (U.S. Patent No. 7,662,943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (U.S. Patent No. 10,584,338 B2), O2 promoter (U.S. Patent No. 10,273,491 B2), tkt promoter, and yccA promoter.

[0068] The modification of the base sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide (3) may be, for example, a substitution with a base sequence encoding another start codon that has a higher polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0069] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) above may be, but is not limited to, a mutation in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, such as deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to enhance the activity of the polypeptide, or an amino acid sequence or polynucleotide sequence modified to have stronger activity or to have increased activity. Specifically, the modification may be performed by inserting the polynucleotide into a chromosome via homologous recombination, but is not limited to this. The vector used in this case may further contain a selection marker to confirm the presence or absence of chromosomal insertion. The selection marker is as described above.

[0070] The introduction of an exogenous polynucleotide that exhibits the activity of a polypeptide (6) above may be the introduction of an exogenous polynucleotide that encodes a polypeptide that exhibits the same or similar activity as the polypeptide into a host cell. The exogenous polynucleotide is not limited in its origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The method used for the introduction can be any publicly known transformation method appropriately selected by those skilled in the art. Expression of the introduced polynucleotide in a host cell can result in the production of a polypeptide and its activity being increased.

[0071] The codon optimization of the polynucleotide encoding the polypeptide (7) above may be an optimization of the codons of an endogenous polynucleotide to increase transcription or translation in a host cell, or an optimization of the codons of an exogenous polynucleotide to optimize transcription and translation in a host cell.

[0072] The above 8) analyzing the tertiary structure of a polypeptide and selecting and modifying or chemically modifying exposed sites may involve, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins to determine candidate template proteins according to the degree of sequence similarity, confirming the structure based on the candidate template proteins, and selecting and modifying exposed sites to be modified or chemically modified.

[0073] Such enhancement of polypeptide activity may be, but is not limited to, an increase in the activity or expression level of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in a wild-type or untransformed microbial strain, or an increase in the amount of a product produced from the polypeptide.

[0074] Modification of a portion or the entire polynucleotide in the microorganism of the present application may be induced by, but is not limited to, (a) homologous recombination using a vector for chromosomal insertion in the microorganism or genome editing using engineered nucleases (e.g., CRISPR-Cas9) and / or (b) treatment with light and / or chemicals, such as ultraviolet light and radiation. Methods for modifying a portion or the entire gene may include DNA recombination techniques. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene may be injected into the microorganism to cause homologous recombination, resulting in the deletion of a portion or the entire gene. The injected nucleotide sequence or vector may contain, but is not limited to, a dominant selectable marker.

[0075] In the microorganism of the present application, the mutant, polynucleotide, L-isoleucine, etc. are as described above in other aspects.

[0076] Another aspect of the present application provides a method for producing L-isoleucine, comprising culturing in a medium a Corynebacterium strain comprising a variant of the present application or a polynucleotide of the present application.

[0077] The method for producing L-isoleucine of the present application may include the step of culturing a Corynebacterium strain comprising a mutant of the present application, a polynucleotide of the present application, or a vector of the present application in a medium.

[0078] In the present application, the term "culturing" refers to growing the Corynebacterium strain of the present application under appropriately controlled environmental conditions. The culturing process of the present application is carried out using appropriate media and culture conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing may be, but is not limited to, a batch, continuous, and / or fed-batch culture.

[0079] As used herein, the term "culture medium" refers to a mixture of nutrients, primarily those required for culturing the Corynebacterium strain of the present application, which provides nutrients and growth factors, including water, essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the Corynebacterium strain of the present application may be any medium used for culturing conventional microorganisms without any particular limitations. The Corynebacterium strain of the present application may be cultured in a conventional culture medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins under aerobic conditions, with temperature, pH, etc., being adjusted.

[0080] Specifically, culture media for Corynebacterium strains can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0081] In the present application, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid may also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugars) may be used. A variety of other carbon sources may also be used in appropriate amounts without limitation. These carbon sources may be used alone or in combination, and are not limited thereto.

[0082] Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more, and are not limited thereto.

[0083] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and other compounds, including amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the medium in a batch or continuous manner. However, the present invention is not limited to these.

[0084] During the cultivation of the Corynebacterium sp. strain of the present application, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the medium in an appropriate manner. Furthermore, during cultivation, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, oxygen or an oxygen-containing gas can be injected into the medium to maintain an aerobic state, or nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection or to maintain an anaerobic or microaerobic state, but this is not limitative.

[0085] In the culture of the present application, the culture temperature can be maintained at 20 to 45°C, specifically 25 to 40°C, and the culture can be performed for about 10 to 160 hours, but is not limited to this.

[0086] The L-isoleucine produced by the culture of the present application may be secreted into the medium or may remain intracellularly.

[0087] The method for producing L-isoleucine of the present application may further include a step of preparing the Corynebacterium strain of the present application, a step of preparing a medium for culturing the strain, or a combination thereof (in any order), for example, before the culturing step.

[0088] The method for producing L-isoleucine of the present application may further include a step of recovering L-isoleucine from the culture medium (culture medium) or the Corynebacterium strain. The recovery step may be performed after the culturing step.

[0089] The recovery may involve collecting the target L-isoleucine using an appropriate method known in the art through the microbial culture method of the present application, such as a batch, continuous, or fed-batch culture method. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination of these methods may be used, and the target L-isoleucine can be recovered from the medium or the microorganism using an appropriate method known in the art.

[0090] Furthermore, the method for producing L-isoleucine of the present application may further include a purification step. The purification may be carried out using an appropriate method known in the art. In one example, when the method for producing L-isoleucine of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be carried out continuously or discontinuously, in any order, simultaneously, or integrated into one step, but are not limited thereto.

[0091] In the methods of the present application, the variants, polynucleotides, vectors, strains, etc. are as described in other aspects above.

[0092] Another aspect of the present application is to provide a composition for producing L-isoleucine, comprising: a mutant of the present application; a polynucleotide encoding the mutant; a vector comprising the polynucleotide; or a Corynebacterium strain comprising the polynucleotide of the present application; a medium in which the strains are cultured; or a combination of two or more of these.

[0093] The composition of the present application may further contain any suitable excipient commonly used in compositions for producing amino acids, and such excipients may be, for example, but are not limited to, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, or an isotonic agent.

[0094] In the compositions of the present application, the variants, polynucleotides, vectors, strains, media, L-isoleucine, etc. are as described above in other aspects.

[0095] Another aspect of the present application provides use of a variant of the present application; a polynucleotide encoding the variant; or a Corynebacterium strain comprising the variant or a polynucleotide encoding the variant for the production of L-isoleucine.

[0096] The present application will be described in more detail below with reference to examples. However, the following examples are merely preferred embodiments for illustrating the present application, and are not intended to limit the scope of the present application. Meanwhile, technical matters not described in this specification can be fully understood and easily implemented by those of ordinary skill in the technical field of the present application or a similar technical field.

[0097] Example 1: Construction of an L-isoleucine-producing strain Wild-type Corynebacterium glutamicum has the ability to produce L-isoleucine but does not overproduce it. To identify the genetic traits that increase L-isoleucine production, we constructed a strain with increased L-isoleucine production compared to the wild-type.

[0098] First, to eliminate the feedback inhibition of threonine, the precursor of isoleucine, in the L-isoleucine biosynthetic pathway of wild-type Corynebacterium glutamicum ATCC13032, the gene hom encoding homoserine dehydrogenase was mutated, and the 407th amino acid, arginine, of homoserine dehydrogenase was replaced with histidine (Korean Patent Registration No. 10-1996769).

[0099] Specifically, to construct a vector for introducing the hom(R407H) mutation into the chromosome, PCR was performed using the chromosome of wild-type Corynebacterium glutamicum ATCC13032 as a template and the primer pair of SEQ ID NO: 14 and SEQ ID NO: 15 or the primer pair of SEQ ID NO: 16 and SEQ ID NO: 17. The primer sequences are shown in Table 1 below.

[0100] [Table 1]

[0101] PfuUltra is used as the polymerase for PCR reactions. TM High-fidelity DNA polymerase (Stratagene) was used, and the PCR conditions were denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute. These denaturation, annealing, and polymerization reactions were repeated 28 times to obtain a 1000 bp DNA fragment at the 5' upper end and a 1000 bp DNA fragment at the 3' lower end, centered on the hom gene mutation.

[0102] Using the two amplified DNA fragments as templates, PCR was performed using the primer pair of SEQ ID NO: 14 and SEQ ID NO: 17. 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.

[0103] As a result, a 2-kb DNA fragment (SEQ ID NO: 13) containing a mutation in the hom gene encoding a homoserine dehydrogenase mutant in which arginine at position 407 was replaced with histidine was amplified. The amplified product was purified using a PCR Purification kit (QIAGEN) and used as an insert DNA fragment for vector construction.

[0104] The purified amplified product was treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes. The molar concentration (M) ratio of the pDCM2 vector (Korean Patent Publication No. 10-2020-0136813) to the amplified insert DNA fragment was adjusted to 1:2, and the vector pDCM2-R407H for introducing the hom(R407H) mutation into the chromosome was produced by cloning using an Infusion Cloning Kit (TaKaRa) according to the provided instructions.

[0105] 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 designated Corynebacterium glutamicum ATCC13032 hom(R407H).

[0106] To increase the feedback loop and activity of L-isoleucine in the constructed ATCC13032 hom (R407H) strain, the ilvA gene encoding L-threonine dehydratase was mutated, substituting alanine for the 381st amino acid threonine and alanine for the 383rd amino acid phenylalanine.

[0107] Specifically, to construct a vector for introducing the ilvA (T381A, F383A) mutation into the chromosome, PCR was performed using the chromosome of wild-type Corynebacterium glutamicum ATCC13032 as a template and the primer pair of SEQ ID NO: 19 and SEQ ID NO: 20 or the primer pair of SEQ ID NO: 21 and SEQ ID NO: 22. The primer sequences are shown in Table 2 below.

[0108] [Table 2]

[0109] PfuUltra is used as a polymerase for PCR reactions. TM High-fidelity DNA polymerase (Stratagene) was used, and the PCR conditions were denaturation at 95°C for 30 seconds, denaturation at 55°C for 30 seconds, and polymerization at 72°C for 1 minute. These denaturation, annealing, and polymerization reactions were repeated 28 times to obtain a 1126-bp DNA fragment at the 5'-top and a 286-bp DNA fragment at the 3'-bottom, centered on the ilvA gene mutation.

[0110] PCR was performed using the two amplified DNA fragments as templates and the primer pair of SEQ ID NO: 19 and SEQ ID NO: 22. 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.

[0111] As a result, a 1.4-kb DNA fragment (SEQ ID NO: 18) containing a mutation in the ilvA gene encoding an L-threonine dehydratase mutant in which threonine at position 381 was replaced with alanine and phenylalanine at position 383 was replaced with alanine was amplified. The amplified product was purified using a PCR purification kit and used as the insert DNA fragment for vector construction. The purified amplified product was treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes. The molar ratio of pDCM2 to the amplified insert DNA fragment was adjusted to 1:2, and the vector pDCM2-ilvA(T381A, F383A) for introducing the ilvA(T381A, F383A) mutation into the chromosome was constructed by cloning using the TaKaRa Infusion Cloning Kit according to the supplied instructions.

[0112] The constructed vector was transformed into Corynebacterium glutamicum ATCC13032 hom (R407H) by electroporation, and a strain containing the ilvA (T381A, F383A) mutation on the chromosome was obtained through a second crossover process and designated Corynebacterium glutamicum CA10-3101.

[0113] The above strain CA10-3101 was internationally deposited with the Korea Center for Microorganisms (KCCM), an international depository under the Budapest Treaty, on May 27, 2020, and was assigned the deposit number KCCM12739P.

[0114] Next, the following experiment was performed to confirm whether the introduction of the above ilvA (T381A, F383A) mutation into an L-isoleucine-producing strain actually increases L-isoleucine productivity by releasing the feedback loop and increasing activity of L-isoleucine.

[0115] Specifically, the ilvA (T381A, F383A) mutation was introduced into the KCJI-38 strain (KCCM11248P, Korean Patent No. 10-1335789) treated with NTG (N-Methyl-N'-nitro-N-nitrosoguanidine), an L-isoleucine-producing strain, by electroporation to create the KCCM11248P / pECCG117-ilvA (T381A, F383A) strain. The fermentation activity of the strain was then evaluated as follows.

[0116] The parent strain and the mutant strain were inoculated into a 250 ml corner baffle flask containing 25 ml of isoleucine production medium, and then cultured at 32°C for 60 hours with shaking at 200 rpm to produce L-isoleucine. The composition of the production medium is as follows:

[0117] <Production medium> Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, monobasic potassium phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, ferrous sulfate heptahydrate 10 mg / l, manganese sulfate monohydrate 10 mg / l, biotin 200 μg / l, pH 7.2

[0118] After the cultivation, the L-isoleucine and L-threonine concentrations in the culture medium for each strain tested were measured using high performance liquid chromatography (HPLC), and the results are shown in Table 3 below.

[0119] [Table 3]

[0120] As shown in Table 3, the KCCM11248P / pECCG117-ilvA(T381A, F383A) strain, which had the ilvA(T381A, F383A) mutation introduced, exhibited significantly increased L-isoleucine production and a higher L-threonine degradation rate than the parent strain, KCCM11248P. This confirmed that introducing the ilvA(T381A, F383A) mutation into the strain released the feedback loop for L-isoleucine and increased activity.

[0121] Example 2: Construction of mutant ilvN library vectors A mutation library of the ilvN gene encoding the small subunit of acetohydroxy acid synthase (AHAS) was constructed using an error-prone PCR kit (clontech Diversify® PCR Random Mutagenesis Kit). PCR was performed using the chromosome of wild-type Corynebacterium glutamicum ATCC 13032 as a template and the primer pair of SEQ ID NO: 7 and SEQ ID NO: 8. The primer sequences are shown in Table 4 below.

[0122] [Table 4]

[0123] Specifically, the transformation was performed under conditions that would result in 0–3 mutations per 1,000 ml, with preheating at 94°C for 30 seconds followed by 25 cycles of 94°C for 30 seconds and 68°C for 1 minute 30 seconds. The resulting product was then subjected to 25 cycles of 95°C for 50 seconds, 60°C for 50 seconds, and 68°C for 12 minutes using megaprimer (500–125 ng). It was then digested with DpnI and transformed into E. coli DH5α and plated on LB solid medium containing kanamycin (25 mg / L). Twenty transformed colonies were selected, and the plasmids were isolated and analyzed for polynucleotide sequence. Mutations were introduced at distinct positions at a frequency of 2 mutations / kb. Plasmids were extracted from approximately 20,000 transformed E. coli colonies and designated pTOPO-ilvN-library.

[0124] Example 3: Construction of an L-isoleucine-producing strain incorporating the ilvN library The pTOPO-ilvN-library prepared in Example 2 was transformed into the L-isoleucine-producing strain CA10-3101 (KCCM12739P) prepared in Example 1 by electroporation, and then the transformed strains were plated on a nutrient medium containing 25 mg / L kanamycin to obtain 5,000 colonies of the strains into which the mutant gene had been inserted. The colonies were named CA10-3101 / pTOPO-ilvNm1 to CA10-3101 / pTOPO-ilvNm5000.

[0125] To identify colonies with increased L-isoleucine production ability among the 5,000 colonies obtained, the fermentation titer of each colony was evaluated as follows. Specifically, the parent strain and the mutant strain were inoculated into a 250 ml corner baffle flask containing 25 ml of isoleucine production medium, and then cultured at 32°C for 60 hours with shaking at 200 rpm to produce L-isoleucine. The composition of the production medium is as follows:

[0126] <Production medium> Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, monobasic potassium phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, ferrous sulfate heptahydrate 10mg / l, manganese sulfate monohydrate 10mg / l, biotin 200μg / l, pH 7.2

[0127] After the cultivation, the L-isoleucine concentration in the culture medium for each strain tested was measured using high performance liquid chromatography (HPLC), and the results are shown in Table 5 below.

[0128] [Table 5]

[0129] As shown in Table 5, the mutant strain CA10-3101 / pTOPO-ilvNm2991 was confirmed to have increased L-isoleucine production ability compared to the parent strain Corynebacterium glutamicum CA10-3101, which has ilvN WT. Sequencing of the mutant strain and comparison with the ilvN gene of wild-type Corynebacterium glutamicum ATCC13032 confirmed that the mutant strain contained a mutation in which asparagine at position 17 in the amino acid sequence of ilvN was replaced with alanine (D17A).

[0130] Based on the above results, it was confirmed that the ilvN(D17A) mutant strain can produce L-isoleucine at a higher yield than the parent strain, and that the increase in L-isoleucine concentration was approximately 39% compared to the parent strain.

[0131] Example 4: Construction of an L-isoleucine-producing strain carrying a mutant ilvN The ilvN(D17A) mutation confirmed in Example 3 above was introduced into the Corynebacterium glutamicum CA10-3101 strain constructed in Example 1 above.

[0132] Specifically, to prepare a vector for introducing the mutant ilvN gene (D17A) into the chromosome, PCR was performed using the chromosome of CA10-3101 / pTOPO-ilvNm2991(D17A) as a template and the primer pair of SEQ ID NO: 7 and SEQ ID NO: 8. The polymerase used for the PCR reaction was PfuUltra TMHigh-fidelity DNA polymerase (Stratagene) was used for PCR, with the following conditions: denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and polymerization at 72°C for 1 minute. This denaturation, annealing, and polymerization cycle was repeated 28 times to obtain three 545-bp DNA fragments, each containing the mutant ilvN gene (D17A). The amplified products were purified using a QIAGEN PCR purification kit and used as insert DNA fragments for vector construction. The purified amplified products were digested with the restriction enzyme SmaI and then heat-treated at 65°C for 20 minutes. The molar ratio of the amplified insert DNA fragments to the pDCM2 vector (previously heat-treated) was adjusted to 1:2. The vector pDCM2-ilvN(D17A) for chromosomal integration of the mutant ilvN gene of Corynebacterium glutamicum was constructed using the Takara Infusion Cloning Kit (TaKaRa) according to the instructions provided.

[0133] The above-constructed vector was transformed into Corynebacterium glutamicum CA10-3101 by electroporation, and a strain with mutant ilvN replaced on the chromosome through a second crossover process was obtained. CA10-3101::ilvN(D17A) was named CA10-3128.

[0134] To confirm the effect of increasing L-isoleucine productivity in the parent strain (CA10-3101) and the mutant strain CA10-3128 prepared in this example, the fermentation titer of each strain was evaluated by the following method.

[0135] The parent strain and the mutant strain were inoculated into a 250 ml corner baffle flask containing 25 ml of isoleucine production medium, and then cultured at 32°C for 60 hours with shaking at 200 rpm to produce L-isoleucine. The composition of the production medium is shown below.

[0136] <Production medium> Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, monobasic potassium phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, ferrous sulfate heptahydrate 10mg / l, manganese sulfate monohydrate 10mg / l, biotin 200μg / l, pH 7.2

[0137] After the cultivation, the L-isoleucine concentration in the culture medium for each strain tested was measured using high performance liquid chromatography (HPLC), and the results are shown in Table 6 below.

[0138] [Table 6]

[0139] As shown in Table 6 above, the L-isoleucine concentration was confirmed to be increased in the CA10-3128 strain into which ilvN(D17A) was introduced compared to the parent strain (CA10-3101), which is an L-isoleucine-producing strain having ilvN WT. The increase in L-isoleucine concentration in the strain into which ilvN(D17A) was introduced was confirmed to be approximately 53% compared to the parent strain.

[0140] Example 5: Construction of combinatorially mutant ilvN plasmids In order to introduce the combined mutations including the ilvN mutant confirmed in Example 3 into the L-isoleucine strain, a vector for introducing the combined mutant ilvN into the chromosome was constructed.

[0141] Specifically, PCR was performed using pDCM2-ilvN(D17A) as a template and a primer pair of SEQ ID NOs: 7 and 9 or a primer pair of SEQ ID NOs: 10 and 8, and PCR was performed using pDCM2-ilvN(H47L) as a template and a primer pair of SEQ ID NOs: 7 and 11 or a primer pair of SEQ ID NOs: 12 and 8. The primer sequences are as shown in Table 7 below.

[0142] [Table 7]

[0143] PfuUltra is used as a polymerase for PCR reactions. TM High-fidelity DNA polymerase (Stratagene) was used for PCR, with denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute. These denaturation, annealing, and polymerization reactions were repeated 28 times to obtain 149-bp and 423-bp DNA fragments and 166-bp and 405-bp DNA fragments, respectively. The amplified products were purified using a QIAGEN PCR purification kit and used as insert DNA fragments for vector construction. The purified amplified product was treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes. The molar ratio of pDCM2 to the amplified insert DNA fragment was adjusted to 1:2, and the vectors pDCM2-ilvN(D17A, A42V) and pDCM2-ilvN(D17A, H47L) for introducing the mutant ilvN into the chromosome were constructed by cloning using the TaKaRa infusion cloning kit according to the supplied instructions.

[0144] Furthermore, PCR was performed using pDCM2-ilvN(D17A, H47L) as a template with the primer pair of SEQ ID NO:7 and SEQ ID NO:9 and the primer pair of SEQ ID NO:10 and SEQ ID NO:8 to obtain 149-bp and 423-bp DNA fragments. The amplified product was purified using a QIAGEN PCR purification kit and used as the insert DNA fragment for vector construction. The purified amplified product was treated with the restriction enzyme smaI and then heat-treated at 65°C for 20 minutes. The molar ratio of the amplified insert DNA fragment to the pDCM2 vector was adjusted to 1:2. The vector pDCM2-ilvN(D17A, A42V, H47L) for introducing the mutant ilvN of Corynebacterium glutamicum into the chromosome was constructed by cloning using the Takara Infusion Cloning Kit according to the instructions provided.

[0145] Example 6: Construction of an L-isoleucine-producing strain incorporating a combined mutant ilvN The vector constructed in Example 5 was transformed into the Corynebacterium glutamicum CA10-3101 strain constructed in Example 1 by electroporation, and a second crossover process was performed to obtain strains in which the recombinant mutant ilvN was replaced on the chromosome. CA10-3101::ilvN(D17A, A42V) was designated CA10-3131, CA10-3101::ilvN(D17A, H47L) was designated CA10-3133, and CA10-3101::ilvN(D17A, A42V, H47L) was designated CA10-3134.

[0146] To confirm the effect of increasing L-isoleucine productivity of the three strains constructed, the fermentation titer was evaluated as follows.

[0147] The parent strain and the mutant strain were inoculated into a 250 ml corner baffle flask containing 25 ml of isoleucine production medium, and then cultured at 32°C for 60 hours with shaking at 200 rpm to produce L-isoleucine. The composition of the production medium is as follows:

[0148] <Production medium> Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, monobasic potassium phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, ferrous sulfate heptahydrate 10mg / l, manganese sulfate monohydrate 10mg / l, biotin 200μg / l, pH 7.2

[0149] After the cultivation was completed, the L-isoleucine concentration in the culture medium for each strain tested was measured, and the results are shown in Table 8 below.

[0150] [Table 8]

[0151] As shown in Table 8 above, the L-isoleucine concentration was confirmed to be increased in the mutant strains containing the ilvN single mutation (ilvN(D17A)) or the ilvN combination mutations (ilvN(D17A, A42V), ilvN(D17A, H47L), ilvN(D17A, A42V, H47L)) compared to the parent strain (CA10-3101), an L-isoleucine-producing strain containing ilvN WT.

[0152] From the above results, it was confirmed that the ilvN mutations alone or in combination increased the L-isoleucine-producing ability of the strain.

[0153] Example 7: Construction of mutant ilvN-substituted strain in L-isoleucine-producing Corynebacterium glutamicum KCCM11248P strain One ilvN single mutation and three combination mutations confirmed to be effective in increasing L-isoleucine productivity in Example 6 above were introduced into the NTG (N-Methyl-N'-nitro-N-nitrosoguanidine)-treated L-isoleucine-producing strain KCJI-38 (KCCM11248P, Korean Patent No. 10-1335789) by electroporation, followed by transformation by plating on a selection medium containing 25 mg / L kanamycin. After a second crossover process, mutant ilvNs and combination ilvN-substituted strains with substituted ilvNs on the chromosome were obtained. Fermentation titers were then evaluated as follows.

[0154] The parent strain and the mutant strain were inoculated into a 250 ml corner-baffled flask containing 25 ml of isoleucine production medium, and then cultured at 32°C for 60 hours with shaking at 200 rpm to produce L-isoleucine. The composition of the production medium is as follows:

[0155] <Production medium> Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, monobasic potassium phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, ferrous sulfate heptahydrate 10mg / l, manganese sulfate monohydrate 10mg / l, biotin 200μg / l, pH 7.2

[0156] After the cultivation, the L-isoleucine concentration in the culture medium for each strain tested was measured using high performance liquid chromatography (HPLC), and the results are shown in Table 9 below.

[0157] [Table 9]

[0158] As shown in Table 9 above, the L-isoleucine concentration was confirmed to be increased in the mutant strains containing the ilvN single mutation (ilvN(D17A)) or the ilvN combination mutations (ilvN(D17A, A42V), ilvN(D17A, H47L), ilvN(D17A, A42V, H47L)) compared to the parent strain (KCCM11248P), an L-isoleucine-producing strain containing ilvN WT. In particular, the KCCM11248P△ilvN::ilvN(D17A) strain showed a high rate of increase in L-isoleucine concentration compared to the parent strain, and it was confirmed that the KCCM11248P△ilvN::ilvN(D17A, A42V), KCCM11248P△ilvN::ilvN(D17A, H47L), and KCCM11248P△ilvN::ilvN(D17A, A42V, H47L) strains all showed increased L-isoleucine production compared to the parent strain.

[0159] From the above description, those skilled in the art to which the present application pertains will understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present application should be interpreted as including all modifications and variations derived from the meaning and scope of the claims below, and equivalent concepts thereof, rather than the above detailed description.

[0160] [Table 10] The present application provides the following aspects of the invention. (Aspect 1) Acetohydroxy acid synthase (AHAS) mutant in which the amino acid corresponding to the 17th position in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid. (Aspect 2) 2. The variant of embodiment 1, wherein the amino acid corresponding to position 17 is substituted with alanine. (Aspect 3) 2. The variant of embodiment 1, wherein the amino acid corresponding to position 17 is asparagine. (Aspect 4) The variant of embodiment 1, wherein the variant further comprises one or more substitutions selected from the following: a substitution of the amino acid corresponding to position 42 in the amino acid sequence of SEQ ID NO: 1 with another amino acid; a substitution of the amino acid corresponding to position 47 with another amino acid; or a combination thereof. (Aspect 5) 5. The variant of embodiment 4, wherein the amino acid corresponding to position 42 is substituted with valine. (Aspect 6) 5. The variant of embodiment 4, wherein the amino acid corresponding to position 47 is substituted with leucine. (Aspect 7) The variant of embodiment 4, wherein the amino acid corresponding to position 42 is alanine. (Aspect 8) The variant of embodiment 4, wherein the amino acid corresponding to position 47 is histidine. (Aspect 9) A polynucleotide encoding the mutant according to any one of aspects 1 to 8. (Aspect 10) A Corynebacterium strain comprising the mutant according to any one of aspects 1 to 8; or a polynucleotide encoding the mutant. (Aspect 11) 11. The strain according to claim 10, wherein the strain has increased L-isoleucine production ability compared to a Corynebacterium strain containing a wild-type acetohydroxyacid synthase having the amino acid sequence of SEQ ID NO: 1 or a polynucleotide encoding the same. (Aspect 12) 11. The strain according to aspect 10, wherein the strain is Corynebacterium glutamicum. (Aspect 13) A method for producing L-isoleucine, comprising the step of culturing in a medium a Corynebacterium strain comprising the mutant according to any one of aspects 1 to 8; or a polynucleotide encoding the mutant. (Aspect 14) A composition for producing L-isoleucine, comprising: a Corynebacterium strain comprising a mutant according to any one of aspects 1 to 8, a polynucleotide encoding the mutant, a vector comprising the polynucleotide, or a polynucleotide of the present application; a medium in which the strain is cultured; or a combination of two or more of these. (Aspect 15) A mutant according to any one of aspects 1 to 8; a polynucleotide encoding the mutant; or use of a Corynebacterium strain comprising the mutant or a polynucleotide encoding the mutant for producing L-isoleucine.

Claims

1. An acetohydroxyacid synthase (AHAS) mutant comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, The above mutant, wherein the amino acid corresponding to the 17th position in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine.

2. The mutant has the amino acid sequence of SEQ ID NO:

1. The amino acid corresponding to position 42 is replaced with another amino acid; a substitution of the amino acid corresponding to position 47 with another amino acid; or combinations of these; The variant of claim 1 further comprising one or more substitutions selected from:

3. The mutant of claim 2 , wherein the amino acid corresponding to position 42 is substituted with valine.

4. The mutant of claim 2 , wherein the amino acid corresponding to position 47 is substituted with leucine.

5. The mutant of claim 2 , wherein the amino acid corresponding to position 42 is alanine.

6. The mutant of claim 2 , wherein the amino acid corresponding to the 47th position is histidine.

7. A polynucleotide encoding the variant according to any one of claims 1 to 6.

8. A Corynebacterium strain comprising the mutant according to any one of claims 1 to 6; or a polynucleotide encoding said mutant.

9. The strain according to claim 8, wherein the strain has increased L-isoleucine productivity compared to a Corynebacterium strain containing a wild-type acetohydroxyacid synthase having the amino acid sequence of SEQ ID NO: 1 or a polynucleotide encoding the same.

10. The strain of claim 8, wherein the strain is Corynebacterium glutamicum.

11. A method for producing L-isoleucine, comprising the step of culturing in a medium a Corynebacterium strain containing the mutant according to any one of claims 1 to 6; or a polynucleotide encoding said mutant.

12. A composition for producing L-isoleucine, comprising: a mutant according to any one of claims 1 to 6; a polynucleotide encoding the mutant; a vector comprising the polynucleotide; a Corynebacterium strain comprising the polynucleotide of the present application; a culture medium in which the strain is cultured; or a combination of two or more of these.

13. A mutant according to any one of claims 1 to 6; a polynucleotide encoding said mutant; or use of a Corynebacterium strain comprising said mutant or a polynucleotide encoding said mutant for the production of L-isoleucine.

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