RU2846710C2 - Novel variant of acetohydroxy acid synthase and method of producing l-isoleucine using same - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to acetohydroxy acid synthase (AHAS) and use thereof in the production of L-isoleucine. Disclosed is acetohydroxy acid synthase (AHAS), in which an amino acid corresponding to position 17 in the amino acid sequence SEQ ID NO: 1, is replaced with alanine. Also disclosed are a polynucleotide encoding said acetohydroxy acid synthase (AHAS), microorganism of Corynebacterium genus for producing L-isoleucine containing said acetohydroxy acid synthase (AHAS) or polynucleotide, a composition for producing L-isoleucine containing said acetohydroxy acid synthase (AHAS), a polynucleotide, a vector containing said polynucleotide, or a microorganism of genus Corynebacterium, including said polynucleotide. Invention also discloses a method of producing L-isoleucine using said microorganism and the use of said acetohydroxy acid synthase (AHAS), a polynucleotide or a microorganism of the genus Corynebacterium in the production of L-isoleucine.

EFFECT: invention provides high-yield production of L-isoleucine.

9 cl, 9 tbl, 7 ex

Description

AREA OF TECHNOLOGY

The present disclosure relates to a novel acetohydroxy acid synthase (AHAS) variant that improves the ability to produce L-isoleucine, a microorganism comprising the variant, and a method for producing L-isoleucine using the microorganism.

PRIOR ART

L-isoleucine is one of the 20 branched-chain amino acids. It is classified as an essential amino acid and is used in animal feed, dietary supplements, and pharmaceuticals. Because L-isoleucine has functions such as energy generation after metabolism, hemoglobin production, blood sugar control, muscle formation and repair, and more, its use is increasing in animal feed, infusion solutions, dietary supplements, and sports nutrition supplements.

Based on this trend, various microorganisms and their variants are used to produce L-amino acids (U.S. Patent No. 10,113,190). Even then, many byproducts other than L-isoleucine are produced. These byproducts are substances that greatly affect the purity of L-isoleucine during the purification step. Therefore, a method capable of removing these byproducts is required. In this regard, the methods for purifying L-isoleucine that have been developed to increase the purity of L-isoleucine have the disadvantage of requiring an additional, separate purification process (U.S. Patent No. 6,072,083). Therefore, a method for increasing the purity of L-isoleucine needs to be developed.

DESCRIPTION OF THE INVENTION

Technical problem

Regarding acetohydroxy acid synthase (AHAS), which is one of the proteins in the L-isoleucine producing pathway, the present inventors studied a variant thereof and found that the L-isoleucine producing ability of the strain can be improved by the variant, thereby completing the present disclosure.

Technical solution

The purpose of the present disclosure is to provide a variant of acetohydroxy acid synthase (AHAS) in which the amino acid corresponding to position 17 in the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid.

Another object of the present disclosure is to provide a polynucleotide encoding a variant of the present disclosure.

Yet another object of the present disclosure is to provide a strain of the genus Corynebacterium comprising a variant of the present disclosure, or a polynucleotide encoding the variant.

Yet another object of the present disclosure is to provide a method for producing L-isoleucine comprising the step of culturing in a medium a strain of the genus Corynebacterium comprising a variant according to the present disclosure; or a polynucleotide encoding this variant.

Beneficial effects

A microorganism expressing the acetohydroxy acid synthase variant according to the present disclosure can significantly improve L-isoleucine production compared to a strain that does not express this variant, and thus, its use can efficiently produce L-isoleucine. Accordingly, a wide range of industrial applications can be expected, such as in food, feed, pharmaceuticals, and other applications where L-isoleucine is used.

Detailed description of preferred embodiments

The present disclosure will be described in detail as follows. However, each description and embodiment disclosed in this disclosure may also be applied to other descriptions and embodiments. That is, all combinations of various elements disclosed in this disclosure fall within the scope of the present disclosure. Furthermore, the scope of the present disclosure is not limited to the specific description described below. Furthermore, throughout this specification, reference is made to and cited by numerous articles and patent documents. The disclosures of the cited articles and patent documents are incorporated herein by reference in their entirety to further clarify the level and scope of the subject matter to which the present disclosure pertains.

According to one aspect of the present disclosure, there is provided a variant of acetohydroxy acid synthase (AHAS) in which the amino acid corresponding to position 17 in the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid.

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

A variant of the present disclosure may comprise an amino acid sequence having an alanine substituted for an asparagine, which is the amino acid corresponding to position 17, based on the amino acid sequence represented by SEQ ID NO: 1, which is the parent sequence, and having at least 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, 99.5% or greater, or 99.7% or greater and less than 100% homology or identity with the amino acid sequence represented by SEQ ID NO: 1. It is also evident that a variant having an amino acid sequence having a deletion, modification, substitution, conservative substitution or addition of some amino acids, also falls within the scope of the present disclosure, provided that the amino acid sequence has such homology or identity and exhibits performance corresponding to the performance of the variant of the present disclosure.

Examples thereof include variants having an addition or deletion of a sequence that does not alter the function of the variant of the present disclosure, at the N-terminus or C-terminus of the amino acid sequence and/or within the amino acid sequence, a naturally occurring mutation, a silent mutation or a conservative substitution.

The term "conservative substitution" refers to the replacement of one amino acid with another amino acid that has similar structural and/or chemical properties. Such amino acid substitutions are typically based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues. Typically, a conservative substitution has little or no effect on the activity of proteins or polypeptides.

The term "variant," as used herein, refers to a polypeptide that has a different amino acid sequence from the sequence of the variant before alteration by conservative substitution and/or modification of one or more amino acids, but retains functions or properties. Such a variant can generally be identified by making a modification of one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. In other words, the ability of the variant may be increased, unchanged, or decreased compared to the ability of the polypeptide before the alteration. Some variants may include variants in which one or more parts, such as the N-terminal leader sequence or the transmembrane domain, have been removed. Other variants may include variants in which a portion of the N- and/or C-terminus has been removed from the mature protein. The term "variant" may be used interchangeably with terms such as modification, modified polypeptide, modified protein, mutant, mutein, and divergent, and is not limited to them, provided that it is a term used with the meaning of alteration.

A variant may include deletions or additions of amino acids that have minimal impact on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence that participates cotranslationally or posttranslationally in protein translocation may be conjugated to the N-terminus of a variant. The variant may be conjugated to other sequences or linkers so that it can be identified, purified, or synthesized.

The term "parent sequence," as used herein, refers to the reference sequence that becomes the modified polypeptide by introducing a modification. In other words, the parent sequence is the original sequence and may be the target for introducing a change, such as a substitution, insertion, and/or deletion, etc. The parent sequence may be a naturally occurring or wild-type sequence, or a variant in which one or more substitutions, insertions, or deletions have occurred in the naturally occurring or wild-type sequence, or it may be an artificially synthesized sequence.

The term "homology" or "identity," as used here, refers to the degree of similarity between two given amino acid or base sequences, and can be expressed as a percentage. The terms "homology" and "identity" are often used interchangeably.

Homology or sequence identity of a conserved polynucleotide or polypeptide is determined using standard alignment algorithms, which can be combined with the default gap penalty of the program used. Substantially homologous or identical sequences are typically capable of hybridizing to all or at least approximately 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the entire sequence under moderately or highly stringent conditions. Hybridization also includes hybridization to a polynucleotide containing a universal codon or a codon with codon degeneracy.

Whether or not any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using known computer algorithms such as the FASTA program, for example, using default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444. Alternatively, homology, similarity, or identity 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) (including the GCG software package (Devereux, J., et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. E, 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, NCBI's BLAST or ClustalW can be used to determine homology, similarity, or identity.

Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using, for example, the GAP computer program, such as Needleman et al. (1970) J Mol Biol. 48:443, as announced, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the output of the GAP program can be defined as the value obtained by dividing the number of similarly aligned characters (i.e., nucleotides or amino acids) by the total number of characters in the shorter of the two sequences. Default parameters for the GAP program may include: (1) a binary comparison matrix (containing the values 1 for identity and 0 for non-identity) and a weighted comparison matrix Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (EMBOSS version NCBI NUC4.4) substitution matrix), as disclosed in 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 penalty of 0.10 for each character in each gap (or a gap-opening penalty of 10, a gap-extending penalty of 0.5); and (3) no penalty for terminal gaps.

The variant of the present disclosure may have acetohydroxyacid synthase (AHAS) activity. Furthermore, the variant of the present disclosure may have activity to increase the ability to produce L-isoleucine compared to a wild-type polypeptide having acetohydroxyacid synthase activity.

Acetohydroxyacid synthase (AHAS), as the term is used here, is the first enzyme in the L-valine biosynthetic pathway and is also called acetolactate synthase. Acetohydroxyacid synthase catalyzes the decarboxylation of pyruvate and its condensation reaction with other pyruvic acid molecules to form acetolactate, a precursor of valine, or the decarboxylation of pyruvate and its condensation reaction with 2-ketobutyrate to form acetohydroxybutyrate, a precursor of isoleucine.

Acetohydroxyacid synthase is encoded by two genes: ilvB and ilvN. The ilvB gene encodes the large subunit of acetohydroxyacid synthase, and the ilvN gene encodes the small subunit of acetohydroxyacid synthase. Of these, the small subunit, encoded by the ilvN gene, is considered to be critically involved in feedback inhibition. The term "feedback inhibition" means that the end product of an enzymatic system inhibits a reaction at an early stage of the enzymatic system. For the purposes of this disclosure, acetohydroxyacid synthase may be acetohydroxyacid synthase encoded by the ilvN gene.

The sequence of the acetohydroxy acid synthase encoded by the ilvN gene can be obtained from the NCBI GenBank, which is a well-known database and may in particular have the amino acid sequence SEQ ID NO: 1, but is not limited thereto.

The term "corresponding" as used herein refers to amino acid residues at positions listed in the polypeptide, or to amino acid residues that are similar, identical, or homologous to residues listed in the polypeptide. Identifying an amino acid at a corresponding position can determine the specific amino acid in a sequence that relates to the specific sequence. The term "corresponding region" as used herein generally refers to an analogous or corresponding position in a related protein or reference protein.

For example, an arbitrary amino acid sequence is aligned with SEQ ID NO: 1 and, based on this, each amino acid residue of this amino acid sequence can be numbered with reference to the numerical position of the amino acid residue corresponding to the amino acid residue of SEQ ID NO: 1. For example, a sequence alignment algorithm as described in the present disclosure can determine the position of an amino acid or the position at which a modification, such as a substitution, insertion or deletion, occurs by comparison with an amino acid in a query sequence (also referred to as a "reference sequence").

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

The variant of the present disclosure may further comprise one or more substitutions selected from the substitution of the amino acid corresponding to position 42 with another amino acid; the substitution of the amino acid corresponding to position 47 with another amino acid, or combinations thereof, based on the amino acid sequence presented in SEQ ID NO: 1.

In one embodiment, the amino acid corresponding to position 42 may be replaced by valine.

In another embodiment, the amino acid corresponding to position 47 may be replaced by leucine.

In particular, a variant of the present disclosure may have, include, or consist of, or essentially consist of, the amino acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6.

In particular, SEQ ID NO: 3 may be an amino acid sequence in which asparagine, which is the amino acid corresponding to position 17, is replaced by alanine, SEQ ID NO: 4 may be an amino acid sequence in which asparagine, which is the amino acid corresponding to position 42, is replaced by valine, SEQ ID NO: 5 may be an amino acid sequence in which asparagine, which is the amino acid corresponding to position 17, is replaced by alanine, and the amino acid corresponding to position 47 is replaced by leucine, SEQ ID NO: 6 may be an amino acid sequence in which asparagine, which is the amino acid corresponding to position 17, is replaced by alanine, the amino acid corresponding to position 42 is replaced by valine, and the amino acid corresponding to position 47 is replaced by leucine based on the amino acid sequence represented by SEQ ID NO: 1.

According to another aspect of the present disclosure, a polynucleotide encoding a variant of the present disclosure is provided.

The term "polynucleotide" as used herein means a strand of DNA or RNA of a given or greater length, in the form of a polymer of nucleotides in which the nucleotide monomers are linked into a long chain by covalent bonds, and more particularly means a fragment of a polynucleotide encoding a given variant.

A polynucleotide encoding a variant of the present disclosure may include a nucleotide sequence encoding an amino acid sequence represented by 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 disclosure, a polynucleotide of the present disclosure may have or include the sequence of SEQ ID NO: 23 or SEQ ID NO: 24 or SEQ ID NO: 25 or SEQ ID NO: 26. Furthermore, a polynucleotide of the present disclosure may consist of or essentially consist of the sequence of SEQ ID NO: 23 or SEQ ID NO: 24 or SEQ ID NO: 25 or SEQ ID NO: 26.

Various modifications can be made in the coding region of the polynucleotide of the present disclosure, provided that the amino acid sequence of the variant of the present disclosure is not changed, taking into account codon degeneracy or codon preferences in organisms that are intended to express the variant of the present disclosure. In particular, a polynucleotide of the present disclosure may have or include a base sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, or 98% or greater and less than 100% homology or identity to the sequence of SEQ ID NO: 2, or may consist of or consist essentially of a base sequence having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, or 98% or greater and less than 100% homology or identity to the sequence of SEQ ID NO: 2, but is not limited to it. Here, in a sequence having homology or identity, the codon encoding the amino acid corresponding to position 17 of SEQ ID NO: 1 may be one of the codons encoding alanine, the codon encoding the amino acid corresponding to position 42 of SEQ ID NO: 1 may be one of the codons encoding valine, and the codon encoding the amino acid corresponding to position 47 of SEQ ID NO: 1 may be one of the codons encoding leucine.

Furthermore, a polynucleotide of the present disclosure may include a probe that can be derived from a known gene sequence, such as, but not limited to, a sequence that is capable of hybridizing to a complementary sequence, to all or part of a polynucleotide sequence of the present disclosure, under stringent conditions. The term "stringent conditions" means conditions that ensure specific hybridization between polynucleotides. These conditions are specifically described in the documents (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, 9.50-9.51, 11.7-11.8). Examples thereof include conditions under which polynucleotides having higher homology or identity, i.e., polynucleotides having 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater homology or identity hybridize with each other, while polynucleotides having less homology or identity do not hybridize with each other, or conditions in which washing is carried out once, specifically two to three times at a salt concentration and temperature equivalent to 60°C, 1x SSC (sodium chloride citrate solution), 0.1% SDS (sodium dodecyl sulfate), specifically at 60°C, 0.1x SSC, 0.1% SDS, more specifically at 68°C, 0.1x SSC, 0.1% SDS, which represents the washing conditions for conventional Southern hybridization.

Hybridization requires two nucleic acids to have complementary sequences, although mismatches between bases are permitted, depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases capable of hybridizing with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotide of the present disclosure may also include substantially similar nucleic acid sequences, as well as isolated nucleic acid fragments that are complementary to the entire sequence.

In particular, a polynucleotide having homology or identity to a polynucleotide of the present disclosure can be detected using hybridization conditions comprising a hybridization step at a Tm value of 55°C and the conditions described above. Furthermore, the Tm value can be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by those skilled in the art according to the purpose.

The appropriate stringency for polynucleotide hybridization depends on the length and degree of complementarity of the polynucleotide, and these variables are well known in the art (e.g., J. Sambrook et al., supra).

According to yet another aspect of the present disclosure, a vector comprising a polynucleotide according to the present disclosure is provided. This vector may be, but is not limited to, an expression vector for expressing the polynucleotide in a host cell.

A "vector" according to the present disclosure may comprise a DNA construct comprising a polynucleotide sequence encoding a polypeptide of interest, which is operably linked to a suitable expression regulatory region (or expression control sequence) such that the polypeptide of interest can be expressed in a suitable host. The expression regulatory region may include a promoter capable of initiating transcription, any operator sequence for controlling transcription, a sequence encoding a suitable mRNA-ribosome binding site, and a sequence controlling the termination of transcription and translation. This vector can be transformed into a suitable host cell and then replicate or function independently of the host genome, or can integrate into the genome itself.

The vector used in the present disclosure is not particularly limited, but any vector known in the art can be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as a phage vector or a cosmid vector, and the pDZ system, the pBR system, the pUC system, the pBluescript II system, the pGEM system, the pTZ system, the pCL system, the pET system, etc. can be used as a plasmid vector. In particular, the following vectors can be used: pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCClBAC, etc.

For example, a polynucleotide encoding a polypeptide of interest can be inserted into a chromosome using a vector for intracellular chromosomal insertion. Insertion of the polynucleotide into the chromosome can be accomplished by any method known in the art, such as, but not limited to, homologous recombination. The vector may further include a selectable marker to confirm insertion into the chromosome. The selectable marker serves to select cells transformed by the vectors, i.e., to confirm insertion of the nucleic acid molecule of interest. Markers that confer selectable phenotypes, such as drug resistance, auxotrophy, cytotoxic resistance, or expression of surface polypeptides, can be used. In a medium treated with a selective agent, only cells expressing the selectable marker survive or exhibit other phenotypic characteristics, and thus, transformed cells can be selected.

The term "transformation," as used herein, means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism in such a way that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide can be located, regardless of position, either within the host cell's chromosome or outside the chromosome, as long as it can be expressed in the host cell. The polynucleotide includes DNA and/or RNA encoding the polypeptide of interest. The polynucleotide can be introduced in any form, as long as it can be introduced into the host cell and expressed. 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 the elements required for autonomous expression. This expression cassette may typically include a promoter operably linked to a polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of autonomous replication. This polynucleotide may be introduced into the host cell in its native form and may be operably linked to, but not limited to, a sequence required for expression in the host cell.

Furthermore, the term "operably linked" means that the polynucleotide sequence is operably linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the variant of interest of the present disclosure.

According to yet another aspect of the present disclosure, there is provided a strain of the genus Corynebacterium comprising a variant of the present disclosure or a polynucleotide of the present disclosure.

A strain of the present disclosure may include a modified polypeptide of the present disclosure, a polynucleotide encoding the polypeptide, or a vector including a polynucleotide of the present disclosure.

The term "strain (or microorganism)" as used herein includes all wild-type microorganisms or naturally or artificially genetically modified microorganisms, and it may be a microorganism in which a specific mechanism is weakened or strengthened due to the insertion of a foreign gene or the enhancement or inactivation of an endogenous gene, and it may be a microorganism incorporating genetic modification for the production of a polypeptide, protein or product of interest.

A strain of the present disclosure may be, but is not limited to, a strain comprising any one or more variants of the present disclosure, a polynucleotide of the present disclosure, and a vector comprising a polynucleotide of the present disclosure; a strain modified to express a variant of the present disclosure or a polynucleotide of the present disclosure; a strain (e.g., a recombinant strain) expressing a variant of the present disclosure or a polynucleotide of the present disclosure; or a strain (e.g., a recombinant strain) having the activity of a variant of the present disclosure.

The strain of the present disclosure may be a strain having the ability to produce L-isoleucine.

The strain of the present disclosure may be a microorganism that naturally has acetohydroxy acid synthase or the ability to produce L-isoleucine, or a microorganism in which a variant of the present disclosure or a polynucleotide encoding the variant (or a vector comprising the polynucleotide) is introduced into a parent strain that does not have acetohydroxy acid synthase or the ability to produce L-isoleucine, and/or in which the parent strain is given the ability to produce L-isoleucine, but is not limited thereto.

For example, a strain according to the present disclosure is a cell or microorganism that has been transformed with a polynucleotide according to the present disclosure or a vector comprising a polynucleotide encoding a variant according to the present disclosure, for the expression of the variant according to the present disclosure. With respect to the subjects of the present disclosure, a strain according to the present disclosure may include all microorganisms that comprise a variant according to the present disclosure and are capable of producing L-isoleucine. For example, a strain according to the present disclosure may be a recombinant strain in which a polynucleotide encoding a variant according to the present disclosure is introduced into a naturally occurring wild-type microorganism or into a microorganism producing L-isoleucine, for the expression of an acetohydroxy acid synthase variant and for the increased ability to produce L-isoleucine. The recombinant strain having an increased ability to produce L-isoleucine may be a microorganism having an increased ability to produce L-isoleucine compared to a naturally occurring wild-type microorganism or a microorganism not modified with acetohydroxy acid synthase (i.e., a microorganism expressing wild-type acetohydroxy acid synthase (SEQ ID NO: 1) or a microorganism that does not express a modified protein (SEQ ID NO: 3 or SEQ ID NO: 4, or SEQ ID NO: 5 or SEQ ID NO: 6), but is not limited to them. For example, a microorganism not modified with acetohydroxy acid synthase, which is a target strain for comparing whether or not the ability to produce L-isoleucine is increased, may be Corynebacterium glutamicum strain ATCC13032 (CA10-3101, KCCM12739P) into which hom(R407H) and ilvA(T381A,F383A) changes were introduced, or the KCJI-38 strain (KCCM11248P, Korean Patent No. 10-1335789), which is a strain producing L-isoleucine treated with N-methyl-N'-nitro-N-nitrosoguanidine (NTG), but not limited to them.

For example, a recombinant strain having this increased production capacity may have an L-isoleucine producing capacity increased by about 1% or more, in particular 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, about 57% or more, about 59% or more, about 60% or more, by about 62% or more, by about 63% or more, by about 64% or more, or by about 73% or more (the upper limit is not particularly limited, but 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) compared to the L-isoleucine-producing ability of the parent strain before modification or with an unmodified microorganism, but this increased amount is not limited thereto, as long as the producing ability has an increased amount of a plus value compared to the producing ability of the parent strain before modification or with an unmodified microorganism. In another example, a recombinant strain having an increased production capacity may have an L-isoleucine producing capacity increased by about 1.01 times or more, about 1.02 times or more, about 1.05 times or more, about 1.10 times or more, about 1.15 times or more, about 1.20 times or more, about 1.25 times or more, about 1.30 times or more, about 1.32 times or more, about 1.34 times or more, about 1.35 times or more, about 1.39 times or more, about 1.40 times or more, about 1.45 times or more, about 1.46 times or more, 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 (the upper limit is not particularly limited, but may be, for example, about 10 times or less, about 5 times or less, about 3 times or less, about 2 times or less) compared to the L-isoleucine producing ability of the parent strain before modification or with the unmodified microorganism, but not limited to.

The term "unmodified microorganism," as used herein, does not exclude strains incorporating a mutation that may occur naturally in microorganisms and may be the wild-type or natural strain itself, or may be a strain before a trait has been altered through genetic change due to natural or artificial factors. For example, an unmodified microorganism may be a strain in which the acetohydroxyacid synthase variant described herein is not introduced or has not yet been introduced. The term "unmodified microorganism" may be used interchangeably with the terms "strain before modification," "microorganism before modification," "unmodified strain," "unmodified strain," "unmodified microorganism," or "reference microorganism."

In another example of the present disclosure, the microorganism of the present disclosure may be of the genus Corynebacterium (Corynebacterium stationis), Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotoleram, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens, in particular, but not limited to, Corynebacterium glutamicum.

As used herein, the term "enhancement" of a polypeptide's activity means that the activity of the polypeptide is increased relative to the endogenous activity. The term "enhancement" can be used interchangeably with terms such as activation, upregulation, overexpression, and enhancement. Here, activation, enhancement, upregulation, overexpression, and enhancement can include both the demonstration of an activity that was not originally possessed and the demonstration of an activity that is improved relative to the endogenous activity or the activity before modification. The term "endogenous activity" refers to the activity of a particular polypeptide that was originally possessed by the parent strain before the change in the trait or by the unmodified microorganism when the trait is changed through genetic alteration due to natural or artificial factors. This term can be used interchangeably with the term "activity before modification." The fact that the activity of a polypeptide is "enhanced," "up-regulated," "overexpressed," or "increased" compared to the endogenous activity means that the activity of the polypeptide is improved compared to the activity and/or concentration (expression level) of the particular polypeptide originally possessed by the parent strain before the trait change or the unmodified microorganism.

Enhancement can be achieved by introducing a foreign polypeptide or by increasing the endogenous activity and/or concentration (expression level) of the polypeptide. Enhancement of polypeptide activity can be confirmed by increasing the activity level and expression level of the corresponding polypeptide or the amount of product produced from the corresponding polypeptide.

To enhance the activity of a polypeptide, various methods well known in the art can be used, and this method is not limited, provided that the activity of the polypeptide of interest can be enhanced compared to the activity of the microorganism before modification. In particular, genetic engineering and/or protein engineering, well known to those skilled in the art, which are traditional methods of molecular biology, can be used, but this method is not limited to them (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

In particular, the enhancing activity of the polypeptide of the present disclosure may be:

1) an increase in the number of intracellular copies of the polynucleotide encoding the polypeptide;

2) replacement of the gene expression regulatory region on the chromosome encoding the polypeptide with a sequence demonstrating strong activity;

3) modification of the initiation codon of the transcript of the gene encoding the polypeptide, or the nucleotide sequence encoding the 5'-UTR (untranslated) region;

4) modification of the amino acid sequence of a polypeptide to enhance the activity of this polypeptide;

5) modifying the sequence of a polynucleotide encoding a polypeptide to enhance the activity of the polypeptide (e.g., modifying the sequence of a polynucleotide of a polypeptide gene encoding a polypeptide that has been modified to enhance the activity of the polypeptide);

6) the introduction of a foreign polypeptide demonstrating the activity of the given polypeptide, or a foreign polynucleotide encoding the given polypeptide;

7) optimization of codons of the polynucleotide encoding the polypeptide;

8) analysis of the tertiary structure of the polypeptide for selection and for modification or chemical modification of the exposed site; or

9) a combination of two or more than two selected from (1) to (8), but not specifically limited to them.

More specifically:

1) An increase in the number of intracellular copies of a polynucleotide encoding a polypeptide can be achieved by introducing into the host cell a vector that can replicate and function independently of the host and to which the polynucleotide encoding the corresponding polypeptide is functionally linked. Alternatively, the increase can be achieved by introducing one copy or two or more copies of the polynucleotide encoding the corresponding polypeptide into the chromosome of the host cell. Introduction into the chromosome can be accomplished by introducing into the host cell a vector capable of inserting the polynucleotide into the chromosome of the host cell, but is not limited thereto. This vector is as described above.

2) The replacement of a gene expression regulatory region (or expression control sequence) on a chromosome encoding a polypeptide with a sequence exhibiting strong activity may be, for example, a deletion, an insertion, a non-conservative or conservative substitution, or the occurrence of a change in the sequence due to a combination thereof, or a replacement with a sequence exhibiting stronger activity, such that the activity of the expression regulatory region is further enhanced. The expression regulatory region may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence controlling the termination of transcription and translation, etc., but is not particularly limited to them. For example, the replacement may be the replacement of the original promoter with a strong promoter, but is not limited to it.

Examples of known strong promoters include, but are not limited to, the cj1-cj7 promoters (US Patent No. US 7,662,943 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 1,0584,338 B2), the O2 promoter (US Patent No. US 1,0273,491 B2), the tkt promoter, the yccA promoter, etc.

3) Modification of the initiation codon of a transcript of a gene encoding a polypeptide or of a nucleotide sequence encoding the 5'-UTR region may be, for example, a replacement with a base sequence encoding another initiation codon having a higher rate of expression of the polypeptide compared to the endogenous initiation codon, but is not limited to it.

4) and 5) The modification of the amino acid sequence or the polynucleotide sequence may be, but is not limited to, a deletion, an insertion, a non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the sequence of the polynucleotide encoding this polypeptide, or the occurrence of a change in the sequence due to a combination thereof, or a replacement with an amino acid sequence or a polynucleotide sequence modified to exhibit a stronger activity, or an amino acid sequence or a polynucleotide sequence modified to be more active, such that the activity of this polypeptide is enhanced. This substitution can be specifically carried out by inserting the polynucleotide into the chromosome by homologous recombination, but is not limited thereto. The vector used herein may further include a selectable marker to confirm the insertion into the chromosome. The selectable marker is as described above.

6) Introduction of a foreign polynucleotide exhibiting polypeptide activity may involve the introduction into a host cell of a foreign polynucleotide encoding a polypeptide exhibiting identical or similar activity to the given polypeptide. There is no limitation on its origin or sequence, provided that it exhibits the same or similar activity to the given polypeptide. Introduction may be accomplished by appropriately selecting a transformation method known to those skilled in the art. Since the introduced polynucleotide is expressed in the host cell, the polypeptide may be produced and its activity may be increased.

7) Optimization of codons of a polynucleotide encoding a polypeptide may be optimization of codons of an endogenous polynucleotide in such a way as to increase transcription or translation in the host cell, or optimization of codons of a foreign polynucleotide in such a way as to carry out optimized transcription and translation in the host cell.

8) Analysis of the tertiary structure of a polypeptide for selection and for modification or chemical modification of an exposed site can be carried out, for example, to determine a candidate template protein according to the degree of sequence similarity by comparing the sequence information of the polypeptide to be analyzed with a database of sequence information of known proteins to confirm the structure based on this and to modify or chemically modify the exposed site to be modified or chemically modified.

Such enhancement of the activity of the polypeptide may be, but is not limited to, an increase in the activity or concentration, or expression level of the corresponding polypeptide based on the activity or concentration of the polypeptide expressed in the wild-type microbial strain or in the microbial strain before modification, or an increase in the amount of product produced from the corresponding polypeptide.

In the microorganism of the present disclosure, partial or complete modification of a polynucleotide can be induced by (a) homologous recombination using a vector for insertion into a chromosome in the microorganism or genome editing using a genetically modified nuclease (e.g., CRISPR-Cas9), and/or (b) treatment with light, such as ultraviolet rays and radiation, and/or chemical agents, but not limited to. The method for modifying part or all of a gene can include a method using recombinant DNA technology. For example, by introducing a nucleotide sequence or a vector comprising a nucleotide sequence homologous to a gene of interest into a microorganism to induce homologous recombination, a part or all of the gene can be deleted. The nucleotide sequence or vector to be introduced can include, but is not limited to, a dominant selectable marker.

In the microorganism of the present disclosure, the variant, polynucleotide, L-isoleucine, etc. are as described in other aspects.

According to yet another aspect of the present disclosure, there is provided a method for producing L-isoleucine, comprising the step of culturing in a medium a strain of the genus Corynebacterium comprising a variant of the present disclosure or a polynucleotide of the present disclosure.

The method for producing L-isoleucine according to the present disclosure may include the step of culturing in a medium a strain of the genus Corynebacterium comprising a variant according to the present disclosure or a polynucleotide according to the present disclosure, or a vector according to the present disclosure.

The term "cultivating," as used herein, refers to growing a strain of the genus Corynebacterium according to the present disclosure under suitably controlled environmental conditions. The culturing method according to the present disclosure can be carried out in accordance with suitable media and culture conditions known in the art. Such a culturing method can be easily adjusted and utilized by those skilled in the art according to the selected strain. In particular, culturing can be of the batch, continuous, and/or fed-batch type, but is not limited to these.

The term "medium" as used herein means a mixed substance containing the nutrients required for cultivating the strain of the genus Corynebacterium according to the present disclosure as the main component, and this medium supplies nutrients, growth factors, etc., including water, which are essential for survival and development. In particular, any medium and other culture conditions used for cultivating the strain of the genus Corynebacterium according to the present disclosure can be used without any particular limitation, provided that it is a medium used for the conventional cultivation of microorganisms. The strain of the genus Corynebacterium according to the present disclosure can be cultivated in a conventional medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and/or vitamins, etc., while simultaneously controlling temperature, pH, etc. under aerobic conditions.

For example, a culture medium for a strain of the genus Corynebacterium can be found in the document "Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington, D.C., USA, 1981).

In the present disclosure, carbon sources include carbohydrates such as glucose, sucrose, 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. Natural organic nutrients such as starch hydrolysate, molasses, raw molasses, rice bran, cassava, sugarcane residue, and liquid corn steep liquor can be used. In particular, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted into reducing sugar) can be used, and suitable amounts of other carbon sources can be used in various ways without limitation. These carbon sources can be used singly or in combination of two or more than two of them, but are not limited to them.

Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, liquid corn steep liquor, casein hydrolysate, fish or its degradation products, and defatted soybean meal or its degradation products. These nitrogen sources can be used singly or in combination of two or more, but are not limited to them.

Phosphorus sources may include monopotassium phosphate, dipotassium phosphate, or their corresponding sodium salts. Inorganic compounds that can be used include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, calcium carbonate, etc. Additionally, amino acids, vitamins, and/or suitable precursors may be included. These components or precursors may be added to the medium in batches or continuously, but are not limited to these.

During cultivation of the Corynebacterium strain according to the present disclosure, the pH of the medium can be adjusted by properly adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. During cultivation, foaming can be suppressed by 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 of the medium, or no gas can be injected, or nitrogen, hydrogen, or carbon dioxide gas can be injected to maintain an anaerobic and microaerobic state, but is not limited to these.

In the culturing according to the present disclosure, the culturing temperature can be maintained at 20°C to 45°C, in particular 25°C to 40°C, and the strain can be cultured for about 10 to 160 hours, but not limited thereto.

L-isoleucine produced by the culture according to the present disclosure may be secreted into the medium or may remain in the cells.

The method for producing L-isoleucine according to the present disclosure may further comprise the steps of obtaining a strain of the genus Corynebacterium according to the present disclosure, preparing a medium for culturing the strain, or a combination thereof (in any order), for example, before the culturing step.

The method for producing L-isoleucine according to the present disclosure may further comprise a step of isolating L-isoleucine from a culture medium (culture-exposed medium) or from a strain of the genus Corynebacterium. An isolation step may further be included after the culture step.

The isolation can serve to collect the L-isoleucine of interest by a suitable method known in the art, according to the microorganism culturing method of the present disclosure, for example, a batch, continuous, or fed-batch culturing method. For example, centrifugation, filtration, treatment with a precipitant of crystallized protein (salting out), extraction, sonication, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography and affinity chromatography, etc., HPLC (high performance liquid chromatography), or a combination thereof can be used. The L-isoleucine of interest can be isolated from the medium or microorganism by a suitable method known in the art.

The method for producing L-isoleucine according to the present disclosure may further include a purification step. Purification can be accomplished using a suitable method known in the art. In one example, when the method for producing L-isoleucine according to the present disclosure includes both an isolation step and a purification step, these isolation steps and purification steps can be performed continuously or intermittently, regardless of order, or can be performed simultaneously, or by combining them into a single step, but are not limited to these.

In the method of the present disclosure, the variant, polynucleotide, vector, strain, etc. are as described in other aspects.

According to yet another aspect of the present disclosure, there is provided a composition for producing L-isoleucine, comprising a variant of the present disclosure, a polynucleotide encoding the variant, a vector comprising the polynucleotide, a strain of the genus Corynebacterium comprising the polynucleotide of the present disclosure; a culture medium thereof, or a combination of two or more of them.

The composition according to the present disclosure may further comprise any suitable excipients commonly used in compositions for producing amino acids. Such excipients may include, but are not limited to, a preservative, a humectant, a dispersing agent, a suspending agent, a buffering agent, a stabilizer, or an isotonic agent, etc.

In the composition of the present disclosure, the variant, polynucleotide, vector, strain, medium, L-isoleucine, etc. are as described in other aspects.

According to yet another aspect of the present disclosure, there is provided the use of a variant of the present disclosure; a polynucleotide encoding the variant, or a strain of the genus Corynebacterium comprising the variant, or a polynucleotide encoding the variant, in the production of L-isoleucine. A method for carrying out the invention

The present disclosure will be described in more detail below using exemplary embodiments. However, the following exemplary embodiments represent merely preferred embodiments for illustrating the present disclosure and, therefore, are not intended to limit the scope of the present disclosure to them. Meanwhile, technical issues not described in the present specification can be sufficiently understood and easily implemented by those skilled in the technical field of the present disclosure or similar technical fields.

Example 1. Obtaining a strain producing L-isoleucine

Wild-type Corynebacterium glutamicum has the ability to produce L-isoleucine, but does not produce L-isoleucine in excess. Therefore, to confirm the genetic trait that increases L-isoleucine production, a strain with increased L-isoleucine production compared to the wild-type was obtained.

First, in order to release the feedback inhibition of threonine, which is a precursor of isoleucine in the L-isoleucine biosynthetic pathway of wild-type Corynebacterium glutamicum ATCC13032, the horn gene encoding homoserine dehydrogenase was mutated to replace arginine, which is an amino acid at position 407 of homoserine dehydrogenase, with histidine (Korean Patent No. 10-1996769).

In detail, to construct a vector for introducing the hom(R407H) change on the chromosome, PCR (polymerase chain reaction) was performed using the primer pair SEQ ID NO: 14 and SEQ ID NO: 15 or the primer pair SEQ ID NO: 16 and SEQ ID NO: 17, using the wild-type Corynebacterium glutamicum ATCC13032 chromosome as a template, respectively. The primer sequences are as shown in Table 1 below.

PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as the polymerase for PCR amplification, and the PCR conditions were as follows: 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 for 28 cycles to obtain a 1,000 bp DNA fragment of the 5' upstream region and a 1,000 bp DNA fragment of the 3' downstream region, focusing on the alteration of the hom gene, respectively.

PCR was performed using the primer pair SEQ ID NO: 14 and SEQ ID NO: 17 using these two amplified PCR fragments as a template. After denaturation at 95°C for 5 minutes, PCR was performed for 28 cycles under the conditions of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 2 minutes, and then polymerization at 72°C for 5 minutes.

As a result, a 2-kb DNA fragment (SEQ ID NO: 13) was amplified, containing a mutation in the hom gene encoding a homoserine dehydrogenase variant in which the arginine at position 407 was replaced by histidine. This amplification product was purified using a PCR purification kit (QUIAGEN) and used as the insert DNA fragment for vector construction.

After treating this purified amplified product with a smal restriction enzyme, the pDCM2 vector (Korean Patent Publication No. 10-2020-0136813) heated at 65°C for 20 minutes and the insert DNA fragment, which is the amplified product, were kept at a molar concentration (M) ratio of 1:2 and cloned using an infusion cloning kit (TaKaRa) according to the provided manual to construct the pDCM2-R407H vector to introduce the hom (R407H) change on the chromosome.

This constructed vector was transformed into Corynebacterium glutamicum ATCC13032 by electroporation, and a strain containing the hom(R407H) change on the chromosome was obtained by the secondary crossing method, which was named Corynebacterium glutamicum ATCC13032 hom(R407H).

In order to enhance the release of feedback inhibition and the activity toward L-isoleucine in the obtained strain ATCC13032 hom(R407H), ilvA, which is a gene encoding L-threonine dehydratase, was mutated to replace threonine, which is the amino acid at position 381, with alanine and phenylalanine, which is the amino acid at position 383, with alanine in L-threonine dehydratase.

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

PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as the polymerase for PCR amplification, and the PCR conditions were as follows: 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 for 28 cycles to obtain a 1126 bp DNA fragment of the 5' upstream region and a 286 bp DNA fragment of the 3' downstream region, focusing on the alteration of the ilvA gene, respectively.

PCR was performed using the primer pair SEQ ID NO: 19 and SEQ ID NO: 22 using these two amplified PCR fragments as a template. After denaturation at 95°C for 5 minutes, PCR was performed for 28 cycles under the conditions of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 2 minutes, and then polymerization at 72°C for 5 minutes.

As a result, a 1.4-kb DNA fragment (SEQ ID NO: 18) was amplified, containing a mutation in the ilvA gene encoding a variant of L-threonine dehydratase in which threonine at position 381 was replaced by alanine, and phenylalanine at position 383 was replaced by alanine. This amplification product was purified using a PCR purification kit and used as the insert DNA fragment for vector construction. After treating this purified amplified product with the restriction enzyme smal, the pDCM2 vector, which was heated at 65°C for 20 minutes, and the insert DNA fragment, which was the amplified product, were kept at a molar concentration (M) ratio of 1:2 and cloned using an infusion cloning kit (TaKaRa) according to the provided manual to construct the pDCM2-ilvA(T381A,F383A) vector to introduce the ilvA(T381A,F383A) changes on the chromosome.

This constructed vector was transformed into Corynebacterium glutamicum ATCC13032 hom(R407H) by electroporation, and a strain containing ilvA(T381A,F383A) changes on the chromosome was obtained by the secondary crossing method, which was named Corynebacterium glutamicum CA10-3101.

The CA10-3101 strain was deposited with the Korea Center for Culture of Microorganisms (KCCM), an international depository authority operating under the Budapest Treaty, on May 27, 2020, and assigned accession number KCCM12739P.

Then, the following experiment was conducted to confirm whether introducing the ilvA(T381A,F383A) changes into the L-isoleucine-producing strain actually increases the L-isoleucine-producing ability by increasing the release of feedback inhibition and the activity toward L-isoleucine.

In detail, ilvA(T381A,F383A) was modified into the KCJI-38 strain (KCCM11248P, Korean Patent No. 10-1335789), which is an N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-treated L-isoleucine-producing strain, by the electric pulse method to obtain the KCCM11248P/pECCG117-ilvA(T381A,F383A) strain. Then, fermentation titration was performed as follows.

The parental strain and the variant strain were inoculated into a 250-ml baffled flask containing 25 ml of isoleucine production medium and cultured at 32°C for 60 hours with shaking at 200 rpm to produce L-isoleucine, respectively. The composition of the production medium was as follows.

Production environment

10% glucose, 0.2% yeast extract, 1.6% ammonium sulfate, 0.1% monobasic potassium phosphate, 0.1% magnesium sulfate heptahydrate, 10 mg/L ferrous sulfate heptahydrate, 10 mg/L manganese sulfate monohydrate, 200 µg/L biotin, pH 7.2.

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

As shown in Table 3, it was confirmed that the KCCM11248P/pECCG117-ilvA(T381A,F383A) strain, into which ilvA(T381A,F383A) alterations were introduced, had a significantly increased L-isoleucine producing ability and a high L-threonine degradation rate compared with the parental strain KCCM11248P. Accordingly, it was confirmed that the introduction of ilvA(T381A,F383A) alterations into this strain increased the release of feedback inhibition and the activity towards L-isoleucine.

Example 2: Generation of a Vector Library with ilvN Variants A library of variants of the ilvN gene encoding the small subunit of acetohydroxy acid synthase (AHAS) was generated. The library was generated using an error-tolerant PCR kit (clontech Diversify® Random PCR Mutagenesis Kit), and the PCR reaction was performed using the wild-type Corynebacterium glutamicum ATCC13032 chromosome as a template and the primer pair SEQ ID NO: 7 and SEQ ID NO: 8. The primer sequences are as shown in Table 4 below.

In detail, under conditions where 0 to 3 changes per 1000 bp could occur, preheating was performed at 94°C for 30 seconds, followed by 25 cycles of 94°C for 30 seconds and 68°C for 1 minute and 30 seconds. The PCR product obtained in this way was subjected to 25 cycles of 95°C for 50 seconds, 60°C for 50 seconds and 68°C for 12 minutes using the megaprimer (500 ng to 125 ng), then treated with DpnI, transformed into E. coli DH5a and spread on solid LB medium containing kanamycin (25 mg/L). Twenty transformed colonies were selected, and plasmids were then prepared, followed by polynucleotide sequence analysis. As a result, it was confirmed that changes were introduced at different sites with a frequency of 2 mutations/kb. Approximately 20,000 transformed E. coli colonies were collected, plasmids were isolated from them, and they were designated as the pTOPO-ilvN library.

Example 3: Generation of an L-isoleucine-producing strain with an introduced ilvN library

The pTOPO-ilvN library obtained in Example 2 was transformed into CA10-3101 (KCCM12739P), which is the L-isoleucine-producing strain obtained in Example 1, by electroporation and then spread on a nutrient medium containing 25 mg/L kanamycin to obtain 5000 colonies of this strain into which the variant gene was inserted. Each colony was named CA10-3101/pTOPO-ilvNm1 to CA10-3101/pTOPO-ilvNm5000.

To confirm colonies with enhanced L-isoleucine production capacity among the 5,000 colonies obtained, each colony was subjected to fermentation titration as follows. In detail, the parent strain and variant strain were inoculated into a 250-mL baffled flask containing 25 mL of isoleucine production medium, respectively, and cultured at 32°C for 60 hours with shaking at 200 rpm to produce L-isoleucine. The composition of the production medium was as follows.

Production environment

10% glucose, 0.2% yeast extract, 1.6% ammonium sulfate, 0.1% monobasic potassium phosphate, 0.1% magnesium sulfate heptahydrate, 10 mg/L ferrous sulfate heptahydrate, 10 mg/L manganese sulfate monohydrate, 200 µg/L biotin, pH 7.2.

After completion of the culture, L-isoleucine concentrations in the culture medium for each analyzed strain were measured using high-performance liquid chromatography (HPLC), and the results are shown in Table 5 below.

As shown in Table 5, the variant strain CA10-3101/pTOPO-ilvNm2991 was confirmed to have an increased ability to produce L-isoleucine compared with Corynebacterium glutamicum CA10-3101, which is the parent strain with the wild-type ilvN gene. This variant strain was sequenced and, when compared with the wild-type ilvN gene of Corynebacterium glutamicum ATCC13032, it was confirmed that the variant strain contains a change (D17A) in which asparagine at position 17 in the amino acid sequence of ilvN was replaced by alanine.

Based on this result, it was confirmed that the ilvN(D17A) variant strain was able to produce L-isoleucine with a high yield compared with the parent strain, and the increase rate of L-isoleucine concentration was approximately 39% compared with the parent strain.

Example 4: Generation of an L-isoleucine-producing strain variant with introduced ilvN

The ilvN(D17A) change confirmed in Example 3 was introduced into the Corynebacterium glutamicum strain CA10-3101 obtained in Example 1.

In detail, PCR was performed using the CA10-3101/pTOPO-ilvNm2991(D17A) chromosome as a template and the primer pair of SEQ ID NO: 7 and SEQ ID NO: 8 to construct a vector for introducing the ilvN(D17A) gene variant into the chromosome. PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as the polymerase for PCR amplification, and the PCR conditions were as follows: 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 for 28 cycles to obtain three 545 bp DNA fragments, each including the ilvN(D17A) gene variant. This amplification product was purified using a PCR purification kit (QUIAGEN) and used as the insert DNA fragment for vector construction. After treatment of this purified amplification product with the smal restriction enzyme, the pDCM2 vector, heated at 65°C for 20 minutes, and the PCR-amplified insert DNA fragment were kept at a molar concentration (M) ratio of 1:2 and cloned using an infusion cloning kit (TaKaRa) according to the provided manual with the construction of the pDCM2-ilvN(D17A) vector for the introduction of the ilvN gene variant of Corynebacterium glutamicum into the chromosome.

This constructed vector was transformed into Corynebacterium glutamicum CA10-3101 hom(R407H) by electroporation, and a strain having the ilvN variant substitution on the chromosome was obtained by the secondary crossing method, and CA10-3101::ilvN(D17A) was named CA10-3128.

In order to test the effect of increasing the L-isoleucine producing ability, the parent strain (CA10-3101) and the variant strain CA10-3128 obtained in this Example were subjected to fermentation titration as follows, respectively.

The parental strain and the variant strain were inoculated into a 250-ml baffled flask containing 25 ml of isoleucine production medium and cultured at 32°C for 60 hours with shaking at 200 rpm to produce L-isoleucine. The composition of the production medium was as follows.

Production environment

10% glucose, 0.2% yeast extract, 1.6% ammonium sulfate, 0.1% monobasic potassium phosphate, 0.1% magnesium sulfate heptahydrate, 10 mg/L ferrous sulfate heptahydrate, 10 mg/L manganese sulfate monohydrate, 200 µg/L biotin, pH 7.2.

After completion of the culture, L-isoleucine concentrations in the culture medium for each analyzed strain were measured using high-performance liquid chromatography (HPLC), and the results are shown in Table 6 below.

As shown in Table 6, it was confirmed that the ilvN(D17A)-introduced strain CA10-3128 had an increased concentration of L-isoleucine compared with the parent strain (CA10-3131), which is an L-isoleucine-producing strain with ilvN DT. The increase in L-isoleucine concentration in the ilvN(D17A)-introduced strain was approximately 53% compared with the parent strain.

Example 5: Construction of a combined variant of the ilvN plasmid

In order to introduce a combination variant including the ilvN variant identified in Example 3 into an L-isoleucine producing strain, a vector was constructed to introduce the combination variant ilvN onto the chromosome.

In detail, PCR was performed using pDCM2-ilvN(D17A) as a template and the primer pair of SEQ ID NO: 7 and SEQ ID NO: 9 or the primer pair of SEQ ID NO: 10 and SEQ ID NO: 8, and PCR was performed using pDCM2-ilvN(H47L) as a template and the primer pair of SEQ ID NO: 7 and SEQ ID NO: 11 or the primer pair of SEQ ID NO: 12 and SEQ ID NO: 8. The sequences of the primers are as shown in Table 7 below.

PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as the polymerase for PCR amplification, and the PCR conditions were as follows: 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 conditions were repeated for 28 cycles to obtain DNA fragments of 149 bp and 423 bp, and DNA fragments of 166 bp and 405 bp, respectively. Each amplification product was purified using a PCR purification kit (QUIAGEN) and used as an insert DNA fragment for vector construction. After treating each purified amplified product with the smal restriction enzyme, the pDCM2 vector, which was heated at 65°C for 20 minutes, and the insert DNA fragment, which was the amplified product, were kept at a molar concentration (M) ratio of 1:2 and cloned using the infusion cloning kit (TaKaRa) according to the provided manual to construct the pDCM2-ilvN(D17A,A42V) vector and the pDCM2-ilvN(D17A,H47L) vector to introduce the ilvN variant onto the chromosome.

In addition, PCR was performed using pDCM2-ilvN(D17N,H47L) as a template and the primer pair SEQ ID NO: 7 and SEQ ID NO: 9 or the primer pair SEQ ID NO: 10 and SEQ ID NO: 8 to obtain DNA fragments of 149 bp and 423 bp. Each amplification product was purified using a PCR purification kit (QUIAGEN) and used as an insert DNA fragment for vector construction. After treating each purified amplification product with the smal restriction enzyme, the pDCM2 vector, which was heat-treated at 65°C for 20 minutes, and the insert DNA fragment amplified by PCR were kept at a molar concentration (M) ratio of 1:2 and cloned using the infusion cloning kit (TaKaRa) according to the provided manual to construct the pDCM2-ilvN(D17A,A42V,H47L) vector to introduce the ilvN variant into the chromosome of Corynebacterium glutamicum.

Example 6: Production of an L-isoleucine-producing strain with an introduced ilvN combination variant

The vector obtained in Example 5 was transformed into the Corynebacterium glutamicum strain CA10-3101 obtained in Example 1 by electroporation to obtain a strain in which the combined variant of ilvN was replaced on the chromosome by the secondary crossing method. CA10-3101::ilvN(D17A,A42V) was named CA10-3131, CA10-3101::ilvN(D17A,H47L) was named CA10-3133, and CA10-3101::ilvN(D17A,A42V,H47L) was named CA10-3134.

To test the effect of increasing the productivity of L-isoleucine in the three strains obtained, fermentation titration was carried out as follows.

The parental strain and the variant strain were inoculated into a 250-ml baffled flask containing 25 ml of isoleucine production medium, respectively, and cultured at 32°C for 60 hours with shaking at 200 rpm to produce L-isoleucine. The composition of the production medium was as follows.

Production environment

10% glucose, 0.2% yeast extract, 1.6% ammonium sulfate, 0.1% monobasic potassium phosphate, 0.1% magnesium sulfate heptahydrate, 10 mg/L ferrous sulfate heptahydrate, 10 mg/L manganese sulfate monohydrate, 200 µg/L biotin, pH 7.2.

After completion of the culture, L-isoleucine concentrations in the culture medium were measured for each strain analyzed, and the results are shown in Table 8 below.

As shown in Table 8, the variant strains each introduced with a single ilvN change (ilvN(D17A) or a combined ilvN change (ilvN(D17A,A42V), ilvN(D17A,H47L), ilvN(D17A,A42V,H47L)) showed an increased concentration of L-isoleucine compared with the parent strain (CA10-3101), which is an L-isoleucine-producing strain having ilvN WT.

These results confirmed that single or combined ilvN alteration could enhance the L-isoleucine producing ability of this strain.

Example 7: Obtaining a strain with an ilvN substitution variant from the L-isoleucine-producing Corynebacterium glutamicum strain KCCM11248P

One single ilvN change and three combined changes, which were confirmed to be effective in increasing the L-isoleucine producing ability in Example 6, were introduced into the L-isoleucine-producing strain KCJI-38 (KCCM11248P, Korean Patent No. 10-1335789) treated with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) by the electric pulse method and spread on a selective medium containing 25 mg/L kanamycin to transform both the ilvN substitution variant strains and the ilvN substitution combined variant strains, each of which has a substitution of

culture medium for each strain analyzed using high performance liquid chromatography (HPLC), and the results are shown in Table 9 below.

chromosome, were obtained by the secondary crossing-over method. Then, enzymatic titration was carried out as follows.

The parental strain and the variant strain were inoculated into a 250-ml baffled flask containing 25 ml of isoleucine production medium, respectively, and cultured at 32°C for 60 hours with shaking at 200 rpm to produce L-isoleucine. The composition of the production medium was as follows.

Production environment

10% glucose, 0.2% yeast extract, 1.6% ammonium sulfate, 0.1% monobasic potassium phosphate, 0.1% magnesium sulfate heptahydrate, 10 mg/L ferrous sulfate heptahydrate, 10 mg/L manganese sulfate monohydrate, 200 µg/L biotin, pH 7.2.

After completion of the cultivation, L-isoleucine concentrations were measured in

As shown in Table 9, it was confirmed that the variant strains each introduced with a single ilvN change (ilvN(D17A)) or a combined ilvN change (ilvN(D17A,A42V), ilvN(D17A,H47L), ilvN(D17A,A42V,H47L)) showed an increased concentration of L-isoleucine compared with the parent strain (KCCM11248P), which is an L-isoleucine-producing strain having ilvN WT. In particular, it was confirmed that the strain KCCM11248PAilvN::ilvN(D17A) demonstrated a high increase in the concentration of L-isoleucine compared with the parent strain, and all strains KCCM11248PAilvN::ilvN(D17A,A42V), KCCM11248PMvN::ilvN(D17A,H47L), KCCM11248PAilvN::ilvN(D17A,A42V,H47L)

demonstrated an increased capacity to produce L-isoleucine compared to the parent strain.

Based on the above description, those skilled in the art will appreciate that the present disclosure may be embodied in various specific forms without altering its technical essence or essential characteristics. In this regard, it should be understood that the above embodiment is not limiting, but illustrative in all respects. The scope of this disclosure is defined by the appended claims, not the preceding description, and therefore, it is intended that all changes and modifications that fall within the boundaries of the claims or equivalents thereof be encompassed by the claims.

--->

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<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>aagaccgaaacactcggcatcaaccgcatc</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="13">

<INSDSeq>

<INSDSeq_length>1338</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..1338</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q76">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>hom(R407H)</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..1338</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q77">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atgacctcagcatctgccccaagctttaaccccggcaagggtcccggct

cagcagtcggaattgcccttttaggattcggaacagtcggcactgaggtgatgcgtctgatgaccgagta

cggtgatgaacttgcgcaccgcattggtggcccactggaggttcgtggcattgctgtttctgatatctca

aagccacgtgaaggcgttgcacctgagctgctcactgaggacgcttttgcactcatcgagcgcgaggatg

ttgacatcgtcgttgaggttatcggcggcattgagtacccacgtgaggtagttctcgcagctctgaaggc

cggcaagtctgttgttaccgccaataaggctcttgttgcagctcactctgctgagcttgctgatgcagcg

gaagccgcaaacgttgacctgtacttcgaggctgctgttgcaggcgcaattccagtggttggcccactgc

gtcgctccctggctggcgatcagatccagtctgtgatgggcatcgttaacggcaccaccaacttcatctt

ggacgccatggattccaccggcgctgactatgcagattctctttggctgaggcaactcgtttgggttacgcc

gaagctgatccaactgcagacgtcgaaggccatgacgccgcatccaaggctgcaattttggcatccatcg

ctttccacacccgtgttaccgcggatgatgtgtactgcgaaggtatcagcaacatcagcgctgccgacat

tgaggcagcacagcaggcaggccacaccatcaagttgttggccatctgtgagaagttcaccaacaaggaa

ggaaagtcggctatttctgctcgcgtgcacccgactctattacctgtgtcccacccactggcgtcggtaa

acaagtcctttaatgcaatctttgttgaagcagaagcagctggtcgcctgatgttctacggaaacggtgc

aggtggcgcgccaaccgcgtctgctgtgcttggcgacgtcgttggtgccgcacgaaacaaggtgcacggt

ggccgtgctccaggtgagtccacctacgctaacctgccgatcgctgatttcggtgagaccaccactcgtt

accacctcgacatggatgtggaagatcgcgtggggggttttggctgaattggctagcctgttctctgagca

aggaatctccctgcgtacaatccgacaggaagagcgcgatgatgatgcacatctgatcgtggtcaccac

tctgcgctggaatctgatctttcccgcaccgttgaactgctgaaggctaagcctgttgttaaggcaatca

acagtgtgatccgcctcgaaagggactaa</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="14">

<INSDSeq>

<INSDSeq_length>35</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..35</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q78">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer 1</INSDQualifier_value>

<NonEnglishQualifier_value>primer

1</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..35</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q79">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>tcgagctcggtaccccgcttttgcactcatcgagc</INSDSeq_sequ

ence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="15">

<INSDSeq>

<INSDSeq_length>23</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..23</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q80">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer 2</INSDQualifier_value>

<NonEnglishQualifier_value>primer

2</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..23</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q81">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>cacgatcagatgtgcatcatcat</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="16">

<INSDSeq>

<INSDSeq_length>23</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..23</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q82">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer 3</INSDQualifier_value>

<NonEnglishQualifier_value>primer

3</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..23</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q83">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atgatgatgcacatctgatcgtg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="17">

<INSDSeq>

<INSDSeq_length>35</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..35</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q84">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer 4</INSDQualifier_value>

<NonEnglishQualifier_value>primer

4</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..35</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q85">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ctctagaggatccccgagcatcttccaaaaccttg</INSDSeq_sequ

ence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="18">

<INSDSeq>

<INSDSeq_length>1311</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..1311</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q86">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>ilvA(T381A, F383A)</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..1311</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q87">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atgagtgaaacatacgtgtctgagaaaagtccaggagtgatggctagcg

gagcggagctgattcgtgccgccgacattcaaacggcgcaggcacgaatttcctccgtcattgcaccaac

tccattgcagtattgccctcgtctttctgaggaaaccggagcggaaatctaccttaagcgtgaggatctg

caggatgttcgttcctacaagatccgcggtgcgctgaactctggagcgcagctcactcaggagcagcgcg

atgcaggtatcgttgccgcatctgcaggtaaccatgcccagggcgtggcctatgtgtgcaagtccttggg

cgttcagggacgcatctatgttcctgtgcagactccaaagcaaaagcgtgaccgcatcatggttcacggc

ggagagtttgtctccttggtggtcactggcaataacttcgacgaagcatcggctgcagcgcatgaagatg

cagagcgcaccggcgcaacgctgatcgagcctttcgatgctcgcaacaccgtcatcggtcagggtacagt

ggctgctgagatcttgtcgcagctgacttccatgggcaagagtgcagatcacgtgatggttccagtcggc

ggtggcggacttcttgcaggtgtggtcagctacatggctgatatggcacctcgcactgcgatcgttggta

tcgaaccagcgggagcagcatccatgcaggctgcattgcacaatggtggaccaatcactttggagactgt

tgatccctttgtggacggcgcagcagtcaaacgtgtcggagatctcaactacaccatcgtggagaagaac

cagggtcgcgtgcacatgatgagcgcgaccgagggcgctgtgtgtactgagatgctcgatctttaccaaa

acgaaggcatcatcgcggagcctgctggcgcgctgtctatcgctgggttgaaggaaatgtcctttgcacc

tggttctgtcgtggtgtgcatcatctctggtggcaacaacgatgtgctgcgttatgcggaaatcgctgag

cgctccttggtgcaccgcggtttgaagcactacttcttggtgaacttcccgcaaaagcctggtcagttgc

gtcacttcctggaagatatcctgggaccggatgatgacatcgcgctggcagagtacctcaagcgcaacaa

ccgtgagaccggtactgcgttggtgggtattcacttgagtgaagcatcaggattggattctttgctggaa

cgtatggaggaatcggcaattgattcccgtcgcctcgagccgggcacccctgagtacgaatacttgacct

aa</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="19">

<INSDSeq>

<INSDSeq_length>35</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..35</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q88">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer 5</INSDQualifier_value>

<NonEnglishQualifier_value>primer

5</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..35</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q89">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>tcgagctcggtacccatgagtgaaacatacgtgtc</INSDSeq_sequ

ence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="20">

<INSDSeq>

<INSDSeq_length>39</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..39</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q90">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer 6</INSDQualifier_value>

<NonEnglishQualifier_value>primer

6</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..39</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q91">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>gcgcttgaggtactctgccagcgcgatgtcatcatccgg</INSDSeq_

sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="21">

<INSDSeq>

<INSDSeq_length>39</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..39</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q92">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer 7</INSDQualifier_value>

<NonEnglishQualifier_value>primer

7</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..39</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q93">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ccggatgatgacatcgcgctggcagagtacctcaagcgc</INSDSeq_

sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="22">

<INSDSeq>

<INSDSeq_length>34</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q94">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer 8</INSDQualifier_value>

<NonEnglishQualifier_value>primer

8</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q95">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ctctagaggatcccccgtcaccgacacctccaca</INSDSeq_seque

nce>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="23">

<INSDSeq>

<INSDSeq_length>519</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..519</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q96">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>ilvN(D17A)</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..519</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q97">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggctaattctgacgtcacccgccacatcctgtccgtactcgttcagg

ccgtagacggaatcatttcccgcgtatcaggtatgttcacccgacgcgcattcaacctcgtgtccctcgt

gtctgcaaagaccgaaacacacggcatcaaccgcatcacggttgttgtcgacgccgacgagctcaacatt

gagcagatcaccaagcagctcaacaagctgatccccgtgctcaaagtcgtgcgacttgatgaagagacca

ctatcgcccgcgcaatcatgctggttaaggtctctgcggacagcaccaaccgtccgcagatcgtcgacgc

cgcgaacatcttccgcgcccgagtcgtcgacgtggctccagactctgtggttattgaatccacaggcacc

ccaggcaagctccgcgcactgcttgacgtgatggaaccattcggaatccgcgaactgatccaatccggac

agattgcactcaaccgcggtccgaagaccatggctccggccaagatctaa</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="24">

<INSDSeq>

<INSDSeq_length>519</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..519</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q98">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>ilvN(D17A, A42V)</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..519</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q99">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggctaattctgacgtcacccgccacatcctgtccgtactcgttcagg

ccgtagacggaatcatttcccgcgtatcaggtatgttcacccgacgcgcattcaacctcgtgtccctcgt

gtctgttaagaccgaaacacacggcatcaaccgcatcacggttgttgtcgacgccgacgagctcaacatt

gagcagatcaccaagcagctcaacaagctgatccccgtgctcaaagtcgtgcgacttgatgaagagacca

ctatcgcccgcgcaatcatgctggttaaggtctctgcggacagcaccaaccgtccgcagatcgtcgacgc

cgcgaacatcttccgcgcccgagtcgtcgacgtggctccagactctgtggttattgaatccacaggcacc

ccaggcaagctccgcgcactgcttgacgtgatggaaccattcggaatccgcgaactgatccaatccggac

agattgcactcaaccgcggtccgaagaccatggctccggccaagatctaa</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="25">

<INSDSeq>

<INSDSeq_length>519</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..519</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q100">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>ilvN(D17A, H47L)</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..519</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q101">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggctaattctgacgtcacccgccacatcctgtccgtactcgttcagg

ccgtagacggaatcatttcccgcgtatcaggtatgttcacccgacgcgcattcaacctcgtgtccctcgt

gtctgcaaagaccgaaacactcggcatcaaccgcatcacggttgttgtcgacgccgacgagctcaacatt

gagcagatcaccaagcagctcaacaagctgatccccgtgctcaaagtcgtgcgacttgatgaagagacca

ctatcgcccgcgcaatcatgctggttaaggtctctgcggacagcaccaaccgtccgcagatcgtcgacgc

cgcgaacatcttccgcgcccgagtcgtcgacgtggctccagactctgtggttattgaatccacaggcacc

ccaggcaagctccgcgcactgcttgacgtgatggaaccattcggaatccgcgaactgatccaatccggac

agattgcactcaaccgcggtccgaagaccatggctccggccaagatctaa</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="26">

<INSDSeq>

<INSDSeq_length>519</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..519</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q102">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>ilvN(D17A, A42V,

H47L)</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..519</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q103">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>synthetic

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atggctaattctgacgtcacccgccacatcctgtccgtactcgttcagg

ccgtagacggaatcatttcccgcgtatcaggtatgttcacccgacgcgcattcaacctcgtgtccctcgt

gtctgttaagaccgaaacactcggcatcaaccgcatcacggttgttgtcgacgccgacgagctcaacatt

gagcagatcaccaagcagctcaacaagctgatccccgtgctcaaagtcgtgcgacttgatgaagagacca

ctatcgcccgcgcaatcatgctggttaaggtctctgcggacagcaccaaccgtccgcagatcgtcgacgc

cgcgaacatcttccgcgcccgagtcgtcgacgtggctccagactctgtggttattgaatccacaggcacc

ccaggcaagctccgcgcactgcttgacgtgatggaaccattcggaatccgcgaactgatccaatccggac

agattgcactcaaccgcggtccgaagaccatggctccggccaagatctaa</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (11)

1. An acetohydroxy acid synthase (AHAS) in which the amino acid corresponding to position 17 in the amino acid sequence of SEQ ID NO: 1 is replaced by alanine. 2. The acetohydroxy acid synthase (AHAS) of claim 1, further comprising any one or more substitutions selected from a substitution of the amino acid corresponding to position 42 with valine, a substitution of the amino acid corresponding to position 47 with leucine, or combinations thereof, based on the amino acid sequence of SEQ ID NO: 1. 3. A polynucleotide encoding acetohydroxy acid synthase (AHAS) according to any one of claims 1 and 2. 4. A microorganism of the genus Corynebacterium for producing L-isoleucine, containing acetohydroxy acid synthase (AHAS) according to any one of claims 1 and 2 or a polynucleotide according to claim 3. 5. The microorganism according to claim 4, having an increased ability to produce L-isoleucine compared to a microorganism of the genus Corynebacterium , comprising a wild-type acetohydroxy acid synthase having the amino acid sequence SEQ ID NO: 1, or a polynucleotide encoding it. 6. The microorganism according to claim 4, which is Corynebacterium glutamicum . 7. A method for producing L-isoleucine, comprising the step of culturing in a medium a microorganism of the genus Corynebacterium , comprising an acetohydroxy acid synthase (AHAS) according to any one of claims 1 and 2 or a polynucleotide according to claim 3. 8. A composition for producing L-isoleucine, comprising: (1) the acetohydroxy acid synthase (AHAS) of any one of claims 1 and 2, the polynucleotide of claim 3, a vector comprising the polynucleotide, or a microorganism of the genus Corynebacterium comprising the polynucleotide of the present disclosure; and (2) excipient. 9. The use of an acetohydroxy acid synthase (AHAS) according to any one of claims 1 and 2; a polynucleotide according to claim 3 or a microorganism of the genus Corynebacterium according to claim 4 in the production of L-isoleucine.