Isopropylmalate synthase variant and method for producing L-leucine using the same

Abstract

The present application relates to an isopropylmalate synthase mutant and a method for producing L-leucine using the same.

Description

Technical Field

[0001] The present application relates to an isopropylmalate synthase mutant and a method for producing L-leucine using the same.

Background Art

[0002] L-leucine is an essential amino acid and is an expensive amino acid widely used in pharmaceuticals, foods, feed additives, industrial chemicals, etc., and is mainly produced using microorganisms. The fermentative production of branched-chain amino acids containing L-leucine is mainly carried out by microorganisms of the genus Escherichia or Corynebacterium, and it is known that 2-ketoisocaproate is biosynthesized as a precursor from pyruvic acid through various steps (Patent Documents 1 and 2).

[0003] Isopropylmalate synthase, an enzyme involved in the above L-leucine biosynthesis, is the first-stage enzyme of leucine biosynthesis that converts 2-ketoisovalerate produced in the valine biosynthesis pathway into isopropylmalate required for leucine biosynthesis, rather than converting it into valine, and is an important enzyme in the leucine biosynthesis process. However, the isopropylmalate synthase is subject to feedback inhibition by the final product L-leucine or its derivatives. Therefore, although there are various prior arts regarding isopropylmalate synthase mutants with feedback inhibition released for the purpose of high-concentration leucine production (Patent Documents 3 and 4), research to find better mutants is still ongoing.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

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

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

[0006] As a result of intensive efforts to find an isopropylmalate synthase mutant that can be used for the production of high-concentration L-leucine, the present inventors have found a novel isopropylmalate synthase mutant and confirmed that L-leucine can be produced from a microorganism containing the same in a high yield, thus completing the present application. [Means for Solving the Problems]

[0007] The present application aims to provide a mutant polypeptide having isopropylmalate synthase activity.

[0008] In addition, the present application aims to provide a polynucleotide encoding the mutant polypeptide of the present application.

[0009] Furthermore, the present application aims to provide a vector containing the polynucleotide of the present application.

[0010] Furthermore, the present application aims to provide a microorganism of the genus Corynebacterium that produces L-leucine and contains the mutant polypeptide of the present application, a polynucleotide encoding the same, or a vector containing the same.

[0011] Furthermore, the present application aims to provide a method for producing L-leucine, which includes a step of culturing a Corynebacterium microorganism capable of producing L-leucine in a medium, the microorganism containing a variant polypeptide of the present application, a polynucleotide encoding the same, or a vector containing the same.

[0012] Furthermore, the present application aims to provide a composition for producing L-leucine, which contains a Corynebacterium glutamicum strain containing a variant polypeptide of the present application or a polynucleotide of the present application, or a medium in which the strain has been cultured.

Effect of the Invention

[0013] In the present application, the variant polypeptide having the activity of isopropylmalate synthase has improved activity compared to the wild-type isopropylmalate synthase, and L-leucine can be mass-produced at a high yield using the same.

Modes for Carrying Out the Invention

[0014] These will be specifically described below. Note that each description and embodiment disclosed in the present application is also applicable to other descriptions and embodiments. That is, any combination of various elements disclosed in the present application is included in the present application. Also, the present application is not limited to the following specific descriptions. Furthermore, those having ordinary knowledge in the relevant technical field will be able to recognize and confirm many equivalents of the specific embodiments of the present application described in the present application using only ordinary experiments. Furthermore, such equivalents are also intended to be included in the present application.

[0015] One aspect of the present application for achieving the above object is to provide a variant polypeptide having the activity of isopropylmalate synthase.

[0016] Specifically, the variant polypeptide may include at least one substitution selected from the group consisting of: i) substitution of the amino acid residue at the position corresponding to the 138th amino acid residue in the amino acid sequence of SEQ ID NO: 1 with another amino acid residue; ii) substitution of the amino acid residue at the position corresponding to the 162nd amino acid residue with another amino acid residue; iii) substitution of the amino acid residue at the position corresponding to the 211th amino acid residue with another amino acid residue; iv) substitution of the amino acid residue at the position corresponding to the 245th amino acid residue with another amino acid residue; and v) substitution of the amino acid residue at the position corresponding to the 588th amino acid residue with another amino acid residue.

[0017] In the present application, "isopropylmalate synthase (IPMS)" refers to an enzyme that reacts 2-ketoisovalerate with acetyl-CoA to convert it into isopropylmalate, which is one of the precursors of L-leucine. In the present application, isopropylmalate synthase may be used interchangeably with isopropylmalate synthetase, IPMS, LeuA protein, or LeuA.

[0018] The LeuA in the present application has a sequence obtained from GenBank of NCBI, a well-known database. Specifically, it is a protein having isopropylmalate synthase activity encoded by the leuA gene, but is not limited thereto.

[0019] The LeuA may be an enzyme derived from a microorganism of the genus Corynebacterium, specifically, may be isopropylmalate synthase derived from Corynebacterium glutamicum.

[0020] The LeuA of the present application includes, but is not limited to, the amino acid sequence of SEQ ID NO: 1. Further, the LeuA may include a polypeptide having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% homology with the amino acid sequence of SEQ ID NO: 1. Furthermore, as long as it has such homology or identity and shows an activity corresponding to isopropylmalate synthase, it goes without saying that proteins having an amino acid sequence in which some sequences are deleted, modified, substituted or added are also included in the present application.

[0021] For example, those having an addition or deletion of a sequence that does not change the function of the protein of the present application at the N-terminus, C-terminus and / or inside of the amino acid sequence, naturally occurring mutations, silent mutations or conservative substitutions can be mentioned.

[0022] The "conservative substitution" means that an amino acid is substituted with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can generally occur based on the similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues. Usually, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0023] The LeuA of the present application may have the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 90% or more identity therewith, may consist of the amino acid sequence, or may consist essentially of the amino acid sequence.

[0024] As used herein, the term "variant polypeptide" means a polypeptide in which at least one amino acid is different from the amino acid sequence of the polypeptide prior to the mutation due to conservative substitution and / or modification, but its functions or properties are maintained. Such variant polypeptides can generally be identified by modifying at least one amino acid in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant polypeptide can be improved, unchanged, or decreased compared to the polypeptide before the mutation. In addition, some variant polypeptides may also include variant polypeptides in which at least one part, such as an N-terminal leader sequence or a transmembrane domain, has been removed. Other variant polypeptides may also include variant polypeptides in which a part has been removed from the N- and / or C-terminus of the mature protein. The term "variant polypeptide" is used interchangeably with terms such as variant, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc. (in English expressions), and can be any term that means mutation.

[0025] In addition, the variant polypeptide may include deletions or additions of amino acids that have a minimal impact on the properties and secondary structure of the polypeptide. For example, at the N-terminus of the variant polypeptide, a signal (or leader) sequence involved in the translocation of the protein may be attached co-translationally or post-translationally. In addition, the variant polypeptide may be attached to other sequences or linkers so that it can be identified, purified, or synthesized.

[0026] The variant polypeptide of the present application may have isopropylmalate synthase activity. Further, the variant polypeptide of the present application may have isopropylmalate synthase activity enhanced compared to the wild-type polypeptide having isopropylmalate synthase activity.

[0027] The variant polypeptide of the present application may contain at least one substitution selected from the group consisting of: i) substitution of the amino acid residue at the position corresponding to the 138th amino acid residue in the amino acid sequence of SEQ ID NO: 1 with another amino acid residue; ii) substitution of the amino acid residue at the position corresponding to the 162nd amino acid residue in the amino acid sequence of SEQ ID NO: 1 with another amino acid residue; iii) substitution of the amino acid residue at the position corresponding to the 211th amino acid residue in the amino acid sequence of SEQ ID NO: 1 with another amino acid residue; iv) substitution of the amino acid residue at the position corresponding to the 245th amino acid residue in the amino acid sequence of SEQ ID NO: 1 with another amino acid residue; and v) substitution of the amino acid residue at the position corresponding to the 588th amino acid residue in the amino acid sequence of SEQ ID NO: 1 with another amino acid residue. Specifically, in the amino acid sequence of SEQ ID NO: 1, it may contain at least one substitution selected from the group consisting of: i) substitution of leucine, which is the amino acid residue at the position corresponding to the 138th amino acid residue, with another amino acid residue other than leucine; ii) substitution of histidine, which is the amino acid residue at the position corresponding to the 162nd amino acid residue, with another amino acid residue other than histidine; iii) substitution of serine, which is the amino acid residue at the position corresponding to the 211th amino acid residue, with another amino acid residue other than serine; iv) substitution of asparagine, which is the amino acid residue at the position corresponding to the 245th amino acid residue, with another amino acid residue other than asparagine; and v) substitution of isoleucine, which is the amino acid residue at the position corresponding to the 588th amino acid residue, with another amino acid residue other than isoleucine. More specifically, in the amino acid sequence of SEQ ID NO: 1, it may contain at least one substitution selected from the group consisting of: i) substitution of leucine, which is the amino acid residue at the position corresponding to the 138th amino acid residue, with glycine; ii) substitution of histidine, which is the amino acid residue at the position corresponding to the 162nd amino acid residue, with glutamate; iii) substitution of serine, which is the amino acid residue at the position corresponding to the 211th amino acid residue, with leucine; iv) substitution of asparagine, which is the amino acid residue at the position corresponding to the 245th amino acid residue, with serine; and v) substitution of isoleucine, which is the amino acid residue at the position corresponding to the 588th amino acid residue, with proline. Even more specifically, it may contain at least one, at least two, at least three, at least four, or at least five substitutions. The at least two substitutions may be, but are not limited to, the combination of i) and v), the combination of ii) and v), the combination of iii) and v), or the combination of iv) and v).The at least 4 substitutions are a combination of i), ii), iii) and iv), but are not limited thereto. The at least 5 substitutions may be a combination of i), ii), iii), iv) and v).

[0028] The variant polypeptide of the present application may have / contain the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14, and may consist of / be essentially composed of the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14. The variant polypeptide of the present application has at least 80%, 90%, 95%, 96%, 97%, 98% or 99% or more, and less than 100% identity or homology with the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14, and in the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14, i) the amino acid residue at the position corresponding to the 138th position is glycine, ii) the amino acid residue at the position corresponding to the 162nd position is glutamate, iii) the amino acid residue at the position corresponding to the 211st position is leucine, iv) the amino acid residue at the position corresponding to the 245th position is serine, or v) the amino acid residue at the position corresponding to the 588th position is proline. Specifically, SEQ ID NO: 6 may be an amino acid sequence in which leucine, which is the amino acid residue at the position corresponding to the 138th position in the amino acid sequence of SEQ ID NO: 1, is substituted with glycine, SEQ ID NO: 8 may be an amino acid sequence in which histidine, which is the amino acid residue at the position corresponding to the 162nd position, is substituted with glutamate, SEQ ID NO: 10 may be an amino acid sequence in which serine, which is the amino acid residue at the position corresponding to the 211st position, is substituted with leucine, SEQ ID NO: 12 may be an amino acid sequence in which isoleucine, which is the amino acid residue at the position corresponding to the 588th position, is substituted with proline, and SEQ ID NO: 14 may be an amino acid sequence in which asparagine, which is the amino acid residue at the position corresponding to the 245th position, is substituted with serine.

[0029] Moreover, any amino acid sequence having such identity or homology and exhibiting an efficacy corresponding to the mutant polypeptide of the present application, as long as it has an amino acid sequence in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, in which, in addition to positions i) 138, ii) 162, iii) 211, iv) 245, or v) 588, some sequences are deleted, modified, substituted, conservatively substituted, or added, mutant polypeptides are also included in the present application. Specifically, the substitutions include (1) a mutation (R558H) in which G, the 1673rd nucleotide of the leuA gene encoding isopropylmalate synthase, is substituted with A, and arginine, the amino acid at the position corresponding to the 558th position of the LeuA protein, is substituted with histidine; (2) a mutation (G561D) in which GC, the 1682nd and 1683rd nucleotides of the leuA gene, is substituted with AT, and glycine, the amino acid at the position corresponding to the 561st position, is substituted with aspartic acid; and (3) a mutation (P247C) in which CC, the 739th and 740th nucleotides of the leuA gene, is substituted with TG, and proline, the 247th amino acid, is substituted with cysteine, and at least one of these is included, as described above.

[0030] More specifically, the variant polypeptide includes, but is not limited to, the following polypeptides: (i) a polypeptide further including the mutations at positions 247, 558, and 561 in addition to the mutation at position 138 (SEQ ID NO: 38); (ii) a polypeptide further including the mutations at positions 247, 558, and 561 in addition to the mutation at position 162 (SEQ ID NO: 40); (iii) a polypeptide further including the mutations at positions 247, 558, and 561 in addition to the mutation at position 211 (SEQ ID NO: 42); (iv) a polypeptide further including the mutations at positions 247, 558, and 561 in addition to the mutation at position 245 (SEQ ID NO: 44); (v) a polypeptide further including the mutations at positions 247, 558, and 561 in addition to the mutation at position 588 (SEQ ID NO: 46); (iii) a polypeptide further including the mutations at positions 247, 558, and 561 in addition to the mutations at positions 211 and 588 (SEQ ID NO: 48); a polypeptide further including the mutations at positions 247, 558, and 561 in addition to the mutations at positions (i) 138, (ii) 162, (iii) 211, and (iv) 245 (SEQ ID NO: 50); or a polypeptide further including the mutations at positions 247, 558, and 561 in addition to the mutations at positions (i) 138, (ii) 162, (iii) 211, (iv) 245, and (v) 588 (SEQ ID NO: 52).

[0031] In this application, "corresponding to" means an amino acid residue at the position listed in the polypeptide, or an amino acid residue similar to, identical to, or corresponding to the residue listed in the polypeptide. Identifying the amino acid at the corresponding position determines the specific amino acid of the sequence with reference to the specific sequence. In this application, "corresponding region" generally means a similar or corresponding position in a related protein or a reference protein.

[0032] For example, when any amino acid sequence is aligned with SEQ ID NO: 1, based on this, each amino acid residue of the amino acid sequence can be numbered with reference to the number and position of the amino acid residue corresponding to the amino acid residue of SEQ ID NO: 1. For example, the sequence alignment algorithm in the present application can confirm the position of an amino acid or the position where modifications such as substitution, insertion, and deletion occur when compared with a query sequence (also referred to as a "reference sequence").

[0033] For such alignment, for example, the Needleman-Wunsch algorithm (Non-Patent Document 1), the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2), etc. can be used, but it is not limited thereto, and sequence alignment programs, pairwise sequence comparison algorithms, etc. known in the art can be appropriately used.

[0034] The "homology" or "identity" in the present application means the degree to which two given amino acid sequences or nucleotide sequences are similar, and is expressed as a percentage. Homology and identity are often used interchangeably.

[0035] The sequence homology or identity of a conserved polynucleotide or polypeptide is determined by a standard sequence algorithm, and the default gap penalty established by the program used may also be used together. Substantially, homologous or identical sequences generally hybridize with all or part of the sequence under moderately or highly stringent conditions. It goes without saying that hybridization also includes hybridization with a polynucleotide having a codon considering general codons or codon degeneracy in the polynucleotide.

[0036] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as default parameters like those in Non-Patent Document 3 and the "FASTA" program. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 1) as performed by the needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later versions) (including the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, FASTA (Non-Patent Documents 5, 6 and 7)). For example, homology, similarity or identity can be determined using BLAST or Clustal W of the National Center for Biotechnology Information.

[0037] The homology, similarity or identity of a polynucleotide or polypeptide can be determined by comparing sequence information using a GAP computer program such as Non-Patent Document 1, as disclosed in Non-Patent Document 8 for example. Briefly, the GAP program defines it as the value obtained by dividing the number of similar sequence symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. The default parameters for the GAP program include: (1) a binary comparison matrix (with a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Non-Patent Document 10 (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 9, (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5), and (3) no penalty for terminal gaps.

[0038] Another aspect of this application provides a polynucleotide encoding a variant polypeptide of this application.

[0039] In this application, the "polynucleotide" refers to a polymer of nucleotide monomers covalently linked in a long chain, meaning a DNA or RNA chain longer than a predetermined length, and more specifically refers to a polynucleotide fragment encoding the mutant protein.

[0040] The polynucleotide encoding the mutant polypeptide of this application includes a base sequence encoding the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50 or SEQ ID NO: 52, but is not limited thereto. Specifically, the polynucleotide of this application may have a base sequence of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 or SEQ ID NO: 53, or may include the above base sequence.

[0041] Due to the degeneracy of codons or considering the preferred codons in the organism in which the polypeptide is to be expressed, various modifications can be made to the coding region within the range where the amino acid sequence of the polypeptide does not change. Specifically, it consists of a base sequence having 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 or SEQ ID NO: 53, but is not limited thereto.

[0042] In addition, the polynucleotide of the present application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a complementary sequence to all or part of the polynucleotide sequence of the present application. The "stringent condition" means a condition that enables specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Documents 11 and 12). For example, conditions for hybridizing polynucleotides having high homology or identity, such as polynucleotides having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity, and not hybridizing polynucleotides having lower homology or identity, or washing conditions for ordinary Southern hybridization, such as 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS, and washing once, specifically 2 to 3 times, at the corresponding salt concentration and temperature can be mentioned.

[0043] Hybridization requires that two nucleic acids have complementary sequences, even if base mismatches are possible depending on the stringency of hybridization. "Complementary" is used to represent the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotide of the present application may include not only nucleic acid base sequences that are substantially similar, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0044] Specifically, a polynucleotide having homology or identity with the polynucleotide of the present application can be detected using hybridization conditions in which the hybridization step is performed at a Tm value of 55°C and the conditions described above. Further, the Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.

[0045] An appropriate stringency for hybridizing the polynucleotide depends on the length and degree of complementarity of the polynucleotide, and the variables are known in the art (for example, Non-Patent Document 11).

[0046] For example, the polynucleotide of the present application may include any sequence as long as it encodes the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, or SEQ ID NO: 52.

[0047] Regarding the variant polypeptide in the polynucleotide of the present application, it is as described above.

[0048] Still another aspect of the present application provides a vector containing the polynucleotide of the present application.

[0049] The vector of the present application means a DNA product containing the base sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable expression regulatory region (or expression regulatory sequence) so that the target polypeptide can be expressed in a suitable host. The expression regulatory region may include a promoter that initiates transcription, any operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating the termination of transcription and translation. When transformed into a suitable host cell, the vector can replicate or function regardless of the host genome and may be integrated into the genome itself.

[0050] The vector used in this application is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant states. For example, as phage vectors or cosmid vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used, and as plasmid vectors, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, pET series, etc. can be used. Specifically, pDCM2 (Patent Document 5), pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector, etc. can be used.

[0051] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome by a vector for intracellular chromosome introduction. The insertion of the polynucleotide into the chromosome can be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. A selection marker for confirming whether it has been introduced into the chromosome may be further included. The selection marker is for selecting cells transformed with the vector, that is, for confirming whether the target nucleic acid molecule has been inserted, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, and expression of surface polypeptides is used. In an environment treated with a selective agent, only cells expressing the selection marker survive or show different phenotypes, so transformed cells can be selected.

[0052] As used in this application, "transformation" means introducing a vector containing a polynucleotide encoding a target protein into a host cell or microorganism to express the protein encoded by the polynucleotide in the host cell. The transformed polynucleotide may be any polynucleotide as long as it is expressed in the host cell, regardless of whether it is inserted into the host cell chromosome or located extrachromosomally. Further, the polynucleotide includes DNA and RNA encoding the target protein. The polynucleotide may be introduced into the host cell in any form as long as it is introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a gene construct containing all the elements necessary for its own expression. Usually, the expression cassette may include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of a self-replicating expression vector. Further, the polynucleotide may be introduced into the host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0053] Further, the "operably linked" means that the promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the target mutant protein of this application is functionally linked to the gene sequence.

[0054] Regarding the polynucleotide in the vector of this application, it is as described above.

[0055] Still another aspect of this application provides a microorganism of the genus Corynebacterium that produces L-leucine and contains the mutant polypeptide of this application, the polynucleotide encoding the same, or a vector containing the same.

[0056] As used herein, the term "microorganism" encompasses all wild-type microorganisms and those that have been genetically modified either naturally or artificially. Due to reasons such as the insertion of foreign genes or the enhancement or inactivation of the activity of endogenous genes, the microorganism has a weakened or enhanced specific mechanism and has been genetically modified (modification) for the production of a target polypeptide, protein, or product.

[0057] The microorganism of the present application includes a microorganism that contains at least one of a variant of the present application, a polynucleotide of the present application, and a vector containing the polynucleotide of the present application, a microorganism that has been modified to express a variant of the present application or a polynucleotide of the present application, a microorganism (e.g., a recombinant strain) that expresses a variant of the present application or a polynucleotide of the present application, or a microorganism (e.g., a recombinant strain) that has the activity of a variant of the present application, but is not limited thereto.

[0058] The microorganism of the present application is a microorganism that naturally has isopropylmalate synthase activity or the ability to produce L-leucine, or a parent strain that does not have isopropylmalate synthase activity or the ability to produce L-leucine, and expresses the mutant polypeptide of the present application or has been given the ability to produce L-leucine, but is not limited thereto.

[0059] Specifically, the microorganism of the present application is a cell or microorganism that is transformed with a vector containing the polynucleotide of the present application or a gene encoding the variant polypeptide of the present application and expresses the variant polypeptide of the present application. For the purpose of the present application, the microorganism of the present application may be any microorganism that contains the variant polypeptide of the present application and produces L-leucine. For example, the microorganism of the present application may be a recombinant microorganism in which the variant polypeptide of the present application is expressed and the L-leucine-producing ability is improved by introducing the polynucleotide encoding the variant polypeptide of the present application into a natural wild-type microorganism or a microorganism that produces L-leucine. The recombinant microorganism with improved L-leucine-producing ability is a microorganism with improved L-leucine-producing ability compared to a natural wild-type microorganism or an unmodified microorganism, but is not limited thereto.

[0060] The "unmodified microorganism" in the present application does not exclude strains containing mutations that can occur naturally in microorganisms, but means the wild-type strain or the natural strain itself, or the strain before genetic mutation and phenotypic change due to natural or artificial factors. For example, the unmodified microorganism means a strain in which the protein variant in the present specification has not been introduced or before it is introduced. The "unmodified microorganism" is used interchangeably with "strain before modification", "microorganism before modification", "non-mutant strain", "unmodified strain", "non-mutant microorganism" or "reference microorganism".

[0061] Specifically, the microorganism of the present application may be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens.

[0062] The microorganism of the present application may be a microorganism comprising a nucleotide sequence encoding isopropylmalate synthase in which at least one amino acid residue other than (i) the 138th, (ii) the 162nd, (iii) the 211th, (iv) the 245th or (v) the 588th amino acid residue in the amino acid sequence of SEQ ID NO: 1 constituting isopropylmalate synthase of the present application is substituted with another amino acid. Specifically, the substitution may include (1) a mutation (R558H) in which G, the 1673rd nucleotide of the leuA gene encoding isopropylmalate synthase, is substituted with A, and arginine, the 558th amino acid of the LeuA protein, is substituted with histidine; (2) a mutation (G561D) in which GC, the 1682nd and 1683rd nucleotides of the leuA gene, is substituted with AT, and glycine, the 561st amino acid, is substituted with aspartic acid; or (3) a mutation (P247C) in which CC, the 739th and 740th nucleotides of the leuA gene, is substituted with TG, and proline, the 247th amino acid, is substituted with cysteine. As described above, at least one of these mutations is included.

[0063] Specifically, the microorganism producing L-leucine of the present application may be a microorganism in which the activity of isopropylmalate synthase is enhanced by expressing isopropylmalate synthase containing such a mutation.

[0064] The "enhancement" of polypeptide activity in this application means improving the activity of the polypeptide over its endogenous activity. The said enhancement is sometimes used interchangeably with up-regulation, overexpression, increase, etc. Here, an increase includes all cases where the polypeptide shows an activity that it originally did not have, or where the activity is improved compared to the endogenous activity or the activity before modification. The "endogenous activity" means the activity of a specific polypeptide that the parental strain or unmodified microorganism had before the trait change when the trait changes due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with the "activity before modification". That the activity of a polypeptide is "enhanced" or "increased" compared to the endogenous activity means that it is improved compared to the activity of the specific polypeptide that the parental strain or unmodified microorganism had before the trait change.

[0065] The said enhancement may be achieved by introducing a foreign polypeptide or by enhancing the activity of an endogenous polypeptide. Whether the activity of the said polypeptide is enhanced can be confirmed by an increase in the degree of activity, the expression level of the polypeptide, or the amount of the product produced from the polypeptide.

[0066] For the enhancement of the activity of the said polypeptide, various methods well-known in the art can be applied, and any method can be used as long as it can enhance the activity of the target polypeptide over that of the microorganism before modification. Specifically, it is a common method in molecular biology and uses genetic engineering and / or protein engineering well-known to those with ordinary knowledge in the technical field, but is not limited thereto (for example, Non-Patent Documents 13, 14, etc.).

[0067] Specifically, the enhancement of the polypeptide activity of the present application is achieved by: 1) increasing the intracellular copy number of the polynucleotide encoding the polypeptide; 2) replacing the gene expression regulatory region on the chromosome encoding the polypeptide with a sequence having strong activity; 3) modifying the nucleotide sequence of the start codon or 5' UTR region of the gene encoding the polypeptide; 4) modifying the amino acid sequence of the polypeptide so as to improve the polypeptide activity; 5) modifying the polynucleotide sequence encoding the polypeptide so as to improve the polypeptide activity; 6) introducing a foreign polynucleotide showing the activity of the polypeptide; 7) optimizing the codons of the polynucleotide encoding the polypeptide; 8) analyzing the tertiary structure of the polypeptide, selecting and modifying the exposed portions, or chemically modifying them; or 9) by a combination of the above 1) to 8), but is not particularly limited thereto.

[0068] More specifically, the above-mentioned 1) increasing the intracellular copy number of the polynucleotide encoding the polypeptide is achieved by introducing into the host cell a vector that replicates and functions regardless of the host and to which the polynucleotide encoding the polypeptide is operably linked. Alternatively, it may be achieved by introducing one copy or two or more copies of the polynucleotide encoding the polypeptide into the chromosome in the host cell. The introduction into the chromosome is achieved by introducing into the host cell a vector capable of inserting the polynucleotide into the chromosome in the host cell, but is not limited thereto. The vector is as described above.

[0069] Replacing the gene expression regulatory region (or expression regulatory sequence) on the chromosome encoding the polypeptide in 2) above with a sequence having strong activity can be achieved by introducing sequence mutations through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, for example, such that the activity of the expression regulatory region is further enhanced, or by replacing it with a sequence having higher activity. The expression regulatory region includes, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating the termination of transcription and translation, and the like. For example, it can be achieved by replacing the original promoter with a strong promoter, but is not limited thereto.

[0070] Examples of known strong promoters include, but are not limited to, the cj1-cj7 promoters (Patent Document 6), 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 (Patent Document 7), the O2 promoter (Patent Document 8), the tkt promoter, the yccA promoter, and the like.

[0071] Modifying the nucleotide sequence of the start codon or the 5'UTR region of the gene encoding the polypeptide in 3) above can be achieved, for example, by replacing it with another start codon having a higher polypeptide expression rate compared to the endogenous start codon, but is not limited thereto.

[0072] Modifying the amino acid sequence or polynucleotide sequence of 4) and 5) above can be achieved by introducing deletions, insertions, non-conservative or conservative substitutions, or combinations thereof in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide so as to enhance the activity of the polypeptide, or substituting with an improved amino acid sequence or polynucleotide sequence having higher activity, or an amino acid sequence or polynucleotide sequence improved for enhanced activity, but is not limited thereto. Specifically, the substitution is performed by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited thereto. Here, the vector used may further contain a selection marker for confirming whether it has been introduced into the chromosome. The selection marker is as described above.

[0073] Introducing a foreign polynucleotide that exhibits the activity of the polypeptide of 6) above is performed by introducing into a host cell a foreign polynucleotide encoding a polypeptide that exhibits the same / similar activity as the polypeptide. The origin and sequence of the foreign polynucleotide may be any as long as it exhibits the same / similar activity as the polypeptide. The introduction can be appropriately selected by those skilled in the art using known transformation methods, and the polypeptide may be produced and its activity improved by the expression of the polynucleotide introduced as described above in the host cell.

[0074] Optimizing the codons of the polynucleotide encoding the polypeptide of 7) above is performed by optimizing the codons of the endogenous polynucleotide so as to increase transcription or translation in the host cell, or by optimizing the codons of the foreign polynucleotide so as to perform optimized transcription and translation in the host cell.

[0075] In addition, analyzing the tertiary structure of the polypeptide described in 8), selecting and modifying the exposed portions, or chemically modifying them may be performed, for example, by comparing the sequence information of the polypeptide to be analyzed with a database in which the sequence information of known proteins is stored, determining candidates for template proteins according to the degree of sequence similarity, confirming the structure based on this, and selecting and modifying or chemically modifying the exposed portions to be modified.

[0076] Such enhancement of polypeptide activity is achieved by the activity or concentration of the corresponding polypeptide being improved compared to the activity or concentration of the polypeptide expressed in the wild-type or unmodified microbial strain, or by an increase in the amount of the product produced from the polypeptide, but is not limited thereto.

[0077] In the microorganism of the present application, partial or total modification of the polynucleotide (for example, the modification for encoding the protein variant described above) can be induced by (a) homologous recombination using a chromosomal introduction vector in the microorganism, or genome editing using an engineered nuclease (e.g., CRISPR-Cas9), and / or (b) treatment with light such as ultraviolet rays or radiation and / or chemical substances, but is not limited thereto. The method for partial or total modification of the gene includes methods by DNA recombination technology. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene is injected into the microorganism to cause homologous recombination, thereby deleting part or all of the gene. The introduced nucleotide sequence or vector may include a dominant selection marker, but is not limited thereto.

[0078] More specifically, the microorganism for producing L-leucine of the present application may be a microorganism further comprising a polypeptide comprising SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50 or SEQ ID NO: 52, a polynucleotide encoding a polypeptide comprising SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50 or SEQ ID NO: 52, or a polynucleotide comprising SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51 or SEQ ID NO: 53.

[0079] In the microorganism of the present application, the mutant polypeptide, polynucleotide, vector, L-leucine and the like are as described above.

[0080] Still another aspect of the present application provides a method for producing L-leucine, which includes the step of culturing a Corynebacterium microorganism for producing L-leucine containing the mutant polypeptide of the present application, a polynucleotide encoding the same, or a vector containing the same in a medium.

[0081] "Culturing" in the present application means growing the Corynebacterium strain of the present application under appropriately adjusted environmental conditions. The culturing process of the present application can be carried out in a suitable medium and culturing conditions known in the art. Such a culturing process can be easily adjusted and used according to the selected strain by those skilled in the art. Specifically, the culturing is batch, continuous and fed-batch culturing, but is not limited thereto.

[0082] In the present application, the "medium" refers to a substance obtained by mixing, as the main components, the nutrients necessary for culturing the Corynebacterium strains of the present application, and supplies nutrients such as water, which is essential for survival and growth, and growth factors. Specifically, the medium and other culture conditions used for culturing the Corynebacterium glutamicum strains of the present application may be any medium and conditions that are used for culturing ordinary microorganisms, and the temperature, pH, etc. are adjusted under aerobic conditions in an ordinary medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin, etc., to culture the Corynebacterium glutamicum strains of the present application. Specifically, the culture medium for Corynebacterium strains is disclosed in Non-Patent Document 15.

[0083] Examples of the carbon source in the present application include carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, maltose, sugar alcohols such as mannitol and sorbitol, organic acids such as pyruvic acid, lactic acid, and citric acid, and amino acids such as glutamic acid, methionine, and lysine. Further, natural organic nutrient sources such as starch hydrolyzates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steep liquor can be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugar) can be used, and any other appropriate amount of carbon source can be used. These carbon sources can be used alone or in combination of two or more, but are not limited thereto.

[0084] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc., amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. can be used. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0085] Examples of the phosphorus source include potassium dihydrogen phosphate, dipotassium hydrogen phosphate or sodium-containing salts corresponding thereto. As inorganic compounds, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. can be used. In addition, amino acids, vitamins and / or suitable precursors, etc. can be used. These components or precursors can be added to the medium in a batch or continuous manner. However, it is not limited thereto.

[0086] Also, during the culture of the Corynebacterium glutamicum strain of the present application, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the medium in a suitable manner. Furthermore, during the culture, defoaming agents such as fatty acid polyglycol esters can be used to suppress the generation of bubbles. Furthermore, in order to maintain the aerobic state of the medium, oxygen or oxygen-containing gas can be injected into the medium. In order to maintain the anaerobic and microaerobic states, it is not necessary to inject gas, and nitrogen, hydrogen or carbon dioxide gas can be injected, but it is not limited thereto.

[0087] In the culture of the present application, the culture temperature is maintained at 20 to 45 °C, specifically 25 to 40 °C, and cultured for about 10 to 160 hours, but is not limited thereto.

[0088] The L-leucine produced by culturing in the present application may be secreted into the culture medium or remain intracellularly.

[0089] The method for producing L-leucine in the present application may further include the step of preparing the Corynebacterium glutamicum strain of the present application or the step of preparing a culture medium for culturing the strain.

[0090] The method for producing L-leucine in the present application may further include the step of recovering L-leucine from the culture medium cultured as described above or the Corynebacterium glutamicum strain of the present application.

[0091] The recovery may be to recover the target L-leucine using a suitable method known in the art according to the method for culturing the microorganism of the present application, such as batch, continuous, fed-batch culture methods, etc. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting-out method), extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography and other various chromatographies, HPLC or a combination thereof can be used, and the target L-leucine can be recovered from the culture medium or the microorganism using a suitable method known in the art.

[0092] In addition, the method for producing L-leucine in the present application may further include a purification step. The purification can be carried out by a suitable method known in the art. For example, when the method for producing L-leucine in the present application includes both a recovery step and a purification step, the recovery step and the purification step may be carried out continuously or discontinuously regardless of the order, or simultaneously or integrated as one step, but are not limited thereto.

[0093] In the method of the present application, the mutant polypeptide, polynucleotide, L-leucine, etc. are as described above.

[0094] Still other aspects of the present application provide a composition for producing L-leucine, which comprises a variant polypeptide of the present application, a Corynebacterium glutamicum strain containing the polynucleotide of the present application, or a medium in which the strain has been cultured.

[0095] The composition of the present application may further contain any suitable excipient commonly used in compositions for amino acid production. Such excipients include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffering agents, stabilizing agents, isotonic agents, etc.

[0096] In the composition of the present application, the variant polypeptide, polynucleotide, L-leucine, etc. are as described above.

Examples

[0097] Hereinafter, the present application will be described in more detail with reference to examples. However, these examples are merely preferred embodiments for illustrating the present application, and the present application is not limited thereto. Technical matters not described in this specification can be fully understood and easily implemented by those skilled in the art in the technical field of the present application or similar technical fields.

Examples

[0098] Preparation of a DNA library encoding a mutated isopropylmalate synthase 1-1. Preparation of a vector containing leuA To create a library of leuA mutants with isopropylmaleate synthase activity, first a recombinant vector containing leuA was constructed. To amplify the leuA gene (SEQ ID NO: 2) encoding the LeuA protein (SEQ ID NO: 1, Uniprot accession code: P42455) from wild-type Corynebacterium glutamicum, the chromosomal DNA of Corynebacterium glutamicum ATCC13032 wild strain was used as a template, and PCR was performed with the primers of SEQ ID NOs: 3 and 4 for 25 cycles of denaturation at 94°C for 1 minute, annealing at 58°C for 30 seconds, and polymerization with Pfu DNA polymerase at 72°C for 1 minute. The sequences of the primers used are shown in Table 1.

[0099] [Table 1]

[0100] The PCR product was cloned into the E. coli vector pCR2.1 using the TOPO Cloning Kit (Invitrogen) to obtain "pCR-leuA".

[0101] 1-2. Preparation of the leuA mutant library Based on the vector prepared in Example 1-1, a leuA mutant library was prepared using an error-prone PCR kit (error-prone PCR kit, clontech Diversify® PCR Random Mutagenesis Kit). PCR reactions were performed with the primers of SEQ ID NO: 3 and SEQ ID NO: 4 shown in Table 1 under conditions where 0 to 3 mutations occur per 1000 bp.

[0102] Specifically, after pre-heating at 94°C for 30 seconds, PCR was performed by conducting 25 cycles of denaturation at 94°C for 30 seconds and polymerization at 68°C for 1 minute and 30 seconds. Here, for the obtained PCR product, 25 cycles of denaturation at 95°C for 50 seconds, binding at 60°C for 50 seconds, and polymerization at 68°C for 12 minutes were performed using a megaprimer (50 - 125 ng). Then, it was treated with DpnI and transformed into Escherichia coli DH5α by the heat shock method, and spread on an LB solid medium containing 25 mg / L of kanamycin. Twenty transformed colonies were selected to obtain plasmids, and as a result of analyzing the nucleotide sequences, it was confirmed that mutations were introduced at different positions at a frequency of 2 mutations / kb. Approximately 20,000 transformed Escherichia coli colonies were collected and plasmids were extracted. This was named "pTOPO-leuA-library".

Example

[0103] Evaluation of the prepared library and selection of mutants 2-1. Selection of mutant strains with increased L-leucine production The pTOPO- leuA -library prepared in Example 1-2 was transformed into wild-type Corynebacterium glutamicum ATCC13032 by electroporation, and then spread on a nutrient medium (Table 2) containing 25 mg / L of kanamycin to select 10,000 colonies of strains into which the mutant gene was inserted. Each selected colony was named ATCC13032 / pTOPO_ leuA (mt)1 to ATCC13032 / pTOPO_ leuA (mt)10,000.

[0104] Among the 10,000 colonies secured, in order to identify colonies with increased L-leucine production and increased and decreased production of L-phenylalanine, an aromatic amino acid, the fermentation titer of each colony was evaluated by the following method.

[0105]

Table 2

[0106] Each colony was inoculated with a platinum loop into 25 ml of the production medium in Table 2 and 250 ml of a conical baffle flask containing 25 μg / ml of kanamycin, and then cultured with shaking at 30 °C and 200 rpm for 60 hours. After the cultivation was completed, the L-leucine production amount was measured by a method using high performance liquid chromatography (HPLC, SHIMAZDU LC20A).

[0107] As a result, out of 10,000 colonies, five strains (ATCC13032 / pTOPO_leuA(mt)3847, ATCC13032 / pTOPO_leuA(mt)4708, ATCC13032 / pTOPO_leuA(mt)5109, ATCC13032 / pTOPO_leuA(mt)7563, ATCC13032 / pTOPO_leuA(mt)8459) with the most improved L-leucine production ability compared to the wild-type Corynebacterium glutamicum ATCC13032 were selected. The concentrations of L-leucine produced from the selected strains are shown in Table 3.

[0108]

Table 3

[0109] As shown in Table 3, Corynebacterium glutamicum ATCC13032 / pTOPO_leuA(mt)3847 with a mutation in the leuA gene was confirmed to have about 1.41-fold improved L-leucine production ability compared to the parent strain Corynebacterium glutamicum ATCC13032. Also, ATCC13032 / pTOPO_leuA(mt)4708, ATCC13032 / pTOPO_leuA(mt)5109, ATCC13032 / pTOPO_leuA(mt)7563, and ATCC13032 / pTOPO_leuA(mt)8459 were confirmed to have improved by about 1.45-fold, 1.59-fold, 1.36-fold, and 1.38-fold, respectively, compared to the parent strain.

[0110] Confirmation of Mutation in Mutant Strains with Increased L-Leucine Production To confirm the leuA gene mutations in the five selected mutant strains, using the primers of SEQ ID NO: 3 and SEQ ID NO: 4 shown in Table 1, with the DNA of each mutant strain as a template, after denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute and 30 seconds were performed for 30 cycles, and then polymerization at 72°C for 5 minutes was carried out under the conditions of PCR, and DNA sequencing was performed.

[0111] As a result of sequencing, it was confirmed that in the ATCC13032 / pTOPO_leuA(mt)3847 strain, both C and T, the 412th and 413th nucleotides of the leuA gene of SEQ ID NO: 2, were replaced by G. This means that it encodes a mutant in which leucine, the 138th amino acid (when based on a known document where the translation start codon is represented 35 later and the LeuA protein consists of 581 amino acids (SEQ ID NO: 5), it is the 103rd amino acid; hereinafter referred to as the 138th amino acid) of the LeuA protein, is replaced by glycine (hereinafter referred to as L138G). The amino acid sequence of the LeuA mutant (L138G) and the nucleotide sequence of the leuA mutant encoding the same are shown in SEQ ID NO: 6 and SEQ ID NO: 7.

[0112] In the ATCC13032 / pTOPO_leuA(mt)4708 strain, it was confirmed that C, the 484th and 486th nucleotides of the leuA gene, were all replaced by G. This means that it encodes a mutant in which histidine, the 162nd amino acid (when based on a known document where the translation start codon is represented 35 later and the LeuA protein consists of 581 amino acids (SEQ ID NO: 5), it is the 127th amino acid; hereinafter referred to as the 162nd amino acid) of the LeuA protein, is replaced by glutamate (hereinafter referred to as H162E). The amino acid sequence of the LeuA mutant (H162E) and the nucleotide sequence of the leuA mutant encoding the same are shown in SEQ ID NO: 8 and SEQ ID NO: 9.

[0113] It was confirmed that in the strain ATCC13032 / pTOPO_leuA(mt)5109, the TCC at nucleotides 631 to 633 of the leuA gene was replaced by CTT. This means that it encodes a mutant (hereinafter referred to as S211L) in which serine, the 211st amino acid (when expressed 35 positions after the translation start codon, it is the 176th position based on a known document where the LeuA protein consists of 581 amino acids (SEQ ID NO: 5); hereinafter referred to as the 211st position) of the LeuA protein, is replaced by leucine. The amino acid sequence of the LeuA mutant (S211L) and the nucleotide sequence of the leuA mutant encoding the same are shown in SEQ ID NO: 10 and SEQ ID NO: 11.

[0114] It was confirmed that in the strain ATCC13032 / pTOPO_leuA(mt)7563, the AT at nucleotides 1762 to 1763 of the leuA gene was replaced by CC. This means that it encodes a mutant (hereinafter referred to as I588P) in which isoleucine, the 588th amino acid (when expressed 35 positions after the translation start codon, it is the 553rd position based on a known document where the LeuA protein consists of 581 amino acids (SEQ ID NO: 5); hereinafter referred to as the 588th position) of the LeuA protein, is replaced by proline. The amino acid sequence of the LeuA mutant (I588P) and the nucleotide sequence of the leuA mutant encoding the same are shown in SEQ ID NO: 12 and SEQ ID NO: 13.

[0115] In addition, it was confirmed that in the ATCC13032 / pTOPO_leuA(mt)8459 strain, the AA at nucleotides 733 to 734 of the leuA gene was replaced by TC. This means that it encodes a mutant in which asparagine, which is the amino acid at position 245 of the LeuA protein (when expressed 35 codons after the translation start codon, it is position 210 based on a known document stating that the LeuA protein consists of 581 amino acids (SEQ ID NO: 5); hereinafter referred to as position 245), is replaced by serine (hereinafter referred to as N245S). The amino acid sequence of the LeuA mutant (N245S) and the nucleotide sequence of the leuA mutant encoding the same are shown in SEQ ID NO: 14 and SEQ ID NO: 15.

[0116] In the following examples, it is confirmed whether the above mutations (L138G, H162E, S211L, N245S, I588P) affect the L-leucine production amount of Corynebacterium genus microorganisms.

Example

[0117] Confirmation of L-leucine production ability of the selected mutant strains 3-1. Preparation of an insertion vector containing the leuA mutation To introduce the mutations selected in Example 2 into the strain, an insertion vector was prepared. For the preparation of the vector for introducing the leuA (L138G, H162E, S211L, N245S, I588P) mutation, the site-directed mutagenesis method was used. Specifically, using the chromosome of the wild-type Corynebacterium glutamicum ATCC13032 strain as a template, for generating the L138G mutation, the primer pairs of SEQ ID NO: 16 and SEQ ID NO: 17, and the primer pairs of SEQ ID NO: 18 and SEQ ID NO: 19 were used; for generating the H162E mutation, the primer pairs of SEQ ID NO: 16 and SEQ ID NO: 20, and the primer pairs of SEQ ID NO: 19 and SEQ ID NO: 21 were used to perform PCR. For generating the S211L mutation, the primer pairs of SEQ ID NO: 16 and SEQ ID NO: 22, and the primer pairs of SEQ ID NO: 19 and SEQ ID NO: 23 were used; for generating the N245S mutation, the primer pairs of SEQ ID NO: 16 and SEQ ID NO: 24, and the primer pairs of SEQ ID NO: 19 and SEQ ID NO: 25 were used to perform PCR. For generating the I588P mutation, the primer pairs of SEQ ID NO: 16 and SEQ ID NO: 26, and the primer pairs of SEQ ID NO: 19 and SEQ ID NO: 27 were used to perform PCR. More specifically, after denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute and 30 seconds were performed for 30 cycles, and then PCR was performed under the condition of polymerization at 72°C for 5 minutes. The specific sequences of the primers used are shown in Table 4.

[0118]

Table 4

[0119] Using the PCR product, a linear pDCM2 vector digested with SmaI restriction enzyme, and In-Fusion enzyme, the terminal 15-base homologous sequences between DNA fragments were fused and cloned to generate vectors "pDCM2-leuA(L138G)", "pDCM2-leuA(H162E)", "pDCM2-leuA(S211L)", "pDCM2-leuA(N245S)", "pDCM2-leuA(I588P)" that substitute the amino acids of LeuA. Additionally, by combining the mutants, vectors "pDCM2-leuA(S211L,I588P)", "pDCM2-leuA(L138G,H162E,S211L,N245S)", "pDCM2-leuA(L138G,H162E,S211L,N245S,I588P)" that substitute the amino acids of leuA were generated.

[0120] 3-2. Introduction and Evaluation of Mutants into Corynebacterium glutamicum ATCC13032 Strain The pDCM2-leuA(L138G), pDCM2-leuA(H162E), pDCM2-leuA(S211L), pDCM2-leuA(N245S), pDCM2-leuA(I588P), pDCM2-leuA(S211L,I588P), pDCM2-leuA(L138G,H162E,S211L,N245S), and pDCM2-leuA(L138G,H162E,S211L,N245S,I588P) vectors prepared in Example 3-1 were transformed into Corynebacterium glutamicum ATCC13032 strain by electroporation. Strains in which the vector was inserted into the chromosome by homologous sequence recombination were selected from a medium containing 25 mg / L of kanamycin. The selected primary strains were further subjected to a secondary cross to select strains into which mutations in the target gene were introduced. Whether or not the leuA gene mutation was introduced into the finally transformed strains was determined by performing PCR using the primers of SEQ ID NO: 3 and SEQ ID NO: 4 and then analyzing the nucleotide sequence. It was confirmed that mutations were introduced into the strains. A total of 8 types of strains were prepared, and they were named "ATCC13032_leuA_L138G", "ATCC13032_leuA_H162E", "ATCC13032_leuA_S211L", "ATCC13032_leuA_N245S", "ATCC13032_leuA_I588P", "ATCC13032_leuA_(S211L,I588P)", "ATCC13032_leuA_(L138G,H162E,S211L,N245S)", and "ATCC13032_leuA_(L138G,H162E,S211L,N245S,I588P)", respectively.

[0121] To evaluate the L-leucine-producing ability of all eight strains prepared as described above, flask fermentation titer evaluation was performed. In 250 ml conical baffle flasks each containing 25 ml of production medium, 1 platinum loop of Corynebacterium glutamicum ATCC13032, the parental strain, and ATCC13032_leuA_L138G, ATCC13032_leuA_H162E, ATCC13032_leuA_S211L, ATCC13032_leuA_N245S, ATCC13032_leuA_I588P, ATCC13032_leuA_(S211L,I588P), ATCC13032_leuA_(L138G,H162E,S211L,N245S), ATCC13032_leuA_(L138G,H162E,S211L,N245S,I588P) prepared as described above were each inoculated, and then cultured with shaking at 30 °C and 200 rpm for 60 hours to produce L-leucine. After the cultivation was completed, the production amount of L-leucine was measured by HPLC. The leucine concentration in the culture broth of each strain in which the experiment was conducted is shown in Table 5.

[0122]

Table 5

[0123] As shown in Table 5, ATCC13032_leuA_L138G with the L138G mutation in the leuA gene had a yield of L-leucine improved by about 1.45-fold compared to the parent strain Corynebacterium glutamicum ATCC13032. ATCC13032_leuA_H162E with the H162E mutation had a yield of L-leucine improved by about 1.49-fold compared to the parent strain Corynebacterium glutamicum ATCC13032. ATCC13032_leuA_S211L with the S211L mutation had a yield of L-leucine improved by about 1.58-fold compared to the parent strain Corynebacterium glutamicum ATCC13032. ATCC13032_leuA_N245S with the N245S mutation had a yield of L-leucine improved by about 1.40-fold compared to the parent strain Corynebacterium glutamicum ATCC13032. ATCC13032_leuA_I588P with the I588P mutation had a yield of L-leucine improved by about 1.37-fold compared to the parent strain Corynebacterium glutamicum ATCC13032. ATCC13032_leuA_(S211L,I588P) had a yield of L-leucine improved by about 1.51-fold compared to the parent strain. It was confirmed that ATCC13032_leuA_(L138G,H162E,S211L,N245S) and ATCC13032_leuA_(L138G,H162E,S211L,N245S,I588P) had a yield of L-leucine improved by about 1.56-fold compared to the parent strain Corynebacterium glutamicum.

Example

[0124] Confirmation of the leucine production ability of selected leuA mutations in leucine-producing strains Wild-type strains of the genus Corynebacterium produce only trace amounts of leucine even if they produce it. Therefore, an experiment was conducted to prepare a leucine-producing strain derived from wild-type Corynebacterium glutamicum ATCC13032 and introduce the selected mutations to confirm the leucine production ability. The specific experimental methods and results are as follows.

[0125] 4-1. Preparation of L-leucine-producing strain CJL-8109 strain As a strain for high-concentration L-leucine production, a strain derived from wild-type Corynebacterium glutamicum ATCC13032 containing (1) a mutation (R558H) in which G, the 1673rd nucleotide of the leuA gene, is substituted with A, and arginine, the 558th amino acid of the LeuA protein, is substituted with histidine; (2) a mutation (G561D) in which GC, the 1682nd and 1683rd nucleotides of the leuA gene, are substituted with AT, and glycine, the 561st amino acid, is substituted with aspartic acid; and (3) a mutation (P247C) in which CC, the 739th and 740th nucleotides of the leuA gene, are substituted with TG, and proline, the 247th amino acid, is substituted with cysteine was prepared.

[0126] Specifically, the pDCM2-leuA(R558H,G561D) vector (Patent Document 9) containing the leuA gene mutations (R558H,G561D) was transformed into Corynebacterium glutamicum ATCC13032 by electroporation, and a strain in which the vector was inserted into the chromosome by homologous sequence recombination was selected from a medium containing 25 mg / L of kanamycin. The selected primary strain was further subjected to a secondary cross to select a strain into which the mutation of the leuA gene was introduced. Whether or not the mutation was introduced into the finally transformed strain was determined by performing PCR (5 minutes at 94°C, then 30 cycles of 30 seconds at 94°C / 30 seconds at 55°C / 90 seconds at 72°C, and then 5 minutes at 72°C) using the primers of SEQ ID NOs: 28 and 55 and analyzing the nucleotide sequence. It was confirmed that the R558H and G561D mutations were introduced. The specific sequences of the primers used are shown in Table 6. The ATCC13032_leuA_(R558H,G561D) strain transformed with the pDCM2-leuA(R558H,G561D) vector was named "CJL-8100".

[0127]

Table 6

[0128] To introduce a mutation (P247C) into CJL-8100, the L-leucine-producing strain, an insertion vector was prepared.

[0129] Specifically, using the chromosome of the CJL-8100 strain as a template, PCR was performed with the primer pairs of SEQ ID NO: 28 and 29, and SEQ ID NO: 54 and 55. The PCR was carried out with denaturation at 94°C for 5 minutes, followed by 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute and 30 seconds, and then a polymerization reaction at 72°C for 5 minutes. The resulting PCR product was cloned by fusing the 15-base homologous sequences at the ends between DNA fragments using a linear pDCM2 vector cleaved with SmaI restriction enzyme and In-Fusion enzyme, to obtain a vector pDCM2-leuA(P247C,R558H,G561D) containing a leuA mutation encoding a LeuA variant in which arginine, the 558th amino acid in the wild-type strain's LeuA amino acid sequence, is substituted with histidine, and glycine, the 561st amino acid, is substituted with aspartic acid, and proline (Pro), the 247th amino acid of LeuA, is substituted with cysteine (Cys).

[0130] The pDCM2-leuA(P247C,R558H,G561D) vector was transformed into wild-type Corynebacterium glutamicum ATCC13032 by electroporation, and strains in which the vector was inserted into the chromosome by homologous sequence recombination were selected from a medium containing 25 mg / L of kanamycin. The selected primary strains were further subjected to a secondary cross to select strains into which mutations in the leuA gene were introduced. Whether mutations were introduced into the finally transformed strains was determined by performing PCR (denaturation at 94°C for 5 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 90 seconds, and then polymerization at 72°C for 5 minutes) using the primers of SEQ ID NO: 3 and SEQ ID NO: 4 and analyzing the nucleotide sequence. It was confirmed that the P247C, R558H, and G561D mutations were introduced. The ATCC13032_leuA_(P247C,R558H,G561D) strain transformed with the pDCM2-leuA(P247C,R558H,G561D) vector was named "CA13-8105".

[0131] The above CA13-8105 was deposited with the Korean Collection for Microorganisms, a depository institution under the Budapest Treaty, on April 29, 2020, under the accession number KCCM12709P.

[0132] In order to improve the L-leucine-producing ability of the prepared CA13-8105 strain, a strain was prepared in which an ilvE mutant (V156A), a gene encoding branched-chain amino acid aminotransferase, was introduced (Patent Document 10). Specifically, the pDCM2-ilvE(V156A) vector containing the ilvE gene mutation was transformed into Corynebacterium glutamicum CJL-8100 by electroporation, and a strain in which the vector was inserted into the chromosome by recombination of homologous sequences was selected from a medium containing 25 mg / L of kanamycin. The selected primary strain was further subjected to a secondary cross to select a strain into which the ilvE gene mutation was introduced. Whether or not the mutation was introduced into the finally transformed strain was determined by performing PCR (5 minutes at 94°C, then 30 cycles of 30 seconds at 94°C / 30 seconds at 55°C / 90 seconds at 72°C, and then 5 minutes at 72°C) using the primer pair of SEQ ID NO: 30 and SEQ ID NO: 31 in Table 7 and analyzing the nucleotide sequence. It was confirmed that the V156A mutation was introduced. The strain transformed with the pDCM2-ilvE(V156A) vector was named "CJL-8108".

[0133]

Table 7

[0134] In order to improve the L-leucine-producing ability of the prepared CJL-8108 strain, a strain was prepared in which a gltA mutant (M312I) with weakened citrate synthase activity was introduced.

[0135] Specifically, for the preparation of the vector for introducing the gltA(M312I) mutation, the site-directed mutagenesis method was used. Using the chromosome of wild-type Corynebacterium glutamicum ATCC13032 as a template, PCR was performed using the primers shown in Table 8. PCR was carried out with denaturation at 94°C for 5 minutes, followed by 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute and 30 seconds, and then a polymerization reaction at 72°C for 5 minutes. The resulting gene fragment was cloned by ligating the 15-base homologous sequences at the ends between DNA fragments using a linear pDCM2 vector cleaved with the SmaI restriction enzyme and In-Fusion enzyme, and a vector pDCM2-gltA(M312I) was prepared in which methionine, the 312th amino acid, was substituted with isoleucine.

[0136]

Table 8

[0137] The pDCM2-gltA(M312I) vector containing the gltA gene mutation was transformed into Corynebacterium glutamicum CJL-8108 by electroporation, and strains in which the vector was inserted onto the chromosome by recombination of homologous sequences were selected from a medium containing 25 mg / L of kanamycin. The selected primary strains were further subjected to a secondary cross to select strains in which the gltA gene mutation was introduced. Whether or not the mutation was introduced into the finally transformed strain was determined by performing PCR (94°C for 5 minutes, then 30 cycles of 94°C for 30 seconds / 55°C for 30 seconds / 72°C for 90 seconds, and then 72°C for 5 minutes) using the primers of SEQ ID NO: 36 and SEQ ID NO: 37 in Table 9 and analyzing the nucleotide sequence. It was confirmed that the M312I mutation was introduced. The strain transformed with the pDCM2-gltA(M312I) vector was named "CJL-8109".

[0138]

Table 9

[0139] Preparation of Insertion Vector Containing 4-2.leuA Mutation To introduce the mutations (L138G, H162E, S211L, N245S, I588P) selected in Example 2 into CJL-8109, the L-leucine-producing strain prepared in Example 4-1, an insertion vector was prepared.

[0140] Using the chromosome of the CJL-8109 strain as a template, PCR was performed using the primer pairs shown in Table 4. The PCR was carried out with denaturation at 94°C for 5 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute and 30 seconds, and then polymerization at 72°C for 5 minutes. The resulting PCR product was cloned by fusing the 15-base homologous sequences at the ends between DNA fragments using a linear pDCM2 vector cut with SmaI restriction enzyme and an infusion enzyme, and a total of 8 vectors, "pDCM2-leuA(L138G, P247C, R558H, G561D)", "pDCM2-leuA(H162E, P247C, R558H, G561D)", "pDCM2-leuA(S211L, P247C, R558H, G561D)", "pDCM2-leuA(N245S, P247C, R558H, G561D)", "pDCM2-leuA(P247C, R558H, G561D, I588P)", "pDCM2-leuA(S211L, P247C, I588P)", "pDCM2-leuA(L138G, H162E, S211L, N245S, P247C, R558H, G561D)", "pDCM2-leuA(L138G, H162E, S211L, N245S, P247C, R558H, G561D, I588P)" were prepared.

[0141] 4-3. Introduction and Evaluation of leuA Mutants into CJL-8109 Strain The L - leucine - producing strain CJL - 8109 was transformed with the vector prepared in Example 4 - 2, and a strain in which the vector was inserted into the chromosome by recombination of homologous sequences was selected from a medium containing 25 mg / L of kanamycin. The selected primary strain was further subjected to a secondary cross to select a strain into which a mutation of the target gene was introduced. Finally, whether the leuA gene mutation was introduced into the transformed strain was determined by performing PCR using the primers of SEQ ID NO: 3 and SEQ ID NO: 4 and then analyzing the nucleotide sequence, and it was confirmed that the leuA mutation was introduced into the strain. The eight strains produced were named as shown in Table 11. The amino acid sequences of the mutants containing the mutations and the nucleotide sequences of the leuA mutants encoding them are shown in Table 10.

[0142]

Table 10

[0143] Thereafter, the L - leucine - producing abilities of the wild - type Corynebacterium glutamicum ATCC13032, the prepared CJL - 8109, CJL - 8117, CJL - 8118, CA13 - 8119, CJL - 8120, CJL - 8121, CJL - 8122, CJL - 8123, and CJL - 8125 strains were evaluated. Specifically, flask culture was performed in the same manner as in Example 2 - 1, and after the completion of the culture, the L - leucine production amounts of the parent strain and the mutant strains were measured using HPLC. The results are shown in Table 11.

[0144]

Table 11

[0145] As shown in Table 11, CJL-8117, CJL-8118, CA13-8119, CJL-8120, CJL-8121, CJL-8122, CJL-8124, and CJL-8125, which are L-leucine-producing strains further containing the L138G, H162E, S211L, N245S, I588P, S211L / I588P, L138G / H162E / S211L / N245S, or L138G / H162E / S211L / N245S / I588P mutations in the leuA gene, were confirmed to have about 4 to 5 times improved L-leucine production ability compared to the parental strain, wild-type Corynebacterium glutamicum ATCC13032. In addition, L-leucine-producing strains Corynebacterium glutamicum CJL-8117, CJL-8118, CA13-8119, CJL-8120, CJL-8121, CJL-8122, CJL-8123, and CJL-8125 were confirmed to have about 1.2 to 1.6 times improved L-leucine production ability compared to the parental strain, Corynebacterium glutamicum CJL-8109.

[0146] From these results, it was confirmed that the amino acids at positions 138, 162, 211, 245, and 588 in the amino acid sequence of the LeuA protein are important positions for L-leucine production activity.

[0147] 4-4. Measurement of Isopropylmalate Synthase Activity in Strains Introduced with LeuA Mutants In order to measure the activity of isopropylmalate synthase in CJL-8109, CJL-8117, CJL-8118, CA13-8119, CJL-8120, CJL-8121, CJL-8122, CJL-8123, and CJL-8125, which are L-leucine-producing strains prepared in Example 4-3, the experiment was conducted by the following method.

[0148] In a 250 ml corner baffle flask containing 25 ml of the production medium in Table 2, the above-mentioned strains (CJL-8109, CJL-8117, CJL-8118, CA13-8119, CJL-8120, CJL-8121, CJL-8122, CJL-8123, CJL-8125) and wild-type Corynebacterium glutamicum ATCC13032 were each inoculated with one platinum loop, and then cultured with shaking at 30 °C and 200 rpm for 16 hours. After the culture was completed, the culture solution was centrifuged, the supernatant was discarded, the pellet was washed with lysis buffer and made turbid, and the cells were disrupted. Protein quantification of the lysate was performed by the Bradford method, and a lysate containing 100 μg / ml of protein was used. Here, the absorbance change at 412 nm due to thionitrobenzoic acid (TNB) formed from DTNB (5,5'-dithiobis(2-nitrobenzoic acid), Ellman's reagent) by reduction using the generated CoA was measured to determine the activity of isopropylmalate synthase enzyme. The measurement results of the isopropylmalate synthase activity in each strain are shown in Table 12.

[0149]

Table 12

[0150] Next, in order to confirm the degree of relief of feedback inhibition of the enzyme against leucine, the activity of isopropylmalate synthase was measured by measuring the CoA generated when using a lysate containing 100 μg / ml of protein under the condition of adding 2 g / L of leucine. The measurement results of the isopropylmalate synthase activity in each strain are shown in Table 13.

[0151]

Table 13

[0152] As shown in Table 12 and Table 13, for the L-leucine-producing strains CJL-8109, CJL-8117, CJL-8118, CA13-8119, CJL-8120, CJL-8121, CJL-8122, CJL-8123, and CJL-8125 transformed with the LeuA mutant expression vector, it was confirmed that the activity of isopropylmalate synthase was improved by about 1.18 to 1.38 times compared to the wild-type Corynebacterium glutamicum ATCC13032, which is the control group. Further, since the above L-leucine-producing strains maintained 83% to 93% of the isopropylmalate synthase enzyme activity even under the condition of adding 2 g / L of leucine, it was confirmed that the feedback inhibition by leucine was released.

[0153] The above CA13-8119 was deposited with the Korean Collection for Type Cultures, which is a depository institution under the Budapest Treaty, on February 8, 2021, under the accession number KCCM12949P.

[0154] From the above description, those skilled in the technical field to which the present application pertains will understand that the present application can be implemented in other specific forms without changing its technical idea and essential features. It should be understood that the above embodiments are merely illustrative and not restrictive. The present application should be construed as including all changes and modified forms derived from the meaning and scope of the claims and their equivalent concepts, rather than the description of the specification.

[0155] JPEG0007683011000014.jpg204144

[0156] JPEG0007683011000015.jpg208144

Claims

1. In the amino acid sequence of SEQ ID NO: 1, i) substitution of the amino acid residue at the position corresponding to the 138th position with glycine, ii) substitution of the amino acid residue at the position corresponding to the 162nd position with glutamate, iii) substitution of the amino acid residue at the position corresponding to the 211th position with leucine, iv) substitution of the amino acid residue at the position corresponding to the 245th position with serine, and v) substitution of the amino acid residue at the position corresponding to the 588th position with proline, a mutant polypeptide having isopropylmalate synthase activity and having an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

1.

2. The mutant polypeptide according to claim 1, wherein the mutant polypeptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO:

14.

3. A polynucleotide encoding the mutant polypeptide according to claim 1 or 2.

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

5. A microorganism of the genus Corynebacterium that produces L-leucine, comprising the mutant polypeptide according to claim 1, a polynucleotide encoding the same, or a vector comprising the same.

6. The microorganism of the genus Corynebacterium according to claim 5, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.

7. A method for producing L-leucine, comprising culturing a microorganism of the genus Corynebacterium that produces L-leucine in a medium, the microorganism comprising the mutant polypeptide according to claim 1, a polynucleotide encoding the same, or a vector comprising the same.

8. The method for producing L-leucine according to claim 7, further comprising recovering L-leucine from the medium or the microorganism after the culturing step.

9. A composition for producing L-leucine, comprising the mutant polypeptide according to claim 1, a polynucleotide encoding the same, or a vector comprising the same, a microorganism of the genus Corynebacterium that produces L-leucine, or a medium obtained by culturing the microorganism, the medium comprising the microorganism.

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