RU2811433C1 - New polypeptide and method of producing l-leucine using its - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: following is proposed: a mutated polypeptide with isopropyl malate synthase activity containing the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 16, where the proline at position 247 or 212, respectively, is replaced by cysteine. Also the following is proposed: a mutated polypeptide having isopropyl malate synthase activity containing the amino acid sequence SEQ ID NO: 1, where (1) proline at position 247 is replaced by cysteine and (2) where arginine at position 558 is replaced by histidine and/or where glycine at position 561 is replaced by aspartic acid. Besides, the following is proposed: a polynucleotide encoding the said mutated polypeptide, an expression vector containing the said polynucleotide, a microorganism capable of producing L-leucine, a composition for producing L-leucine and a method of producing L-leucine.

EFFECT: invention makes it possible to obtain L-leucine with high yield.

13 cl, 10 tbl, 4 ex

Description

TECHNICAL FIELD

Cross reference to related application(s)

This application claims the benefit of Korean Patent Application No. 10-2020-0060578, filed May 20, 2020, and the entire contents disclosed in the related Korean patent application documents are incorporated as part of this specification.

The present invention relates to a new mutated polypeptide having isopropyl malate synthase activity; a polynucleotide encoding it; a vector containing a polynucleotide; a microorganism comprising a mutated polypeptide, polynucleotide, vector or combination thereof; and a method for producing L-leucine by cultivating a microorganism.

BACKGROUND ART

L-Amino acids are industrially produced through fermentation methods using amino acid-producing bacteria belonging to coryneform bacteria or the family Enterobacteriaceae, which have the ability to produce L-amino acid. As such amino acid-producing bacteria, to increase productivity, strains isolated in nature are used, as well as artificial mutants of such strains and recombinant strains in which the L-amino acid biosynthesis enzyme is enhanced through genetic recombination.

Among L-amino acids, L-leucine is a type of essential amino acid that is expensive and widely used in drugs, food products, feed additives, industrial chemicals and the like. In addition, L-leucine is mainly produced using microorganisms. The fermentation production of L-leucine is mainly carried out using a microorganism of the genus Escherichia or a microorganism of the genus Corynebacterium, which are known to biosynthesize 2-ketoisocaproate in several stages as a precursor from pyruvic acid. However, the enzymes involved in the biosynthesis of L-leucine cause feedback inhibition by the final product, i.e. L-leucine or its derivative, thus making large-scale industrial production of L-leucine difficult.

Thus, the inventors of the present invention have discovered that a mutant strain obtained by changing a specific position of isopropyl malate synthase can optimize the activity of the enzyme and thus can be used to produce L-leucine in high yield, thereby creating the present invention.

Prior Art Literature

Patent literature

Patent Literature 1: US 2018-0251772 A1

DISCLOSURE OF INVENTION

Technical challenge

An object of the present invention is to provide a mutated polypeptide having isopropyl malate synthase activity in which the proline amino acid residue at position 247 in the amino acid sequence SEQ ID NO: 1 is replaced by an amino acid residue other than proline.

Another object of the present invention is to provide a polynucleotide encoding a mutated polypeptide.

Another object of the present invention is to provide a vector containing a polynucleotide.

Another object of the present invention is to provide a microorganism containing at least one selected from the group consisting of a mutated polypeptide, a polynucleotide and a vector containing the polynucleotide.

Another object of the present invention is to provide a method for producing L-leucine, which includes the step of cultivating a microorganism in a medium.

Another object of the present invention is to provide a composition for producing L-leucine containing at least one selected from the group consisting of a mutated polypeptide, a polynucleotide encoding the mutated polypeptide, a vector containing the polynucleotide, and a microorganism (for example, the mutated polypeptide of the present the invention, a polynucleotide of the present invention and/or a microorganism (recombinant cell) containing a vector of the present invention).

Another object of the present invention is to provide the use of at least one selected from the group consisting of a mutated polypeptide, a polynucleotide encoding the mutated polypeptide, a vector containing the polynucleotide, and a microorganism (for example, a mutated polypeptide of the present invention, a polynucleotide of the present invention and/or a microorganism (recombinant cell) containing the vector of the present invention) to produce L-leucine.

Technical solution

In one aspect of the present invention, there is provided a mutated polypeptide having isopropyl malate synthase activity in which the amino acid residue at position 247 from the N-terminus in the amino acid sequence of the isopropyl malate synthase protein SEQ ID NO: 1 or at the corresponding position is replaced by another amino acid residue.

As used herein, the term “isopropyl malate synthase (2-isopropyl malate synthase, α-isopropyl malate synthase)” refers to the enzyme that catalyzes the condensation of the acetyl group of acetyl CoA and 2-ketoisovalerate (3-methyl-2-oxobutanoate, 2-exoisovalerate) to form isopropyl malate (3-carboxy-3-hydroxy-4-methylpentanoate), which is a precursor to L-leucine. Isopropyl malate synthase may include an enzyme having conversion activity, regardless of the origin of the microorganism. For example, isopropyl malate synthase may be an enzyme derived from a microorganism of the genus Corynebacterium.

The isopropyl malate synthase may contain the amino acid sequence of SEQ ID NO: 1 or may consist of the amino acid sequence of SEQ ID NO: 1. Additionally, if the enzyme has the same or similar activity to isopropyl malate synthase as a polypeptide having at least 60% or greater homology, 70% or more, 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 96% or more, 98% or more, or 99% or more, 99.5% or more , or 99.8% or more of the amino acid sequence SEQ ID NO: 1, regardless of the origin of the microorganism, then a polypeptide having an amino acid sequence in which certain sequences are deleted, modified, replaced or added to the amino acid sequence SEQ ID NO: 1, also may be included within the scope of the present invention as isopropyl malate synthase.

That is, even if it is described herein as “a polypeptide (or protein) comprising an amino acid sequence represented by a particular sequence number” or “a polypeptide (or protein) consisting of an amino acid sequence represented by a particular sequence number”, then the polypeptide ( or protein) having an amino acid sequence in which some sequences are deleted, modified, replaced or added, can also be used as a protein or polypeptide to be mutated in the present invention when it has the same or corresponding activity as the polypeptide (or protein ), consisting of an amino acid sequence with a corresponding sequence number. For example, a polypeptide having the same or similar activity as “a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1” may refer to “a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1”.

The amino acid sequence of isopropyl malate synthase and the nucleotide sequence of the gene encoding isopropyl malate synthase can be easily obtained from databases known in the art, such as the National Center for Biotechnology Information (NCBI) and the DNA Data Bank of Japan (DDBJ).

In this specification, a polynucleotide or polypeptide "comprising a specific nucleic acid sequence (nucleotide sequence) or amino acid sequence" may mean that the polynucleotide or polypeptide consists of or essentially contains a specific nucleic acid sequence (nucleotide sequence) or amino acid sequence and can be interpreted as containing sequences in which a particular nucleic acid sequence or amino acid sequence is mutated (deleted, replaced, modified and/or added) (or as not excluding mutation) within the range of maintaining the original function and/or desired function of the polynucleotide or polypeptide. In one embodiment, a polynucleotide or polypeptide "containing a specific nucleic acid sequence (nucleotide sequence) or amino acid sequence" may mean that the polynucleotide or polypeptide (1) consists of or essentially contains a specific nucleic acid sequence (nucleotide sequence) or amino acid sequence or ( 2) consists of or essentially contains a nucleic acid sequence or amino acid sequence having homology of 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98% or more, 99.5% or more or 99.9% or more with a specific nucleic acid sequence (nucleotide sequence) or amino acid sequence and retains the original function and/or desired function.

In this specification, the amino acid sequence of an isopropyl malate synthase derived from wild type Corynebacterium glutamicum (e.g., ATCC13032) is provided under SEQ ID NO: 1, and the nucleotide sequence of the leuA gene encoding isopropyl malate synthase derived from this wild type strain is provided under SEQ ID NO: 2. Isopropyl malate synthase has been shown to consist of 616 amino acids in SEQ ID NO: 1, but from some literature it is known to consist of 581 amino acids, since the translation initiation codon is given after 35 amino acids, and the amino acid sequence of isopropyl malate synthase consisting of of 581 amino acids, is provided under SEQ ID NO: 16. Isopropyl malate synthase, consisting of 581 amino acids, can be included within the scope of the present invention as isopropyl malate synthase, in which case position 247 is interpreted as position 212, which thus can be included in scope of the present invention.

The mutated polypeptide, according to one embodiment, may be a polypeptide wherein:

the amino acid residue of arginine at position 558 (or its corresponding position) in the amino acid sequence SEQ ID NO: 1 is replaced by an amino acid residue other than arginine, the amino acid residue of glycine at position 561 (or its corresponding position) in the amino acid sequence SEQ ID NO: 1 is replaced to an amino acid residue other than glycine, or

the polypeptide is further modified by these two substitutions.

In one embodiment, in the case of the 581 amino acid isopropyl malate synthase of SEQ ID NO: 16, position 558 is interpreted as position 523 and position 561 as position 526, which is thus within the scope of the present invention.

In this specification, the term “homology” refers to the percentage of similarity between two polynucleotides or polypeptide fragments. Homology between sequences from a fragment to another fragment can be determined using techniques known in the art. For example, homology can be determined by arranging the sequence information and directly arranging the sequence information, that is, parameters such as score, identity and similarity, etc., of two polynucleotide molecules or two polypeptide molecules using a readily available computer program. The computer program may be BLAST (NCBI), CLC Main Workbench (CLC bio), Megalign (DNASTAR Inc), or the like. In addition, homology between polynucleotides can be determined by hybridization of polynucleotides under conditions of formation of a stable double strand between homologous regions, cleavage using a single-strand specific nuclease, and subsequent confirmation of the size of the cleaved fragments.

In one aspect, a mutated polypeptide having isopropyl malate synthase activity may be provided in which the proline amino acid residue at position 247 (or its corresponding position) in the amino acid sequence SEQ ID NO: 1 is replaced by an amino acid residue other than proline.

In another aspect, a mutated polypeptide having isopropyl malate synthase activity may be provided in which the proline amino acid residue at position 212 in the amino acid sequence SEQ ID NO: 16 is replaced by an amino acid residue other than proline.

In this specification, “position ~ in the amino acid sequence SEQ ID NO: ~” can be used interchangeably with “position ~ from the N-terminus of a polypeptide consisting of (or containing) the amino acid sequence SEQ ID NO: ~”.

Amino acids other than proline may include arginine, alanine, leucine, isoleucine, valine, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, histidine, aspartic acid, and glutamic acid.

In one embodiment, the mutated polypeptide may be a polypeptide in which the proline at position 247 (or its corresponding position) in the amino acid sequence SEQ ID NO: 1 is replaced by cysteine.

In one embodiment, the mutated polypeptide may comprise the amino acid sequence of SEQ ID NO: 3 or may consist of the amino acid sequence of SEQ ID NO: 3.

The mutated polypeptide according to one embodiment may be a polypeptide wherein the amino acid residue at a position corresponding to position 247 (in the case of SEQ ID NO: 16, position 212) in the amino acid sequence of the polypeptide having 60% or more homology, 70% or more, 80 % or more, 85% or more, 90% or more, 92% or more, 94% or more, 96% or more, 98% or more, or 99% or more, 99.5% or more, or 99, 8% or more of the amino acid sequence SEQ ID NO: 1 (or SEQ ID NO: 16) is replaced by another amino acid residue.

The mutated polypeptide, according to one embodiment, may have increased L-leucine producing activity than a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (or SEQ ID NO: 16) by substituting a proline at position 247 in the amino acid sequence of SEQ ID NO: 1 ( or proline at position 212 in the amino acid sequence SEQ ID NO: 16) or an amino acid at a position corresponding thereto to another amino acid.

In one embodiment, a mutated polypeptide wherein the amino acid residue proline at position 247, or an amino acid residue at its corresponding position in the amino acid sequence of the polypeptide, has 70% or more, 80% or more, 85% or more, 90% or more, 92% homology or more, 94% or more, 96% or more, 98% or more, or 99% or more with the amino acid sequence SEQ ID NO: 1 or in the amino acid sequence SEQ ID NO: 1 replaced by another amino acid residue, may have isopropyl malate synthase activity . Said “other amino acid residue” may mean another type of amino acid residue other than the amino acid (eg, proline) that was present at the specified position (position 247 in the amino acid sequence of SEQ ID NO: 1 or a position corresponding thereto) before the substitution.

The mutated polypeptide according to one embodiment may have increased L-leucine production activity than that of an isopropyl malate synthase derived from a wild-type strain (e.g., an isopropyl malate synthase containing (or consisting of) the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 16) by substitution proline at position 247 (or position 212) in the amino acid sequence SEQ ID NO: 1 (or SEQ ID NO: 16) or an amino acid residue at its corresponding position to another residue.

The mutated polypeptide according to one embodiment may have increased isopropyl malate synthase enzymatic activity than that of an isopropyl malate synthase derived from a wild-type strain (e.g., an isopropyl malate synthase consisting of the amino acid sequence SEQ ID NO: 1 (or SEQ ID NO: 16)) by substituting a proline at position 247 (or at position 212) in the amino acid sequence of SEQ ID NO: 1 (or SEQ ID NO: 16) or an amino acid residue at its corresponding position to another residue.

In this specification, the term “increasing isopropyl malate synthase activity” refers to increasing the activity of conversion to isopropyl malate. Thus, a mutated polypeptide according to one embodiment may have a higher level of isopropyl malate conversion activity compared to a polypeptide having isopropyl malate synthase activity comprising the amino acid sequence of SEQ ID NO: 1 (or SEQ ID NO: 16).

In this specification, the term “enhanced activity” may be used interchangeably with “enhanced activity.” In addition, isopropyl malate is one of the precursors of L-leucine, and thus, use of the mutated polypeptide according to one embodiment results in a higher level of L-leucine compared to a polypeptide having isopropyl malate synthase activity comprising the amino acid sequence SEQ ID NO: 1 (or SEQ ID NO: 16).

Isopropyl malate conversion activity can be confirmed directly by measuring the level of isopropyl malate formed or can be confirmed indirectly by measuring the level of CoA formed. The enzymatic activity of isopropyl malate synthase can be measured in a known manner, for example it can be measured according to the method described in Kohlhaw et al. (Methods in Enzymology 166:423-9 (1988)), and the change in absorbance at 412 nm due to thionitrobenzoate (TNB) derived from DTNB (5,5'-dithiobis-(2-nitrobenzoic acid), Ellman's reagent can be measured ) by reduction using the resulting CoA, thereby determining the activity of the enzyme isopropyl malate synthase.

The mutated polypeptide according to one embodiment may reduce feedback inhibition by L-leucine and/or derivatives thereof compared to isopropyl malate synthase derived from a wild-type strain (e.g., isopropyl malate synthase consisting of the amino acid sequence SEQ ID NO: 1) by replacing proline at position 247 (or position 212) in the amino acid sequence of SEQ ID NO: 1 (or SEQ ID NO: 16) or the amino acid sequence at a position corresponding thereto to another amino acid.

In this specification, the term “feedback inhibition” means that the end product of the enzyme system inhibits a reaction in the initial stage of the enzyme system. For example, feedback inhibition may mean that L-leucine or a derivative thereof inhibits the activity of isopropyl malate synthase, which mediates the first step of its biosynthetic pathway. Therefore, when feedback inhibition of isopropyl malate synthase is reduced (or attenuated), L-leucine productivity can be increased compared to when it is not present.

In this specification, the term “derivative” may refer to compounds that are known to be capable of inhibiting the ability to produce L-leucine from microorganisms by inducing feedback inhibition on the biosynthesis of L-leucine, which is the end product of the present invention. Examples thereof may include isoleucine, terleucine, norleucine and/or cycloleucine, etc.

In one embodiment, the mutated polypeptide may be a polypeptide at which a position other than position 247 (or position 212) in the amino acid sequence SEQ ID NO: 1 (or SEQ ID NO: 16) is further mutated, thereby synergistically enhancing one or more effects selected from the group consisting of (1) increasing L-leucine production activity; (2) increasing the enzymatic activity of isopropyl malate synthase; and (3) reducing feedback inhibition by L-leucine and/or its derivatives.

According to one embodiment, the mutated polypeptide may further comprise mutations that may exhibit one or more effects selected from the group consisting of (1) increasing L-leucine production activity; (2) increasing the enzymatic activity of isopropyl malate synthase; and (3) reducing feedback inhibition by L-leucine and/or its derivatives.

The mutated polypeptide may be a polypeptide in which:

the amino acid residue of arginine at position 558 (or its corresponding position) in the amino acid sequence SEQ ID NO: 1 is replaced by an amino acid residue other than arginine,

the glycine amino acid residue at position 561 (or its corresponding position) in the amino acid sequence SEQ ID NO: 1 is replaced by an amino acid residue other than glycine, or the polypeptide is further modified by these two substitutions.

The mutated polypeptide may be a polypeptide in which:

the amino acid residue of arginine at position 523 (or its corresponding position) in the amino acid sequence SEQ ID NO: 16 is replaced by an amino acid residue other than arginine,

the glycine amino acid residue at position 526 (or its corresponding position) in the amino acid sequence SEQ ID NO: 16 is replaced by an amino acid residue other than glycine, or the polypeptide is further modified by these two substitutions.

An amino acid other than arginine may include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, histidine, aspartic acid and glutamic acid; and an amino acid other than glycine may include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine, arginine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, histidine, aspartic acid and glutamic acid; but not limited to them.

In one embodiment, the amino acid other than arginine may be histidine.

In one embodiment, the amino acid other than glycine may be aspartic acid.

In one embodiment, the mutated polypeptide may be a polypeptide wherein:

the amino acid residue of arginine at position 558 in the amino acid sequence SEQ ID NO: 1 is replaced by the amino acid residue of histidine,

the glycine amino acid residue at position 561 in the amino acid sequence SEQ ID NO: 1 is replaced by an aspartic acid amino acid residue, or

the polypeptide is further modified by these two substitutions.

In one embodiment, the mutated polypeptide may comprise the amino acid sequence of SEQ ID NO: 5 or may consist of the amino acid sequence of SEQ ID NO: 5.

A mutated polypeptide in which the amino acid residue at position 247 (or position 212) in the amino acid sequence SEQ ID NO: 1 (or SEQ ID NO: 16) is replaced by an amino acid residue other than proline,

a mutated polypeptide in which the amino acid residue arginine at position 558 (or position 523) in the amino acid sequence SEQ ID NO: 1 (or SEQ ID NO: 16) is replaced by an amino acid residue other than arginine, a glycine amino acid residue at position 561 (or position 526) is replaced by an amino acid residue other than glycine, or the polypeptide is further modified by these two substitutions, may be a polypeptide in which:

as compared to a mutated polypeptide in which (1) an isopropyl malate synthase derived from a wild-type strain (e.g., an isopropyl malate synthase containing the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 16), or (2) a proline amino acid residue at position 247 (or position 212) in the amino acid sequence of SEQ ID NO: 1 (or SEQ ID NO: 16) is replaced by an amino acid residue other than proline,

one or more effects selected from the group consisting of the following (1)-(3) may be increased:

1) increased activity of L-leucine production;

2) an increase in the enzymatic activity of isopropyl malate synthase; And

3) reduction of feedback inhibition by L-leucine and/or its derivatives.

According to one embodiment, depending on the combination of mutations in which the amino acid at positions 247, 558 and/or 561 (or positions 212, 523 and/or 526) in the amino acid sequence SEQ ID NO: 1 (or SEQ ID NO: 16) is replaced to another type of amino acid, the mutated polypeptide according to one embodiment may have a synergistically enhanced one or more effects selected from the group consisting of the following (1)-(3):

1) increased activity of L-leucine production;

2) an increase in the enzymatic activity of isopropyl malate synthase; And

3) reduction of feedback inhibition by L-leucine and/or its derivatives.

Effects (1)-(3) above are as described above.

In another aspect, a polynucleotide encoding a mutated polypeptide may be provided. In this specification, the term "polynucleotide" is a polymer of nucleotides in which the nucleotide monomers are covalently linked in the form of a long chain, and is a DNA or RNA chain of a certain length or longer. More specifically, it may mean a polynucleotide fragment encoding a mutated polypeptide.

The mutated polypeptide is as described above.

The polynucleotide may contain, without limitation, a polynucleotide sequence encoding a mutated polypeptide of the present invention.

In one embodiment, it may be a polynucleotide encoding a polypeptide in which the amino acid residue proline at position 247 (or position 212) in the amino acid sequence SEQ ID NO: 1 (or SEQ ID NO: 16) is replaced by an amino acid residue other than proline, or a polynucleotide encoding a polypeptide that has homology of 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 96% or more, 98 % or more, or 99% or more, 99.5% or more, or 99.8% or more of the amino acid sequence of SEQ ID NO: 1 (or SEQ ID NO: 16), in which the amino acid residue at a position corresponding to the position 247 (or position 212) in the amino acid sequence SEQ ID NO: 1 (or SEQ ID NO: 16), is replaced by another amino acid residue, or encoding a polypeptide, in which some of the sequences are deleted, modified, replaced or added thereto.

In one embodiment, the polynucleotide may comprise, without limitation, if it is a probe that can be derived from a known gene sequence, for example, a probe capable of hybridizing to a complementary sequence to all or part of the polynucleotide sequence under stringent conditions and containing a sequence corresponding to the sequence , encoding a protein variant in which the amino acid at position 247 (or position 212) or corresponding thereto in the amino acid sequence SEQ ID NO: 1 (or SEQ ID NO: 16) is replaced by another amino acid. The term "stringent conditions" refers to conditions under which a so-called specific hybrid is formed, while non-specific hybrids are not formed. Examples of such conditions include conditions in which genes having high degrees of homology, such as genes having 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 92% or more homology , 94% or more, 96% or more, 98% or more, or 99% or more, 99.5% or more, or 99.8% or more hybridize with each other, while genes having low degrees of homology, do not hybridize with each other, or conditions under which genes are washed 1 time, specifically 2 and 3 times, at a temperature and salt concentration equivalent to 60°C, 1× SSC and 0.1% SDS, particularly 60 °C, 0.1× SSC, and 0.1% SDS, and more specifically 68 °C, 0.1× SSC, and 0.1% SDS, which are wash conditions for traditional Southern hybridization (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).

The probe used in hybridization may be a complementary sequence portion of a nucleotide sequence. Such a probe can be designed by PCR using an oligonucleotide derived from a known sequence as a primer and using a gene fragment containing such a nucleotide sequence as a template. For example, a gene fragment that is about 300 bp in length can be used as a probe. More specifically, when using a probe having a length of approximately 300 bp, 50°C, 2× SSC and 0.1% SDS can be recommended for washing conditions in hybridization.

In one embodiment, the polynucleotide may comprise the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 6, or may consist of the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 6, and a polynucleotide that can be translated into a mutated polypeptide according to one embodiment due to codon degeneracy may also be included within the scope of the present invention.

According to another aspect, a vector comprising a polynucleotide may be provided.

In this specification, the term “vector” refers to any carrier for cloning and/or transferring nucleotides into a host cell. The vector may be a replicon that allows replication of fragments combined with other DNA fragments. "Replicon" refers to any genetic unit (eg plasmid, phage, cosmid, chromosome and virus) that acts as a self-replicating DNA replication in vivo, that is, replicating by self-regulation.

In one embodiment, the vector may be a DNA construct containing a polynucleotide sequence encoding a target protein that is operably linked to a suitable regulatory sequence so that the target protein can be expressed in a suitable host. The regulatory sequence may comprise a promoter capable of initiating transcription, any operator sequence for controlling transcription, a sequence encoding the corresponding mRNA ribosome binding domain, and a sequence controlling the termination of transcription and translation. Once transformed into a suitable host cell, the vector can replicate or function independently of the host genome, and it can be integrated into the host genome itself.

In this specification, the term “operably linked” means that the gene sequence is operably linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the polypeptide.

The vector is not particularly limited as long as it is capable of replicating in a host cell, and any vector known in the art can be used. For example, the vector may be natural or recombinant plasmids, cosmids, viruses and bacteriophages. MBL3, MBL4, LXII, ASHII, APII, t10, t11, pWE15, M13, λEMBL3, λEMBL4, λFIXII, λDASHII, λZAPII λgt10, λgt11, Charon4A and/or Charon21A, etc. can be used as a phage vector or cosmid vector. , and also as a plasmid vector, pDZ vectors, pBR-based vectors, pUC-based vectors, pBluescript II-based vectors, pGEM-based vectors, pTZ-based vectors, pCL-based vectors and/or pET-based vectors can be used. etc. For example, the vectors pCR2.1, pDC, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118 and/or pCC1BAC, etc. can be used.

In another aspect, a microorganism (or recombinant cell) may be provided comprising at least one selected from the group consisting of a mutated polypeptide, a polynucleotide encoding the mutated polypeptide, and a vector containing the polynucleotide.

Details regarding the mutated polypeptide, polynucleotide and vector are as described above.

The microorganism (or recombinant cell) may further contain a mutation that increases the production of L-leucine, and the position of the mutation and/or the type of gene to be mutated can be specified without limitation as long as it increases the production of L-leucine.

The microorganism (or recombinant cell) may additionally contain a mutation that enhances the activity of an enzyme involved in the biosynthesis of L-leucine. The enhancement of enzyme activity is as described above.

The microorganism (recombinant cell) can be used without restrictions as long as it is a cell capable of transformation.

The microorganism (or recombinant cell) may have the ability to produce L-leucine. The microorganism (or recombinant cell) may have an improved ability to produce L-leucine or may have an ability to produce L-leucine that the cell, parental strain, and/or wild-type strain does not possess prior to recombination.

The microorganism (or recombinant cell) may be a polypeptide in which the amino acid sequence corresponding to isopropyl malate synthase is mutated so as to increase its activity relative to the wild type strain, the parental strain and/or the pre-recombination cell, or to free it from inhibition by L-leucine and its derivative by feedback type, or both an increase in enzyme activity and release from feedback inhibition can be achieved.

The microorganism (or recombinant cell) may be one in which the polynucleotide according to one embodiment is integrated into a chromosome, and, for example, the polynucleotide according to one embodiment may be replaced by a native leuA gene (a gene encoding isopropyl malate synthase) at a gene site on the chromosome or integrated into additional gene site.

In this specification, “having the ability to produce L-leucine” refers to microorganisms in which the ability to produce leucine is imparted to cells and/or microorganisms that do not have the ability to produce leucine, or to cells and/or microorganisms that have the natural ability to produce leucine. For example, microorganisms of the genus Corynebacterium having the ability to produce L-leucine refers to a microorganism of the genus Corynebacterium that has an improved ability to produce L-leucine as a result of the insertion of an external gene associated with the leucine production mechanism, or the enhancement or inactivation of the activity of the native gene or itself natural microorganism. The microorganism may be a microorganism of the genus Corynebacterium.

The microorganism of the genus Corynebacterium may be Corynebacterium glutamicum, Corynebacterium ammoniagenes, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium alkanolyticum, Corynebacterium callunae, Corynebacterium lilium, Corynebacterium melassecola, Corynebacterium Thermoaminogenes, Corynebacterium efficiens and/ or Corynebacterium Herculis, etc.

The microorganism (or recombinant cell) can be artificially obtained through transformation and/or naturally occurring. For example, a microorganism (or recombinant cell) can be transformed with a polynucleotide encoding a mutated polypeptide according to one embodiment, or a vector containing it.

In this specification, the term “transformation” refers to the introduction of a gene or polynucleotide into a host cell so that it can be expressed in the host cell, and the transformed gene or polynucleotide may contain both a form inserted into the host cell chromosome and a form that located outside the chromosome, without restrictions as long as it can be expressed in the host cell.

In this specification, the transformation method includes, without limitation, as long as it is a method of introducing a gene into a cell and can be performed by selecting a suitable standard technique known in the art depending on the host cell. For example, electroporation, calcium phosphate precipitation (CaPO ₄ ), calcium chloride precipitation (CaCl ₂ ), microinjection, retroviral infection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and/or lithium acetate method can be used. -dimethyl sulfoxide (DMSO), but not limited to these.

The microorganism (or recombinant cell) may be one in which the polynucleotide sequence according to one embodiment is integrated into a chromosome. Homologous recombination allows for the exchange of DNA fragments on a chromosome with a polynucleotide according to one embodiment, which is transferred by a vector into a cell, while using a vector according to one embodiment. For efficient recombination between a circular vector DNA molecule and a target DNA on the chromosome, the exchangeable DNA region containing the polynucleotide according to one embodiment is provided with a nucleotide sequence homologous to the target site at the end; and this defines the vector integration site and the DNA exchange site. For example, a polynucleotide according to one embodiment may be replaced by a native leuA gene at a native gene site on a chromosome or may be included at an additional gene site.

According to another aspect, a method for producing L-leucine may be provided which includes the step of culturing the microorganism (or recombinant cell) in a medium.

In one embodiment, the method may further include the step of collecting L-leucine from the culture medium or microorganism (or recombinant cell).

Cultivation can be performed in accordance with suitable media and culture conditions known in the art, should suitably suit the needs of the particular strain, and can be suitably modified by one skilled in the art.

The culture method may include, for example, batch culture, continuous culture, fed-batch culture or combined culture, but is not limited to it.

The medium for culturing the microorganism (or recombinant cell) may be known from the literature (Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington D.C., USA, 1981), but is not limited to this.

According to one embodiment, the medium may contain various carbon sources, nitrogen sources and micronutrient components, and the recombinant cells can be cultured under temperature and/or pH control in a conventional medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin and the like. The carbon source contains sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and/or cellulose, oils and fats such as soybean oil, sunflower oil, castor oil and/or coconut oil, fatty acids such as palmitic acid, stearic acid and/or linoleic acid, alcohols such as glycerin and/or ethyl alcohol, organic acids such as acetic acid. These substances may be used alone or in combination of two or more, but are not limited to this. The nitrogen source that can be used may contain peptone, yeast extract, meat juice, malt extract, corn decoction, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen source may also be used alone or in combination of two or more, but is not limited to this. The source of phosphorus that can be used may include, but is not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium salts. In addition, the medium may contain a metal salt, such as magnesium sulfate or ferrous sulfate, necessary for growth, but is not limited to this. In addition, they may contain substances necessary for growth, such as amino acids and vitamins. In addition, you can use predecessors that are appropriate for your environment. The medium or individual components may be added to the batch in a batchwise or continuous manner during, but not limited to, the culture process.

According to one embodiment, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture medium of the microorganism appropriately during cultivation to adjust the pH of the culture medium. In addition, bubble formation can be suppressed by using an antifoam agent such as a polyglycol fatty acid ester during culture. In addition, in order to maintain the aerobic state of the culture medium, oxygen or an oxygen-containing gas (for example, air) can be introduced into the culture medium. The temperature of the culture medium can be from 20°C to 45°C, from 25°C to 40°C or from 30°C to 37°C. The culture period may be continued until the beneficial substance (eg, L-leucine) is produced in the required amount of production, and may be, for example, from 10 to 160 hours.

The step of isolating or collecting L-leucine from the cultured microorganism (or recombinant cell) and/or culture medium can be performed using a suitable method known in the art, depending on the culture method. For example, centrifugation, filtration, extraction, atomization, drying, distillation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), and/or chromatography (e.g., ion exchange, affinity, hydrophobic, and size exclusion) may be used, but is not limited to this. Culture medium refers to the medium in which the microorganism (or recombinant cell) was cultured.

In one embodiment, the L-leucine isolation or collection step can be performed by centrifuging the culture at low speed to remove biomass and recovering the resulting supernatant using ion exchange chromatography.

The method for producing L-leucine may further include the step of purifying L-leucine.

In another aspect, a composition for producing L-leucine may be provided comprising at least one selected from the group consisting of a mutated polypeptide of the present invention, a polynucleotide encoding the mutated polypeptide, a vector containing the polynucleotide, and a microorganism (e.g., a mutated polypeptide of the present invention, a polynucleotide of the present invention and/or a microorganism (recombinant cell) containing a vector of the present invention).

The composition of the present invention may further contain any suitable excipient commonly used in compositions for the production of amino acids, and the excipient may, for example, be a preservative, wetting agent, dispersing agent, suspending agent, buffering agent, stabilizing agent or isotonic agent. this.

In the composition of the present invention, the mutated polypeptides, polynucleotides, vectors, microorganisms and media are the same as those described in other aspects above.

In another aspect, the use of at least one selected from the group consisting of a mutated polypeptide, a polynucleotide encoding a mutated polypeptide, a vector containing the polynucleotide, and a microorganism (e.g., a mutated polypeptide of the present invention, a polynucleotide of the present invention, and/or microorganism (recombinant cell) containing the vector of the present invention) to produce L-leucine.

When using the present invention, the mutated polypeptides, polynucleotides, vectors, microorganisms and media are the same as those described in other aspects above.

BENEFITIVE EFFECTS

By using the mutated polypeptide according to the embodiment, L-leucine can be produced in high yield.

DETAILED DESCRIPTION OF EMBODIMENTS

The present invention will now be described in more detail with reference to the following examples, but the scope of the present invention is not intended to be limited to these examples.

Example 1: Construction of a DNA library encoding a mutated isopropyl malate synthase

Example 1-1. Construction of a vector containing leuA

In order to construct a library of leuA mutants encoding a variant having isopropyl malate synthase activity, a recombinant vector containing leuA was first constructed.

To amplify the leuA gene (SEQ ID NO: 2) encoding the LeuA (2-isopropyl malate synthase) protein (SEQ ID NO: 1, Uniprot accession code: P42455) originating from wild-type Corynebacterium glutamicum, a PCR method was performed using the chromosome of Corynebacterium glutamicum ATCC13032 wild strain as a template and using primers SEQ ID NO: 7 and 8 under the following conditions of repeated 25 cycles: denaturation at 94°C for 1 min, annealing at 58°C for 30 sec and polymerization at 72°C for 1 min using Pfu DNA polymerase. The specific primer sequences used are shown in Table 1 below.

The amplified PCR product was cloned into the E. coli vector pCR2.1 using the TOPO cloning kit (Invitrogen) according to the manufacturer's instructions to obtain “pCR-leuA.”

Example 1-2. Construction of a leuA mutant library

Based on the vector constructed in Example 1-1, a leuA mutant library was constructed using a PCR random mutation kit (clontech Diversify® PCR Random Mutagenesis Kit). Under conditions where between O and 3 mutations per 1000 bp occurred, the PCR reaction was performed using the pCR-leuA vector as a template and using primers SEQ ID NO: 7 and SEQ ID NO: 8. Specifically, as conditions where there were 0 to 3 mutations per 1000 bp, the PCR reaction was performed under the following conditions: preheating at 94°C for 30 seconds, and then 25 cycles at 94°C for 30 seconds and 68° C for 1 min and 30 sec. The resulting PCR product was used as a megaprimer (50-125 ng), and the PCR reaction was performed by repeating 25 cycles at 95°C for 50 s, 60°C for 50 s, and 68°C for 12 m s subsequent treatment with DpnI. The Dpnl-treated PCR product was transformed into E. coli DH5α by heat shock and applied to solid LB medium containing kanamycin (25 mg/L). After selecting 20 transformed colonies, plasmids were obtained and the nucleotide sequence was analyzed. As a result, it was confirmed that the mutations were introduced at mutually different positions with a frequency of 2 mutations/kb. Approximately 20,000 transformed E. coli colonies were collected and plasmids were extracted and called the pTOPO-leuA library.

Example 2. Evaluation of the constructed library and selection of options

Example 2-1. Selection of mutant strains with quantitatively increased L-leucine production

The pTOPO-leuA library constructed in Example 1-2 was transformed into wild-type Corynebacterium glutamicum ATCC13032 by electroporation treatment, the transformed strain was distributed on a nutrient medium (Table 2) containing 25 mg/L kanamycin, and colonies were selected from 10,000 strains, which a mutant gene was inserted. Each selected colony was named ATCC13032/pTOPO_leuA(mt) 1 to ATCC13032/pTOPO_leuA(mt) 10000. To identify colonies with quantitatively increased L-leucine production, among the 10,000 obtained colonies, the fermentation titer was assessed for each colony as follows.

Each colony was inoculated into a 250 ml corner baffled flask containing 25 µg/ml kanamycin in 25 ml autoclaved production medium (Table 2) using a platinum loop and then subjected to shaking culture at 30°C with a shaking speed of 200 rpm /min for 60 hours. After completion of cultivation, the amount of L-leucine produced was measured by a high performance liquid chromatography (HPLC, SHIMAZDU LC20A) method.

From 10,000 colonies obtained, one strain (ATCC13032/pTOPO_leuA(mt)5306) was selected as having the most improved ability to produce L-leucine compared to the wild-type Corynebacterium glutamicum strain (ATCC13032). The concentration of L-leucine produced in the selected strain (ATCC13032/pTOPO_leuA(mt)5306) is shown in Table 3 below.

As shown in Table 3, Corynebacterium glutamicum strain ATCC13032/pTOPO_leuA(mt) 5306, which has a mutation in the leuA gene, increased the ability to produce L-leucine by approximately 1.5 times compared to the parent Corynebacterium glutamicum strain ATCC13032.

Example 2-2. Confirmation of mutations in mutant strains with increased production of L-leucine To confirm the mutation of the leuA gene of Corynebacterium glutamicum strain ATCC13032/pTOPO_leuA(mt) 5306, PCR was performed using DNA of strain ATCC13032/pTOPO_leuA(mt)5306 as a template and using primers SEQ ID NO: 9 and SEQ ID NO: 10 listed in Table 4 under the following conditions: denaturation at 94°C for 5 minutes, then 30 cycles at 94°C for 30 seconds, at 55°C for 30 seconds and at 72 °C for 1 min and 30 sec, then at 72 °C for 5 min, and DNA sequencing was performed.

As a result of sequencing, it was confirmed that in strain ATCC13032/pTOPO_leuA(mt)5306, CC, which represents the 739th and 740th nucleotides of the leuA gene, was replaced by TG. This means the ability to encode a variant (hereinafter P247C) in which proline, which is the amino acid at position 247 (position 212 if the LeuA protein consists of 581 amino acids (SEQ ID NO: 16), since the translation initiation codon is specified after 35 amino acids, based on known literature; further presented only as position 247) of the LeuA protein, replaced by cysteine. The amino acid sequence of the LeuA variant (P247C) and the nucleotide sequence of the leuA variant encoding it are the same as in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

The following examples attempt to confirm whether the mutation (P247C) affects the amount of L-leucine produced by a microorganism of the genus Corynebacterium.

Example 3: Confirmation of the ability of selected mutant strains to produce L-leucine

Example 3-1. Construction of insertion vectors containing the leuA mutation

In this example, in order to introduce the selected mutation (P247C) into the strains using the site-directed mutagenesis method, an attempt was made to construct an insertion vector. PCR was performed using the wild type Corynebacterium glutamicum chromosome (ATCC13032) as a template and using primers SEQ ID NOs: 11 and 12 and primer pairs SEQ ID NOs: 13 and 14. Specifically, PCR was performed under the following conditions: denaturation at 94°C for 5 min, then 30 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 1 min and 30 sec, then 72°C for 5 min. The specific sequences of the primers used are shown in Table 5.

The resulting PCR product was digested with the restriction enzyme Smal to obtain the linear vector pDZ, and the homologous sequence of the terminal 15th base between the DNA fragments was fused and cloned using the In-Fusion enzyme, thereby obtaining the vector "pDZ-leuA (P247C)" , in which proline (Pro), which is the 247th amino acid of leuA, is replaced by cysteine (Cys).

Example 3-2. Introduction of leuA gene mutation into strain ATCC13032 The vector pDZ-leuA (P247C) constructed in Example 3-1 was transformed into ATCC13032 by electroporation treatment, and strains having the vector inserted into the chromosome by recombination of the homologous sequence were selected in medium containing 25 mg /l kanamycin. The selected primary strain was again subjected to secondary crossover, and a strain in which a mutation was introduced in the leuA gene was selected. Confirmation of whether or not a mutation of the leuA gene was introduced into the final transformed strain was carried out by analysis of nucleotide sequences after performing PCR using primers SEQ ID NO: 9 and SEQ ID NO: 15 under the following conditions: at 94°C for 5 min, then 30 cycles at 94°C for 30 seconds / at 55°C for 30 seconds / at 72°C for 90 seconds, then at 72°C for 5 minutes. As a result of the analysis of the nucleotide sequence, it was confirmed that CC, where the 739th and 740th nucleotides of the leuA gene in the strain chromosome was replaced by TG, and the mutation of leuA, encoding LeuA, in which the 247th amino acid proline (Pro) was replaced by cysteine (Cys) were introduced into this strain. The resulting strain was named "ATCC 13032_1еиА_Р247С". The specific primer sequences used are shown in Tables 4 and 6.

Example 3-3. Evaluation of the ability of mutant strains to produce L-leucine

To evaluate the ability of strain ATCC13032_leuA_P247C to produce L-leucine obtained in Example 3-2, the fermentation titer in the flask was assessed in a manner similar to the method of Example 2. The parent strains, Corynebacterium glutamicum ATCC13032 and ATCC13032_leuA_P247C, respectively, were inoculated into a 250 ml flask with corner baffles containing 25 ml production medium using a platinum loop, and then subjected to shaking culture at 30°C with a shaking speed of 200 rpm for 60 hours to obtain L-leucine. After completion of cultivation, the amount of L-leucine produced was measured by HPLC, and the concentration of L-leucine in the culture medium for each strain is shown in Table 7 below.

As shown in Table 7, ATCC 13032_leuA_P 247 C increased the yield of L-leucine by approximately 1.55 times compared to the original Corynebacterium glutamicum strain ATCC13032.

Example 4 Evaluation of Leucine Producing Capacity of Mutant Leucine-Producing Strains Since the wild-type strain of the genus Corynebacterium produces trace amounts of L-leucine, a leucine-producing strain originating from ATCC13032 was prepared, and the mutation (P247C) selected in Example 2 was introduced to confirmation of the ability to produce L-leucine. The specific experiment was carried out as follows.

Example 4-1. Preparation of an L-leucine-producing strain (strain CJL-8100)

As a strain for producing L-leucine at a high concentration, a strain originating from ATCC13032 was obtained containing the following mutations: (1) mutation (R558H), in which arginine, which is the 558th amino acid of the LeuA protein, is replaced by histidine through replacing G, which is the 1673rd nucleotide of the leuA gene, with A, and (2) a mutation (G561D) in which glycine, which is the 561st amino acid, is replaced by aspartic acid by replacing GC, which is the 1682nd oh and 1683rd nucleotides of the leuA gene, on AT.

Specifically, the vector pDZ-leuA (R558H, G561D) containing a mutation of the leuA gene (KR10-2018-0077008 A) was transformed into Corynebacterium glutamicum ATCC13032 by electroporation treatment, and a strain having the vector inserted into the chromosome by recombination of the homologous sequence was selected in medium containing 25 mg/l kanamycin. The selected primary strain was again subjected to secondary crossing over, and a strain into which the leuA gene mutation was introduced was selected. Finally, it was confirmed whether mutations were introduced or not introduced into the transformed strain by performing PCR using primers SEQ ID NO: 7 and SEQ ID NO: 13 under the following conditions: at 94°C for 5 min, then 30 cycles at 94°C for 30 seconds/55°C for 30 seconds/72°C for 90 seconds, then 72°C for 5 minutes, and the nucleotide sequence was analyzed, thereby confirming that the R558H and G561D mutations had been introduced. The specific sequences of the primers used are presented in Tables 1 and 5. Strain ATCC13032 leuA (R558H, G561D) transformed with the vector pDZ-leuA (R558H, G561D) was named “CJL-8100”.

Example 4-2. Construction of an insertion vector containing the leuA mutation

In this example, in order to introduce the mutation (P247C) selected in Example 2 into CJL-8100, which is an L-leucine producing strain in which two mutations (R558H, G561D) are introduced in LeuA, an attempt was made to construct a vector to insert.

PCR was performed using the chromosome of strain CJL-8100 as a template and using primers SEQ ID NO: 9 and 10 and primer pairs SEQ ID NO: 11 and 12. PCR was performed under the following conditions: denaturation at 94°C for 5 min, then 30 cycles at 94°C for 30 sec, at 55°C for 30 sec and at 72°C for 1 min and 30 sec, followed by polymerization at 72°C for 5 min. The resulting PCR product was digested with the restriction enzyme SmaI to obtain the linear vector pDZ, and the homologous sequence of the terminal 15 base between the DNA fragments was fused and cloned using the In-Fusion enzyme, thereby constructing the vector pDZ-leuA(P247C, R558H, G561D), which contains a leuA mutation encoding a LeuA variant in which arginine, which is the 558th amino acid, is replaced by histidine, and glycine, which is the 561st amino acid, is replaced by aspartic acid in the amino acid sequence of wild-type strain LeuA, and proline ( Pro), the 247th amino acid of LeuA, is replaced by cysteine (Cys).

Example 4-3. Introduction and Evaluation of the LeuA Mutant (P247C) in Strain CLJ-8100 CJL-8100, which is an L-leucine-producing strain, was transformed with the vector pDZ-leuA (P247C, R558H, G561D) obtained in Example 4-2, and strains , in which the vector was inserted into the chromosome by recombination of homologous sequences, were selected in a medium containing 25 mg/l of kanamycin. The selected primary strain was again subjected to secondary crossover and then a strain was selected into which the target gene mutation had been introduced. Confirmation of whether or not the leuA gene mutation was introduced into the final transformed strain was carried out by analysis of nucleotide sequences after PCR using primers SEQ ID NO: 9 and SEQ ID NO: 15 under the following conditions: at 94°C for 5 min, then 30 cycles of 94°C for 30 seconds/55°C for 30 seconds/72°C for 90 seconds, then 72°C for 5 minutes. As a result of nucleotide sequence analysis, it was confirmed that the leuA mutation encoding the LeuA variant (P247C, R558H, G561D), in which arginine, which is the 558th amino acid of the LeuA protein, is replaced by histidine, glycine, which is the 561st amino acid, is replaced by aspartic acid, and proline (Pro), which is the 247th amino acid, is replaced by cysteine (Cys) by replacing G, which is the 1673rd nucleotide of the leuA gene, with A, GC, which is the 1682nd and the 1683rd nucleotides on AT, and CC, which are the 739th and 740th nucleotides on TG in the strain chromosome, were introduced into this strain. The resulting CJL8100 leuA P247C was named "CA13-8105" and deposited at the Korean Microbial Culture Center (KCMC), which is an international depository under the Budapest Treaty, on April 29, 2020 under the specified deposit number. The amino acid sequence of the LeuA variant (P247C, R558H, G561D) containing these three mutations and the nucleotide sequence of the leuA variant encoding it are as shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

Strains ATCC 13032, produced by CJL-8100 and CA 13-8105, were evaluated for their ability to produce L-leucine. Specifically, flask culture was performed in the same manner as in Example 2-2, and after completion of culture, the amount of L-leucine production of the parent strain and mutant strain was measured by HPLC, and the results are shown in Table 8 below.

As shown in Table 8, the L-leucine-producing strain Corynebacterium glutamicum CJL8100 improved the ability to produce L-leucine by approximately 130% compared with the parent strain ATCC13032. Strain CA13-8105, which additionally included the leuA_P247C mutation in strain CJL8100, improved the ability to produce L-leucine by approximately 150% compared to the parental strain CJL8100. Based on the above results, it can be confirmed that the amino acid at position 247 in the amino acid sequence of the LeuA protein is an important position for L-leucine production activity.

Example 4-4. Measurement of isopropyl malate synthase activity in a strain with an introduced LeuA variant

In order to measure the isopropyl malate synthase activity in CJL-8100 and CA13-8105, which are the L-leucine-producing strains obtained in Example 4-3, an experiment was performed in the following manner.

The wild type strains (CJL-8100, CA13-8105) and ATCC13032 were respectively inoculated into a 250 ml corner baffled flask containing 25 ml of each seed medium (production medium in Table 2) using a platinum loop and then subjected to shaking culture at 30°C with a shaking speed of 200 rpm for 16 hours. After completion of cultivation, the culture solution was centrifuged, the supernatant was discarded and the pellet was suspended and washed with lysis buffer, and the cells were disrupted using a bead homogenizer. Quantification of protein in the lysate was based on the Bradford assay, and a lysate containing 100 μg/ml protein was used. Isopropyl malate synthase enzyme activity was measured by measuring the change in absorbance at 412 nm due to thionitrobenzoate (TNB) formed from DTNB (5,5'-dithiobis-(2-nitrobenzoic acid), Ellman's reagent) by reduction using currently produced CoA.

The results of isopropyl malate synthase activity measurements in each strain are shown in Table 9 below.

Next, to confirm the degree of release from feedback inhibition of the above enzyme by leucine, the CoA obtained by using a solution containing 100 μg/L protein under conditions in which 2 g/L leucine was added, thereby measuring the activity of isopropyl malate synthase.

The results of isopropyl malate synthase activity measurements in each strain are shown in Table 10 below.

As shown in Tables 9 and 10, it was confirmed that L-leucine-producing strains CJL-8100 and CA13-8105 transformed with the LeuA mutant expression vector increased isopropyl malate synthase activity by 1.13-fold and 1.21-fold, respectively, compared with control Corynebacterium glutamicum ATCC 13032. In addition, it was confirmed that L-leucine-producing strains maintained isopropyl malate synthase enzyme activity at 78% and 88%, respectively, even when supplemented with 2 g/L leucine, indicating that inhibition by type of feedback leucine was released.

Based on the foregoing, one skilled in the art to which the present invention relates will appreciate that the present invention may be embodied in other specific forms without changing the technical concepts or essential characteristics of the present invention. In this regard, the exemplary embodiments disclosed herein are for illustrative purposes only and should not be construed as limiting the scope of the present invention. On the contrary, the present invention is intended to cover not only exemplary embodiments, but also various alternatives, modifications, equivalents and other embodiments that may fall within the spirit and scope of the present invention as defined in the appended claims.

Access number

Depository name: Korean Microbial Culture Center

Accession number: KCCM12709P

Deposit date: 20200429

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LIST OF SEQUENCES

<110>CJ CheilJedang Corporation

<120> New polypeptide and method for producing L-leucine from it

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Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly

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Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val

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Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp

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Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln

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Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr

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Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala

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gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac

1560

ggcaaggacg tcaccgtcga tggccgcggc aacggcccac tggccgctta cgccaacgcg

1620

ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccgcacctcg

1680

ggcgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg

1740

ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta

1800

aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a

1851

<210> 3

<211> 616

<212>PRT

<213> Artificial Sequence

<220>

<223> LeuA P247C AA (616 amino acids)

<400> 3

Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr

1 5 10 15

Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg

20 25 30

Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val

35 40 45

Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr

50 55 60

Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala

65 70 75 80

Leu Ile Asp Pro Met Ser Pro Glu Arg Lys Arg Arg Met Phe Glu Leu

85 90 95

Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala

100 105 110

Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Gly Met

115 120 125

Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His

130 135 140

Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile

145 150 155 160

Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe

165 170 175

Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu

180 185 190

Leu Ile Lys Thr Ile Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp

195 200 205

Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys

210 215 220

Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn

225 230 235 240

Pro Met Ile Ile Asn Leu Cys Ser Thr Val Glu Met Ile Thr Pro Asn

245 250 255

Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg

260 265 270

Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly

275 280 285

Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu

290 295 300

Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val

305 310 315 320

Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp

325 330 335

Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln

340 345 350

Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr

355 360 365

Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala

370 375 380

Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp

385 390 395 400

Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp

405 410 415

Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser

420 425 430

Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly

435 440 445

Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln

450 455 460

Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp

465 470 475 480

Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln

485 490 495

Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser

500 505 510

Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly

515 520 525

Arg Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu

530 535 540

Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala Arg Thr Ser

545 550 555 560

Gly Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly

565 570 575

Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser

580 585 590

Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His

595 600 605

Glu Ala Val Leu Ala Gly Gly Val

610 615

<210> 4

<211> 1851

<212> DNA

<213> Artificial Sequence

<220>

<223> LeuA P247C NT

<400> 4

atgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct

60

cgcaacgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc

120

tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat

180

aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct

240

ctgattgatc cgatgtctcc tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg

300

ggcttcaaag aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt

360

cgtgagatca tcgaaaaggg catgatccct gacgatgtca ccattcaggt tctggttcag

420

gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc

480

gtgcacttct acaactccac ctccatcctg cagcgcaacg tggtgttccg catggacaag

540

gtgcaggtga agaagctggc taccgatgcc gctgaactaa tcaagaccat cgctcaggat

600

tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt

660

gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac

720

ccaatgatca tcaacctgtg ttccaccgtt gagatgatca cccctaacgt ttacgcagac

780

tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac

840

ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct

900

gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc

960

accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata

1020

cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca

1080

tacggcggtg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag

1140

ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg

1200

gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc

1260

ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct

1320

tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc

1380

accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg

1440

gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc

1500

gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac

1560

ggcaaggacg tcaccgtcga tggccgcggc aacggcccac tggccgctta cgccaacgcg

1620

ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccgcacctcg

1680

ggcgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg

1740

ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta

1800

aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a

1851

<210> 5

<211> 616

<212>PRT

<213> Artificial Sequence

<220>

<223> LeuA P247C R558H G561D AA (616 amino acids)

<400> 5

Met Ser Pro Asn Asp Ala Phe Ile Ser Ala Pro Ala Lys Ile Glu Thr

1 5 10 15

Pro Val Gly Pro Arg Asn Glu Gly Gln Pro Ala Trp Asn Lys Gln Arg

20 25 30

Gly Ser Ser Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val

35 40 45

Glu Asp Ile Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr

50 55 60

Val Ala Pro Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala

65 70 75 80

Leu Ile Asp Pro Met Ser Pro Glu Arg Lys Arg Arg Met Phe Glu Leu

85 90 95

Leu Val Gln Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala

100 105 110

Ser Gln Thr Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Gly Met

115 120 125

Ile Pro Asp Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His

130 135 140

Leu Ile Arg Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile

145 150 155 160

Val His Phe Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe

165 170 175

Arg Met Asp Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu

180 185 190

Leu Ile Lys Thr Ile Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp

195 200 205

Gln Tyr Ser Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys

210 215 220

Glu Val Val Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn

225 230 235 240

Pro Met Ile Ile Asn Leu Cys Ser Thr Val Glu Met Ile Thr Pro Asn

245 250 255

Val Tyr Ala Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg

260 265 270

Asp Ser Ile Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly

275 280 285

Val Gly Ala Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu

290 295 300

Gly Cys Leu Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val

305 310 315 320

Thr Leu Ala Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp

325 330 335

Phe Thr Asp Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln

340 345 350

Leu Arg Val Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr

355 360 365

Ala Phe Ser Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala

370 375 380

Met Ala Ala Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp

385 390 395 400

Glu Gln Leu Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp

405 410 415

Pro Lys Asp Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser

420 425 430

Gln Ser Gly Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly

435 440 445

Leu Gln Ile Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln

450 455 460

Asn Val Thr Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp

465 470 475 480

Asp Ile Phe Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln

485 490 495

Ile Ala Leu Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser

500 505 510

Ile Thr Ala Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly

515 520 525

Arg Gly Asn Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu

530 535 540

Gly Ile Asp Val Glu Ile Gln Glu Tyr Asn Gln His Ala His Thr Ser

545 550 555 560

Asp Asp Asp Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly

565 570 575

Arg Lys Val Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser

580 585 590

Leu Lys Ala Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His

595 600 605

Glu Ala Val Leu Ala Gly Gly Val

610 615

<210> 6

<211> 1851

<212> DNA

<213> Artificial Sequence

<220>

<223> LeuA P247C R558H G561D NT

<400> 6

atgtctccta acgatgcatt catctccgca cctgccaaga tcgaaacccc agttgggcct

60

cgcaacgaag gccagccagc atggaataag cagcgtggct cctcaatgcc agttaaccgc

120

tacatgcctt tcgaggttga ggtagaagat atttctctgc cggaccgcac ttggccagat

180

aaaaaaatca ccgttgcacc tcagtggtgt gctgttgacc tgcgtgacgg caaccaggct

240

ctgattgatc cgatgtctcc tgagcgtaag cgccgcatgt ttgagctgct ggttcagatg

300

ggcttcaaag aaatcgaggt cggtttccct tcagcttccc agactgattt tgatttcgtt

360

cgtgagatca tcgaaaaggg catgatccct gacgatgtca ccattcaggt tctggttcag

420

gctcgtgagc acctgattcg ccgtactttt gaagcttgcg aaggcgcaaa aaacgttatc

480

gtgcacttct acaactccac ctccatcctg cagcgcaacg tggtgttccg catggacaag

540

gtgcaggtga agaagctggc taccgatgcc gctgaactaa tcaagaccat cgctcaggat

600

tacccagaca ccaactggcg ctggcagtac tcccctgagt ccttcaccgg cactgaggtt

660

gagtacgcca aggaagttgt ggacgcagtt gttgaggtca tggatccaac tcctgagaac

720

ccaatgatca tcaacctgtg ttccaccgtt gagatgatca cccctaacgt ttacgcagac

780

tccattgaat ggatgcaccg caatctaaac cgtcgtgatt ccattatcct gtccctgcac

840

ccgcacaatg accgtggcac cggcgttggc gcagctgagc tgggctacat ggctggcgct

900

gaccgcatcg aaggctgcct gttcggcaac ggcgagcgca ccggcaacgt ctgcctggtc

960

accctggcac tgaacatgct gacccagggc gttgaccctc agctggactt caccgatata

1020

cgccagatcc gcagcaccgt tgaatactgc aaccagctgc gcgttcctga gcgccaccca

1080

tacggcggtg acctggtctt caccgctttc tccggttccc accaggacgc tgtgaacaag

1140

ggtctggacg ccatggctgc caaggttcag ccaggtgcta gctccactga agtttcttgg

1200

gagcagctgc gcgacaccga atgggaggtt ccttacctgc ctatcgatcc aaaggatgtc

1260

ggtcgcgact acgaggctgt tatccgcgtg aactcccagt ccggcaaggg cggcgttgct

1320

tacatcatga agaccgatca cggtctgcag atccctcgct ccatgcaggt tgagttctcc

1380

accgttgtcc agaacgtcac cgacgctgag ggcggcgagg tcaactccaa ggcaatgtgg

1440

gatatcttcg ccaccgagta cctggagcgc accgcaccag ttgagcagat cgcgctgcgc

1500

gtcgagaacg ctcagaccga aaacgaggat gcatccatca ccgccgagct catccacaac

1560

ggcaaggacg tcaccgtcga tggccgcggc aacggcccac tggccgctta cgccaacgcg

1620

ctggagaagc tgggcatcga cgttgagatc caggaataca accagcacgc ccacacctcg

1680

gatgacgatg cagaagcagc cgcctacgtg ctggctgagg tcaacggccg caaggtctgg

1740

ggcgtcggca tcgctggctc catcacctac gcttcgctga aggcagtgac ctccgccgta

1800

aaccgcgcgc tggacgtcaa ccacgaggca gtcctggctg gcggcgttta a

1851

<210> 7

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> pCR-leuA_F

<400> 7

ctaatctcga ggtcacccat gtctcctaac

thirty

<210> 8

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> pCR-leuA_R

<400> 8

ggctggcggc gtttaaaacc ggttgat

27

<210> 9

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> leuA_sequencing_F

<400> 9

aacacgaccg gcatcccgtc gc

22

<210> 10

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> leuA_sequencing_R

<400> 10

aaatcatttg agaaaactcg agg

23

<210> 11

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> leuA_P247C_Up_F

<400> 11

gtgaattcga gctcggtacc caaatcattt gagaaaactc gaggc

45

<210> 12

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> leuA_P247C_Up_R

<400> 12

ggtgatcatc tcaacggtgg aacacaggtt gatgatcatt gggtt

45

<210> 13

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> leuA_P247C_Down_F

<400> 13

aacccaatga tcatcaacct gtgttccacc gttgagatga tcacc

45

<210> 14

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> leuA_P247C_Down_R

<400> 14

ggtcgactct agaggatccc caagaaggca acatcggaca gc

42

<210> 15

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> leuA_sequencing_R

<400> 15

atccattcaa tggagtctgc g

21

<210> 16

<211> 581

<212>PRT

<213> Artificial Sequence

<220>

<223> LeuA AA (581 amino acids)

<400> 16

Met Pro Val Asn Arg Tyr Met Pro Phe Glu Val Glu Val Glu Asp Ile

1 5 10 15

Ser Leu Pro Asp Arg Thr Trp Pro Asp Lys Lys Ile Thr Val Ala Pro

20 25 30

Gln Trp Cys Ala Val Asp Leu Arg Asp Gly Asn Gln Ala Leu Ile Asp

35 40 45

Pro Met Ser Pro Glu Arg Lys Arg Arg Met Phe Glu Leu Leu Val Gln

50 55 60

Met Gly Phe Lys Glu Ile Glu Val Gly Phe Pro Ser Ala Ser Gln Thr

65 70 75 80

Asp Phe Asp Phe Val Arg Glu Ile Ile Glu Lys Gly Met Ile Pro Asp

85 90 95

Asp Val Thr Ile Gln Val Leu Val Gln Ala Arg Glu His Leu Ile Arg

100 105 110

Arg Thr Phe Glu Ala Cys Glu Gly Ala Lys Asn Val Ile Val His Phe

115 120 125

Tyr Asn Ser Thr Ser Ile Leu Gln Arg Asn Val Val Phe Arg Met Asp

130 135 140

Lys Val Gln Val Lys Lys Leu Ala Thr Asp Ala Ala Glu Leu Ile Lys

145 150 155 160

Thr Ile Ala Gln Asp Tyr Pro Asp Thr Asn Trp Arg Trp Gln Tyr Ser

165 170 175

Pro Glu Ser Phe Thr Gly Thr Glu Val Glu Tyr Ala Lys Glu Val Val

180 185 190

Asp Ala Val Val Glu Val Met Asp Pro Thr Pro Glu Asn Pro Met Ile

195 200 205

Ile Asn Leu Pro Ser Thr Val Glu Met Ile Thr Pro Asn Val Tyr Ala

210 215 220

Asp Ser Ile Glu Trp Met His Arg Asn Leu Asn Arg Arg Asp Ser Ile

225 230 235 240

Ile Leu Ser Leu His Pro His Asn Asp Arg Gly Thr Gly Val Gly Ala

245 250 255

Ala Glu Leu Gly Tyr Met Ala Gly Ala Asp Arg Ile Glu Gly Cys Leu

260 265 270

Phe Gly Asn Gly Glu Arg Thr Gly Asn Val Cys Leu Val Thr Leu Ala

275 280 285

Leu Asn Met Leu Thr Gln Gly Val Asp Pro Gln Leu Asp Phe Thr Asp

290 295 300

Ile Arg Gln Ile Arg Ser Thr Val Glu Tyr Cys Asn Gln Leu Arg Val

305 310 315 320

Pro Glu Arg His Pro Tyr Gly Gly Asp Leu Val Phe Thr Ala Phe Ser

325 330 335

Gly Ser His Gln Asp Ala Val Asn Lys Gly Leu Asp Ala Met Ala Ala

340 345 350

Lys Val Gln Pro Gly Ala Ser Ser Thr Glu Val Ser Trp Glu Gln Leu

355 360 365

Arg Asp Thr Glu Trp Glu Val Pro Tyr Leu Pro Ile Asp Pro Lys Asp

370 375 380

Val Gly Arg Asp Tyr Glu Ala Val Ile Arg Val Asn Ser Gln Ser Gly

385 390 395 400

Lys Gly Gly Val Ala Tyr Ile Met Lys Thr Asp His Gly Leu Gln Ile

405 410 415

Pro Arg Ser Met Gln Val Glu Phe Ser Thr Val Val Gln Asn Val Thr

420 425 430

Asp Ala Glu Gly Gly Glu Val Asn Ser Lys Ala Met Trp Asp Ile Phe

435 440 445

Ala Thr Glu Tyr Leu Glu Arg Thr Ala Pro Val Glu Gln Ile Ala Leu

450 455 460

Arg Val Glu Asn Ala Gln Thr Glu Asn Glu Asp Ala Ser Ile Thr Ala

465 470 475 480

Glu Leu Ile His Asn Gly Lys Asp Val Thr Val Asp Gly Arg Gly Asn

485 490 495

Gly Pro Leu Ala Ala Tyr Ala Asn Ala Leu Glu Lys Leu Gly Ile Asp

500 505 510

Val Glu Ile Gln Glu Tyr Asn Gln His Ala Arg Thr Ser Gly Asp Asp

515 520 525

Ala Glu Ala Ala Ala Tyr Val Leu Ala Glu Val Asn Gly Arg Lys Val

530 535 540

Trp Gly Val Gly Ile Ala Gly Ser Ile Thr Tyr Ala Ser Leu Lys Ala

545 550 555 560

Val Thr Ser Ala Val Asn Arg Ala Leu Asp Val Asn His Glu Ala Val

565 570 575

Leu Ala Gly Gly Val

580

<---

Claims (16)

1. A mutated polypeptide having isopropyl malate synthase activity, containing the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 16, where the proline at position 247 or 212, respectively, is replaced by cysteine. 2. Mutated polypeptide according to claim 1, containing the amino acid sequence SEQ ID NO: 3. 3. Mutated polypeptide having isopropyl malate synthase activity, containing the amino acid sequence SEQ ID NO: 1, (1) where the proline at position 247 is replaced by cysteine; And (2) where the arginine at position 558 is replaced by histidine, and/or where the glycine at position 561 is replaced by aspartic acid. 4. Mutated polypeptide according to claim 3, containing the amino acid sequence SEQ ID NO: 5. 5. A polynucleotide encoding a mutated polypeptide according to any one of claims. 1-4. 6. An expression vector containing the polynucleotide according to claim 5. 7. A microorganism having the ability to produce L-leucine, containing at least one selected from the group consisting of a mutated polypeptide according to any one of paragraphs. 1-4, a polynucleotide encoding the mutated polypeptide, and a vector containing the polynucleotide. 8. The microorganism according to claim 7, which is a microorganism of the genus Corynebacterium. 9. The microorganism according to claim 8, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum. 10. A method for producing L-leucine, including the stage of cultivating the microorganism according to claim 7 in a medium. 11. The method for producing L-leucine according to claim 10, further comprising the step of collecting L-leucine from a culture medium or microorganism. 12. A composition for producing L-leucine, containing at least one selected from the group consisting of a mutated polypeptide according to any one of paragraphs. 1-4, a polynucleotide encoding the mutated polypeptide, a vector containing the polynucleotide, and a microorganism containing the polypeptide, the polynucleotide, or the vector; and excipient. 13. The use of at least one selected from the group consisting of a mutated polypeptide according to any one of paragraphs. 1-4, a polynucleotide encoding the mutated polypeptide, a vector containing the polynucleotide, and a microorganism containing the polypeptide, the polynucleotide, or the vector to produce L-leucine.