RU2831447C1 - Mutant atp-dependent protease and method of producing l-amino acid using same - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a polypeptide involved in the production of a branched-chain amino acid, containing deletion of amino acids corresponding to positions 431–433 based on amino acid sequence presented by SEQ ID NO: 5, where the branched chain amino acid is L-valine or L-isoleucine. Also disclosed is a microorganism of the genus Corynebacterium for producing said branched-chain amino acids, in which activity of ATP (adenosine triphosphate)-dependent protease is weakened in comparison with unmodified microorganism, wherein said microorganism contains said polypeptide or polynucleotide having a deletion of nucleotides corresponding to positions 1,291–1,299 based on the sequence presented by SEQ ID NO: 6. Also disclosed is a method of producing said branched-chain amino acids using said microorganism, as well as the use of a microorganism or polypeptide for producing said branched-chain amino acids.

EFFECT: invention enables to obtain L-valine or L-isoleucine with high output.

11 cl, 11 tbl, 4 ex

Description

1. Field of technology

The present invention relates to a variant of ATP (adenosine triphosphate)-dependent protease and a method for producing L-amino acids using it.

2. Description of the prior art

L-amino acids are the basic building blocks of proteins and have various important applications, such as pharmaceutical raw materials and food additives, animal feed, nutrients, pesticides, bactericidal agents and so on. In particular, branched-chain amino acids (BCAAs) collectively include L-valine, L-leucine and L-isoleucine, which are essential amino acids, and branched-chain amino acids are also known to have antioxidant effects and direct protein synthesis promotion effects in muscle cells.

However, the production of branched-chain amino acids using microorganisms is mainly carried out using microorganisms of the genus Escherichia or microorganisms of the genus Corynebacterium, and it is also known that branched-chain amino acids are obtained by biosynthesis from pyruvic acid in several stages using 2-ketoisocaproate as a precursor (US Patent No. 9885093, US Patent No. 10351859, US Patent No. 8465962). However, there is a problem in the production of branched-chain amino acids using microorganisms, which is that industrial large-scale production is difficult.

However, with the increasing demand for branched chain amino acids, there remains a need to find ways to efficiently produce branched chain amino acids.

The present inventors have made significant efforts to increase the possibilities of producing branched chain amino acids, and as a result, they have developed a method for producing branched chain amino acids in high concentrations using a variant of ATP-dependent protease, which became the basis of the present invention.

SUMMARY OF THE INVENTION

The aim of the present invention is to provide a microorganism of the genus Corynebacterium, in which the activity of the ATP-dependent protease is weakened compared to an unmodified microorganism, preferably a microorganism of the genus Corynebacterium, having the ability to produce branched-chain amino acids.

Another object of the present invention is to provide a ClpC variant in which the amino acid sequence corresponding to positions 431-433 based on the amino acid sequence represented by SEQ ID NO: 5 is deleted.

Another object of the present invention is to provide a polynucleotide encoding a ClpC variant according to the present invention.

Another object of the present invention is to provide a method for producing branched chain amino acids comprising the step of culturing in a medium a microorganism of the genus Corynebacterium according to the present invention or a microorganism of the genus Corynebacterium containing any one or more of the ClpC variants according to the present invention, or a polynucleotide according to the present invention, and a vector containing it.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention will be described in detail below. However, each description and embodiment disclosed in the present invention can also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in the present invention are included in the scope of the present invention. In addition, the scope of the present invention is not limited to the specific description provided below. Also, those skilled in the art will recognize or be able to ascertain by no more than routine experimental work many equivalents to the specific embodiments of the present invention described herein. In turn, these equivalents should be considered as included in the scope of the present invention.

In one aspect of the present invention, a microorganism of the genus Corynebacterium is provided, in which the activity of the ATP-dependent protease is weakened compared to an unmodified microorganism.

In this document, the term "ATP-dependent protease (EC 3.4.21.92)" refers to an enzyme that hydrolyzes protein to produce small peptides in the presence of ATP and Mg2+. The term "ATP-dependent protease" according to the present invention can be used interchangeably with the terms Clp endopeptidase, ATP-dependent Clp protease, ClpP or Clp protease.

The attenuation of the ATP-dependent protease according to the present invention may be an attenuation of the activity of the ATP-binding subunit of the ATP-dependent Clp protease.

The term "ATP-binding subunit of ATP-dependent Clp protease" may be used interchangeably with the term "AAA family ATPase" or "ClpC". In the present invention, the sequence of the ATP-binding subunit of ATP-dependent Clp protease can be obtained from GenBank of NCBI, which is a known database. Specifically, said subunit may be a polypeptide having the activity of the ATP-binding subunit of ATP-dependent Clp protease encoded by clpC, but not limited thereto.

The ATP-binding subunit of the ATP-dependent Clp protease according to the present invention may be derived from the genus Corynebacterium. For example, the ATP-binding subunit of the ATP-dependent Clp protease according to the present invention may be derived from Corynebacterium glutamicum.

The ATP-binding subunit of the ATP-dependent Clp protease according to the present invention may comprise a polypeptide represented by SEQ ID NO: 5 or an amino acid sequence having 90% or more identity thereto.

For example, an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 5 according to the present invention may be an amino acid sequence having at least 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 96.26% or more, 97% or more, 97.5% or more, 97.7% or more, 97.8% or more, 98% or more, 98.5% or more, 98.7% or more, 98.8% or more, 99% or more, 99.5% or more, 99.7% or more, 99.8% or more, or less than 100% homology or identity to the amino acid sequence of SEQ ID NO: 5 according to the present invention. Furthermore, it is obvious that proteins having amino acid sequences in which some sequences are deleted, modified, replaced or added are also included within the scope of the protein to be attenuated in the present invention, provided that said amino acid sequences have the said homology or identity and exhibit an activity identical to or corresponding to the activity of the ATP-binding subunit of the ATP-dependent Clp protease.

In this case, the ATP-binding subunit of the ATP-dependent Clp protease according to the present invention may have a polypeptide represented by the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having 90% or more identity thereto, or may consist or essentially consist of said polypeptide. The ATP-binding subunit of the ATP-dependent Clp protease according to the present invention may include subunits having an addition of an insignificant sequence before or after SEQ ID NO: 5 or an amino acid sequence having 90% or more identity thereto (i.e., an addition of a sequence that does not change the function of the protein, from the N-terminus and/or C-terminus of the amino acid sequence), or a natural mutation, a silent mutation or a conservative substitution.

"Conservative substitution" means the replacement of one amino acid with another amino acid having similar structural and/or chemical properties. Such amino acid substitution may typically occur based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic character of the residues. Typically, a conservative substitution may have little or no effect on the activity of proteins or polypeptides.

The microorganism according to the present invention may comprise a polypeptide having a deletion of amino acids corresponding to positions 431-433 based on the sequence presented by SEQ ID NO: 5, or a polynucleotide having a deletion of nucleotides corresponding to positions 1291-1299 based on the sequence presented by SEQ ID NO: 6.

As used herein, the term "corresponding" refers to amino acid residues or nucleotide residues at positions designated in a polypeptide or polynucleotide, or amino acid residues or nucleotide residues that are similar, identical, or homologous to residues designated in a polypeptide or polynucleotide. Identification of an amino acid or nucleotide at a corresponding position may be the determination of a specific amino acid or nucleotide in a sequence that is assigned to the specific sequence. As used herein, the term "corresponding region" generally refers to a similar or corresponding position in a related protein or reference protein.

For example, an arbitrary amino acid sequence is aligned with SEQ ID NO: 5 or SEQ ID NO: 6, and based on this, each amino acid residue of said amino acid sequence can be numbered relative to the amino acid residue corresponding to the amino acid residue of SEQ ID NO: 6, or the numbered position of the nucleotide residue corresponding to the nucleotide residue of SEQ ID NO: 6. For example, the sequence alignment algorithm described in the present invention can determine the position of an amino acid or the position at which a modification such as a substitution, insertion or deletion occurs by comparison with a position in a query sequence (also referred to as a "reference sequence").

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

In this document, the term "attenuation" of the activity of a polypeptide is a concept that includes both the case where the activity is reduced compared to the endogenous activity and the case where the activity is absent. The term "attenuation" may be used interchangeably with terms such as inactivation, deficiency, down-regulation, reduction, decrease, attenuation, etc.

Attenuation may also include the case where the activity of the polypeptide as such is reduced or eliminated due to a variation in the polynucleotide encoding said polypeptide, etc., compared to the activity of said polypeptide originally inherent in the microorganism, the case where the overall level of polypeptide activity and/or the concentration (expression level) in the cell is low due to inhibition of expression of the gene of the polynucleotide encoding the polypeptide or due to inhibition of translation into the polypeptide, compared to those of the natural strain, the case where the polynucleotide is not expressed at all, and/or the case where the activity of the polypeptide is absent even if the polynucleotide is expressed. "Endogenous activity" means the activity of a particular polypeptide originally inherent in the parent strain before the change in the trait, or in a wild-type microorganism or an unmodified microorganism, in the case where the trait is changed as a result of genetic variability caused by natural or artificial factors. The term "endogenous activity" may be used interchangeably with the term "activity before modification". The fact that the activity of a polypeptide is "inactivated, deficient, reduced, down-regulated, diminished, or attenuated" compared to the endogenous activity means that the activity of the polypeptide is lower compared to the activity of the particular polypeptide originally present in the parent strain prior to the trait change or in the unmodified microorganism.

Such attenuation of the activity of the polypeptide can be accomplished by any method known in the art, but the method is not limited thereto, and the attenuation can be achieved by using various methods well known in the art (e.g., Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).

In particular, the weakening of the activity of the polypeptide according to the present invention may be:

1) complete or partial deletion of the gene encoding the polypeptide;

2) modification of the expression regulatory region (or expression regulatory sequence) to reduce the expression of the gene encoding the polypeptide;

3) modification of the amino acid sequence constituting the polypeptide to eliminate or weaken the activity of the polypeptide (e.g. deletion/replacement/addition of one or more amino acids in the amino acid sequence);

4) modification of the sequence of a gene encoding a polypeptide to eliminate or weaken the activity of the polypeptide (e.g. deletion/replacement/addition of one or more nucleic acid bases in the nucleic acid base sequence of a polypeptide gene encoding a polypeptide that has been modified to eliminate or weaken the activity of the polypeptide);

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

6) introduction of an antisense oligonucleotide (eg, antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide;

7) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding the polypeptide to form a secondary structure to which the ribosome cannot attach;

8) addition of a reverse transcribed promoter to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (reverse transcription engineering (RTE)); or

9) a combination of two or more approaches selected from 1) - 8), but without being specifically limited to these. For example,

1) a partial or complete deletion of a gene encoding a polypeptide may be a complete removal of the polynucleotide encoding the original polypeptide of interest from the chromosome, a substitution with a polynucleotide in which some nucleotides are deleted, or a substitution with a marker gene.

In addition, 2) the modification of the expression regulatory region (or expression regulatory sequence) may be a deletion, an insertion, a non-conservative or conservative substitution, or the occurrence of a variation in the expression regulatory region (or expression regulatory sequence) due to a combination thereof, or a substitution with a sequence exhibiting weaker activity. The expression regulatory region comprises a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating termination of transcription and translation, but is not limited to this.

Also 3) and 4) the modification of the amino acid sequence or polynucleotide sequence may be a deletion, insertion or non-conservative or conservative substitution in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, or the occurrence of a variation in the sequence due to a combination thereof, or a substitution with an amino acid sequence or polynucleotide sequence modified to provide a weaker activity, or with an amino acid sequence or polynucleotide sequence modified to provide a lack of activity, as a result of which the activity of the polypeptide is weakened, but not limited to this. For example, gene expression may be inhibited or weakened by introducing a variation in the polynucleotide sequence and forming a stop codon, but not limited to this.

Also 5) modification of the initiation codon of the transcript of the gene encoding the polypeptide, or the base sequence encoding the 5'-UTR region, may be, for example, a substitution with a base sequence encoding another initiation codon that provides a lower rate of expression of the polypeptide compared to the original initiation codon, but is not limited to this.

Also, with regard to 6) the introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of a gene encoding a polypeptide, reference may be made to documents such as [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews -Trends in Genetics, Vol. 1(1) 1986].

Also 7) the addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide to form a secondary structure to which a ribosome cannot be attached may be to make translation of mRNA (messenger RNA) impossible or to slow down the rate of translation of mRNA.

Also 8) addition of a reverse transcribed promoter to the 3' end of the open reading frame (ORF) of the polypeptide-encoding gene sequence (reverse transcription engineering (RTE)) can be used to attenuate activity by creating an antisense nucleotide complementary to the polypeptide-encoding gene transcript.

The microorganism according to the present invention may have the ability to produce a branched-chain amino acid. In particular, the branched-chain amino acid according to the present invention may be L-valine or L-isoleucine.

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

In particular, the strain according to the present invention may be a microorganism for obtaining which a parent strain lacking the ability to produce branched-chain amino acids is given the ability to produce branched-chain amino acids, or a microorganism for obtaining which an attenuated ATP-dependent protease according to the present invention or a polynucleotide encoding it (or a vector containing such a polynucleotide) is introduced into a microorganism having the ability to produce branched-chain amino acids instead of a natural ATP-dependent protease, or a natural ATP-dependent protease is modified to obtain an attenuated ATP-dependent protease according to the present invention, but not limited thereto.

In the present invention, a microorganism that expresses a variant of the ATP-binding subunit of the ATP-dependent Clp protease and produces a branched-chain amino acid may be a microorganism characterized in that it includes a polynucleotide according to the present invention and expresses a variant of the ATP-binding subunit of the ATP-dependent Clp protease, and thus increases its ability to produce a branched-chain amino acid. In particular, in the present invention, a microorganism expressing a variant of the ATP-binding subunit of the ATP-dependent Clp protease and producing a branched-chain amino acid, or a microorganism having the ability to express a variant of the ATP-binding subunit of the ATP-dependent Clp protease and the ability to produce a branched-chain amino acid, may be a microorganism in which the activity of the ATP-binding subunit of the ATP-dependent Clp protease is weakened, and some genes of the branched-chain amino acid biosynthesis pathway are enhanced or weakened, or the activity of the ATP-binding subunit of the ATP-dependent Clp protease is weakened, and some genes of the branched-chain amino acid degradation pathway are enhanced or weakened.

The microorganism of the genus Corynebacterium according to the present invention may be a microorganism further having an enhanced activity of the small subunit of acetolactate synthase isoenzyme 1, an enhanced activity of aspartokinase, a reduced activity of homoserine dehydrogenase and/or an enhanced activity of biosynthetic IlvA L-threonine dehydratase.

As used herein, the term "enhancement" of the activity of a polypeptide means that the activity of the polypeptide is increased compared to its original activity. The term "enhancement" may be used interchangeably with terms such as activation, up-regulation, overexpression, enhancement, etc. As used herein, activation, enhancement, up-regulation, overexpression, and enhancement may include both the expression of an activity that was not originally present and the expression of an improved activity compared to the original activity or the activity before modification. The term "original activity" means the activity of a particular polypeptide originally present in the parent strain before the change in the trait or in the unmodified microorganism, in the case where the trait is changed by genetic variation due to natural or artificial factors. It may be used interchangeably with the term "activity before modification". The fact that the activity of a polypeptide is "enhanced," "up-regulated," "overexpressed," or "increased" compared to the original activity means that the activity of the polypeptide is improved compared to the activity and/or concentration (expression level) of the particular polypeptide originally present in the parent strain prior to the trait change or in the unmodified microorganism.

The enhancement may be achieved by introducing a foreign polypeptide or enhancing the initial activity and/or concentration (expression level) of the polypeptide. The enhancement of the activity of the polypeptide may be confirmed by increasing the degree of activity and expression level of the corresponding polypeptide or the amount of product obtained by means of the corresponding polypeptide.

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

In particular, the enhancement of the activity of the polypeptide according to the present invention may be:

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

2) replacement of the region of gene expression regulation in the chromosome encoding the polypeptide with a sequence exhibiting strong activity;

3) modification of the initiation codon of the transcript of the gene encoding the polypeptide, or the base sequence encoding the 5'-UTR;

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

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

6) the introduction of a foreign polypeptide exhibiting the activity of the said polypeptide, or a foreign polynucleotide encoding the said polypeptide;

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

8) analysis of the tertiary structure of the polypeptide to select the site to be targeted and to perform modification or chemical modification of the site to be targeted; or

9) a combination of two or more approaches selected from 1) - 8), but without any specific limitation to these.

Such an increase in the activity of the polypeptide may be an increase in the activity or concentration of the expression level of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild-type strain of the microorganism or the strain of the microorganism before modification, or an increase in the amount of a product obtained by means of the corresponding polypeptide, but is not limited to this.

In the microorganism of the present invention, partial or complete modification of a polynucleotide can be caused by (a) homologous recombination using a vector for insertion into a chromosome in the microorganism or genome editing using an engineered nuclease (e.g., CRISPR-Cas9) and/or (b) treatment with radiation, such as ultraviolet radiation and radioactive radiation, and/or chemicals, but not limited thereto. The method of partially or completely modifying a gene can include a method using DNA recombination technology. For example, by introducing a nucleotide sequence or a vector containing a nucleotide sequence homologous to a gene of interest into a microorganism, which causes homologous recombination, the gene can be partially or completely deleted. The introduced nucleotide sequence or vector can include a dominant selectable marker, but not limited thereto.

The microorganism of the genus Corynebacterium according to the present invention 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.

In another aspect of the present invention, there is provided a ClpC variant in which the amino acid sequence corresponding to positions 431-433 based on the amino acid sequence represented by SEQ ID NO: 5 is deleted.

The ClpC variant may be a variant of the ATP-binding subunit of the ATP-dependent Clp protease. "ClpC" or "ATP-binding subunit of the ATP-dependent Clp protease" is as described above.

For example, a ClpC variant of the present invention may comprise a polypeptide represented by the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 90% or more identity thereto.

A ClpC variant according to the present invention may have a polypeptide represented by the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 90% or more identity thereto, as described above, or may consist or consist essentially of said polypeptide. In particular, a ClpC variant according to the present invention may comprise an amino acid sequence having 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity with the amino acid sequence represented by SEQ ID NO: 1. Furthermore, it is obvious that variants having amino acid sequences in which some sequences are deleted, modified, replaced, conservatively replaced or added are also included within the scope of the present invention, provided that said amino acid sequences have said homology or identity and exhibit an efficacy corresponding to the efficacy of a ClpC variant according to the present invention.

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

In addition, the variant may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence that is co- or post-translationally involved in protein translocation may be conjugated to the N-terminus of the variant. The variant may be conjugated to other sequences or linkers in a manner that allows it to be identified, purified, or synthesized.

As used herein, the term "homology" or "identity" means the degree of similarity between two given amino acid sequences or base sequences and may be expressed as a percentage. The terms "homology" and "identity" may often be used interchangeably.

Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, and may be combined with a default gap penalty imposed by the program in use. As such, homologous or identical sequences are generally capable of hybridizing to all or part of the sequence under moderately stringent or very stringent conditions. Hybridization obviously also includes hybridization of a polynucleotide to a polynucleotide containing a common codon or a codon with respect to codon degeneracy.

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

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

As one example of the present invention, a variant according to the present invention may have an activity of an ATP-binding subunit of an ATP-dependent Clp protease. In addition, a variant according to the present invention may have an activity that increases the production of branched-chain amino acids compared to a wild-type polypeptide having an activity of an ATP-binding subunit of an ATP-dependent Clp protease.

In another aspect, the present invention provides a polynucleotide encoding a ClpC variant of the present invention.

"ClpC variant" and "ATP-dependent Clp protease ATP-binding subunit variant" are as described above.

As used herein, the term "polynucleotide" is a DNA or RNA chain of a given or greater length, which is a polymer of nucleotides in which the nucleotide monomers are linked into a long chain by covalent bonds, and more specifically, it means a polynucleotide fragment encoding the said variant.

A polynucleotide encoding a ClpC variant according to the present invention may comprise a base sequence encoding a polypeptide represented by the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 90% or more identity thereto. As an example of the present invention, a polynucleotide according to the present invention may comprise or include a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having 90% or more identity thereto. Furthermore, a polynucleotide according to the present invention may consist of or essentially consist of a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 2.

In the polynucleotide of the present invention, various modifications may be made in the coding region, provided that the amino acid sequence of the variant of the present invention is not changed, taking into account the degeneracy of codons or the preferred codons in organisms intended to express the variant of the present invention.

In addition, the polynucleotide of the present invention may include a probe that can be derived from a known gene sequence, such as a sequence without limitation, as long as it is a sequence that can hybridize with a complementary sequence in whole or in part to the polynucleotide sequence of the present invention under stringent conditions. "Stringent conditions" mean conditions that ensure specific hybridization between polynucleotides. These conditions are described in detail in the documents (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). Examples thereof include conditions under which polynucleotides having a higher homology or identity, namely 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, hybridize with each other, while polynucleotides having a lower homology or identity do not hybridize with each other, or washing conditions for conventional Southern hybridization in which washing is performed once, specifically two to three times, at a salt concentration and temperature equivalent to 60°C, 1X SSC, 0.1% SDS, specifically 60°C, ODX SSC, 0.1% SDS, more specifically 68°C, 0.1X SSC, 0.1% SDS.

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

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

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

In another aspect of the present invention, there is provided a vector comprising a polynucleotide according to the present invention.

The said vector may be an expression vector for providing expression of the polynucleotide in host cells, but is not limited thereto.

The vector of the present invention refers to a DNA construct comprising a polynucleotide sequence of interest operably linked to a suitable regulatory sequence such that the gene of interest can be introduced into a suitable host. The regulatory sequence may comprise a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding a suitable ribosome binding site in mRNA, and a sequence regulating termination of transcription and translation. The vector can be transformed into a suitable host cell and then replicated or operated independently of the host genome, or can be integrated into the genome itself. For example, the polynucleotide of interest in a chromosome can be replaced by a modified polynucleotide using a vector for intracellular insertion into the chromosome. The insertion of the polynucleotide into the chromosome can be accomplished by any method known in the art, such as, but not limited to, homologous recombination.

The vector used in the present invention is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors may include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, or the like can be used as the phage vector or the cosmid vector. As the plasmid vector, the pDZ system, the pBR system, the pUC system, the pBluescript II system, the pGEM system, the pTZ system, the pCL system, the pET system, or the like can be used. In particular, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC or the like vectors can be used.

For example, a polynucleotide encoding a polypeptide of interest can be inserted into a chromosome using a vector for intracellular insertion into a chromosome. The polynucleotide can be inserted into a chromosome by any method known in the art, such as, but not limited to, homologous recombination. The vector can further comprise a selectable marker for detecting the insertion into the chromosome. The selectable marker is intended for selecting cells transformed by the vectors, i.e., for detecting the insertion of the nucleic acid molecule of interest, and markers can be used that provide selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface polypeptides. In a medium treated with a selectable agent, only cells expressing the selectable marker survive or exhibit other phenotypic characteristics, and thus transformed cells can be selected.

As used herein, the term "transformation" means that a vector containing a polynucleotide encoding a protein of interest is introduced into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide can be localized by insertion into a chromosome of the host cell or localized outside the chromosome, provided that it can be expressed in the host cell. In this case, the polynucleotide includes DNA and RNA encoding the protein of interest. The polynucleotide can be introduced in any form, provided that it can be introduced into the host cell and then expressed. For example, the polynucleotide can be introduced into the host cell as an expression cassette, which is a genetic construct containing all the components necessary for independent expression. The expression cassette can typically contain 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 an expression vector capable of self-replication. In addition, the polynucleotide may be introduced into the host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited to this.

The method for transforming the vector according to the present invention includes any method for introducing a nucleic acid into a cell, and can be carried out by selecting an appropriate standard technique known in the art depending on the host cell. For example, the method may include electroporation, calcium phosphate ( _CaPO4 ) precipitation, calcium chloride ( _CaCl2 ) precipitation, microinjection, a polyethylene glycol (PEG) method, a DEAE-dextran method, a cationic liposome method, and a lithium acetate-DMSO method, etc., but not limited thereto.

In this document, the term “operably linked” means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the protein of interest according to the present invention.

In another aspect, the present invention provides a strain of the genus Corynebacterium comprising any one or more of the variants according to the present invention, a polynucleotide according to the present invention, or a vector according to the present invention.

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

For example, the strain of the present invention is a cell or a microorganism transformed with a vector containing a polynucleotide of the present invention or a polynucleotide encoding a variant ClpC of the present invention, and expresses the variant ClpC of the present invention. For the purposes of the present invention, the strain of the present invention may include all microorganisms having the ability to produce branched-chain amino acids due to the inclusion of the variant ClpC of the present invention. For example, the strain of the present invention may be a recombinant strain for which a polynucleotide encoding a variant ClpC of the present invention is introduced into a microorganism producing branched-chain amino acids to express the variant ClpC, thereby enhancing the ability to produce branched-chain amino acids. The recombinant strain having an enhanced ability to produce branched-chain amino acids may be, but is not limited to, a microorganism having an enhanced ability to produce branched-chain amino acids compared with a natural wild-type microorganism or a microorganism without ClpC modification (that is, a microorganism expressing wild-type aldehyde dehydrogenase (SEQ ID NO: 5) or a microorganism that does not express the modified protein (SEQ ID NO: 1)). For example, the microorganism without ClpC modification, which is a target strain in the comparison of the increase in the ability to produce branched-chain amino acids, may be, but is not limited to, the strain CA08-0072 (KCCM11201P, US 8465962 B2) or KCJI-38 (KCCM11248P, Korean Patent No. 10-1335789).

As used herein, the term "unmodified microorganism" does not exclude strains containing mutations that may naturally occur in microorganisms and may refer to a wild-type strain or a natural strain per se, or may refer to a strain before a trait has been altered by genetic variation due to natural or artificial factors. For example, an unmodified microorganism may be a strain into which a ClpC variant described herein has not been introduced or has not yet been introduced. The term "unmodified microorganism" may be used interchangeably with the terms "pre-modification strain," "pre-modification microorganism," "unmodified strain," "unmodified microorganism," or "reference microorganism."

In another aspect of the present invention, there is provided a method for producing branched chain amino acids comprising the step of culturing in a medium a microorganism of the genus Corynebacterium in which the activity of the ATP-dependent protease according to the present invention is weakened compared to an unmodified microorganism, or a microorganism of the genus Corynebacterium comprising a ClpC variant according to the present invention, a polynucleotide encoding a ClpC variant according to the present invention, or a vector according to the present invention.

In the present document, the term "cultivation" means culturing a microorganism of the genus Corynebacterium according to the present invention under appropriately controlled environmental conditions. The culturing process according to the present invention can be carried out using a suitable medium and culturing conditions known in the art. Such a culturing process can be easily adjusted and applied by those skilled in the art depending on the selected strain. In particular, the culturing may be a batch type culturing, a continuous type culturing, and/or a fed-batch type culturing, but is not limited thereto.

In the present document, the term "medium" means a mixture of substances containing nutrients necessary for culturing the microorganism of the genus Corynebacterium according to the present invention as a main component, and the medium provides nutrients and growth factors including water, which are necessary for survival and development. In particular, any medium without particular limitations can be used as the medium and other culture conditions used for culturing the microorganism of the genus Corynebacterium according to the present invention, as long as it is a medium used for conventional microorganism culturing. The microorganism of the genus Corynebacterium according to the present invention can be cultured in a conventional medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and/or vitamins, etc., while controlling temperature, pH, etc., under aerobic conditions.

In particular, the culture medium for the strain of the genus Corynebacterium is described in the document [“Manual of Methods for General Bacteriology”, American Society for Bacteriology (Washington D.C., USA, 1981)].

In the present invention, carbon sources include carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc., organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc.; and the like. Organic nutrients of natural origin such as starch hydrolysate, molasses, raw molasses, rice bran, cassava, sugar cane residues, and corn steep liquor can be used. In particular, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted into reducing sugar) can be used, and suitable amounts of other carbon sources can be used in various ways without limitation. These carbon sources may be used individually or in combination of two or more of them, but are not limited thereto.

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

Phosphorus sources may include monobasic potassium phosphate, dibasic potassium phosphate or their corresponding sodium salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition to these compounds, amino acids, vitamins and/or suitable precursors, etc. may be included. These components or precursors may be added to the medium periodically or continuously, but are not limited to this.

Also, during the cultivation of the microorganism of the genus Corynebacterium according to the present invention, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid or sulfuric acid to the medium appropriately. During the cultivation, foaming can be suppressed by using an antifoaming agent such as a polyglycol fatty acid ester. Oxygen or an oxygen-containing gas may be supplied to the medium to maintain an aerobic state of the medium, or gas may not be supplied, or nitrogen, hydrogen or carbon dioxide gas may be supplied to maintain anaerobic and microaerobic conditions, but not limited thereto.

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

The branched chain amino acids produced during the culture according to the present invention may be secreted into the medium or may remain in the cells.

The method for producing branched chain amino acids according to the present invention may further include a step of obtaining a microorganism of the genus Corynebacterium according to the present invention, a step of preparing a medium for culturing said microorganism, or a combination of these steps (in any order), for example, before the culturing step.

The method for producing branched-chain amino acids according to the present invention may further comprise a step of isolating branched-chain amino acids from a medium depending on the culture (cultured medium) or from a microorganism of the genus Corynebacterium according to the present invention. After the culturing step, a step of isolating may further be included.

The isolation may be collecting the branched chain amino acids of interest by a suitable method known in the art, depending on the method of culturing the microorganism according to the present invention, for example, a batch culture method, a continuous culture method, or a fed-batch culture method. For example, centrifugation, filtration, treatment with a precipitant for crystallizing protein (salting out), extraction, ultrasonic disintegration, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC (high performance liquid chromatography), or a combination thereof may be used. The branched chain amino acids of interest can be isolated from the medium or from the microorganism by a suitable method known in the art.

In addition, the method for producing branched chain amino acids according to the present invention may further include a purification step. The purification may be performed by a suitable method known in the art. For example, in a case where the method for producing branched chain amino acids according to the present invention includes both an isolation step and a purification step, the isolation step and the purification step may be performed intermittently (or continuously) regardless of their order, or they may be performed simultaneously or by combining into one step, but not limited thereto.

In the method according to the present invention, the variant, polynucleotide, vector, microorganism and branched chain amino acid and the like are as described in other aspects.

In another aspect of the present invention, there is provided a composition for producing branched chain amino acids comprising a variant according to the present invention, a polynucleotide encoding said variant, a vector comprising said polynucleotide, or a microorganism according to the present invention; a medium in which the microorganism according to the present invention has been cultured; or a combination of two or more of said.

The composition according to the present invention may further comprise arbitrarily selected suitable excipients commonly used in compositions for producing branched chain amino acids. Such excipients may be, for example, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffering agent, a stabilizer or an isotonic agent, but are not limited thereto.

In the composition according to the present invention, the variant, polynucleotide, vector, strain, medium, branched chain amino acids and the like are as described in other aspects.

In another aspect of the present invention, there is provided the use of a microorganism of the genus Corynebacterium, in which the activity of the ATP-dependent protease is weakened compared to an unmodified microorganism, for the production of branched-chain amino acids.

In another aspect of the present invention, there is provided the use of a ClpC variant in which the amino acid sequence corresponding to positions 431-433 based on the amino acid sequence represented by SEQ ID NO: 5 has been deleted, or a polynucleotide encoding said ClpC variant, for producing branched chain amino acids.

The present invention will now be described in more detail with reference to illustrative embodiments. However, the following illustrative embodiments are merely preferred embodiments for the purpose of illustrating the present invention and are thus not intended to limit the scope of the present invention. However, technical aspects not described herein can be sufficiently understood and easily implemented by those skilled in the art to which the present invention pertains or related art fields.

Example 1. Selection of variants with enhanced ability to produce L-valine by artificial mutation

1-1. Artificial mutagenesis using UV (ultraviolet) irradiation

To select a variant strain having an enhanced ability to produce L-valine, Corynebacterium glutamicum CA08-0072 (KCCM11201P, US 8465962 B2), which is an L-valine-producing NTG strain, was applied to an agar-containing nutrient medium and cultured at 30°C for 36 hours.

Hundreds of colonies obtained in this way were subjected to UV irradiation at room temperature to induce random mutations in the strain genome.

The composition of the nutrient medium is as follows.

<Nutrient medium (pH 7.2)>

10 g glucose, 5 g meat extract, 10 g polypeptone, 2.5 g sodium chloride, 5 g yeast extract, 20 g agar, 2 g urea (per 1 liter of distilled water).

1-2. Determination of the titer of the fermentation product of the strain obtained by mutagenesis and selection of the strain

To select variant strains with an enhanced ability to produce L-valine compared to Corynebacterium glutamicum CA08-0072, which was used as the parent strain in Example 1-1, the titer of the fermentation product was determined for strains with induced random mutations.

Specifically, each colony was subcultured into a nutrient medium, and each strain was seeded into a 250 ml flask with angular partitions containing 25 ml of the production medium, and cultured at 30°C for 72 hours with shaking at 200 rpm. The compositions of the nutrient medium and the production medium, respectively, are as follows.

<Nutrient medium (pH 7.2)>

10 g glucose, 5 g meat extract, 10 g polypeptone, 2.5 g sodium chloride, 5 g yeast extract, 20 g agar, 2 g urea (per 1 liter of distilled water).

<Production medium (pH 7.0)>

100 g glucose, 40 g ammonium sulfate, 2.5 g soy protein, 5 g corn extract, 3 g urea, 1 g dibasic potassium phosphate, 0.5 g magnesium sulfate heptahydrate, 100 mcg biotin, 1 mg thiamine-HCl, 2 mg calcium pantothenate, 3 mg nicotinamide, 30 g calcium carbonate (per 1 liter of distilled water).

Thereafter, the L-valine concentrations were analyzed by HPLC, and the L-valine concentrations determined by the analysis are shown in Table 1 below.

Taking into account the data in Table 1, strain M4 was selected, which demonstrates the greatest increase in L-valine production compared to the control strain CA08-0072.

Example 2. Identification of variation by gene sequencing

The major genes of strain M4, which exhibited enhanced L-valine production ability in Example 1, were sequenced and compared with those of strain CA08-0072 and the wild-type Corynebacterium glutamicum strain ATCC14067. As a result, it was confirmed that strain M4 contains a variation at a specific position of the open reading frame (ORF) of clpC, which is one of the hexameric ATPase-active chaperone subunits constituting the ATP-dependent protease. In particular, it was confirmed that strain M4 had a deletion of nucleotides at positions 1291-1299 (SEQ ID NO: 4) in the sequence represented by SEQ ID NO: 6. The sequence represented by SEQ ID NO: 6 is a sequence typically included in the ORF of clpC of wild-type Corynebacterium glutamicum (ATCC14067, ATCC13032, and ATCC13869).

The following examples attempt to confirm the effect of this variation on amino acid production by microorganisms of the genus Corynebacterium.

Example 3. Obtaining a strain with an introduced variation and studying the ability to produce L-valine

3-1. Obtaining a strain by introducing a variation into Corynebacterium glutamicum CA08-0072 and evaluating the ability to produce L-valine

A vector was constructed for introducing a nucleotide sequence (SEQ ID NO: 2) in which the nucleotides at positions 1291-1299 (SEQ ID NO: 4) in the sequence represented by SEQ ID NO: 6 were deleted into the L-valine-producing NTG strain Corynebacterium glutamicum CA08-0072. Specifically, the genomic DNA of the wild-type Corynebacterium glutamicum strain ATCC14067 was isolated using the G-Spin Total DNA Extraction Mini Kit (Intron, Cat. No. 17045) according to the protocol provided in the kit. PCR (polymerase chain reaction) was performed using each genomic DNA as a template and primers SEQ ID NO: 7 and 8, as well as primers SEQ ID NO: 9 and 10, to obtain DNA fragments (A and B), respectively. The primer sequences used in this case are presented in Table 2 below.

Overlapping primer PCR was performed using the two fragments as templates and primers SEQ ID NO: 7 and 10 to obtain a PCR product of 1040 bp (base pairs) in length (hereinafter referred to as the “variation-introduced fragment”). PCR was performed as follows: denaturation at 94°C for 5 minutes; 25 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 60 seconds; and polymerization at 72°C for 7 minutes. The fragments with the introduced variation were digested with the restriction enzyme XbaI (New England Biolabs, Beverly, MA) and then ligated with the pDZ vector (U.S. Patent No. 9,109,242 and International Patent Publication No. 2008-033001), which is incapable of replicating in Corynebacterium glutamicum, which had been digested with the same restriction enzyme using T4 ligase (New England Biolabs, Beverly, MA). The resulting gene was transformed into E. coli DH5α and selected in LB medium containing 25 mg/L kanamycin, and DNA was prepared using the DNA-spin plasmid DNA purification kit (Intron) to construct the recombinant plasmid pDZ-clpC_M4.

The recombinant plasmid pDZ-clpC_M4 obtained as described above was transformed into the L-valine-producing Corynebacterium glutamicum strain CA08-0072 by homologous recombination in the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). The strain in which the vector was inserted into the chromosome by homologous recombination was selected in a medium containing 25 mg/L kanamycin. Then, PCR was performed on the Corynebacterium glutamicum transformant in which secondary crossing over was completed using the primers SEQ ID NOs: 7 and 10 to obtain a strain in which a variation was introduced in the clpC ORF on the chromosome. The recombinant strain was named Corynebacterium glutamicum CA08-0072-clpC_M4.

To compare the ability to produce L-valine between the obtained L-valine-producing strain Corynebacterium glutamicum CA08-0072 and CA08-0072-clpC_M4, the titer of the fermentation product was determined.

Specifically, each strain was subcultured in a nutrient medium and seeded in a 250 ml flask with angular partitions containing 25 ml of the production medium, and cultured at 30°C for 72 hours with shaking at 200 rpm. The compositions of the nutrient medium and the production medium, respectively, are as follows.

<Nutrient medium (pH 7.2)>

10 g glucose, 5 g meat extract, 10 g polypeptone, 2.5 g sodium chloride, 5 g yeast extract, 20 g agar, 2 g urea (per 1 liter of distilled water).

<Production medium (pH 7.0)>

100 g glucose, 40 g ammonium sulfate, 2.5 g soy protein, 5 g corn extract, 3 g urea, 1 g dibasic potassium phosphate, 0.5 g magnesium sulfate heptahydrate, 100 mcg biotin, 1 mg thiamine-HCl, 2 mg calcium pantothenate, 3 mg nicotinamide, 30 g calcium carbonate (per 1 liter of distilled water).

Thereafter, the L-valine concentrations were analyzed by HPLC, and the L-valine concentrations determined by the analysis are shown in Table 3 below.

As shown in the results shown in Table 3, it was confirmed that the L-valine producing ability of the CA08-0072-clpC_M4 strain increased by 14% compared with the control group. As a result, it was confirmed that the L-valine producing ability could be improved by the variation of clpC ORF.

CA08-0072-clpC_M4 was named CA08-1542 and deposited at the Korea Microbial Culture Center under the Budapest Treaty on July 2, 2020 under the accession number KCCM12755R.

3-2. Strain production by introducing variation into Corynebacterium glutamicum CJ7V and evaluation of L-valine producing ability

To test whether the same effect is observed in other L-valine-producing strains belonging to the species Corynebacterium glutamicum, one type of variation [ilvN(A42V); Biotechnology and Bioprocess Engineering, June 2014, Volume 19, Issue 3, pp 456-467] was introduced into the wild-type Corynebacterium glutamicum strain ATCC14067 to obtain a strain with improved L-valine-producing ability.

Specifically, the genomic DNA of the wild-type Corynebacterium glutamicum strain ATCC14067 was isolated using the G-Spin Total DNA Extraction Mini Kit. To construct a vector for introducing the A42V variation into the ilvN gene, PCR was performed using the genomic DNA as a template and primers SEQ ID NOs: 11 and 12, as well as primers SEQ ID NOs: 13 and 14 to obtain DNA fragments (A and B), respectively. The primer sequences used in this case are presented in Table 4 below.

Overlapping primer PCR was performed using the two fragments as templates and primers SEQ ID NOs: 11 and 14 to obtain a PCR product of 1044 bp in length (hereinafter referred to as the "fragment with introduced variation"). The PCR was performed as follows: denaturation at 94°C for 5 minutes; 25 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 60 seconds; and polymerization at 72°C for 7 minutes. As a result, polynucleotides of 537 bp in length were obtained. for both fragment A and fragment B. The resulting fragments with the introduced variation were treated with the restriction enzyme XbaI and then ligated with the pDZ vector treated with the same restriction enzyme using T4 ligase. The resulting gene was transformed into E. coli DH5α and selection was carried out in LB medium containing 25 mg/L kanamycin, and DNA was prepared using a DNA-spin plasmid DNA purification kit. The vector designed to introduce the A42V variation into the ilvN gene was named pDZ-ilvN(A42V).

Then, the recombinant plasmid pDZ-ilvN(A42V) obtained in the above manner was transformed into the wild-type Corynebacterium glutamicum ATCC14067 by homologous recombination in the chromosome. The strain in which the vector was inserted into the chromosome by homologous recombination was selected in a medium containing 25 mg/L kanamycin. Then, PCR was performed on the Corynebacterium glutamicum transformant in which secondary crossing over was completed using the primers SEQ ID NOs: 11 and 14 to amplify the gene fragment, and then the strain with the introduced variation was identified by gene sequencing analysis. The recombinant strain in which the variation was introduced was named Corynebacterium glutamicum CJ7V.

Corynebacterium glutamicum CJ7V was transformed with pDZ-clpC_M4 in the same manner as in Example 3-1 to obtain a strain in which a variation was introduced in the ORF of clpC, and the resulting strain was named CJ7V-clpC_M4.

In order to compare the ability of the obtained strain to produce L-valine, the strain was cultured in the same manner as in Example 3-1 and the concentration of L-valine was analyzed, and the concentration of L-valine determined in the analysis is shown in Table 5 below.

As shown in the results shown in Table 5, it was confirmed that the ability of the CJ7V-clpC_M4 strain to produce L-valine increased by 14% compared with the control group. In other words, it was also confirmed that the ability of the Corynebacterium genus microorganism to produce L-valine could be improved by varying the ORF of the clpC gene.

3-3. Obtaining a strain by introducing a variation into Corynebacterium glutamicum CJ8V and evaluating the ability to produce L-valine

In order to test whether the same effect is observed in other L-valine-producing strains belonging to the species Corynebacterium glutamicum, one type of variation [ilvN(A42V)] was introduced into the wild-type Corynebacterium glutamicum strain ATCC13869 in the same manner as in Example 3-2 to obtain a variant strain having the ability to produce L-valine. The recombinant strain was named Corynebacterium glutamicum CJ8V.

To obtain a strain in which the clpC variation was introduced into Corynebacterium glutamicum CJ8V, the recombinant vector pDZ-clpC_M4 obtained in Example 3-1 was transformed into CJ8V. The strain in which the vector was inserted into the chromosome by homologous recombination was selected in a medium containing 25 mg/L kanamycin. Then, the Corynebacterium glutamicum transformant in which the secondary crossing over was completed was examined using the primers of SEQ ID NOs: 7 and 10, and the strain in which the clpC variation was introduced was identified. The recombinant strain identified in the above manner was named Corynebacterium glutamicum CJ8V-clpC_M4.

In order to compare the ability of the obtained strain to produce L-valine, the strain was cultured in the same manner as in Example 3-1 and the concentration of L-valine was analyzed, and the concentration of L-valine determined in the analysis is shown in Table 6 below.

As shown in the results shown in Table 6, it was confirmed that the ability of the CJ8V-clpC_M4 strain to produce L-valine increased by 12% compared with the control group. In other words, it was also confirmed that the ability of the Corynebacterium genus microorganism to produce L-valine could be improved by varying the ORF of the clpC gene.

Example 4. Obtaining an isoleucine-producing strain and assessing its production capacity

4-1. Generation of a strain by introducing a clpC ORF variation into the L-isoleucine-producing Corynebacterium glutamicum KCCM11248P strain and its evaluation

A strain was obtained by introducing the recombinant plasmid pDZ-clpC_M4 obtained in Example 3-1 into the L-isoleucine-producing strain Corynebacterium glutamicum KCJI-38 (KCSM11248P, U.S. Patent No. 9885093) in the same manner as in Example 3 by homologous recombination in the chromosome, and the obtained strain was named KCJI-38-clpC_M4. The obtained strain was cultured in the manner described below, and its isoleucine-producing ability was compared.

Specifically, each strain was seeded in a 250-mL angular baffle flask containing 25 mL of seeding medium and cultured at 30°C for 20 hours with shaking at 200 rpm. Then, 1 mL of the seed culture was seeded in a 250-mL angular baffle flask containing 24 mL of production medium and cultured at 30°C for 48 hours with shaking at 200 rpm. The compositions of the seeding medium and the production medium, respectively, are as follows.

<Sowing medium (pH 7.0)>

20 g glucose, 10 g peptone, 5 g yeast extract, 1.5 g urea, 4 g KH ₂ PO ₄ , 8 g K ₂ HPO ₄ , 0.5 g MgSO ₄ 7H ₂ O, 100 mcg biotin, 1000 mcg thiamine HCl, 2000 mcg calcium pantothenate, 2000 mcg nicotinamide (per 1 liter of distilled water).

<Production medium (pH 7.0)>

50 g glucose, 12.5 g (NH ₄ ) ₂ SO ₄ , 2.5 g soy protein, 5 g corn extract, 3 g urea, 1 g KH ₂ PO ₄ , 0.5 g MgSO ₄ 7H ₂ O, 100 μg biotin, 1000 μg thiamine HCl, 2000 μg calcium pantothenate, 3000 μg nicotinamide, 30 g CaCO ₃ (per 1 liter of distilled water).

After completion of the cultivation, the ability to produce L-isoleucine was determined by HPLC. The concentrations of L-isoleucine in the culture of each strain tested are shown in Table 7 below.

As shown in Table 7, it was confirmed that the concentration of L-isoleucine in KCJI-38-clpC_M4 into which the clpC variant was introduced was increased by approximately 55% compared with the L-isoleucine-producing strain KCJI-38. This confirmed that the ability to produce L-isoleucine could be improved by altering the clpC gene. The above results indicate that the introduction of the clpC ORF variation into the L-isoleucine-producing strain of the genus Corynebacterium is effective in producing L-isoleucine.

KCJI-38-c1pC_M4 was named CA10-3123 and deposited at the Korea Microbial Culture Center under the Budapest Treaty on December 1, 2020 under the accession number KCCM12858R.

4-2. Generation of L-isoleucine-producing strain by introducing clpC ORF variation into wild-type Corynebacterium glutamicum strain ATCC13032 and evaluation of L-isoleucine-producing ability

To evaluate the effect of introducing the clpC-M4 variation on the ability to produce L-isoleucine, strains were generated by introducing a known aspartokinase variant (lysC(L377K)) (US 10662450 B2), a homoserine dehydrogenase variant (hom(R407H)) (Appl. Microbiol. Biotechnol. 45, 612-620 (1996)) or an L-threonine dehydratase variant (ilvA(V323A)) (Appl. Enviro. Microbiol., Dec. 1996, p.4345-4351) into the Corynebacterium glutamicum ATCC13032 strain and their ability to produce L-isoleucine was compared.

In particular, PCR was performed using the WT chromosome as a template and primers SEQ ID NO: 15 and 16 or SEQ ID NO: 17 and 18. The primer sequences used in this case are presented in Table 8 below.

The PCR was performed as follows: denaturation at 95°C for 5 minutes; 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds; and polymerization at 72°C for 7 minutes. As a result, an upstream DNA fragment of 509 bp in length from the 5' end and a downstream DNA fragment of 520 bp in length from the 3' end of the gene, respectively, were obtained, with the lysC gene variation located toward the center. PCR was performed using the two amplified DNA fragments as templates and primers SEQ ID NOs: 15 and 18. The PCR was performed as follows: denaturation at 95°C for 5 minutes; 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds; and polymerization at 72°C for 7 minutes. As a result, a 1011 bp DNA fragment containing a variation of the lysC gene (L377K) encoding an aspartokinase variant in which the leucine at position 377 is replaced by lysine was amplified. The pDZ vector and the amplified 1011 bp DNA fragment were digested with the restriction enzyme XbaI, ligated using DNA ligase, and then cloned to obtain a plasmid named pDZ-lysC (L377K).

The pDZ-lysC(L377K) vector obtained in the above manner was introduced into the WT strain by a method involving the use of electrical pulses (Appl. Microbiol. Biothcenol. (1999, 52:541-545)), and then the transformed strain was obtained in a selective medium containing 25 mg/L kanamycin. The WT::lysC(L377K) strain was obtained, in which the nucleotide variation was introduced into the lysC gene using a DNA fragment inserted into the chromosome by secondary recombination.

In addition, to obtain the vector for introducing horn (R407H), PCR was performed using the wild-type genomic DNA as a template and primers SEQ ID NOs: 19 and 20, as well as SEQ ID NOs: 21 and 22. The primer sequences used in this case are presented in Table 9 below.

The PCR was performed as follows: denaturation at 95°C for 5 minutes; 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds; and polymerization at 72°C for 7 minutes. As a result, an upstream DNA fragment of 220 bp in length from the 5' end and a downstream DNA fragment of 220 bp in length from the 3' end of the gene, with the hom gene variation located toward the center, were obtained, respectively. PCR was performed using the two amplified DNA fragments mentioned above as templates and primers SEQ ID NOs: 19 and 20. The PCR was performed as follows: denaturation at 95°C for 5 minutes; 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds; and polymerization at 72°C for 7 minutes. As a result, a 440 bp DNA fragment containing the hom gene variation was amplified. The pDZ vector used in the above manner and the amplified 440 bp DNA fragment were digested with XbaI restriction enzyme, ligated using DNA ligase, and then cloned to obtain a plasmid named pDZ-hom(R407H).

The pDZ-hom(R407H) vector obtained in the above manner was introduced into the WT::lysC(L377K) strain by a method involving the use of electric pulses, and then the transformed strain was obtained in a selective medium containing 25 mg/L kanamycin. The WT::lysC(L377K)-hom(R407H) strain in which a nucleotide variation was introduced into the hom gene by a DNA fragment inserted into the chromosome by secondary recombination was obtained.

In the same manner as in the above Example, a strain was obtained by introducing the recombinant plasmid pDZ-clpC_M4 obtained in Example 3-1 into the WT::lysC(L377K)-hom(R407H) strain by homologous recombination in the chromosome, and the obtained strain was named WT::lysC(L377K)-hom(R407H)-clpC M4.

In this case, to obtain a vector in which the known variation ilvA(V323A) (S. Morbach et al., Appl. Enviro. Microbiol., 62(12): 4345-4351, 1996) was introduced into the ilvA gene, a pair of primers (SEQ ID NOs: 23 and 24) were designed to amplify the upstream fragment from the 5' end and a pair of primers (SEQ ID NOs: 25 and 26) to amplify the downstream fragment from the 3' end, respectively, to the center of which the variation is located. A BamHI restriction site (underlined) was introduced into primers SEQ ID NOs: 23 and 26 at each end. In primers SEQ ID NO: 24 and 25, the nucleotide substitution variation (underlined) was placed in a site that was designed such that the indicated regions overlapped each other. The primer sequences designed in this case are presented in Table 10 below.

PCR was performed using the WT chromosome as a template and the primers SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26. PCR was performed as follows: denaturation at 95°C for 5 minutes; 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds and polymerization at 72°C for 30 seconds; and polymerization at 72°C for 7 minutes. As a result, an upstream DNA fragment of 627 bp in length from the 5'-end and a downstream DNA fragment of 608 bp in length from the 3'-end of the gene were obtained, respectively, with the ilvA gene variation located in the center. PCR was performed using the two amplified DNA fragments as templates and primers SEQ ID NOs: 23 and 26. The PCR was performed as follows: denaturation at 95°C for 5 minutes; 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 60 seconds; and polymerization at 72°C for 7 minutes. As a result, a 1,217 bp DNA fragment containing a variation of the ilvA gene encoding an IlvA variant in which L-valine at position 323 is replaced with alanine was amplified. The pECCG117 vector (Korean Patent No. 10-0057684) and the 1,217 bp DNA fragment were used. were digested with the restriction enzyme BamHI, ligated using DNA ligase, and cloned to yield a plasmid named pECCG117-ilvA(V323A).

The strain ATCC13032::hom(R407H)-lysC(L377K)-clpC/pECCG117-ilvA(V323A) was obtained by introducing the vector pECCG117-ilvA(V323A) into ATCC13032::hom(R407H)-lysC(L377K)-clpC_M4 according to the above Example. Also, as a control, a strain was obtained by introducing only the ilvA(V323A) variation into ATCC13032::-hom(R407H)-lysC(L377K).

The obtained strains were cultured in the same manner as in the flask culture method in Example 4-1, and the concentrations of L-isoleucine in the cultures were determined. The results of the determination of the concentration of L-isoleucine in the cultures of the strains are shown in Table 11 below.

As shown in Table 11, it was confirmed that, compared with ATCC13032::-hom(R407H)-lysC(L377K)/pECCG117-ilvA(V323A), which only introduced the ilvA(V323A) variation into ATCC13032::-hom(R407H)-lysC(L377K), ATCC13032::hom(R407H)-lysC(L377K)-clpC_M4/pECCG117-ilvA(V323A), which further introduced the clpC_M4 variation, showed an increase in the concentration of L-isoleucine by approximately 42%. The above results show that introduction of the clpC variation into an L-isoleucine-producing strain of the genus Corynebacterium is effective in L-isoleucine production.

Based on the above description, it will be clear to those skilled in the art that the present invention can be embodied in another specific form without changing its technical essence or essential characteristics. In this regard, it should be understood that the above embodiment is not limiting, but illustrative in all respects. The scope of the present invention is defined by the appended claims, and not by the description preceding it, and therefore all changes and modifications that fall within the scope of the claims, or the equivalents of such limits, should be considered as covered by the claims.

The effect of invention

The microorganism according to the present invention can produce branched-chain amino acids with high efficiency. In addition, the obtained branched-chain amino acids can be used in various products such as pharmaceutical raw materials and food additives, animal feed, nutrients, pesticides, bactericidal agents, and so on.

--->

SEQUENCE LIST

<110>CJ CheilJedang Corporation

<120> Mutant ATP-dependent protease and

a method for obtaining L-amino acid with its

application

<130>OPA21217

<150> KR 10-2020-0173802

<151> 2020-12-11

<160> 26

<170> KoPatentIn 3.0

<210> 1

<211> 922

<212> PRT

<213> Artificial Sequence

<220>

<223> ClpC ORF AA variant

<400> 1

Met Phe Glu Arg Phe Thr Asp Arg Ala Arg Arg Val Ile Val Leu Ala

1 5 10 15

Gln Glu Glu Ala Arg Met Leu Asn His Asn Tyr Ile Gly Thr Glu His

20 25 30

Ile Leu Leu Gly Leu Ile His Glu Gly Glu Gly Val Ala Ala Lys Ala

35 40 45

Leu Glu Ser Met Gly Ile Ser Leu Asp Ala Val Arg Gln Glu Val Glu

50 55 60

Glu Ile Ile Gly Gln Gly Ser Gln Pro Thr Thr Gly His Ile Pro Phe

65 70 75 80

Thr Pro Arg Ala Lys Lys Val Leu Glu Leu Ser Leu Arg Glu Gly Leu

85 90 95

Gln Met Gly His Lys Tyr Ile Gly Thr Glu Phe Leu Leu Leu Gly Leu

100 105 110

Ile Arg Glu Gly Glu Gly Val Ala Ala Gln Val Leu Val Lys Leu Gly

115 120 125

Ala Asp Leu Pro Arg Val Arg Gln Gln Val Ile Gln Leu Leu Ser Gly

130 135 140

Tyr Glu Gly Gly Gln Gly Gly Ser Pro Glu Gly Gly Gln Gly Ala Pro

145 150 155 160

Thr Gly Gly Asp Ala Val Gly Ala Gly Ala Ala Pro Gly Gly Arg Pro

165 170 175

Ser Ser Gly Gly Pro Gly Glu Arg Ser Thr Ser Leu Val Leu Asp Gln

180 185 190

Phe Gly Arg Asn Leu Thr Gln Ala Ala Lys Asp Gly Lys Leu Asp Pro

195 200 205

Val Val Gly Arg Asp Lys Glu Ile Glu Arg Ile Met Gln Val Leu Ser

210 215 220

Arg Arg Thr Lys Asn Asn Pro Val Leu Ile Gly Glu Pro Gly Val Gly

225 230 235 240

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

245 250 255

Val Pro Glu Thr Leu Lys Asp Lys Gln Val Tyr Ser Leu Asp Leu Gly

260 265 270

Ser Leu Val Ala Gly Ser Arg Tyr Arg Gly Asp Phe Glu Glu Arg Leu

275 280 285

Lys Lys Val Leu Lys Glu Ile Asn Gln Arg Gly Asp Ile Ile Leu Phe

290 295 300

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

305 310 315 320

Ile Asp Ala Ala Ser Leu Leu Lys Pro Lys Leu Ala Arg Gly Glu Leu

325 330 335

Gln Thr Ile Gly Ala Thr Thr Leu Asp Glu Tyr Arg Lys His Ile Glu

340 345 350

Lys Asp Ala Ala Leu Glu Arg Arg Phe Gln Pro Val Gln Val Pro Glu

355 360 365

Pro Ser Val Asp Leu Thr Val Glu Ile Leu Lys Gly Leu Arg Asp Arg

370 375 380

Tyr Glu Ala His His Arg Val Ser Ile Thr Asp Gly Ala Leu Thr Ala

385 390 395 400

Ala Ala Gln Leu Ala Asp Arg Tyr Ile Asn Asp Arg Phe Leu Pro Asp

405 410 415

Lys Ala Val Asp Leu Ile Asp Glu Ala Gly Ala Arg Met Arg Met Thr

420 425 430

Ala Pro Ser Ser Leu Arg Glu Val Asp Glu Arg Ile Ala Asp Val Arg

435 440 445

Arg Glu Lys Glu Ala Ala Ile Asp Ala Gln Asp Phe Glu Lys Ala Ala

450 455 460

Gly Leu Arg Asp Lys Glu Arg Lys Leu Gly Glu Glu Arg Ser Glu Lys

465 470 475 480

Glu Lys Gln Trp Arg Ser Gly Asp Leu Glu Asp Ile Ala Glu Val Gly

485 490 495

Glu Glu Gln Ile Ala Glu Val Leu Ala Asn Trp Thr Gly Ile Pro Val

500 505 510

Phe Lys Leu Thr Glu Ala Glu Ser Ser Arg Leu Leu Asn Met Glu Glu

515 520 525

Glu Leu His Lys Arg Ile Ile Gly Gln Asp Glu Ala Val Lys Ala Val

530 535 540

Ser Arg Ala Ile Arg Arg Thr Arg Ala Gly Leu Lys Asp Pro Lys Arg

545 550 555 560

Pro Ser Gly Ser Phe Ile Phe Ala Gly Pro Ser Gly Val Gly Lys Thr

565 570 575

Glu Leu Ser Lys Ala Leu Ala Gly Phe Leu Phe Gly Asp Asp Asp Ser

580 585 590

Leu Ile Gln Ile Asp Met Gly Glu Phe His Asp Arg Phe Thr Ala Ser

595 600 605

Arg Leu Phe Gly Ala Pro Pro Gly Tyr Val Gly Tyr Glu Glu Gly Gly

610 615 620

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

625 630 635 640

Asp Glu Ile Glu Lys Ala His Lys Glu Ile Tyr Asn Thr Leu Leu Gln

645 650 655

Val Leu Glu Asp Gly Arg Leu Thr Asp Gly Gln Gly Arg Ile Val Asp

660 665 670

Phe Lys Asn Thr Val Leu Ile Phe Thr Ser Asn Leu Gly Thr Ser Asp

675 680 685

Ile Ser Lys Ala Val Gly Leu Gly Phe Ser Gly Ser Ser Glu Thr Asp

690 695 700

Ser Asp Ala Gln Tyr Asp Arg Met Lys Asn Lys Val His Asp Glu Leu

705 710 715 720

Lys Lys His Phe Arg Pro Glu Phe Leu Asn Arg Ile Asp Glu Ile Val

725 730 735

Val Phe His Gln Leu Thr Lys Asp Gln Ile Val Gln Met Val Asp Leu

740 745 750

Leu Ile Gly Arg Val Ser Asn Ala Leu Ala Glu Lys Asp Met Ser Ile

755 760 765

Glu Leu Thr Glu Lys Ala Lys Asp Leu Leu Ala Ser Arg Gly Phe Asp

770 775 780

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

785 790 795 800

Glu Asp Gln Met Ser Glu Lys Ile Leu Phe Gly Glu Ile Gly Ala Gly

805 810 815

Glu Ile Val Thr Val Asp Val Glu Gly Trp Asp Gly Glu Ser Lys Asp

820 825 830

Thr Asp Arg Ala Lys Phe Thr Phe Thr Pro Arg Pro Lys Pro Met Pro

835 840 845

Glu Gly Lys Phe Ser Glu Ile Ser Val Gln Ala Ala Glu Ala Ile Gln

850 855 860

Asp Val Asp Ser Ala Ala Asp Gly Asp Val Pro Glu Thr Asp Ser Leu

865 870 875 880

Ser Asp Ile Asp Leu Glu Thr Leu Glu Lys Phe Glu Glu Asp Val Glu

885 890 895

Asn Gly Thr Asp Ile Asp Gln Val Ser Gly Asp Tyr Tyr Gly Thr Asp

900 905 910

Asp Gln Gly Gly Thr Ala Pro Ser Lys Glu

915 920

<210> 2

<211> 2769

<212> DNA

<213> Artificial Sequence

<220>

<223> clpC ORF NT variant

<400> 2

atgttcgaga ggtttaccga tcgtgcacgc cgcgtgattg tgctcgcgca ggaagaggcg 60

cgcatgctca accacaatta catcggcacg gagcacattc tcctcggcct cattcacgag 120

ggcgagggcg ttgcagccaa ggctttggaa tccatgggaa tttccctgga cgccgtccgc 180

caggaagtcg aagagattat cggccagggc tcccagccca ccaccggcca cattcctttt 240

actccacgtg ccaagaaggt cctggagctc agcctccgcg agggactaca gatgggacac 300

aagtacatcg gtactgagtt cctgcttctc ggtttgatcc gtgagggcga gggcgttgct 360

gcccaggtcc tggtcaagct tggtgctgat ctgccacgcg tgcgtcagca agttattcag 420

cttctctccg gctacgaagg tggccagggc ggatccccag agggcggtca gggcgcccct 480

actggcggtg acgctgttgg tgcaggagct gctcctggcg gtcgtccatc ttcgggcggc 540

ccaggcgagc gttctacctc tttggttctt gaccagttcg gccgcaacct cacccaggcc 600

gcaaaggacg gcaagctgga cccagttgtt ggtcgtgata aggaaatcga gcgcatcatg 660

caggtgctct cccgtcgtac caagaacaac ccagttctta ttggtgagcc aggtgttggt 720

aagaccgcag ttgttgaagg tcttgcacta gacattgtta acggcaaggt tccagagacc 780

ctcaaggaca agcaggttta ctcccttgac ttaggttcac tggttgcagg ttcccgttac 840

cgcggtgact tcgaagagcg cctgaagaag gtcctcaagg agattaacca gcgcggcgac 900

atcatcctgt ttatcgatga gatccacacc ctcgtgggtg caggtgcagc agaaggcgca 960

atcgacgccg cctccctgct taagccaaag cttgcccgcg gtgaactgca gaccattggt 1020

gcaaccacct tggacgagta ccgtaagcac attgaaaagg acgcagctct tgagcgtcgt 1080

ttccagccag tgcaggttcc agagccttcg gttgatctca ccgttgagat cttgaagggt 1140

ctgcgcgacc gctacgaagc tcaccaccgc gtatccatca ccgatggtgc tcttaccgca 1200

gcagctcagc ttgctgatcg ctacattaac gaccgcttct tgccagataa ggccgttgac 1260

ctcatcgatg aggctggcgc ccgcatgcgc atgaccgcac cgtcctccct ccgcgaggtt 1320

gatgagcgca tcgctgatgt tcgccgtgag aaggaagcag cgatcgatgc tcaggacttt 1380

gagaaggcag caggtcttcg cgataaggag cgcaagctcg gcgaagagcg ttcagagaag 1440

gaaaagcagt ggcgctccgg cgacctcgag gacattgctg aggttggcga agagcagatc 1500

gcagaagtac tggccaactg gactggtatt cctgtcttca agctcaccga agctgagtct 1560

tcccgcctgc tcaacatgga agaagagttg cacaagcgca tcatcggaca ggatgaagct 1620

gtcaaggctg tctcccgtgc gatccgtcgt acccgtgcag gtctgaagga tcctaagcgt 1680

ccttccggct ccttcatctt cgctggtcca tccggtgttg gtaagaccga gctgtccaag 1740

gcacttgctg gattcctctt cggtgacgat gattccctca tccaaatcga catgggtgaa 1800

ttccacgacc gcttcaccgc gtcccgactc ttcggtgccc ctccgggata cgttggctac 1860

gaagaaggtg gccagctgac cgagaaggtt cgccgtaagc cattctccgt tgtgcttttc 1920

gacgagatcg agaaggccca caaggagatc tacaacacct tgctgcaggt gttggaagat 1980

ggtcgcctta ccgatggtca gggtcgcatt gtggacttca agaacaccgt cctgatcttc 2040

acctccaacc tgggcacctc tgacatctcc aaggctgttg gcctgggctt ctccggatcc 2100

tctgagactg acagcgatgc tcagtacgac cgcatgaaga acaaggtcca cgacgagctg 2160

aagaagcact tccgccctga gttcctgaac cgtattgatg agatcgtggt cttccaccag 2220

ctcaccaagg atcagatcgt tcagatggtc gaccttctta ttggccgcgt gtccaacgca 2280

ctggctgaga aggacatgag catcgaactg actgagaagg ctaaggatct ccttgccagc 2340

cgcggcttcg atccagttct gggtgcacga ccattgcgtc gcaccattca gcgcgaaatc 2400

gaagaccaga tgtccgagaa gatcctcttc ggagaaatcg gtgctggcga gatcgtcacc 2460

gtcgacgtcg aaggctggga cggcgagtcc aaggacaccg accgtgcgaa gttcaccttc 2520

acaccacgtc caaagccaat gccagaaggt aagttctctg agatctccgt acaggctgca 2580

gaagcaatcc aagatgtaga ttccgcagct gacggcgatg tcccagaaac cgattcactt 2640

tccgacattg accttgaaac ccttgagaag ttcgaggaag atgtagaaaa cggcaccgac 2700

attgatcagg tatccggtga ctactacggc accgatgatc agggaggcac tgctccaagc 2760

aaggagtag 2769

<210> 3

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> 431~433 AA

<400> 3

Ile Lys Arg

1

<210> 4

<211> 9

<212> DNA

<213> Artificial Sequence

<220>

<223> 1291~1299 NT

<400> 4

atcaagcgc 9

<210> 5

<211> 925

<212> PRT

<213> Unknown

<220>

<223> ORF WT AA ClpC

<400> 5

Met Phe Glu Arg Phe Thr Asp Arg Ala Arg Arg Val Ile Val Leu Ala

1 5 10 15

Gln Glu Glu Ala Arg Met Leu Asn His Asn Tyr Ile Gly Thr Glu His

20 25 30

Ile Leu Leu Gly Leu Ile His Glu Gly Glu Gly Val Ala Ala Lys Ala

35 40 45

Leu Glu Ser Met Gly Ile Ser Leu Asp Ala Val Arg Gln Glu Val Glu

50 55 60

Glu Ile Ile Gly Gln Gly Ser Gln Pro Thr Thr Gly His Ile Pro Phe

65 70 75 80

Thr Pro Arg Ala Lys Lys Val Leu Glu Leu Ser Leu Arg Glu Gly Leu

85 90 95

Gln Met Gly His Lys Tyr Ile Gly Thr Glu Phe Leu Leu Leu Gly Leu

100 105 110

Ile Arg Glu Gly Glu Gly Val Ala Ala Gln Val Leu Val Lys Leu Gly

115 120 125

Ala Asp Leu Pro Arg Val Arg Gln Gln Val Ile Gln Leu Leu Ser Gly

130 135 140

Tyr Glu Gly Gly Gln Gly Gly Ser Pro Glu Gly Gly Gln Gly Ala Pro

145 150 155 160

Thr Gly Gly Asp Ala Val Gly Ala Gly Ala Ala Pro Gly Gly Arg Pro

165 170 175

Ser Ser Gly Gly Pro Gly Glu Arg Ser Thr Ser Leu Val Leu Asp Gln

180 185 190

Phe Gly Arg Asn Leu Thr Gln Ala Ala Lys Asp Gly Lys Leu Asp Pro

195 200 205

Val Val Gly Arg Asp Lys Glu Ile Glu Arg Ile Met Gln Val Leu Ser

210 215 220

Arg Arg Thr Lys Asn Asn Pro Val Leu Ile Gly Glu Pro Gly Val Gly

225 230 235 240

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

245 250 255

Val Pro Glu Thr Leu Lys Asp Lys Gln Val Tyr Ser Leu Asp Leu Gly

260 265 270

Ser Leu Val Ala Gly Ser Arg Tyr Arg Gly Asp Phe Glu Glu Arg Leu

275 280 285

Lys Lys Val Leu Lys Glu Ile Asn Gln Arg Gly Asp Ile Ile Leu Phe

290 295 300

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

305 310 315 320

Ile Asp Ala Ala Ser Leu Leu Lys Pro Lys Leu Ala Arg Gly Glu Leu

325 330 335

Gln Thr Ile Gly Ala Thr Thr Leu Asp Glu Tyr Arg Lys His Ile Glu

340 345 350

Lys Asp Ala Ala Leu Glu Arg Arg Phe Gln Pro Val Gln Val Pro Glu

355 360 365

Pro Ser Val Asp Leu Thr Val Glu Ile Leu Lys Gly Leu Arg Asp Arg

370 375 380

Tyr Glu Ala His His Arg Val Ser Ile Thr Asp Gly Ala Leu Thr Ala

385 390 395 400

Ala Ala Gln Leu Ala Asp Arg Tyr Ile Asn Asp Arg Phe Leu Pro Asp

405 410 415

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

420 425 430

Arg Met Thr Ala Pro Ser Ser Leu Arg Glu Val Asp Glu Arg Ile Ala

435 440 445

Asp Val Arg Arg Glu Lys Glu Ala Ala Ile Asp Ala Gln Asp Phe Glu

450 455 460

Lys Ala Ala Gly Leu Arg Asp Lys Glu Arg Lys Leu Gly Glu Glu Arg

465 470 475 480

Ser Glu Lys Glu Lys Gln Trp Arg Ser Gly Asp Leu Glu Asp Ile Ala

485 490 495

Glu Val Gly Glu Glu Gln Ile Ala Glu Val Leu Ala Asn Trp Thr Gly

500 505 510

Ile Pro Val Phe Lys Leu Thr Glu Ala Glu Ser Ser Arg Leu Leu Asn

515 520 525

Met Glu Glu Glu Leu His Lys Arg Ile Ile Gly Gln Asp Glu Ala Val

530 535 540

Lys Ala Val Ser Arg Ala Ile Arg Arg Thr Arg Ala Gly Leu Lys Asp

545 550 555 560

Pro Lys Arg Pro Ser Gly Ser Phe Ile Phe Ala Gly Pro Ser Gly Val

565 570 575

Gly Lys Thr Glu Leu Ser Lys Ala Leu Ala Gly Phe Leu Phe Gly Asp

580 585 590

Asp Asp Ser Leu Ile Gln Ile Asp Met Gly Glu Phe His Asp Arg Phe

595 600 605

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

610 615 620

Glu Gly Gly Gln Leu Thr Glu Lys Val Arg Arg Lys Pro Phe Ser Val

625 630 635 640

Val Leu Phe Asp Glu Ile Glu Lys Ala His Lys Glu Ile Tyr Asn Thr

645 650 655

Leu Leu Gln Val Leu Glu Asp Gly Arg Leu Thr Asp Gly Gln Gly Arg

660 665 670

Ile Val Asp Phe Lys Asn Thr Val Leu Ile Phe Thr Ser Asn Leu Gly

675 680 685

Thr Ser Asp Ile Ser Lys Ala Val Gly Leu Gly Phe Ser Gly Ser Ser

690 695 700

Glu Thr Asp Ser Asp Ala Gln Tyr Asp Arg Met Lys Asn Lys Val His

705 710 715 720

Asp Glu Leu Lys Lys His Phe Arg Pro Glu Phe Leu Asn Arg Ile Asp

725 730 735

Glu Ile Val Val Phe His Gln Leu Thr Lys Asp Gln Ile Val Gln Met

740 745 750

Val Asp Leu Leu Ile Gly Arg Val Ser Asn Ala Leu Ala Glu Lys Asp

755 760 765

Met Ser Ile Glu Leu Thr Glu Lys Ala Lys Asp Leu Leu Ala Ser Arg

770 775 780

Gly Phe Asp Pro Val Leu Gly Ala Arg Pro Leu Arg Arg Thr Ile Gln

785 790 795 800

Arg Glu Ile Glu Asp Gln Met Ser Glu Lys Ile Leu Phe Gly Glu Ile

805 810 815

Gly Ala Gly Glu Ile Val Thr Val Asp Val Glu Gly Trp Asp Gly Glu

820 825 830

Ser Lys Asp Thr Asp Arg Ala Lys Phe Thr Phe Thr Pro Arg Pro Lys

835 840 845

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

850 855 860

Ala Ile Gln Asp Val Asp Ser Ala Ala Asp Gly Asp Val Pro Glu Thr

865 870 875 880

Asp Ser Leu Ser Asp Ile Asp Leu Glu Thr Leu Glu Lys Phe Glu Glu

885 890 895

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

900 905 910

Gly Thr Asp Asp Gln Gly Gly Thr Ala Pro Ser Lys Glu

915 920 925

<210> 6

<211> 2778

<212> DNA

<213> Unknown

<220>

<223> ORF WT NT clpC

<400> 6

atgttcgaga ggtttaccga tcgtgcacgc cgcgtgattg tgctcgcgca ggaagaggcg 60

cgcatgctca accacaatta catcggcacg gagcacattc tcctcggcct cattcacgag 120

ggcgagggcg ttgcagccaa ggctttggaa tccatgggaa tttccctgga cgccgtccgc 180

caggaagtcg aagagattat cggccagggc tcccagccca ccaccggcca cattcctttt 240

actccacgtg ccaagaaggt cctggagctc agcctccgcg agggactaca gatgggacac 300

aagtacatcg gtactgagtt cctgcttctc ggtttgatcc gtgagggcga gggcgttgct 360

gcccaggtcc tggtcaagct tggtgctgat ctgccacgcg tgcgtcagca agttattcag 420

cttctctccg gctacgaagg tggccagggc ggatccccag agggcggtca gggcgcccct 480

actggcggtg acgctgttgg tgcaggagct gctcctggcg gtcgtccatc ttcgggcggc 540

ccaggcgagc gttctacctc tttggttctt gaccagttcg gccgcaacct cacccaggcc 600

gcaaaggacg gcaagctgga cccagttgtt ggtcgtgata aggaaatcga gcgcatcatg 660

caggtgctct cccgtcgtac caagaacaac ccagttctta ttggtgagcc aggtgttggt 720

aagaccgcag ttgttgaagg tcttgcacta gacattgtta acggcaaggt tccagagacc 780

ctcaaggaca agcaggttta ctcccttgac ttaggttcac tggttgcagg ttcccgttac 840

cgcggtgact tcgaagagcg cctgaagaag gtcctcaagg agattaacca gcgcggcgac 900

atcatcctgt ttatcgatga gatccacacc ctcgtgggtg caggtgcagc agaaggcgca 960

atcgacgccg cctccctgct taagccaaag cttgcccgcg gtgaactgca gaccattggt 1020

gcaaccacct tggacgagta ccgtaagcac attgaaaagg acgcagctct tgagcgtcgt 1080

ttccagccag tgcaggttcc agagccttcg gttgatctca ccgttgagat cttgaagggt 1140

ctgcgcgacc gctacgaagc tcaccaccgc gtatccatca ccgatggtgc tcttaccgca 1200

gcagctcagc ttgctgatcg ctacattaac gaccgcttct tgccagataa ggccgttgac 1260

ctcatcgatg aggctggcgc ccgcatgcgc atcaagcgca tgaccgcacc gtcctccctc 1320

cgcgaggttg atgagcgcat cgctgatgtt cgccgtgaga aggaagcagc gatcgatgct 1380

caggactttg agaaggcagc aggtcttcgc gataaggagc gcaagctcgg cgaagagcgt 1440

tcagagaagg aaaagcagtg gcgctccggc gacctcgagg acattgctga ggttggcgaa 1500

gagcagatcg cagaagtact ggccaactgg actggtattc ctgtcttcaa gctcaccgaa 1560

gctgagtctt cccgcctgct caacatggaa gaagagttgc acaagcgcat catcggacag 1620

gatgaagctg tcaaggctgt ctcccgtgcg atccgtcgta cccgtgcagg tctgaaggat 1680

cctaagcgtc cttccggctc cttcatcttc gctggtccat ccggtgttgg taagaccgag 1740

ctgtccaagg cacttgctgg attcctcttc ggtgacgatg attccctcat ccaaatcgac 1800

atgggtgaat tccacgaccg cttcaccgcg tcccgactct tcggtgcccc tccgggatac 1860

gttggctacg aagaaggtgg ccagctgacc gagaaggttc gccgtaagcc attctccgtt 1920

gtgcttttcg acgagatcga gaaggcccac aaggatct acaacacctt gctgcaggtg 1980

ttggaagatg gtcgccttac cgatggtcag ggtcgcattg tggacttcaa gaacaccgtc 2040

ctgatcttca cctccaacct gggcacctct gacatctcca aggctgttgg cctgggcttc 2100

tccggatcct ctgagactga cagcgatgct cagtacgacc gcatgaagaa caaggtccac 2160

gacgagctga agaagcactt ccgccctgag ttcctgaacc gtattgatga gatcgtggtc 2220

ttccaccagc tcaccaagga tcagatcgtt cagatggtcg accttcttat tggccgcgtg 2280

tccaacgcac tggctgagaa ggacatgagc atcgaactga ctgagaaggc taaggatctc 2340

cttgccagcc gcggcttcga tccagttctg ggtgcacgac cattgcgtcg caccattcag 2400

cgcgaaatcg aagaccagat gtccgagaag atcctcttcg gagaaatcgg tgctggcgag 2460

atcgtcaccg tcgacgtcga aggctgggac ggcgagtcca aggacaccga ccgtgcgaag 2520

ttcaccttca caccacgtcc aaagccaatg ccagaaggta agttctctga gatctccgta 2580

caggctgcag aagcaatcca agatgtagat tccgcagctg acggcgatgt cccagaaacc 2640

gattcacttt ccgacattga ccttgaaacc cttgagaagt tcgaggaaga tgtagaaaac 2700

ggcaccgaca ttgatcaggt atccggtgac tactacggca ccgatgatca gggaggcact 2760

gctccaagca aggagtag 2778

<210> 7

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> P1

<400> 7

ctattctaga tccagagacc ctcaaggaca agcag 35

<210> 8

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> P2

<400> 8

ggacggtgcg gtcatgcgca tgcgggcgcc 30

<210> 9

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> P3

<400> 9

ggcgcccgca tgcgcatgac cgcaccgtcc 30

<210> 10

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223> P4

<400> 10

ctattctaga tcgatttgga tgagggaatc atcg 34

<210> 11

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> P5

<400> 11

aatttctaga ggcagaccct attctatgaa gg 32

<210> 12

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> P6

<400> 12

agtgtttcgg tctttacaga cacgagggac 30

<210> 13

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> P7

<400> 13

gtccctcgtg tctgtaaaga ccgaaacact 30

<210> 14

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> P8

<400> 14

aatttctaga cgtgggagtg tcactcgctt gg 32

<210> 15

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> P9

<400> 15

tcctctagag ctgcgcagtg ttgaatacg 29

<210> 16

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> P10

<400> 16

tggaaatctt ttcgatgttc acgttgacat 30

<210> 17

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> P11

<400> 17

acatcgaaaa gatttccacc tctgagattc 30

<210> 18

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> P12

<400> 18

gactctagag ttcacctcag agacgatta 29

<210> 19

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> P13

<400> 19

tcctctagac tggtcgcctg atgttctac 29

<210> 20

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> P14

<400> 20

cacgatcaga tgtgcatcat cat 23

<210> 21

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> P15

<400> 21

atgatgatgc acatctgatc gtg 23

<210> 22

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<223> P16

<400> 22

gactctagat tagtcccttt cgaggcgga 29

<210> 23

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> P17

<400> 23

acggatccca gactccaaag caaaagcg 28

<210> 24

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> P18

<400> 24

acaccacggc agaaccaggt gcaaaggaca 30

<210> 25

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> P19

<400> 25

ctggttctgc cgtggtgtgc atcatctctg 30

<210> 26

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> P20

<400> 26

acggatccaa ccaaacttgc tcacactc 28

<---

Claims (11)

1. A microorganism of the genus Corynebacterium for producing a branched-chain amino acid, in which the activity of the ATP (adenosine triphosphate)-dependent protease is weakened compared to an unmodified microorganism, wherein said microorganism comprises a polypeptide having a deletion of amino acids corresponding to positions 431-433 based on the sequence presented by SEQ ID NO: 5, or a polynucleotide having a deletion of nucleotides corresponding to positions 1291-1299 based on the sequence presented by SEQ ID NO: 6, wherein the branched-chain amino acid is L-valine or L-isoleucine. 2. A microorganism of the genus Corynebacterium according to claim 1, wherein the weakening is a weakening of the activity of the ATP-binding subunit of the ATP-dependent Clp protease. 3. A microorganism of the genus Corynebacterium according to claim 2, wherein the ATP-binding subunit of the ATP-dependent Clp protease is of origin from the genus Corynebacterium . 4. A microorganism of the genus Corynebacterium according to claim 2, wherein the ATP-binding subunit of the ATP-dependent Clp protease comprises a polypeptide represented by SEQ ID NO: 5 or an amino acid sequence having 90% or more identity thereto. 5. A microorganism of the genus Corynebacterium according to claim 1, wherein said microorganism of the genus Corynebacterium is Corynebacterium glutamicum . 6. A polypeptide involved in the production of a branched-chain amino acid comprising a deletion of the amino acids corresponding to positions 431-433 based on the amino acid sequence represented by SEQ ID NO: 5, wherein the branched-chain amino acid is L-valine or L-isoleucine. 7. The polypeptide of claim 6, wherein said polypeptide consists of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 90% or more identity thereto. 8. A method for producing a branched-chain amino acid, comprising a stage of culturing in a medium a microorganism of the genus Corynebacterium according to claim 1, wherein the branched-chain amino acid is L-valine or L-isoleucine. 9. The method according to claim 8, further comprising the step of isolating the branched chain amino acid from the medium or from the microorganism after the culturing step. 10. The use of a microorganism of the genus Corynebacterium according to claim 1 for producing a branched-chain amino acid, wherein the branched-chain amino acid is L-valine or L-isoleucine. 11. Use of a polypeptide comprising a deletion of amino acids corresponding to positions 431-433 based on the amino acid sequence represented by SEQ ID NO: 5, for producing a branched chain amino acid, wherein the branched chain amino acid is L-valine or L-isoleucine.