NOVEL GLUTAMATE GLUCONATE REPRESSOR MUTANT AND METHOD FOR PRODUCING L-ARGININE USING THE SAME

Abstract

The present application relates to novel gluconate repressor mutants, polynucleotides comprising the mutants of the present application, Corynebacterium microorganisms comprising the mutants or polynucleotides of the present application, and methods for producing L-arginine using the microorganisms of the present application.

Description

[Technical field]

[0001] The present application relates to novel gluconate repressor mutants, polynucleotides comprising the mutants of the present application, Corynebacterium microorganisms comprising the mutants of the present application or the polynucleotides of the present application, and methods for producing L-arginine using the microorganisms of the present application. [Background technology]

[0002] L-arginine is used for medicinal purposes such as liver function promoters, brain function improvers, and comprehensive amino acid preparations, and in recent years has also been attracting attention as a food additive such as a kamaboko additive, a health drink additive, and a salt substitute for hypertensive patients. Research is ongoing into the use of microorganisms to produce high-concentration arginine for industrial use.

[0003] Meanwhile, Corynebacterium genus microorganisms, especially Corynebacterium glutamicum, are Gram-positive microorganisms that are widely used for L-amino acid production. To produce L-arginine, target substance-specific approaches are mainly used in Corynebacterium genus strains, such as increasing the expression of genes encoding enzymes involved in L-arginine biosynthesis and removing genes unnecessary for L-arginine biosynthesis (Patent Document 1).

[0004] However, there remains a need for research into methods for producing L-arginine efficiently and in high yield. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Korean Patent No. 10-1102263 [Patent Document 2] Korean Patent Publication No. 10-2020-0136813 [Patent Document 3] U.S. Patent No. 8,034,602

Non-licensed literature

[0006] [Non-licensed document 1] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-licensed document 2] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-licensed document 3] J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

[0007] The present inventors have developed a novel gluconate repressor mutant that increases L-arginine production, a polynucleotide encoding the mutant, an L-arginine-producing microorganism containing the mutant or the polynucleotide, and an L-arginine production method using the microorganism, and have completed the present application. [Means for solving the problem]

[0008] The present application aims to provide a gluconate repressor mutant in which at least one amino acid corresponding to a position in the amino acid sequence of SEQ ID NO:1 is replaced by another amino acid.

[0009] The present application also aims to provide polynucleotides encoding the variants of the present application.

[0010] A further object of the present application is to provide a Corynebacterium microorganism comprising the variant of the present application or a polynucleotide encoding the variant of the present application.

[0011] Furthermore, the present application aims to provide a gluconate repressor mutant in which the amino acid corresponding to the 70th position in the amino acid sequence of SEQ ID NO:1 is replaced with another amino acid, or a Corynebacterium microorganism containing a polynucleotide encoding the mutant of the present application.

[0012] Furthermore, the present application aims to provide a method for producing L-arginine, comprising a step of culturing the microorganism of the present application in a medium.

[0013] Furthermore, the present application aims to provide a composition for producing L-arginine, comprising the microorganism of the present application, a medium in which the microorganism of the present application has been cultured, or a combination of at least two thereof.

[0014] Furthermore, the present application aims to provide a use of the microorganism of the present application for producing L-arginine.

[0015] Furthermore, the present application aims to provide a use of a gluconate repressor mutant in which at least one amino acid selected from the group consisting of the amino acid corresponding to the 36th position, the 59th position, the 60th position, the 63rd position, the 79th position, and the 92nd position of the amino acid sequence of SEQ ID NO:1 is substituted with another amino acid for L-arginine production. Effect of the Invention

[0016] When a Corynebacterium microorganism containing the gluconate repressor mutant of the present application is cultured, L-arginine can be produced in a higher yield than when a microorganism does not contain the mutant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] These will be described in detail below. Each description and embodiment disclosed in this application is also applicable to other descriptions and embodiments. That is, all combinations of various elements disclosed in this application are included in this application. In addition, this application is not limited to the following specific description. Furthermore, many papers and patent documents are referenced throughout this specification, and citations are provided. The disclosure contents of the cited papers and patent documents are incorporated in their entirety into this specification by reference, thereby more clearly explaining the state of the art to which this application pertains and the contents of this application.

[0018] One aspect of the present application provides a gluconate repressor mutant in which at least one amino acid corresponding to a position in the amino acid sequence of SEQ ID NO:1 is replaced with another amino acid.

[0019] In the present application, the term "gluconate repressor mutant" refers to a gluconate repressor mutant that contains at least one amino acid substitution in the amino acid sequence of a polypeptide having gluconate repressor activity.

[0020] In the present application, "gluconate repressor (GntR)" refers to a regulatory protein involved in gluconate metabolism, sugar uptake, etc. The gluconate repressor is also referred to as "gluconate repressor protein."

[0021] The gluconate repressor protein of the present application is a gluconate repressor protein or a variant thereof derived from a Corynebacterium microorganism, specifically Corynebacterium glutamicum, but is not limited thereto. Specifically, the gluconate repressor protein includes, for example, an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having 70% or more homology or identity thereto, but is not limited thereto as long as it has gluconate repressor activity. Specifically, the amino acid sequence includes an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto. The sequence of SEQ ID NO: 1 can be obtained from the publicly known databases NCBI GenBank or KEGG (Kyoto Encyclopedia of Genes and Genomes). For example, the present invention is not limited to those derived from the genus Corynebacterium or Corynebacterium glutamicum, and more specifically, to those polypeptides / proteins that contain the amino acid sequence represented by SEQ ID NO: 1. Needless to say, the present application also includes auxiliary proteins having an amino acid sequence in which a part of the sequence has been deleted, modified, substituted or added, so long as the amino acid sequence has such homology or identity and exhibits an efficacy equivalent to that of the above protein.

[0022] Furthermore, the gluconate repressor protein having the amino acid sequence of SEQ ID NO:1 has or contains the sequence of SEQ ID NO:2, or a base sequence 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, and less than 100% homology or identity to the sequence of SEQ ID NO:2, or consists of the sequence of SEQ ID NO:2, or a base sequence 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, and less than 100% homology or identity to the sequence of SEQ ID NO:2, or is encoded by a polynucleotide that is substantially composed of the base sequence, but is not limited to these.

[0023] In the present application, the term "variant" refers to a polypeptide that differs from the amino acid sequence of the variant before the mutation by conservative substitution and / or modification of at least one amino acid, but maintains its functions or properties. Such variants can generally be identified by modifying at least one amino acid in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant is improved, unchanged, or decreased compared to the polypeptide before the mutation. Some variants also include variants in which at least one portion, such as an N-terminal leader sequence or a transmembrane domain, has been removed. Other variants also include variants in which a portion has been removed from the N- and / or C-terminus of a mature protein. The term "variant" is used interchangeably with terms such as mutant, modification, mutant polypeptide, mutated protein, and mutation (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), but may be any term that means a mutation.

[0024] The variant may also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the N-terminus of the variant may be linked to a signal (or leader) sequence involved in co-translationally or post-translationally protein translocation. The variant may also be linked to other sequences or linkers to allow identification, purification, or synthesis.

[0025] The gluconate repressor mutant of the present application may be, but is not limited to, a gluconate repressor mutant in which at least one amino acid selected from the group consisting of the amino acid corresponding to the 36th position, the 59th position, the 60th position, the 63rd position, the 79th position, and the 92nd position of the amino acid sequence of SEQ ID NO:1 is substituted with another amino acid.

[0026] In one embodiment, the mutant has at least one amino acid selected from the group consisting of the amino acid corresponding to the 36th position, the amino acid corresponding to the 59th position, the amino acid corresponding to the 60th position, the amino acid corresponding to the 63rd position, the amino acid corresponding to the 79th position, and the amino acid corresponding to the 92nd position of the amino acid sequence of SEQ ID NO: 1 replaced with another amino acid, and may further have the amino acid corresponding to the 70th position of the amino acid sequence of SEQ ID NO: 1 replaced with another amino acid, but is not limited to this.

[0027] The "other amino acid" may be any amino acid different from the amino acid before substitution. It goes without saying that "a specific amino acid is substituted" in the present application means that the amino acid before substitution is substituted with an amino acid different from the amino acid before substitution, even if it is not specified that the amino acid is substituted with another amino acid.

[0028] The amino acids are commonly classified based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues.

[0029] As an example of such a classification, the positively charged (basic) amino acids include arginine, lysine, and histidine; the negatively charged (acidic) amino acids include glutamic acid and aspartic acid; the amino acids with a nonpolar side chain (nonpolar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and the amino acids with a polar or hydrophilic side chain (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Other examples include arginine, lysine, histidine, glutamic acid, and aspartic acid, which are electrically charged amino acids, and glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, which are uncharged amino acids (also called neutral amino acids). As further examples, phenylalanine, tryptophan, and tyrosine are classified as aromatic amino acids. As further examples, valine, leucine, and isoleucine are classified as branched amino acids. As another example, the 20 types of amino acids are classified by size into five groups in order of decreasing relative volume: glycine, alanine, serine group, cysteine, proline, threonine, aspartic acid, asparagine group, valine, histidine, glutamic acid, glutamine group, isoleucine, leucine, methionine, lysine, arginine group, phenylalanine, tryptophan, tyrosine group, but are not necessarily limited to these.

[0030] For example, "the amino acid corresponding to the 36th position in SEQ ID NO:1 is substituted with another amino acid" means that the amino acid corresponding to the 36th position in SEQ ID NO:1 is substituted with asparagine, phenylalanine, glycine, alanine, arginine, aspartate, cysteine, glutamate, leucine, glutamine, histidine, proline, serine, tyrosine, lysine, tryptophan, valine, methionine, or threonine, excluding isoleucine, but is not limited to these.

[0031] Even if the present application describes a "protein having an amino acid sequence represented by a specific SEQ ID NO," it goes without saying that a protein having an amino acid sequence in which a part of the sequence has been deleted, modified, substituted, conservatively substituted, or added is also included in the present application, so long as it has the same or corresponding activity as a protein consisting of the amino acid sequence of the SEQ ID NO. For example, it goes without saying that addition of a sequence that does not change the function of the protein before or after the amino acid sequence, naturally occurring mutations, silent mutations, and conservative substitutions are not excluded, and those having such additions or mutations are also included in the present application, so long as they have the same or corresponding activity as the mutant protein.

[0032] In the present application, the "Nth position" includes the Nth position and an amino acid position corresponding to the Nth position. Specifically, it includes an amino acid position corresponding to any amino acid residue in a mature polypeptide represented by a specific amino acid sequence. The specific amino acid sequence may be the amino acid sequence of SEQ ID NO:1.

[0033] "Corresponding to" in this application means the amino acid residue at the recited position in the polypeptide, or an amino acid residue similar, identical or equivalent to the recited residue in the polypeptide. Identifying the amino acid at the corresponding position will determine the particular amino acid of the sequence to which the particular sequence is referenced. "Corresponding region" in this application generally refers to a similar or corresponding position in a related or reference protein.

[0034] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, and based thereon, each amino acid residue of the amino acid sequence can be numbered with reference to the number and position of the amino acid residues corresponding to the amino acid residues in SEQ ID NO: 1. For example, the sequence alignment algorithm in the present application can identify the positions of amino acids or positions where modifications such as substitutions, insertions, deletions, etc. occur when compared to a query sequence (also referred to as a "reference sequence").

[0035] For such alignment, for example, the Needleman-Wunsch algorithm (Non-Patent Document 1) or the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) can be used, but is not limited to these. Sequence alignment programs and pairwise sequence comparison algorithms known in the technical field can be used as appropriate.

[0036] In one embodiment, the variant provided by the present application may include an amino acid sequence in which one, two, three, four, five or six amino acids at positions corresponding to the 36th, 59th, 60th, 63rd, 79th and 92nd positions from the N-terminus of SEQ ID NO: 1 are replaced with other amino acids.

[0037] In any of the above-mentioned embodiments, the variant includes at least one substitution selected from the group consisting of, but not limited to, a substitution of the amino acid corresponding to position 36 of the amino acid sequence of SEQ ID NO:1 with asparagine, a substitution of the amino acid corresponding to position 59 with alanine, a substitution of the amino acid corresponding to position 60 with leucine, a substitution of the amino acid corresponding to position 63 with alanine, a substitution of the amino acid corresponding to position 79 with alanine, and a substitution of the amino acid corresponding to position 92 with leucine.

[0038] In any of the above-mentioned embodiments, the variant comprises at least one substitution selected from the group consisting of: substitution of the amino acid corresponding to position 36 of the amino acid sequence of SEQ ID NO:1 with asparagine, substitution of the amino acid corresponding to position 59 with alanine, substitution of the amino acid corresponding to position 60 with leucine, substitution of the amino acid corresponding to position 63 with alanine, substitution of the amino acid corresponding to position 79 with alanine, and substitution of the amino acid corresponding to position 92 with leucine, and may further comprise substitution of the amino acid corresponding to position 70 of the amino acid sequence of SEQ ID NO:1 with lysine, but is not limited thereto.

[0039] In any of the above-mentioned embodiments, the variant provided by the present application may include a substitution of the amino acid corresponding to the 36th position from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0040] In any of the above-mentioned embodiments, the mutant may be one in which the amino acid corresponding to the 36th position from the N-terminus of SEQ ID NO: 1 is replaced with an amino acid selected from polar amino acids serine, cysteine, tyrosine, asparagine and glutamine. In any of the above-mentioned embodiments, the mutant may be one in which the amino acid corresponding to the 36th position from the N-terminus of SEQ ID NO: 1 is replaced with asparagine (N).

[0041] In any of the above-mentioned embodiments, the variant provided by the present application may include a substitution of the amino acid corresponding to the 59th position from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0042] In any of the above-mentioned embodiments, the mutant may be one in which the amino acid corresponding to the 59th position from the N-terminus of SEQ ID NO: 1 is replaced with a non-polar amino acid. For example, the amino acid may be an amino acid selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. In any of the above-mentioned embodiments, the mutant may be one in which the amino acid corresponding to the 59th position from the N-terminus of SEQ ID NO: 1 is replaced with alanine (A).

[0043] In any of the above-mentioned embodiments, the variant provided by the present application may include a substitution of the amino acid corresponding to the 60th position from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0044] In any of the above-mentioned embodiments, the mutant may be one in which the amino acid corresponding to the 60th position from the N-terminus of SEQ ID NO: 1 is replaced with a non-polar amino acid. For example, the amino acid may be an amino acid selected from glycine, alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. In any of the above-mentioned embodiments, the mutant may be one in which the amino acid corresponding to the 60th position from the N-terminus of SEQ ID NO: 1 is replaced with leucine (L).

[0045] In any of the above-mentioned embodiments, the variant provided by the present application may include a substitution of the amino acid corresponding to the 63rd position from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0046] In any of the above-mentioned embodiments, the mutant may be one in which the amino acid corresponding to the 63rd position from the N-terminus of SEQ ID NO: 1 is replaced with a non-polar amino acid. For example, the amino acid may be an amino acid selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. In any of the above-mentioned embodiments, the mutant may be one in which the amino acid corresponding to the 63rd position from the N-terminus of SEQ ID NO: 1 is replaced with alanine (A).

[0047] In any of the above-mentioned embodiments, the variant provided by the present application may include a substitution of the amino acid corresponding to the 79th position from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0048] In any of the above-mentioned embodiments, the mutant may be one in which the amino acid corresponding to the 79th position from the N-terminus of SEQ ID NO: 1 is replaced with a non-polar amino acid. For example, the amino acid may be an amino acid selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. In any of the above-mentioned embodiments, the mutant may be one in which the amino acid corresponding to the 79th position from the N-terminus of SEQ ID NO: 1 is replaced with alanine (A).

[0049] In any of the above-mentioned embodiments, the variant provided by the present application may include a substitution of the amino acid corresponding to the 92nd position from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0050] In any of the above-mentioned embodiments, the mutant may be one in which the amino acid corresponding to the 92nd position from the N-terminus of SEQ ID NO: 1 is replaced with a non-polar amino acid. For example, the amino acid may be an amino acid selected from glycine, alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. In any of the above-mentioned embodiments, the mutant may be one in which the amino acid corresponding to the 92nd position from the N-terminus of SEQ ID NO: 1 is replaced with leucine (L).

[0051] In any of the above-mentioned embodiments, the variant provided by the present application may include a substitution of the amino acid corresponding to the 70th position from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0052] In any of the above-mentioned embodiments, the variant provided by the present application may be one in which the amino acid corresponding to the 70th position from the N-terminus of SEQ ID NO: 1 is replaced with an amino acid selected from arginine, lysine, histidine, glutamic acid, and aspartic acid, which are amino acids having a charged side chain. For example, the amino acid may be an amino acid selected from basic amino acids arginine, lysine, and histidine. In any of the above-mentioned embodiments, the variant may be one in which the amino acid corresponding to the 70th position from the N-terminus of SEQ ID NO: 1 is replaced with lysine (K).

[0053] Meanwhile, a person skilled in the art can ascertain, by sequence alignment known in the technical field, the amino acids corresponding to the 36th, 59th, 60th, 63rd, 70th, 79th, and 92nd positions in the amino acid sequence of SEQ ID NO: 1 of the present application in an arbitrary amino acid sequence. It goes without saying that, even if not otherwise specified in the present application, "an amino acid at a specific position in a specific SEQ ID NO" includes "an amino acid at a corresponding position" in an arbitrary amino acid sequence.

[0054] The variant of the present application may be an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity to the amino acid sequence represented by SEQ ID NO:1, in which at least one amino acid selected from the group consisting of amino acids corresponding to the 36th, 59th, 60th, 63rd, 79th and 92nd positions from the N-terminus of SEQ ID NO:1 is substituted with another amino acid, or may be an amino acid sequence in which at least one amino acid selected from the group consisting of amino acids corresponding to the 36th, 59th, 60th, 63rd, 79th and 92nd positions from the N-terminus of SEQ ID NO:1 is substituted with another amino acid, and further in which the amino acid corresponding to the 70th position of the amino acid sequence of SEQ ID NO:1 is substituted with another amino acid. Furthermore, it goes without saying that variants having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted or added are also included in the present application, so long as the variant has such homology or identity and exhibits efficacy equivalent to that of the variant of the present application.

[0055] For example, the variant of the present application may have an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 23 to SEQ ID NO: 29, may contain the amino acid sequence, or may essentially consist of the amino acid sequence. Alternatively, the variant may contain an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity to an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 23 to SEQ ID NO: 29.

[0056] For example, the variants may have additions or deletions of sequences at the N-terminus, C-terminus and / or internally of the amino acid sequence that do not alter the function of the variants of the present application, naturally occurring mutations, silent mutations or conservative substitutions.

[0057] In the present application, "conservative substitution" refers to the replacement of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally occur based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine, negatively charged (acidic) amino acids include glutamic acid and aspartic acid, aromatic amino acids include phenylalanine, tryptophan, and tyrosine, and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Amino acids are also classified into those with electrically charged side chains and those with uncharged side chains, with the charged side chains including aspartic acid, glutamic acid, lysine, arginine, and histidine, and the uncharged side chains are further classified into nonpolar and polar amino acids, with the nonpolar amino acids including glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, and the polar amino acids including serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Typically, conservative substitutions have little or no effect on the activity of the resulting protein or polypeptide.

[0058] Variants may also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide may be linked to an N-terminal signal (or leader) sequence of a protein involved in co-translationally or post-translationally protein transfer. The polypeptide may also be linked to other sequences or linkers that allow the polypeptide to be identified, purified or synthesized.

[0059] As an example of the present application, the variant of the present application may have gluconate repressor activity, and may have an activity that improves L-arginine production ability compared to a wild-type polypeptide having gluconate repressor activity.

[0060] Another aspect of the present application provides polynucleotides encoding the variants of the present application.

[0061] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and means a DNA or RNA chain longer than a certain length, and more specifically, means a polynucleotide fragment encoding the mutant.

[0062] A polynucleotide encoding a gluconate repressor variant of the present application can be any polynucleotide sequence that encodes a gluconate repressor variant of the present application, including, but not limited to, a polynucleotide sequence that encodes the amino acid sequence of the variant of the present application.

[0063] For example, a polynucleotide encoding a gluconate repressor mutant of the present application is a polynucleotide sequence selected from the group consisting of polynucleotide sequences represented by SEQ ID NO: 30 to SEQ ID NO: 36, or a polynucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to a polynucleotide sequence selected from the group consisting of polynucleotide sequences represented by SEQ ID NO: 30 to SEQ ID NO: 36, but is not limited thereto. A nucleotide sequence having homology or identity may be one excluding sequences having 100% identity within the above category, or may be a sequence having less than 100% identity.

[0064] The polynucleotide of the present application may be modified in various ways in the coding region, by codon degeneracy or by taking into consideration the codons preferred in the organism in which the variant of the present application is to be expressed, as long as the amino acid sequence of the variant of the present application is not changed. Therefore, it goes without saying that the polynucleotide of the present application includes a polynucleotide that is translated into a polypeptide consisting of the amino acid sequence of the variant of the present application, or a polypeptide having homology or identity thereto, by codon degeneracy.

[0065] Furthermore, the polynucleotide of the present application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a complementary sequence to all or a part of the polynucleotide sequence of the present application.

[0066] The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Documents 3 and 4). For example, the conditions include conditions under which polynucleotides having high homology or identity, such as polynucleotides having a homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, hybridize with each other, and polynucleotides having a lower homology or identity than that do not hybridize with each other, or conditions under which washing is performed once, specifically 2 to 3 times, at a salt concentration and temperature equivalent to 60°C, 1×SSC, and 0.1% SDS, specifically 60°C, 0.1×SSC, and 0.1% SDS, which are washing conditions for normal Southern hybridization, more specifically 68°C, 0.1×SSC, and 0.1% SDS.

[0067] Hybridization requires that two nucleic acids have complementary sequences, even though mismatches between bases are possible depending on the stringency of hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application may include isolated nucleic acid fragments that are complementary to the entire sequence, as well as substantially similar nucleic acid sequences.

[0068] Specifically, polynucleotides having homology or identity can be detected using the hybridization conditions described above, in which the hybridization step is performed at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and may be appropriately adjusted by those skilled in the art depending on the purpose.

[0069] The appropriate stringency for hybridizing the polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables known in the art (eg, Non-Patent Document 3).

[0070] In this application, "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, expressed as a percentage. Homology and identity are often used interchangeably.

[0071] Sequence homology or identity of conserved polynucleotides or polypeptides may be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences will generally hybridize to all or part of the sequence under moderate or high stringent conditions. Hybridization, of course, also includes hybridization to polynucleotides having common codons or codons that take into account the degeneracy of codons in the polynucleotide.

[0072] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program with default parameters as in, for example, Non-Patent Document 5. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 1), as implemented in the Needle program (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later) of the EMBOSS package (which includes the GCG program package (Non-Patent Document 6), BLASTP, BLASTN, FASTA (Non-Patent Documents 7, 8 and 9)). For example, BLAST or Clustal W from the National Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0073] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as that disclosed in, for example, J. Biol. 1999, 143:1311-1354 (2002). Briefly, the GAP program defines the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include (1) a binary comparison matrix (identity takes a value of 1 and non-identity takes a value of 0) and a weighted comparison matrix (or EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in, (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5), and (3) no penalty for terminal gaps. Thus, "homology" or "identity" in this application refers to the relevance between sequences.

[0074] Yet another aspect of the present application provides a vector comprising the polynucleotide of the present application, the vector being, but not limited to, an expression vector for expressing the polynucleotide in a microorganism.

[0075] In this application, the term "vector" refers to a DNA product comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host. The expression control region includes a promoter for initiating transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating the termination of transcription and translation. When transformed into a suitable microorganism, the vector can replicate and function independently of the host genome and is integrated into the genome itself.

[0076] The vector used in the present application is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. may be used as phage or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc. may be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. may be used.

[0077] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome by a vector for intracellular chromosome introduction. The polynucleotide can be inserted into a chromosome by any method known in the art, such as, but not limited to, homologous recombination. A selection marker for confirming whether or not it has been inserted into the chromosome may be further included. The selection marker is for selecting cells transformed with the vector, i.e., for confirming whether or not the target nucleic acid molecule has been inserted, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide is used. In an environment treated with a selection agent, only cells expressing the selection marker survive or show a different phenotype, so that transformed cells can be selected.

[0078] In the present application, "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a microorganism to express the polypeptide encoded by the polynucleotide in the microorganism. The transformed polynucleotide may be any polynucleotide that is expressed in the microorganism, regardless of whether it is inserted into the chromosome of the microorganism or located extrachromosomally. The polynucleotide may include DNA and / or RNA that encodes the target polypeptide. The polynucleotide may be introduced in any form as long as it is introduced into the microorganism and expressed. For example, the polynucleotide may be introduced into the microorganism in the form of an expression cassette, which is a genetic structure containing all elements necessary for its own expression. Typically, the expression cassette contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. The polynucleotide may be introduced into the microorganism in its own form and operably linked to a sequence necessary for expression in the microorganism, but is not limited thereto.

[0079] Additionally, 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 target variant of the present application.

[0080] Yet another aspect of the present application provides a Corynebacterium microorganism comprising a gluconate repressor mutant of the present application, a polynucleotide encoding the gluconate repressor mutant, or a vector comprising the polynucleotide.

[0081] As another example of the present application, the microorganism of the present application may be a Corynebacterium microorganism comprising a gluconate repressor mutant in which the amino acid corresponding to the 70th position of the amino acid sequence of SEQ ID NO:1 is substituted with another amino acid, a polynucleotide encoding the mutant, or a vector containing the polynucleotide.

[0082] The Corynebacterium microorganism of the present application may be a microorganism having the gluconate repressor mutant of the present application or the ability to produce L-arginine.

[0083] Specifically, the microorganism of the present application may be a microorganism naturally having a gluconate repressor or L-arginine producing ability, or a microorganism into which a mutant of the present application or a polynucleotide encoding the mutant (or a vector containing the polynucleotide) has been introduced into a parent strain lacking a gluconate repressor or L-arginine producing ability, and / or which has been imparted with L-arginine producing ability, but is not limited thereto.

[0084] In this application, "L-arginine" refers to a conditionally essential amino acid present in all living organisms. 6 H 14 N 4 O 2 It is known that L-arginine is produced mainly by microorganisms of the genus Corynebacterium, but it is known that these microorganisms are subject to feedback inhibition by arginine in the cells (Non-Patent Document 13), and it is known that there is a limit to the high-yield production of L-arginine.

[0085] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have been genetically modified, either naturally or artificially, and is a microorganism in which a specific mechanism has been weakened or enhanced by, for example, inserting an exogenous gene or enhancing or inactivating the activity of an endogenous gene, and is a microorganism that has been genetically modified to produce a desired polypeptide, protein, or product.

[0086] In the present application, the term "microorganism having L-arginine producing ability" refers to a prokaryotic or eukaryotic microbial strain that produces L-arginine in the organism, and includes a microorganism in which L-arginine producing ability has been imparted to a parent strain that does not have L-arginine producing ability, and a microorganism that has L-arginine producing ability endogenously. L-arginine producing ability can be imparted or improved by breeding.

[0087] In the present application, the term "unmodified microorganism" does not exclude strains containing naturally occurring mutations in microorganisms, but refers to a wild-type strain or a natural strain itself, or a strain before its characteristics are changed due to genetic mutation caused by natural or artificial factors. For example, the unmodified microorganism refers to a strain into which the gluconate repressor mutant in this specification has not been introduced or has not been introduced. The term "unmodified microorganism" is used interchangeably with "strain before modification", "microorganism before modification", "non-mutated strain", "non-modified strain", "non-mutated microorganism" or "reference microorganism".

[0088] The unmodified microorganism may be a microorganism comprising the amino acid sequence of SEQ ID NO:1 or the polynucleotide sequence of SEQ ID NO:2.

[0089] A microorganism capable of producing L-arginine according to the present application is, but is not limited to, a microorganism comprising at least one of a variant according to the present application, a polynucleotide according to the present application, and a vector comprising a polynucleotide according to the present application, a microorganism modified to express a variant according to the present application or a polynucleotide according to the present application, a microorganism (e.g., a recombinant strain) expressing a variant according to the present application or a polynucleotide according to the present application, or a microorganism (e.g., a recombinant strain) having activity of a variant according to the present application.

[0090] For example, the microorganism of the present application may be a cell or a microorganism transformed with a vector containing the polynucleotide of the present application or a polynucleotide encoding the mutant of the present application, and expressing the mutant of the present application. The microorganism of the present application also includes all microorganisms that contain the mutant of the present application and produce L-arginine. For example, the strain of the present application may be a recombinant strain in which a gluconate repressor mutant is expressed and L-arginine production ability is improved by introducing a polynucleotide encoding the mutant of the present application into a natural wild-type microorganism or a microorganism having L-arginine production ability. The recombinant strain with improved L-arginine production ability is a microorganism with improved L-arginine production ability compared to a natural wild-type microorganism or a microorganism with a non-modified gluconate repressor (i.e., a microorganism expressing a wild-type gluconate repressor or a microorganism not expressing the mutant of the present application), but is not limited thereto. For example, a strain with improved L-arginine production ability of the present application is a microorganism with improved L-arginine production ability compared to a microorganism containing the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding same, but is not limited thereto.

[0091] The microorganisms of the present application include all microorganisms that express the gluconate repressor mutants of the present application by various known methods other than the introduction of the nucleic acid or vector.

[0092] For example, the microorganism with improved L-arginine producing ability has an L-arginine producing ability of about 1% or more, specifically about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 10.5% or more, about 11% or more, about 11.5% or more, about 12% or more, about 12.5% ​​or more, about 13% or more, about 13.5% or more, about 14% or more, about 14.5% or more compared to the L-arginine producing ability of the parent strain before mutation or an unmodified microorganism having the endogenous activity of a gluconate repressor protein. or about 15% or more, about 15.5% or more, about 16% or more, about 16.5% or more, about 17% or more, about 17.5% or more, about 18% or more, about 18.5% or more, or about 19% or more (there is no particular limitation on the upper limit, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, or about 25% or less), but any increase in the + value compared to the productivity of the parent strain or unmodified microorganism before the mutation may be used. As another example, the recombinant strain with improved L-arginine production ability has an improved L-arginine production ability of about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, about 1.14 times or more, about 1.15 times or more, about 1.16 times or more, about 1.17 times or more, about 1.18 times or more, or about 1.19 times or more (there is no particular upper limit, for example, about 10 times or less, about 5 times or less, about 3 times or less, about 2 times or less, about 1.5 times or less, or about 1.3 times or less) compared to the parent strain or unmodified microorganism before mutation, but is not limited thereto. The term "about" refers to a range including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and may be any number in the same range as or similar to the number following the term "about", but is not limited thereto.

[0093] The microorganisms of the present application are Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens.

[0094] Specifically, the microorganism is Corynebacterium glutamicum, but is not limited thereto.

[0095] On the other hand, it is already known that Corynebacterium microorganisms produce L-arginine, but their productivity is very low, and the genes and mechanism that affect the production mechanism have not been elucidated.Therefore, the Corynebacterium microorganisms having L-arginine production ability of the present application include all of the following: natural wild-type microorganisms themselves, Corynebacterium microorganisms whose L-arginine production ability has been improved by strengthening or weakening the activity of genes related to the L-arginine production mechanism, and Corynebacterium microorganisms whose L-arginine production ability has been improved by introducing or strengthening the activity of an exogenous gene.

[0096] Yet another aspect of the present application provides a method for producing L-arginine, comprising culturing a microorganism of the present application in a medium.

[0097] Specifically, the method for producing L-arginine of the present application includes, but is not limited to, a step of culturing a Corynebacterium microorganism containing a mutant of the present application, a polynucleotide of the present application, or a vector of the present application in a medium.

[0098] "Culturing" in the present application means growing the microorganism of the present application under appropriately adjusted environmental conditions. The culturing process of the present application can be carried out in a suitable medium and under suitable culture conditions known in the art. Such a culturing process can be easily adjusted and used by a person skilled in the art depending on the strain selected. Specifically, the culturing can be batch, continuous and / or fed-batch culture, but is not limited thereto.

[0099] In the present application, the term "culture medium" refers to a substance that is a mixture of nutrients necessary for culturing the microorganism of the present application as the main components, and that supplies nutrients such as water essential for survival and growth, growth factors, etc. Specifically, the culture medium and other culture conditions used for culturing the microorganism of the present application may be any medium used for culturing ordinary microorganisms, and the microorganism of the present application can be cultured in an ordinary culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, etc., under aerobic conditions by adjusting the temperature, pH, etc.

[0100] For example, the culture medium for Corynebacterium sp. strains is disclosed in Non-Patent Document 14.

[0101] In the present application, the carbon source may be carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc., sugar alcohols such as mannitol, sorbitol, etc., organic acids such as pyruvic acid, lactic acid, citric acid, etc., amino acids such as glutamic acid, methionine, lysine, etc. Also, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, corn steeping liquid, etc. may be used, specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and any other suitable carbon source may be used. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0102] The nitrogen source may be an inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, or ammonium nitrate, or an organic nitrogen source such as an amino acid such as glutamic acid, methionine, or glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its decomposition product, or defatted soybean cake or its decomposition product. These nitrogen sources may be used alone or in combination of two or more kinds, but are not limited thereto.

[0103] The phosphorus source may be potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or a sodium-containing salt equivalent thereto. The inorganic compound may be sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, or other suitable compounds, including amino acids, vitamins, and / or suitable precursors. These components or precursors may be added to the medium in a batch or continuous manner. However, they are not limited thereto.

[0104] In addition, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the medium in a suitable manner during the cultivation of the microorganism of the present application. Furthermore, during the cultivation, foam formation can be suppressed using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, and in order to maintain the anaerobic and microaerobic state, no gas may be injected, and nitrogen, hydrogen, or carbon dioxide gas may be injected, but is not limited thereto.

[0105] In the culture of the present application, the culture temperature is maintained at 20 to 45° C., specifically 25 to 40° C., and the culture is carried out for about 10 to 160 hours, but is not limited thereto.

[0106] The L-arginine produced by the culture of the present application is either secreted into the medium or remains intracellularly.

[0107] The L-arginine production method of the present application may further comprise the step of providing a microorganism of the present application, the step of preparing a medium for culturing said strain, or a combination thereof (in any order), e.g., prior to said culturing step.

[0108] The method for producing L-arginine of the present application may further include a step of recovering L-arginine from the culture medium (culture medium) used in the culture or the microorganism of the present application. The recovery step may be further included after the culture step.

[0109] The recovery may be a collection of L-arginine using a suitable method known in the art depending on the culture method of the microorganism of the present application, such as batch, continuous, fed-batch culture, etc. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographies such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof may be used, and L-arginine may be collected from the medium or the microorganism using a suitable method known in the art.

[0110] In addition, the L-arginine production method of the present application may further include a purification step. The purification can be performed by any suitable method known in the art. For example, when the L-arginine production method of the present application includes both a recovery step and a purification step, the recovery step and the purification step can be performed continuously or discontinuously regardless of the order, and can be performed simultaneously or integrated into one step, but are not limited thereto.

[0111] Yet another aspect of the present application provides a composition for producing L-arginine, comprising a microorganism comprising at least one of a gluconate repressor mutant of the present application, a polynucleotide encoding the mutant, and a vector comprising the polynucleotide, a medium in which the microorganism is cultured, or a combination of at least two thereof.

[0112] The composition of the present application may further contain any suitable excipient that is commonly used in compositions for producing amino acids, including, but not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, isotonicity agents, etc.

[0113] Yet another aspect of the present application provides use of the microorganism of the present application for producing L-arginine.

[0114] Yet another aspect of the present application provides a use of a gluconate repressor mutant in which at least one amino acid selected from the group consisting of an amino acid corresponding to the 36th position, an amino acid corresponding to the 59th position, an amino acid corresponding to the 60th position, an amino acid corresponding to the 63rd position, an amino acid corresponding to the 79th position, and an amino acid corresponding to the 92nd position of the amino acid sequence of SEQ ID NO:1 is substituted with another amino acid for L-arginine production. EXAMPLES

[0115] The present application will be described in more detail below with reference to examples. However, these examples are merely preferred embodiments illustrating the present application, and the present application is not limited thereto. Note that technical matters not described in this specification are fully understood and easily implemented by skilled engineers in the technical field of the present application or a similar technical field. EXAMPLES

[0116] Construction of gluconate repressor (gntR1) mutant vector In order to insert the gntR1(I36N), gntR1(R59A), gntR1(T60L), gntR1(R63A), gntR1(E70K), gntR1(R79A), and gntR1(A92L) mutants represented by SEQ ID NOs: 23 to 29, respectively, into the Corynebacterium glutamicum KCCM10741P strain, vectors containing target mutations were constructed. Specifically, using the genomic DNA of Corynebacterium glutamicum ATCC13869 as a template, PCR was performed using the primer pairs in Table 1 (SEQ ID NOs: 3 and 5, SEQ ID NOs: 4 and 6, SEQ ID NOs: 3 and 7, SEQ ID NOs: 4 and 8, SEQ ID NOs: 3 and 9, SEQ ID NOs: 4 and 10, SEQ ID NOs: 3 and 11, SEQ ID NOs: 4 and 12, SEQ ID NOs: 3 and 13, SEQ ID NOs: 4 and 14, SEQ ID NOs: 3 and 15, SEQ ID NOs: 4 and 16, SEQ ID NOs: 3 and 17, SEQ ID NOs: 4 and 18), and overlapping PCR was performed using the primer pairs of SEQ ID NOs: 3 and 4 to obtain homologous recombination fragments having the respective gntR1-I36N, R59A, T60L, R63A, E70K, R79A, and A92L mutation sequences (hereinafter referred to as "mutation-introduced fragment 1" to "mutation-introduced fragment 7", respectively). Here, the PCR reaction consisted of 30 cycles of denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and extension at 72° C. for 1 minute.

[0117] Next, the pDCM2 vector (Patent Document 2), which is incapable of replicating in Corynebacterium glutamicum, and the PCR-amplified fragments were treated with the restriction enzymes BamHI and XbaI for chromosomal introduction, and then linked using DNA ligase to prepare plasmids into which each mutation-introduced fragment had been inserted. These were then transformed into Escherichia coli DH5α and spread on LB solid medium containing kanamycin (25 mg / l). Colonies transformed with the plasmids were selected from the LB solid medium, and DNA was then obtained using a DNA-spin Plasmid DNA Purification Kit (iNtRON). Vectors (recombinant plasmids) containing the mutation-introduced fragments 1 to 7, respectively, were prepared: pDCM2-gntR1(I36N), pDCM2-gntR1(R59A), pDCM2-gntR1(T60L), pDCM2-gntR1(R63A), pDCM2-gntR1(E70K), pDCM2-gntR1(R79A), and pDCM2-gntR1(A92L).

[0118] [Table 1] EXAMPLES

[0119] Construction of a strain containing a gluconate repressor (gntR1) mutant based on the KCCM10741P strain and evaluation of its L-arginine production ability Example 2-1. Preparation of strains In order to introduce a mutation into the gntR1 gene of Corynebacterium glutamicum KCCM10741P (Patent Document 3), which is an L-arginine producing strain, the strain was transformed using the recombinant plasmids (pDCM2-gntR1(I36N), pDCM2-gntR1(R59A), pDCM2-gntR1(T60L), pDCM2-gntR1(R63A), pDCM2-gntR1(E70K), pDCM2-gntR1(R79A), and pDCM2-gntR1(A92L)) prepared in Example 1 (Non-Patent Document 15). Next, secondary recombination was performed on a solid plate medium containing 4% sucrose, and PCR was performed using a primer pair (SEQ ID NOs: 3 and 4) for the transformed strains after secondary recombination to confirm that each mutation was introduced into the gntR1 gene on the chromosome. Here, the PCR reaction consisted of 30 cycles of denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, and extension at 72° C. for 1 minute.

[0120] The transformed strains were named KCCM10741P-gntR1(I36N), KCCM10741P-gntR1(R59A), KCCM10741P-gntR1(T60L), KCCM10741P-gntR1(R63A), KCCM10741P-gntR1(E70K), KCCM10741P-gntR1(R79A), and KCCM10741P-gntR1(A92L), respectively.

[0121] Example 2-2. Evaluation of L-arginine production ability In order to compare the L-arginine producing ability of Corynebacterium glutamicum KCCM10741P and the transformed strains prepared in Example 2-1 (strains introduced with gntR1 mutants, KCCM10741P-gntR1(I36N), KCCM10741P-gntR1(R59A), KCCM10741P-gntR1(T60L), KCCM10741P-gntR1(R63A), KCCM10741P-gntR1(E70K), KCCM10741P-gntR1(R79A), KCCM10741P-gntR1(A92L)), the L-arginine concentration in the culture medium was analyzed by culturing them in the following manner.

[0122] The parent strain Corynebacterium glutamicum KCCM10741P and the transformants (each gntR1 mutant-introduced strain) prepared in Example 2-1 were inoculated into a 250-ml corner baffle flask containing 25 ml of the following seed medium, and cultured with shaking at 30°C and 200 rpm for 20 hours. Next, 1 ml of the seed culture was inoculated into a 250-ml corner baffle flask containing 24 ml of the production medium, and cultured with shaking at 30°C and 200 rpm for 72 hours. The compositions of the seed medium and production medium are as follows: <Seed medium (pH 7.2)> Glucose 20g, ammonium sulfate 45g, magnesium sulfate heptahydrate 2g, potassium dihydrogen phosphate 2g, ammonium chloride 10g, biotin 0.01mg, thiamine HCl 0.1mg, calcium pantothenate 2mg, nicotinamide 3mg, ferrous sulfate 10mg, manganese sulfate 10mg, zinc sulfate 0.02mg, copper sulfate 0.5mg (in 1 liter of distilled water) <Production medium (pH 7.2)> Glucose 60g, ammonium sulfate 45g, magnesium sulfate heptahydrate 2g, potassium dihydrogen phosphate 2g, ammonium chloride 10g, biotin 0.01mg, thiamine HCl 0.1mg, calcium pantothenate 2mg, nicotinamide 3mg, ferrous sulfate 10mg, manganese sulfate 10mg, zinc sulfate 0.02mg, copper sulfate 0.5mg, calcium carbonate 30g (in 1 liter of distilled water)

[0123] After the cultivation was completed, the L-arginine production ability (concentration) was analyzed by HPLC (Waters 2478). The analyzed L-arginine concentration is shown in Table 2.

[0124] [Table 2]

[0125] As a result, it was confirmed that the L-arginine production ability was improved by an average of about 17% compared to the parent strain in all of the transformed strains (strains into which the gntR1 mutants were introduced, KCCM10741P-gntR1(I36N), KCCM10741P-gntR1(R59A), KCCM10741P-gntR1(T60L), KCCM10741P-gntR1(R63A), KCCM10741P-gntR1(E70K), KCCM10741P-gntR1(R79A), KCCM10741P-gntR1(A92L)). EXAMPLES

[0126] Construction of strains containing a gluconate repressor (gntR1) mutant based on the CJ1R strain and evaluation of their L-arginine production ability Example 3-1. Preparation of CJ1R strain In order to confirm whether the effect of improving L-arginine production is also observed in other Corynebacterium glutamicum strains having L-arginine production ability, one mutation (△argR) was introduced into a wild-type strain (ATCC13869) to further generate Corynebacterium glutamicum strain CJ1R having L-arginine production ability. Specifically, a homologous recombination fragment having an argR deletion mutant sequence was obtained by performing PCR using the primer pairs (SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22) in Table 3 and overlapping PCR using the primer pair of SEQ ID NOs: 19 and 22 using the genomic DNA of Corynebacterium glutamicum ATCC13869 as a template. Here, the PCR reaction was performed by performing 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute. The constructed vector (recombinant plasmid) was named pDCM2-ΔargR.

[0127] [Table 3]

[0128] Next, the wild-type Corynebacterium glutamicum strain (ATCC13869) was transformed with the pDCM2-ΔargR prepared as described above (Non-Patent Document 15), and then secondary recombination was performed on a solid plate medium containing 4% sucrose. For the transformed strains after secondary recombination, PCR was performed using a primer pair (SEQ ID NOs: 19 and 22) to confirm that the argR gene was deleted on the chromosome. Here, the PCR reaction consisted of 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and elongation at 72°C for 1 minute. The transformed strain was named CJ1R.

[0129] Example 3-2. Construction of a strain containing a gluconate repressor (gntR1) mutant based on the CJ1R strain In order to introduce the gntR1 mutation into the CJ1R strain, mutant strains were prepared by introducing the vectors prepared in Example 1 in the same manner as in Example 2-1, and the mutant strains were named CJ1R-gntR1(I36N), CJ1R-gntR1(R59A), CJ1R-gntR1(T60L), CJ1R-gntR1(R63A), CJ1R-gntR1(E70K), CJ1R-gntR1(R79A), and CJ1R-gntR1(A92L), respectively.

[0130] Example 3-3. Evaluation of L-arginine production ability In order to compare the L-arginine producing ability of Corynebacterium glutamicum CJ1R, which is an L-arginine producing strain, and the mutant strains prepared in Example 3-2 (strains with each gntR1 mutant introduced, CJ1R-gntR1(I36N), CJ1R-gntR1(R59A), CJ1R-gntR1(T60L), CJ1R-gntR1(R63A), CJ1R-gntR1(E70K), CJ1R-gntR1(R79A), CJ1R-gntR1(A92L)), the strains were cultured in the same manner as in Example 2-2, and the L-arginine concentration in the culture medium was analyzed.

[0131] After the cultivation was completed, the L-arginine production ability (concentration) was analyzed by HPLC (Waters 2478). The analyzed L-arginine concentration is shown in Table 4.

[0132] [Table 4]

[0133] As a result, it was confirmed that all of the mutant strains (strains into which the gntR1 mutants have been introduced, CJ1R-gntR1(I36N), CJ1R-gntR1(R59A), CJ1R-gntR1(T60L), CJ1R-gntR1(R63A), CJ1R-gntR1(E70K), CJ1R-gntR1(R79A), CJ1R-gntR1(A92L)) had an average L-arginine production ability that was improved by approximately 19% compared to the parent strain.

[0134] From the above description, a person skilled in the art to which the present application pertains will understand that the present application can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. This application should be interpreted as including all modifications or alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. A gluconate repressor mutant in which at least one amino acid selected from the group consisting of the amino acid corresponding to the 36th position, the amino acid corresponding to the 59th position, the amino acid corresponding to the 60th position, the amino acid corresponding to the 63rd position, the amino acid corresponding to the 79th position, and the amino acid corresponding to the 92nd position of the amino acid sequence of SEQ ID NO:1 is replaced with another amino acid.

2. The gluconate repressor mutant of claim 1 , further comprising a substitution of the amino acid at position 70 of the amino acid sequence of SEQ ID NO:1 with another amino acid.

3. The gluconate repressor mutant of claim 1, wherein the mutant comprises at least one substitution selected from the group consisting of a substitution of the amino acid corresponding to the 36th position of the amino acid sequence of SEQ ID NO:1 with asparagine, a substitution of the amino acid corresponding to the 59th position with alanine, a substitution of the amino acid corresponding to the 60th position with leucine, a substitution of the amino acid corresponding to the 63rd position with alanine, a substitution of the amino acid corresponding to the 79th position with alanine, and a substitution of the amino acid corresponding to the 92nd position with leucine.

4. The gluconate repressor mutant of claim 2 , wherein the other amino acid is lysine.

5. The gluconate repressor mutant of claim 1 , which has 80% or more sequence identity with SEQ ID NO:

1.

6. A polynucleotide encoding the gluconate repressor mutant of any one of claims 1 to 5.

7. A Corynebacterium microorganism comprising the gluconate repressor mutant according to any one of claims 1 to 5, or a polynucleotide encoding said mutant.

8. The microorganism of the genus Corynebacterium according to claim 7, which is capable of producing L-arginine.

9. The Corynebacterium microorganism according to claim 7, wherein the Corynebacterium microorganism is Corynebacterium glutamicum.

10. A Corynebacterium microorganism comprising a gluconate repressor mutant in which the amino acid at position 70 of the amino acid sequence of SEQ ID NO:1 is replaced with another amino acid, or a polynucleotide encoding said mutant, The microorganism of the genus Corynebacterium is capable of producing L-arginine.

11. The Corynebacterium microorganism according to claim 10, wherein the Corynebacterium microorganism is Corynebacterium glutamicum.

12. A method for producing L-arginine, comprising the step of culturing the Corynebacterium microorganism according to claim 7 in a medium.

13. A method for producing L-arginine, comprising the step of culturing the Corynebacterium microorganism according to claim 10 in a medium.

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