Novel gluconate repressor variants and method for producing l-arginine using same

Abstract

The present application relates to: a novel gluconate repressor variant, a polynucleotide comprising the variant of the present application, a microorganism of the genus Corynebacterium comprising the variant of the present application or the polynucleotide, and a method for producing L-arginine using the microorganism of the present application.

Description

Technical Field

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

[0002] L-arginine is used for medical purposes such as liver function enhancers, brain function enhancers, and polyamino acid preparations, and is also a substance that has recently attracted attention in food applications such as fish cake additives, health drink additives, and salt substitutes for patients with hypertension. In order to produce high-concentration arginine that is suitable for industrial use, the use of microorganisms has been continuously studied.

[0003] Meanwhile, the microorganism of Corynebacterium, particularly Corynebacterium glutamicum, is the gram-positive microorganism that is widely used in producing L-amino acid.For L-arginic production, mainly use target substance specific method, as increase the method for the expression of the gene of the synthetic enzyme of L-arginine that coding mainly participates in in the Corynebacterium glutamicum strain, or delete the method (Korean Patent No. 10-1102263) of the unnecessary gene of L-arginine biosynthesis.

[0004] However, there is still a growing demand for research on methods that can efficiently produce L-arginine in high yield. Summary of the invention

[0005] [Technical issues]

[0006] The present inventors have developed novel gluconate repressor variants for increasing L-arginine production, polynucleotides encoding the variants, microorganisms for producing L-arginine, microorganisms comprising the variants or polynucleotides, and methods for producing L-arginine using the microorganisms, thereby completing the present disclosure.

[0007] [Technical solution]

[0008] One object of the present disclosure is to provide a gluconate repressor variant, wherein one or more amino acids corresponding to the amino acid sequence positions of SEQ ID NO: 1 are substituted with different amino acids.

[0009] Another object of the present disclosure is to provide polynucleotides encoding the variants of the present disclosure.

[0010] Yet another object of the present disclosure is to provide a microorganism of the genus Corynebacterium comprising a variant of the present disclosure or a polynucleotide encoding the variant of the present disclosure.

[0011] Still another object of the present disclosure is to provide a Corynebacterium microorganism comprising a gluconate repressor variant or a polynucleotide encoding the variant of the present disclosure, wherein the amino acid at position 70 of the amino acid sequence corresponding to SEQ ID NO: 1 is substituted with a different amino acid.

[0012] Still another object of the present disclosure is to provide a method for producing L-arginine, the method comprising the step of culturing the microorganism of the present disclosure in a culture medium.

[0013] Still another object of the present disclosure is to provide a composition for producing L-arginine, the composition comprising the microorganism of the present disclosure, a culture medium in which the microorganism of the present disclosure is cultured, or a combination of two or more thereof.

[0014] Still another object of the present disclosure is to provide use of the microorganism of the present disclosure in producing L-arginine.

[0015] Another object of the present disclosure is to provide use of a gluconate repressor variant in producing L-arginine, wherein one or more amino acids selected from the group consisting of the amino acid corresponding to position 36, the amino acid corresponding to position 59, the amino acid corresponding to position 60, the amino acid corresponding to position 63, the amino acid corresponding to position 79, and the amino acid corresponding to position 92 in the amino acid sequence of SEQ ID NO: 1 are substituted by different amino acids.

[0016] [Beneficial Effects]

[0017] When a microorganism of the genus Corynebacterium containing the gluconate repressor variant of the present disclosure is cultured, L-arginine can be produced at a high yield compared to using a microorganism not containing the variant. DETAILED DESCRIPTION

[0018] The present disclosure will be described in detail below. At the same time, each description and embodiment disclosed in the present disclosure can also be applied to other descriptions and embodiments. That is to say, all combinations of various elements disclosed in the present disclosure fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the specific description described below. In addition, many papers and patent documents are referenced and cited throughout the specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to further illustrate the level and scope of the subject matter to which the present disclosure belongs.

[0019] One aspect of the present disclosure provides a gluconate repressor variant, wherein one or more amino acids at positions corresponding to the amino acid sequence of SEQ ID NO: 1 are substituted with a different amino acid.

[0020] As used herein, the term "gluconate repressor variant" refers to a gluconate repressor variant comprising one or more amino acid substitutions in the amino acid sequence of a polypeptide having gluconate repressor activity.

[0021] As used herein, the term "gluconate repressor (GntR)" refers to a regulatory protein involved in gluconate metabolism or sugar uptake. Gluconate repressor can be used interchangeably with the term "gluconate repressor protein".

[0022] The gluconate repressor protein of the present disclosure may be, but is not particularly limited to, a gluconate repressor protein derived from a microorganism of the genus Corynebacterium (specifically, Corynebacterium glutamicum), or a variant thereof, but is not limited thereto. Specifically, the gluconate repressor protein may include, 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 may include 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 may be obtained from GenBank of NCBI or Kyoto Encyclopedia of Genes and Genomes (KEGG), which is a known database. For example, it may be derived from Corynebacterium or Corynebacterium glutamicum, and more specifically, may be a polypeptide / protein comprising an amino acid sequence shown in SEQ ID NO:1, but is not limited thereto. It is also obvious that auxiliary proteins having amino acid sequences with certain sequences deleted, modified, substituted or added also fall within the scope of the present disclosure, as long as the amino acid sequence has such homology or identity and exhibits the corresponding efficacy of the protein.

[0023] In addition, the gluconate repressor protein having the amino acid sequence of SEQ ID NO:1 can be encoded by a polynucleotide having or comprising the sequence of SEQ ID NO:2 or a nucleotide 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 consisting of the sequence of SEQ ID NO:2 or a nucleotide 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 consisting essentially of the sequence of SEQ ID NO:2 or a nucleotide 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. The sequence of NO:2 has a nucleotide sequence composition 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 and less than 100% homology or identity, but is not limited thereto.

[0024] As used herein, the term "variant" refers to a polypeptide in which one or more amino acids are conservatively substituted and / or modified to be different from the amino acid sequence of the variant before modification, while retaining its function or property. Such variants can generally be identified by modifying one or more amino acids in the polypeptide amino acid sequence and evaluating the properties of the modified polypeptide. In other words, the ability of the variant can be increased, unchanged or reduced compared to the polypeptide before modification. In addition, some variants may include variants in which one or more parts, such as the N-terminal leader sequence or the transmembrane domain have been removed. Other variants may include variants in which a part has been removed from the N- and / or C-terminus of the mature protein. The term "variant" can also be used interchangeably with modifications, modified polypeptides, modified proteins, mutants, mutant proteins, allotypes, etc., and any term is not limited as long as it is used in the sense of being mutated.

[0025] The variants may also include deletions or additions of amino acids that have minimal effects on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence that co-translationally or post-translationally directs protein transfer may be conjugated to the N-terminus of the variant. The variants may also be conjugated to other sequences or linkers for identification, purification or synthesis.

[0026] The gluconate repressor variant disclosed herein may be a gluconate repressor variant in which one or more amino acids selected from the group consisting of the amino acid corresponding to position 36, the amino acid corresponding to position 59, the amino acid corresponding to position 60, the amino acid corresponding to position 63, the amino acid corresponding to position 79, and the amino acid corresponding to position 92 in the amino acid sequence of SEQ ID NO: 1 are substituted with different amino acids, but are not limited thereto.

[0027] In one embodiment, in addition to one or more amino acids selected from the group consisting of the amino acid corresponding to position 36, the amino acid corresponding to position 59, the amino acid corresponding to position 60, the amino acid corresponding to position 63, the amino acid corresponding to position 79 and the amino acid corresponding to position 92 in the amino acid sequence of SEQ ID NO: 1 being replaced by different amino acids, the variant may also replace the amino acid corresponding to position 70 in the amino acid sequence of SEQ ID NO: 1 with a different amino acid, but is not limited to this.

[0028] There is no limitation on the "different amino acid" as long as it is an amino acid different from the amino acid before substitution. Meanwhile, when it is stated that "a specific amino acid is substituted", it is obvious that the amino acid is substituted with an amino acid different from the amino acid before substitution, even if it is not specifically stated that the amino acid is substituted with a different amino acid.

[0029] The amino acids can generally be classified based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues.

[0030] Examples of such classifications may include positively charged (basic) amino acids, such as arginine, lysine, and histidine; negatively charged (acidic) amino acids, such as glutamic acid and aspartic acid; amino acids with nonpolar side chains (nonpolar amino acids), such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; amino acids with polar or hydrophilic side chains (polar amino acids), such as serine, threonine, cysteine, tyrosine, asparagine, and glutamine. As another example, amino acids may be classified into amino acids with charged side chains (charged amino acids) such as arginine, lysine, histidine, glutamic acid, and aspartic acid, and amino acids with uncharged side chains (uncharged amino acids are also called neutral amino acids) such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. For another example, phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. For another example, valine, leucine, and isoleucine can be classified as branched-chain amino acids. For another example, the 20 amino acids are classified according to size, and starting from the relatively small amino acid group, the amino acids can be divided into five groups; glycine, alanine, serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, tyrosine. However, they are not necessarily limited thereto.

[0031] For example, when it is stated that "the amino acid corresponding to position 36 of SEQ ID NO: 1 is replaced by a different amino acid", this may mean that the amino acid is replaced by asparagine, phenylalanine, glycine, alanine, arginine, aspartic acid, cysteine, glutamic acid (glutamate), leucine, glutamine, histidine, proline, serine, tyrosine, lysine, tryptophan, valine, methionine or threonine, excluding the amino acid isoleucine corresponding to position 36 of SEQ ID NO: 1, but is not limited thereto.

[0032] In the present invention, although described as "a protein having an amino acid sequence shown by a specific SEQ ID NO.", it is obvious that any protein having an amino acid sequence with deletion, modification, substitution, conservative substitution or addition of certain sequences can be used in the present disclosure, as long as the protein has the same or corresponding activity as the protein consisting of the amino acid sequence of the corresponding SEQ ID NO. For example, as long as the protein has the same or corresponding activity as the variant protein, it is not excluded to add a sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation or a conservative substitution thereof upstream or downstream of the amino acid sequence, and it is obvious that such sequence addition or mutation also belongs to the scope of the present disclosure.

[0033] As used herein, "position N" may include position N and an amino acid position corresponding to position N. Specifically, it may include an amino acid position corresponding to any amino acid residue in a mature polypeptide disclosed in a specific amino acid sequence. The specific amino acid sequence may be the amino acid sequence of SEQ ID NO: 1.

[0034] As used herein, the term "corresponding to" refers to an amino acid residue at a listed position in a polypeptide, or an amino acid residue that is similar, identical, or homologous to a listed residue in a polypeptide. Identifying an amino acid at a corresponding position may involve determining a specific amino acid in a particular sequence that points to a particular sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related protein or a reference protein.

[0035] For example, any amino acid sequence is aligned with SEQ ID NO: 1, based on which each amino acid residue of the amino acid sequence can be numbered by reference to the numerical position of the amino acid residue corresponding to the amino acid residue of SEQ ID NO: 1. For example, a sequence alignment algorithm as described herein can confirm the position of an amino acid, or the position at which a modification such as a substitution, insertion or deletion occurs, compared to a query sequence (also referred to as a "reference sequence").

[0036] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needleman program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277), etc. can be used, but are not limited thereto, and sequence alignment programs, pairwise sequence comparison algorithms, etc. known in the art can be appropriately used.

[0037] In one embodiment, the variants provided in the present disclosure may comprise an amino acid sequence in which one, two, three, four, five or six amino acids corresponding to positions 36, 59, 60, 63, 79 and 92 from the N-terminus of SEQ ID NO: 1 are substituted with different amino acids.

[0038] In any of the above embodiments, the variant may comprise one or more substitutions selected from the group consisting of: the amino acid corresponding to position 36 in the amino acid sequence of SEQ ID NO:1 is substituted by asparagine, the amino acid corresponding to position 59 is substituted by alanine, the amino acid corresponding to position 60 is substituted by leucine, the amino acid corresponding to position 63 is substituted by alanine, the amino acid corresponding to position 79 is substituted by alanine, and the amino acid corresponding to position 92 is substituted by leucine, but is not limited thereto.

[0039] In any of the above embodiments, the variant may comprise one or more substitutions selected from the group consisting of: the amino acid corresponding to position 36 in the amino acid sequence of SEQ ID NO:1 is substituted by asparagine, the amino acid corresponding to position 59 is substituted by alanine, the amino acid corresponding to position 60 is substituted by leucine, the amino acid corresponding to position 63 is substituted by alanine, the amino acid corresponding to position 79 is substituted by alanine, and the amino acid corresponding to position 92 is substituted by leucine; and the amino acid corresponding to position 70 in the amino acid sequence of SEQ ID NO:1 may be further substituted by lysine, but is not limited thereto.

[0040] In any of the above embodiments, the variants provided in the present disclosure may comprise substitution of the amino acid corresponding to position 36 from the N-terminus of SEQ ID NO: 1 with a different amino acid.

[0041] In any one of the above embodiments, for the variant, the amino acid at position 36 from the N-terminus corresponding to SEQ ID NO: 1 may be substituted with an amino acid selected from serine, cysteine, tyrosine, asparagine and glutamine, which is a polar amino acid. In one embodiment of the above embodiment, the variant may be a variant in which the amino acid at position 36 from the N-terminus corresponding to SEQ ID NO: 1 is substituted with asparagine (N).

[0042] In any of the above embodiments, the variants provided in the present disclosure may comprise substitution of the amino acid corresponding to position 59 from the N-terminus of SEQ ID NO: 1 with a different amino acid.

[0043] In any of the above embodiments, for the variant, the amino acid corresponding to position 59 from the N-terminus of SEQ ID NO: 1 may be substituted 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 embodiments, the variant may be a variant in which the amino acid corresponding to position 59 from the N-terminus of SEQ ID NO: 1 is substituted with alanine (A).

[0044] In any of the above embodiments, the variants provided in the present disclosure may comprise substitution of the amino acid corresponding to position 60 from the N-terminus of SEQ ID NO: 1 with a different amino acid.

[0045] In any of the above embodiments, for the variant, the amino acid corresponding to position 60 from the N-terminus of SEQ ID NO: 1 may be substituted 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 embodiments, the variant may be a variant in which the amino acid corresponding to position 60 from the N-terminus of SEQ ID NO: 1 is substituted with leucine (L).

[0046] In any of the above embodiments, the variants provided in the present disclosure may comprise substitution of the amino acid corresponding to position 63 from the N-terminus of SEQ ID NO: 1 with a different amino acid.

[0047] In any of the above embodiments, for the variant, the amino acid corresponding to position 63 from the N-terminus of SEQ ID NO: 1 may be substituted 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 embodiments, the variant may be a variant in which the amino acid corresponding to position 63 from the N-terminus of SEQ ID NO: 1 is substituted with alanine (A).

[0048] In any of the above embodiments, the variants provided in the present disclosure may comprise substitution of the amino acid corresponding to position 79 from the N-terminus of SEQ ID NO: 1 with a different amino acid.

[0049] In any of the above embodiments, for the variant, the amino acid corresponding to position 79 from the N-terminus of SEQ ID NO: 1 may be substituted with a non-polar amino acid. For example, the amino acid may be selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan and proline. In any of the above embodiments, the variant may be a variant in which the amino acid corresponding to position 79 from the N-terminus of SEQ ID NO: 1 is substituted with alanine (A).

[0050] In any of the above embodiments, the variants provided in the present disclosure may comprise substitution of the amino acid corresponding to position 92 from the N-terminus of SEQ ID NO: 1 with a different amino acid.

[0051] In any of the above embodiments, for the variant, the amino acid corresponding to position 92 from the N-terminus of SEQ ID NO: 1 may be substituted with a non-polar amino acid. For example, the amino acid may be selected from the group consisting of glycine, alanine, isoleucine, methionine, phenylalanine, tryptophan and proline. In any of the above embodiments, the variant may be a variant in which the amino acid corresponding to position 92 from the N-terminus of SEQ ID NO: 1 is substituted with leucine (L).

[0052] In any of the above embodiments, the variants provided in the present disclosure may comprise substitution of the amino acid corresponding to position 70 from the N-terminus of SEQ ID NO: 1 with a different amino acid.

[0053] In any one of the above embodiments, for the variants provided in the present disclosure, the amino acid corresponding to SEQ ID NO:1 at position 70 from the N-terminus can be substituted with an amino acid selected from arginine, lysine, histidine, glutamic acid and aspartic acid, and the amino acid is an amino acid with a charged side chain. For example, the amino acid can be selected from the group consisting of arginine, lysine and histidine, which are basic amino acids. In any one of the above embodiments, the variant can be a variant in which the amino acid corresponding to SEQ ID NO:1 at position 70 from the N-terminus is substituted with lysine (K).

[0054] At the same time, through sequence alignment known in the art, those skilled in the art can identify in any amino acid sequence the amino acid corresponding to position 35, the amino acid corresponding to position 59, the amino acid corresponding to position 60, the amino acid corresponding to position 63, the amino acid corresponding to position 70, the amino acid corresponding to position 79 and the amino acid corresponding to position 92 in the amino acid sequence of SEQ ID NO:1 of the present disclosure, even if not separately described in the present disclosure, when describing "the amino acid at a specific position of a specific SEQ ID NO.", it is obvious that the amino acid includes "the amino acid corresponding to its position" in any amino acid sequence.

[0055] In addition, the variants of the present disclosure may include, in 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 higher homology or identity with the amino acid sequence shown in SEQ ID NO: 1, replacing one or more amino acids selected from the group consisting of amino acids corresponding to positions 36, 59, 60, 63, 79 and 92 from the N-terminus of SEQ ID NO: 1 with different amino acids, or in addition to replacing one or more amino acids selected from the group consisting of amino acids corresponding to positions 36, 59, 60, 63, 79 and 92 from the N-terminus of SEQ ID NO: 1 with different amino acids, it may further include replacing the amino acid at position 70 of the amino acid sequence corresponding to SEQ ID NO: 1 with a different amino acid. It is also obvious that variants of amino acid sequences having deletions, modifications, substitutions, conservative substitutions or additions of certain sequences also fall within the scope of the present disclosure, as long as the amino acid sequence has such homology or identity and exhibits the corresponding efficacy of the variants of the present disclosure.

[0056] For example, the variant of the present disclosure may have and / or include 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, or may consist essentially of the amino acid sequence. Alternatively, the variant may include 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 with 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.

[0057] For example, there are cases where a sequence, a naturally occurring mutation, a silent mutation, or a conservative substitution is added or deleted at the N-terminus, C-terminus, and / or within the amino acid sequence that does not change the function of the variant of the present disclosure.

[0058] As used herein, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structure and / or chemical properties. Such amino acid substitutions generally occur based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathicity 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; hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan. In addition, amino acids can be divided into amino acids with charged side chains and amino acids with uncharged side chains. The amino acids with charged side chains include aspartic acid, glutamic acid, lysine, arginine and histidine, and the amino acids with uncharged side chains can be further divided into non-polar amino acids or polar amino acids. The non-polar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan and proline, and the polar amino acids include serine, threonine, cysteine, tyrosine, asparagine and glutamine. Generally, conservative substitutions have little or no effect on the activity of the resulting polypeptide. Generally, conservative substitutions have little or no effect on the activity of the protein or polypeptide.

[0059] In addition, the variant may also include the deletion or addition of amino acids that have the least effect on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated to a signal (or leader) sequence at the N-terminus of the protein, which co-translationally or post-translationally directs the transfer of the protein. The polypeptide may also be conjugated to another sequence or linker for identification, purification or synthesis of the polypeptide.

[0060] For example, the variants of the present disclosure may have gluconate repressor activity. In addition, compared to the wild-type polypeptide having gluconate repressor activity, the variants of the present disclosure may have an activity of increasing L-arginine production capacity.

[0061] Another aspect of the present disclosure provides polynucleotides encoding the variants of the present disclosure.

[0062] As used herein, the term "polynucleotide" refers to a DNA or RNA chain of a polymer of nucleotides of a certain length or longer, in which nucleotide monomers are linked into a long chain by covalent bonds, and more specifically, refers to a polynucleotide fragment encoding the variant.

[0063] The polynucleotide encoding the gluconate repressor variant of the present disclosure may include any polynucleotide sequence without limitation, as long as it is a polynucleotide sequence encoding the gluconate repressor variant of the present disclosure. For example, the polynucleotide encoding the gluconate repressor variant of the present disclosure may be a polynucleotide sequence encoding the amino acid sequence of the variant of the present disclosure, but is not limited thereto.

[0064] For example, the polynucleotide encoding the gluconate repressor variant of the present disclosure may be, for example, a polynucleotide sequence selected from the group consisting of the polynucleotide sequences shown in 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 higher homology or identity with a polynucleotide sequence selected from the group consisting of the polynucleotide sequences shown in SEQ ID NO: 30 to SEQ ID NO: 36, but is not limited thereto. Nucleotide sequences having homology or identity may be those within the above range, excluding sequences having 100% identity, or may be sequences having less than 100% identity.

[0065] Taking into account codon degeneracy or preferred codons in the organism expressing the disclosed variant, the polynucleotides of the present disclosure may be modified in various ways in the coding region without changing the amino acid sequence of the disclosed variant. Thus, it is apparent that polynucleotides that can be translated into polypeptides consisting of the amino acid sequence of the disclosed variant, or polypeptides that have homology or identity thereto due to codon degeneracy, may also be included.

[0066] In addition, the polynucleotide of the present disclosure may include a probe that can be prepared from a known gene sequence, for example, may include any sequence without limitation as long as it is a sequence that can hybridize with a complementary sequence of all or part of the polynucleotide sequence of the present disclosure under stringent conditions.

[0067] "Stringent conditions" refer to conditions that enable specific hybridization between polynucleotides. These conditions are described in detail in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, conditions under which polynucleotides having higher homology or identity, i.e., 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 to each other, while polynucleotides having lower homology or identity do not hybridize to each other, or conditions under which washing is performed once, specifically, two to three times, at a salt concentration and temperature equivalent to 60°C, 1хSSC, 0.1% SDS, specifically, 60°C, 0.1хSSC, 0.1% SDS, more specifically, 68°C, 0.1хSSC, 0.1% SDS can be listed, which are general washing conditions for Southern hybridization.

[0068] Hybridization requires that the two nucleic acids have complementary sequences, although mismatches between bases are allowed depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to each other. For example, for DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of the present disclosure may also include substantially similar nucleic acid sequences and isolated nucleic acid fragments that are complementary to the entire sequence.

[0069] Specifically, polynucleotides having homology or identity can be detected using hybridization conditions, which include a Tm value of 55° C. and a hybridization step under the above conditions. In addition, the Tm value may be 60° C., 63° C., or 65° C., but is not limited thereto, and can be appropriately adjusted by those skilled in the art according to the purpose.

[0070] The appropriate stringency for hybridization of polynucleotides depends on the length of the polynucleotides and the degree of complementation, and variables are well known in the art (eg, J. Sambrook et al., supra).

[0071] As used herein, the term "homology" or "identity" refers to the degree of similarity between two given amino acid sequences or nucleotide sequences, and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0072] The sequence homology or identity of conservative polynucleotides or polypeptides is determined by standard comparison algorithms, and can be used together with the default gap penalty set up by the program to be used. Basically, homology or identity sequences can hybridize with all or part of the sequence under moderate or highly stringent conditions, usually. Obviously, hybridization also includes hybridization with polynucleotides containing common codons or codons that take into account codon degeneracy.

[0073] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program, for example using the default parameters as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) can be used to determine, such as the Needleman program (version 5.0.0 or later) in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (including the GCG program 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, homology, similarity or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology.

[0074] Homology, similarity or identity between polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, such as Needleman et al., (1970), J Mol Biol. 48: 443), as described in Smith and Waterman, Adv. Appl. Math (1981) 2: 482. Briefly, the GAP program defines identity as the value obtained by dividing the number of similar aligned symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. The default parameters of the GAP program may include: (1) a binary comparison matrix as disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) (containing a value of 1 for identity and a value of 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14: 6745 (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (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 opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps. Therefore, the terms "homology" or "identity" as used herein represent the relatedness between sequences.

[0075] Another aspect of the present disclosure provides a vector comprising the polynucleotide of the present disclosure. The vector may be an expression vector for expressing the polynucleotide in a microorganism, but is not limited thereto.

[0076] As used herein, the term "vector" may include a DNA construct comprising a nucleotide sequence of a polynucleotide encoding a desired polypeptide, which is operably linked to a suitable expression regulatory region (or expression control sequence) so that the desired polypeptide can be expressed in a suitable host. The expression regulatory region may include a promoter capable of initiating transcription, any operator sequence controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling transcription and translation termination. The vector may be transformed into a suitable microorganism and then replicated or functioned independently of the host genome, or may be integrated into the genome itself.

[0077] The vector used in the present disclosure is not particularly limited, but any vector known in the art can be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as phage vectors or cosmid vectors, and pDZ system, pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, pET system, etc. can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

[0078] For example, the polynucleotide encoding the desired polypeptide can be inserted into the chromosome by the vector for intracellular chromosome insertion. The polynucleotide can be inserted into the chromosome by any method known in the art, such as homologous recombination, but is not limited thereto. The carrier can also include a selection marker for confirming chromosome insertion. The selection marker is used to select cells transformed with the carrier, i.e., to confirm the insertion of the desired nucleic acid molecule, and can use markers that confer selectable phenotypes such as drug resistance, auxotrophy, cytotoxic agent resistance or surface polypeptide expression. In the environment of processing with a selection agent, only cells expressing the selection marker survive or show other phenotypic traits, and therefore the cells transformed can be selected.

[0079] As used herein, the term "conversion" refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the microorganism. Regardless of the position, the converted polynucleotide can be located by inserting into the chromosome of the microorganism or outside the chromosome, as long as it can be expressed in the microorganism. In addition, the polynucleotide comprises DNA and / or RNA encoding the desired polypeptide. The polynucleotide can be introduced in any form, as long as it can be introduced into the microorganism and expressed. For example, the polynucleotide can be introduced into the microorganism in the form of an expression cassette, which is a gene construct containing all elements required for self-expression. The expression cassette can generally include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably connected to the polynucleotide. The expression cassette can be in the form of an expression vector capable of self-replication. In addition, the polynucleotide can be introduced into the microorganism in its own form and operably connected to the sequence required for expression in the microorganism, but is not limited thereto.

[0080] As used herein, the term "operably linked" means that a polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a desired variant of the present disclosure.

[0081] Still another aspect of the present disclosure provides a microorganism of the genus Corynebacterium, comprising the gluconate repressor variant of the present disclosure, a polynucleotide encoding the gluconate repressor variant, or a vector comprising the polynucleotide.

[0082] For another example of the present disclosure, the microorganism of the present disclosure can be a microorganism of the genus Corynebacterium, which contains a gluconate repressor variant, a polynucleotide encoding the variant, or a vector containing the polynucleotide, wherein the amino acid at position 70 of the amino acid sequence corresponding to SEQ ID NO: 1 in the gluconate repressor variant is substituted by a different amino acid.

[0083] The Corynebacterium microorganism of the present disclosure may be a microorganism having the gluconate repressor variant of the present disclosure or L-arginine-producing ability.

[0084] Specifically, the microorganism of the present disclosure may be a microorganism that naturally has a gluconate repressor or L-arginine production ability, or a microorganism prepared by introducing a variant of the present disclosure or a polynucleotide encoding the same (or a vector comprising the polynucleotide) into a parent strain that does not have a gluconate repressor or L-arginine production ability, and / or by providing it with L-arginine production ability, but is not limited thereto.

[0085] As used herein, the term "L-arginine" refers to an amino acid having the chemical formula C 6 H 14 N 4 O 2 L-amino acid, which is a conditional essential amino acid present in all organisms. Known L-arginine is mainly produced by Corynebacterium microorganisms in microorganisms; However, it is known that these are subject to feedback inhibition of intracellular arginine (Vehary Sakanyan, et al, Microbiology, 142: 9-108, 1996). Therefore, it is known that these microorganisms are limited in producing L-arginine in high yield.

[0086] As used herein, the term "microorganism (or strain)" includes all wild-type microorganisms or naturally or artificially genetically modified microorganisms, and it may be a microorganism in which a specific mechanism is weakened or enhanced due to the insertion of foreign genes or the enhancement or inactivation of the activity of endogenous genes, and it may be a microorganism comprising a genetic modification for producing a desired polypeptide, protein or product.

[0087] As used herein, the term "microorganism with L-arginine production ability" refers to a prokaryotic or eukaryotic microbial strain that can produce L-arginine in vivo, and may comprise a microorganism prepared by providing L-arginine production ability for a parent strain that does not have L-arginine production ability or a microorganism that inherently has L-arginine production ability. The ability to produce L-arginine can be imparted or enhanced by species improvement.

[0088] As used herein, the term "unmodified microorganism" does not exclude bacterial strains comprising mutations that may occur naturally in microorganisms, and may refer to wild-type strains or natural strains themselves, or may be strains before properties are changed due to natural or artificial factors by genetic variation. For example, an unmodified microorganism may refer to a strain in which no gluconate repressor variants as described herein are introduced or a strain before introduction." unmodified microorganism" may be used interchangeably with "strain before modification", "microorganism before modification", "unvaried strain", "unmodified strain", "unvaried microorganism" or "reference microorganism".

[0089] The unmodified microorganism may be a microorganism comprising an amino acid sequence consisting of SEQ ID NO: 1 or a polynucleotide consisting of SEQ ID NO: 2.

[0090] The microorganism with L-arginine production ability disclosed herein can be a microorganism comprising any one or more of the variants disclosed herein, the polynucleotides disclosed herein, and a vector comprising the polynucleotides disclosed herein; a microorganism modified to express the variants disclosed herein or the polynucleotides disclosed herein; a microorganism (e.g., a recombinant strain) expressing the variants disclosed herein or the polynucleotides disclosed herein; or a microorganism (e.g., a recombinant strain) having the activity of the variants disclosed herein, but is not limited thereto.

[0091] For example, the microorganism of the present invention can be a cell or microorganism transformed with a polynucleotide of the present invention or a vector comprising a polynucleotide encoding a variant of the present invention to express a variant of the present invention. By comprising a variant of the present invention, the microorganism of the present invention can include all microorganisms capable of producing L-arginine. For example, the strain of the present invention can be a recombinant strain with increased L-arginine production capacity, wherein the gluconate repressor variant is expressed by introducing a polynucleotide encoding a variant of the present invention into a natural wild-type microorganism or a microorganism with L-arginine production capacity. Compared with a natural wild-type microorganism or a microorganism unmodified by a gluconate repressor (e.g., a microorganism expressing a wild-type gluconate repressor or a microorganism not expressing a variant of the present invention), the recombinant strain with increased L-arginine production capacity can be a microorganism in which L-arginine production capacity is increased, but is not limited thereto. For example, compared with a microorganism comprising a polypeptide of SEQ ID NO:1 or a polynucleotide encoding it, the strain with increased L-arginine production capacity of the present invention can be a microorganism in which L-arginine production capacity is increased, but is not limited thereto.

[0092] The microorganism of the present disclosure may include all microorganisms capable of expressing the gluconate repressor variant of the present disclosure by various known methods in addition to the introduction of nucleic acids or vectors.

[0093] For example, the microorganism with increased L-arginine productivity can have an increase 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, about 15% or more, about 16% or more, about 17% or more, about 18% or more, about 19% or more, about 20% or more, about 21% or more, about 22% or more, about 23% or more, about 24% or more, about 25% or more, about 26% or more, about 27% or more, about 28% or more, about 29% or more, about 30% or more, about 31% or more, about 32% or more, about 33% or more, about 34% or more, about 35% or more, about 36% or more, about 37% or more, about 38% or more, about 39% or more, about 40% or more, about 41% or more, about 42% or more, about 43% or more, about 44% or more, about 45% or more, about 46% or more, about 47% or more, about 48% or more, about 49% or more, about 50% or more, about 51% 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 (the upper limit is not particularly limited, but can be, 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) L-arginine productivity, but the amount increased is not limited thereto, as long as the productivity has an increased α+ value compared to the productivity of the parent strain before modification or the unmodified microorganism. In another example, compared with the parent strain before modification or unmodified microorganism, the recombinant strain with increased production capacity can have an increase 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 (the upper limit is not particularly limited, but can be, 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) L-arginine production capacity, but not limited thereto.Term "about" refers to the scope comprising whole ± 0.5, ± 0.4, ± 0.3, ± 0.2, ± 0.1 etc., and comprises all values ​​that are equal to or similar to the value thereafter, but the scope is not limited thereto.

[0094] The microorganism of the present disclosure may be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacteriumammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens.

[0095] Specifically, the microorganism may be Corynebacterium glutamicum, but is not limited thereto.

[0096] Meanwhile, it is known that the microorganisms of the genus Corynebacterium can produce L-arginine, but their productivity is very low, and the genes or the mechanism involved in the production mechanism have not been fully elucidated. Therefore, the Corynebacterium microorganism with L-arginine production capacity of the present disclosure may include any natural wild-type microorganism itself, the Corynebacterium microorganism with improved L-arginine production capacity by enhancing or weakening the activity of the gene related to the L-arginine production mechanism, or the Corynebacterium microorganism with improved L-arginine production capacity by introducing or enhancing the activity of exogenous genes.

[0097] Yet another aspect of the present disclosure provides a method for producing L-arginine, the method comprising the step of culturing the microorganism of the present disclosure in a culture medium.

[0098] Specifically, the method for producing L-arginine of the present disclosure may include the step of culturing a microorganism of the genus Corynebacterium comprising the variant of the present disclosure, the polynucleotide of the present disclosure, or the vector of the present disclosure in a culture medium, but is not limited thereto.

[0099] As used herein, the term "culture" refers to culturing the microorganism of the present disclosure under appropriately adjusted environmental conditions. The culture procedure of the present disclosure can be carried out according to suitable culture media or culture conditions known in the art. Those skilled in the art can easily adjust this culture procedure according to the selected strain. Specifically, the culture can be batch type, continuous type and / or fed-batch type, but is not limited thereto.

[0100] As used herein, "culture medium" refers to a mixture containing nutrients required for culturing the microorganism of the present disclosure as the main component, wherein the culture medium provides nutrients including water, growth factors, etc. necessary for survival and growth. Specifically, for the culture medium and other culture conditions for culturing the microorganism of the present disclosure, any culture medium used for conventional culture of microorganisms can be used without particular limitation. However, the microorganism of the present disclosure can be cultured under aerobic conditions in a common culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and / or vitamin, etc., while controlling temperature, pH, etc.

[0101] For example, culture media for Corynebacterium strains can be found in the literature ["Manual of Methods for General Bacteriology" of the American Society of Bacteriology (Washington, DC, USA, 1981)].

[0102] In the present disclosure, the carbon source may include 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.; and amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn syrup may be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and appropriate amounts of various other carbon sources may be used without restriction. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0103] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc., amino acids such as glutamic acid, methionine, glutamine, etc., and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition product, defatted soybean cake or its degradation product, etc. can be used. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0104] The phosphorus source may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate and corresponding sodium salts. For inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. may be used. In addition, amino acids, vitamins and / or suitable precursors may also be included. These components or precursors may be added to the culture medium in batches or continuously. However, the present disclosure is not limited thereto.

[0105] In addition, in the cultivation process of the disclosed microorganism, the pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the culture medium in an appropriate manner. In addition, defoamers such as fatty acid polyethylene glycol esters can be used to suppress foam formation during cultivation. In addition, oxygen or oxygen-containing gas can be injected into the culture medium to maintain the aerobic state of the culture medium, or gas can be not injected or nitrogen, hydrogen or carbon dioxide gas can be injected to maintain the anaerobic or microaerobic state of the culture medium, but is not limited thereto.

[0106] In the culture of the present disclosure, the culture temperature may be maintained at 20° C. to 45° C., specifically, 25° C. to 40° C., and the culture may be performed for about 10 hours to about 160 hours, but is not limited thereto.

[0107] L-arginine produced by the culture of the present disclosure may be released into the medium or may be retained in the cells.

[0108] The method for producing L-arginine of the present disclosure may further include, for example, the step of preparing the microorganism of the present disclosure, preparing a medium for culturing the microorganism, or a combination thereof (regardless of the order, in any order) before the culturing step.

[0109] The method for producing L-arginine of the present disclosure may further include a step of recovering L-arginine from a culture medium produced by the culture (a culture medium in which the culture has been performed) or from the microorganism of the present disclosure. A recovery step may be further included after the culture step.

[0110] Recovery can be a method for cultivating microorganisms according to the present disclosure, such as batches, continuous or fed-batch type cultivation, by using suitable methods known in the art, to collect L-arginine. For example, centrifugal, filtration, processing (salting out) with a crystal protein precipitation agent, extraction, ultrasonic treatment, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography and affinity chromatography, etc.), HPLC and the combination of these methods can be used, and L-arginine can be recovered from culture medium or microorganism by using suitable methods known in the art.

[0111] In addition, the method for producing L-arginine of the present disclosure may further include a purification step. It can be purified by using a suitable method known in the art. In an exemplary embodiment, when the method for producing L-arginine of the present disclosure includes a recovery step and a purification step, the recovery step and the purification step can be performed continuously or discontinuously without considering the order, or can be performed simultaneously or integrated into one step, but is not limited thereto.

[0112] Yet another aspect of the present disclosure provides a composition for producing L-arginine, the composition comprising a microorganism comprising any one or more of the gluconate repressor variants of the present disclosure, a polynucleotide encoding the variant, and a vector comprising the polynucleotide; a culture of the microorganism; or a combination of two or more thereof.

[0113] The composition of the present disclosure may further comprise any suitable excipients commonly used in compositions for producing amino acids, and such excipients may include, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, isotonic agents, etc., but are not limited thereto.

[0114] Yet another aspect of the present disclosure provides use of the microorganism of the present disclosure in producing L-arginine.

[0115] Another aspect of the present disclosure provides use of the gluconate repressor variant in producing L-arginine, wherein one or more amino acids selected from the group consisting of the amino acid corresponding to position 36, the amino acid corresponding to position 59, the amino acid corresponding to position 60, the amino acid corresponding to position 63, the amino acid corresponding to position 79 and the amino acid corresponding to position 92 in the amino acid sequence of SEQ ID NO:1 are substituted by different amino acids.

[0116] [Mode for Carrying Out the Invention]

[0117] Hereinafter, the present disclosure will be described in more detail by way of exemplary embodiments. However, the following exemplary embodiments are only used to illustrate preferred embodiments of the present disclosure, and are therefore not intended to limit the scope of the present disclosure thereto. At the same time, technical matters not described in this specification can be fully understood and easily implemented by those skilled in the art of the present disclosure or similar technical fields.

[0118] Example 1. Construction of gluconate repressor (gntR1) mutation vector

[0119] In order to insert gntR1(I36N), gntR1(R59A), gntR1(T60L), gntR1(R63A), gntR1(E70K), gntR1(R79A) and gntR1(A92L) mutations (each mutation is represented by SEQ ID NO:23 to SEQ ID NO:29) into the Corynebacterium glutamicum KCCM10741P strain, a vector containing each target mutation was constructed. In detail, PCR was performed using genomic DNA of Corynebacterium glutamicum ATCC13869 as a template and the primer pairs of 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 recombinant fragments (hereinafter, "mutation-introduced fragment 1" to "mutation-introduced fragment 7"), each fragment having a gntR1-I36N, R59A, T60L, R63A, E70K, R79A or A92L mutation sequence. At this time, the PCR reaction was performed by repeating the following cycle 30 times: denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and extension at 72°C for 1 minute.

[0120] Subsequently, the pDCM2 (Korean Patent Publication No. 10-2020-0136813) vector that is not replicable in Corynebacterium glutamicum and the above-mentioned fragments amplified by PCR were treated with BamHI and XbaI, which are restriction enzymes for chromosomal insertion, and then ligated using DNA ligase to prepare a plasmid, each mutation-introduced fragment was inserted into the plasmid, and each plasmid was transformed into Escherichia coli DH5α, which was plated on an LB solid medium containing kanamycin (25 mg / l). Colonies transformed with the plasmid were selected from LB solid medium, and each DNA was obtained using a DNA-spin plasmid DNA purification kit (iNtRON) to prepare pDCM2-gntR1(I36N), pDCM2-gntR1(R59A), pDCM2-gntR1(T60L), pDCM2-gntR1(R63A), pDCM2-gntR1(E70K), pDCM2-gntR1(R79A) and pDCM2-gntR1(A92L) vectors (recombinant plasmids), each of which contained fragments 1 to 7 into which mutations were introduced.

[0121] [Table 1]

[0122]

[0123]

[0124] Example 2. Preparation of strains containing a gluconate repressor (gntR1) variant based on the KCCM10741P strain and evaluation of L-arginine production capacity

[0125] Example 2-1 Preparation of strains

[0126] In order to introduce mutations into the gntR1 gene of the L-arginine producing strain Corynebacterium glutamicum KCCM10741P (US 8034602B2), the recombinant plasmids prepared in Example 1 (pDCM2-gntR1(I36N), pDCM2-gntR1(R59A), pDCM2-gntR1(T60L), pDCM2-gntR1(R63A), pDCM2-gntR1(E70K), pDCM2-gntR1(R79A) and pDCM2-gntR1(A92L)) were used for transformation (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Subsequently, secondary recombination was performed on a solid plate containing 4% sucrose, and PCR was performed on the transformants that completed the secondary recombination using a primer pair (SEQ ID NO: 3 and 4) to confirm that each mutation was introduced into the gntR1 gene on the chromosome. At this time, the PCR reaction was performed by repeating the following cycle 30 times: 95°C denaturation for 30 seconds; 55°C annealing for 30 seconds; and 72°C extension for 1 minute.

[0127] The transformants 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.

[0128] Example 2-2 Evaluation of L-arginine production capacity

[0129] In order to compare the L-arginine production ability of the L-arginine producing strain Corynebacterium glutamicum KCCM10741P and the transformants prepared in Example 2-1 (strains introduced with gntR1 variants, namely KCCM10741P-gntR1(I36N), KCCM10741P-gntR1(R59A), KCCM10741P-gntR1(T60L), KCCM10741P-gntR1(R63A), KCCM10741P-gntR1(E70K), KCCM10741P-gntR1(R79A) and KCCM10741P-gntR1(A92L)), the strains were cultured by the following method and the L-arginine concentration in the culture medium was analyzed.

[0130] The parent strain Corynebacterium glutamicum KCCM10741P and the transformed strain prepared in Example 2-1 (strain into which each gntR1 variant was introduced) were inoculated into a 250 mL corner-baffled flask containing 25 mL of the following seed culture medium, followed by shaking culture at 30° C. with 200 rpm for 20 hours. Then, 1 mL of each seed culture was inoculated into a 250 mL corner-baffled flask containing 24 mL of production culture medium, followed by shaking culture at 30° C. with 200 rpm for 72 hours. The compositions of the seed culture medium and the production culture medium are as follows.

[0131] <Seed culture medium (pH 7.2)>

[0132] 20g glucose, 45g ammonium sulfate, 2g magnesium sulfate heptahydrate, 2g potassium dihydrogen phosphate, 10g ammonium chloride, 0.01mg biotin, 0.1mg thiamine hydrochloride, 2mg calcium pantothenate, 3mg niacinamide, 10mg ferrous sulfate, 10mg manganese sulfate, 0.02mg zinc sulfate, 0.5mg copper sulfate (based on 1 liter of distilled water)

[0133] <Production medium (pH 7.2)>

[0134] 60g glucose, 45g ammonium sulfate, 2g magnesium sulfate heptahydrate, 2g potassium dihydrogen phosphate, 10g ammonium chloride, 0.01mg biotin, 0.1mg thiamine hydrochloride, 2mg calcium pantothenate, 3mg niacinamide, 10mg ferrous sulfate, 10mg manganese sulfate, 0.02mg zinc sulfate, 0.5mg copper sulfate, 30g calcium carbonate (based on 1 liter of distilled water)

[0135] After the cultivation was completed, the L-arginine production capacity (concentration) was analyzed using HPLC (Waters 2478), and the analyzed L-arginine concentrations are shown in Table 2 below.

[0136] [Table 2]

[0137]

[0138]

[0139] As a result, it was confirmed that the L-arginine production capacity was increased by an average of about 17% in all transformants (strains introduced with gntR1 variants, namely KCCM10741P-gntR1(I36N), KCCM10741P-gntR1(R59A), KCCM10741P-gntR1(T60L), KCCM10741P-gntR1(R63A), KCCM10741P-gntR1(E70K), KCCM10741P-gntR1(R79A) and KCCM10741P-gntR1(A92L)) compared with the parent strain.

[0140] Example 3. Preparation of strains containing a gluconate repressor (gntR1) variant based on the CJ1R strain and evaluation of L-arginine production capacity

[0141] Example 3-1 Preparation of CJ1R strain

[0142] In order to check whether other Corynebacterium glutamicum strains with L-arginine productivity also show the effect of increasing L-arginine productivity, a type of mutation (△ argR) is introduced into wild type (ATCC13869) to further prepare Corynebacterium glutamicum strain CJ1R with L-arginine productivity. In detail, PCR is carried out using the genomic DNA of Corynebacterium glutamicum ATCC13869 as template and the primers of Table 3 (SEQ ID NO:19 and 20, SEQ ID NO:21 and 22), and the primers of SEQ ID NO:19 and 22 are used to carry out overlapping PCR to obtain the homologous recombination fragment with argR deletion mutation sequence. At this time, PCR reaction is carried out by repeating 30 following cycles: 95 ℃ of denaturation for 30 seconds; 55 ℃ of annealing for 30 seconds; and 72 ℃ of extensions for 1 minute. The carrier (recombinant plasmid) prepared is named as pDCM2-△ argR.

[0143] [Table 3]

[0144]

[0145] Subsequently, the pDCM2-△argR prepared above was transformed into wild-type Corynebacterium glutamicum (ATCC13869) (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999), and then secondary recombination was carried out on a solid plate containing 4% sucrose, and PCR was carried out on the transformant that completed the secondary recombination using primer pairs (SEQ ID NO:19 and 22) to identify the strain in which argR on the chromosome was deleted. At this time, the PCR reaction was carried out by repeating the following cycle 30 times: 95°C denaturation for 30 seconds; 55°C annealing for 30 seconds; and 72°C extension for 1 minute. The transformed strain was named CJ1R.

[0146] Example 3-2 Preparation of strains containing gluconate repressor (gntR1) variants based on CJ1R strain

[0147] In order to introduce the gntR1 mutation into the CJ1R strain, mutant strains were prepared in the same manner as in Example 2-1, wherein each vector prepared in Example 1 was introduced, 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.

[0148] Example 3-3 Evaluation of L-arginine production capacity

[0149] In order to compare the L-arginine production capacity between the L-arginine producing strain Corynebacterium glutamicum CJ1R and the mutant strains prepared in Example 3-2 (strains introduced with gntR1 variants, namely CJ1R-gntR1(I36N), CJ1R-gntR1(R59A), CJ1R-gntR1(T60L), CJ1R-gntR1(R63A), CJ1R-gntR1(E70K), CJ1R-gntR1(R79A) and CJ1R-gntR1(A92L)), the strains were cultured in the same manner as in Example 2-2, and the L-arginine concentration in the culture was analyzed.

[0150] After the cultivation was completed, the L-arginine production capacity (concentration) was analyzed using HPLC (Waters 2478), and the analyzed L-arginine concentrations are shown in Table 4 below.

[0151] [Table 4]

[0152]

[0153] The results confirmed that the L-arginine production capacity increased by an average of about 19% in all mutant strains (strains introduced with gntR1 variants, namely CJ1R-gntR1(I36N), CJ1R-gntR1(R59A), CJ1R-gntR1(T60L), CJ1R-gntR1(R63A), CJ1R-gntR1(E70K), CJ1R-gntR1(R79A) and CJ1R-gntR1(A92L)) compared with the parent strain.

[0154] Based on the above description, it will be appreciated by those skilled in the art that the present disclosure can be implemented in different specific forms without changing its technical essence or basic features. In this regard, it should be understood that the above embodiments are not restrictive, but illustrative in all respects. The scope of the present disclosure is limited by the appended claims rather than by the description thereafter, so all changes and modifications falling within the boundaries and scope of the claims, or the equivalents of these boundaries and scopes, are included by the claims.

Claims

1. A gluconate repressor variant, wherein one or more amino acids selected from the group consisting of the amino acid corresponding to position 36, the amino acid corresponding to position 59, the amino acid corresponding to position 60, the amino acid corresponding to position 63, the amino acid corresponding to position 79 and the amino acid corresponding to position 92 in the amino acid sequence of SEQ ID NO: 1 are substituted by different amino acids.

2. The gluconate repressor variant according to claim 1, wherein the amino acid at position 70 of the amino acid sequence corresponding to SEQ ID NO: 1 is further substituted by a different amino acid.

3. The gluconate repressor variant of claim 1, comprising one or more substitutions selected from the group consisting of: in the amino acid sequence of SEQ ID NO:1, the amino acid corresponding to position 36 is substituted by asparagine, the amino acid corresponding to position 59 is substituted by alanine, the amino acid corresponding to position 60 is substituted by leucine, the amino acid corresponding to position 63 is substituted by alanine, the amino acid corresponding to position 79 is substituted by alanine, and the amino acid corresponding to position 92 is substituted by leucine.

4. The gluconate repressor variant of claim 2, wherein the different amino acid is lysine. 5 . The gluconate repressor variant according to claim 1 , wherein the gluconate repressor variant has 80% or more sequence identity with SEQ ID NO:

1. 6 . A polynucleotide encoding the gluconate repressor variant according to any one of claims 1 to 5 . 7 . A microorganism of the genus Corynebacterium, comprising the gluconate repressor variant according to any one of claims 1 to 5 or a polynucleotide encoding the variant. 8 . The microorganism of the genus Corynebacterium according to claim 7 , wherein the microorganism has an L-arginine-producing ability. 9 . The microorganism of the genus Corynebacterium according to claim 7 , wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum .

10. A microorganism of the genus Corynebacterium, comprising a gluconate repressor variant or a polynucleotide encoding the variant, wherein the variant has a substitution of the amino acid at position 70 of the amino acid sequence corresponding to SEQ ID NO: 1 with a different amino acid, wherein the microorganism of the genus Corynebacterium has the ability to produce L-arginine. 11 . The microorganism of the genus Corynebacterium according to claim 10 , wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum .

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

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

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