Microorganism with attenuated gluconate repressor protein activity and method for producing L-arginine using the same

Abstract

The present application relates to a microorganism having attenuated gluconate repressor protein activity, a method for producing L-arginine using the microorganism of the present application, a composition for producing L-arginine containing the microorganism of the present application, and use of the microorganism of the present application in L-arginine production.

Description

[Technical field]

[0001] The present application relates to a microorganism having attenuated gluconate repressor protein activity, a method for producing L-arginine using the microorganism of the present application, a composition for producing L-arginine containing the microorganism of the present application, and use of the microorganism of the present application in L-arginine production. [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] Vehary Sakanyan, et al, Microbiology, 142:9-108, 1996

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

[0007] The present inventors have developed a Corynebacterium microorganism having attenuated gluconate repressor protein activity, a method for producing L-arginine using said microorganism, a composition for producing L-arginine containing said microorganism, and use of said microorganism in L-arginine production, and have completed the present application. [Means for solving the problem]

[0008] An object of the present application is to provide a Corynebacterium microorganism in which gluconate repressor protein activity is weakened.

[0009] Another object of the present application is to provide a method for producing L-arginine, comprising the step of culturing the Corynebacterium microorganism of the present application in a medium.

[0010] Another object of the present application is to provide a composition for producing L-arginine, comprising the Corynebacterium microorganism of the present application.

[0011] Another object of the present application is to provide a use of the Corynebacterium microorganism of the present application for producing L-arginine. Effect of the Invention

[0012] The Corynebacterium microorganism of the present application in which gluconate repressor protein activity is attenuated can produce L-arginine more efficiently than a Corynebacterium microorganism in which gluconate repressor protein activity is not attenuated. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] 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.

[0014] One aspect of the present application provides a Corynebacterium microorganism in which gluconate repressor protein activity is attenuated.

[0015] The Corynebacterium microorganism of the present application may have an ability to produce L-arginine, or may have an improved ability to produce L-arginine compared to a Corynebacterium microorganism in which the activity of a gluconate repressor protein is not attenuated.

[0016] In the present application, the term "gluconate repressor (GntR) protein" refers to a regulatory protein involved in gluconate metabolism, sugar uptake, and the like.

[0017] Specifically, the gluconate repressor protein is known in the art, and the protein and gene sequence of the gluconate repressor protein can be obtained from a known database, for example, but not limited to, GenBank of NCBI. More specifically, the gluconate repressor protein is a protein consisting of the amino acid sequence of SEQ ID NO: 1. In addition, the gluconate repressor protein of the present application may have the amino acid sequence shown in SEQ ID NO: 1, may include the amino acid sequence, or may essentially consist of the amino acid sequence.

[0018] Furthermore, the gluconate repressor protein of the present application may include not only the amino acid sequence represented by SEQ ID NO: 1, but also 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 SEQ ID NO: 1. Furthermore, it goes without saying that the present application also includes amino acid sequences having such homology or identity and having the same or corresponding biological activity as the gluconate repressor protein of the present application, in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added.

[0019] Furthermore, the gluconate repressor protein having the amino acid sequence of SEQ ID NO:1 has or contains the nucleotide 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 is encoded by a polynucleotide that consists of or essentially consists of the nucleotide 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, but is not limited to these.

[0020] 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 1), and it is known that there is a limit to the high-yield production of L-arginine.

[0021] 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.

[0022] In the present application, the term "Corynebacterium microorganism having L-arginine producing ability" refers to a Corynebacterium 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 intrinsically. L-arginine producing ability can be imparted or improved by breeding.

[0023] 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 trait is changed due to genetic mutation caused by natural or artificial factors. For example, the unmodified microorganism refers to a strain in which the gluconate repressor protein activity in this specification is not weakened compared to its endogenous activity or before it is weakened. 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".

[0024] 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.

[0025] The microorganism having L-arginine production ability of the present application is a genetically modified microorganism or recombinant microorganism in which the activity of the gluconate repressor protein is weakened compared to the endogenous activity, and the L-arginine production ability is improved, but is not limited thereto. Specifically, the recombinant strain having improved L-arginine production ability is a microorganism having improved L-arginine production ability compared to a natural wild-type microorganism or a non-modified microorganism having the endogenous activity of the gluconate repressor protein, but is not limited thereto. For example, the strain having improved L-arginine production ability of the present application is a microorganism having improved L-arginine production ability compared to a microorganism containing the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding the same, but is not limited thereto.

[0026] For example, the microorganism having L-arginine producing ability may be a microorganism that endogenously contains a gluconate repressor protein.

[0027] For example, the microorganism having L-arginine producing ability may be a microorganism that endogenously contains a polynucleotide sequence encoding the gluconate repressor protein.

[0028] 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 the unmodified microorganism before the mutation is acceptable. 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.

[0029] In the present application, the "microorganism of the genus Corynebacterium" may be any microorganism belonging to the genus Corynebacterium. Specifically, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens, and more specifically Corynebacterium glutamicum.

[0030] 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.

[0031] In the present application, the term "attenuation" of gluconate repressor protein activity is a concept that includes all of the following: activity is reduced compared to endogenous activity, and activity is eliminated. The gluconate repressor protein activity is used interchangeably with polypeptide activity, protein activity, etc. The term "attenuation" is used interchangeably with inactivation, deficiency, down-regulation, decrease, reduction, attenuation, etc.

[0032] The attenuation includes, but is not limited to, inactivation, including, but not limited to, gluconate protein activity being less than 100%, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 0% of the endogenous activity.

[0033] The term "inactivated" means that the gluconate repressor protein is not expressed at all, or even if expressed, its activity is absent or reduced, as compared to that in a non-modified microorganism.

[0034] The weakening includes at least one of the following: the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide originally possessed by the microorganism due to, for example, a mutation in the polynucleotide encoding the polypeptide; the overall level and / or concentration (expression amount) of polypeptide activity in the cell is reduced compared to that of a natural strain due to, for example, inhibition of expression of the gene of the polynucleotide encoding it or inhibition of translation into the polypeptide; no expression of the polynucleotide at all; and no polypeptide activity even if the polynucleotide is expressed.

[0035] The "endogenous activity" refers to the activity of a specific polypeptide that a parent strain, wild-type, or unmodified microorganism originally had before the trait change when the trait is changed due to genetic mutation caused by natural or artificial factors. This is used interchangeably with "activity before modification." When the activity of a polypeptide is "inactivated," "lacking," "reduced," "down-regulated," "decreased," or "attenuated" compared to the endogenous activity, it means that the activity of the specific polypeptide is decreased compared to the activity of the specific polypeptide that a parent strain or unmodified microorganism originally had before the trait change.

[0036] Such attenuation of the activity of a polypeptide can be achieved by applying various methods well known in the art, including, but not limited to, those described in Non-Patent Documents 2 and 3.

[0037] Specifically, the attenuation of a polypeptide activity in the present application can be achieved by 1) deleting all or a part of a gene encoding the polypeptide, 2) modifying an expression regulatory region (or an expression regulatory sequence) so as to reduce the expression of a gene encoding the polypeptide, 3) modifying an amino acid sequence constituting the polypeptide so as to delete or weaken the activity of the polypeptide (e.g., deleting / substituting / adding one or more amino acids in the amino acid sequence), or 4) modifying a gene sequence encoding the polypeptide so as to delete or weaken the activity of the polypeptide (e.g., modifying the gene sequence encoding the polypeptide so as to encode a polypeptide modified so as to delete or weaken the activity of the polypeptide). The modification can be carried out by, but is not limited to, the following: deleting / substituting / adding one or more nucleic acid bases in the nucleic acid base sequence of a gene; 5) modifying the base sequence encoding the start codon or 5'UTR region of the gene transcript encoding a polypeptide; 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcript encoding a polypeptide; 7) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding a polypeptide so that a secondary structure that makes ribosome attachment impossible is formed; 8) adding a promoter to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide so that it is reverse transcribed (reverse transcription engineering, RTE); or 9) a combination of two or more selected from 1) to 8) above.

[0038] For example, 1) deleting a part or the whole of the gene encoding the polypeptide may be carried out by deleting the entire polynucleotide encoding the endogenous target polypeptide in a chromosome, or by replacing it with a polynucleotide lacking some nucleotides or a marker gene.

[0039] In addition, the expression regulatory region (or expression regulatory sequence) may be modified by generating a mutation in the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by substituting a sequence having a weaker activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0040] Furthermore, the modification of the amino acid sequence or polynucleotide sequence of 3) and 4) above can be performed by, but is not limited to, generating a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide so as to weaken the activity of the polypeptide, or by replacing the sequence with an amino acid sequence or polynucleotide sequence improved to have a weaker activity, or an amino acid sequence or polynucleotide sequence improved to have no activity. For example, gene expression can be inhibited or weakened by, but is not limited to, introducing a mutation into a polynucleotide sequence to form a stop codon.

[0041] Furthermore, the base sequence encoding the start codon or 5'UTR region of the gene transcription product encoding the polypeptide can be modified, for example, by substituting a base sequence encoding another start codon that has a lower polypeptide expression rate compared to the endogenous start codon, but is not limited to this.

[0042] 6) The introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcription product encoding the polypeptide can be described, for example, in Non-Patent Document 4.

[0043] 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide so that a secondary structure is formed that makes ribosome attachment impossible may be achieved by making mRNA translation impossible or slowing down the rate.

[0044] 8) Adding a promoter to the 3' end of the ORF (open reading frame) of a gene sequence encoding a polypeptide so as to reverse transcribe the promoter (Reverse transcription engineering, RTE) may be carried out by creating an antisense nucleotide complementary to the gene transcript encoding the polypeptide to attenuate the activity.

[0045] Specifically, the Corynebacterium microorganism having L-arginine producing ability of the present application includes, but is not limited to, a gluconate repressor mutant in which the amino acid sequence constituting the gluconate repressor protein has been modified so that the activity of the gluconate repressor protein is deleted or weakened, a polynucleotide encoding the gluconate repressor mutant, or a vector containing the polynucleotide.

[0046] More specifically, the gluconate repressor mutant is 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 substituted with another amino acid, but is not limited thereto.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] The amino acids are commonly classified based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues.

[0051] 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 yet 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.

[0052] 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.

[0053] Even if the present application describes "a polypeptide or protein comprising an amino acid sequence represented by a specific SEQ ID NO," "a polypeptide or protein consisting of an amino acid sequence represented by a specific SEQ ID NO," or "a polypeptide or protein having an amino acid sequence represented by a specific SEQ ID NO," it goes without saying that any protein having an amino acid sequence in which a part of the sequence has been deleted, modified, substituted, conservatively substituted, or added is also used in the present application, so long as it has the same or corresponding activity as the polypeptide consisting of the amino acid sequence of the SEQ ID NO. For example, there are proteins having an addition of a sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation, or a conservative substitution at the N-terminus and / or C-terminus of the amino acid sequence.

[0054] For example, the amino acid sequence may have an addition or deletion of a sequence that does not change the function of the gluconate repressor protein of the present application, a naturally occurring mutation, a silent mutation, or a conservative substitution at the N-terminus, C-terminus, and / or internally.

[0055] 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.

[0056] "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.

[0057] 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").

[0058] For such alignment, for example, the Needleman-Wunsch algorithm (Non-Patent Document 5) or the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 6) 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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 7. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 5), as implemented in the Needle program (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 6) (version 5.0.0 or later) of the EMBOSS package (which includes the GCG program package (Non-Patent Document 8), BLASTP, BLASTN, FASTA (Non-Patent Documents 9, 10 and 11)). For example, BLAST or Clustal W from the National Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0063] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as GAP (1999) (1999) (1999) (1999) (1999) (1999). 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 of GAP (1999) (or EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in GAP (1999) (1999), (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.

[0064] As an example, the variant includes at least one substitution selected from the group consisting of, but is not limited to, 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.

[0065] 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.

[0066] 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).

[0067] 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.

[0068] 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).

[0069] 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.

[0070] 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).

[0071] 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.

[0072] 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).

[0073] 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.

[0074] 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).

[0075] 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.

[0076] 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).

[0077] 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, 79th, and 92nd positions in the amino acid sequence of SEQ ID NO: 1 of the present application in an arbitrary amino acid sequence. In the present application, it goes without saying that "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, even if not otherwise specified.

[0078] Furthermore, 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. Furthermore, it goes without saying that the present application also includes variants having an amino acid sequence in which a part of the sequence has been deleted, modified, substituted, conservatively substituted or added, so long as the amino acid sequence has such homology or identity and exhibits efficacy equivalent to that of the variant of the present application.

[0079] For example, the variant of the present application may have, contain, or essentially consist of an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 25 to 31. 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 represented by SEQ ID NOs: 25 to 31.

[0080] Specifically, the L-arginine-producing Corynebacterium microorganism of the present application has gluconate repressor protein activity inactivated, but is not limited thereto.

[0081] Specifically, in order to inactivate the gluconate repressor protein activity, a method of deleting a part or the whole of the gene encoding the protein may be used. More specifically, the method may be performed by replacing a polynucleotide encoding an endogenous target protein in a chromosome with a polynucleotide having a deleted nucleotide sequence or a marker gene using a vector for chromosomal introduction into a microorganism. One example of a method of deleting a part or the whole of such a polynucleotide is a method of deleting a polynucleotide by homologous recombination, but is not limited thereto. As another example, the method of deleting a part or the whole of the gene may be performed by inducing a mutation using light such as ultraviolet light or a chemical substance, and selecting a strain in which the target gene is deleted from the obtained mutants.

[0082] The gene deletion method includes a method using genetic recombination techniques. For example, the gene deletion may be performed by introducing a polynucleotide sequence or vector containing a polynucleotide sequence having homology to a target gene into the microorganism to cause homologous recombination. The introduced polynucleotide sequence or vector contains a dominant selection marker. However, the gene deletion method is not limited to this.

[0083] 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.

[0084] Specifically, the Corynebacterium microorganism having L-arginine producing ability of the present application comprises, but is not limited to, a polynucleotide encoding a gluconate repressor mutant in which the amino acid sequence constituting the gluconate repressor protein is modified so that the activity of the gluconate repressor protein is deleted or weakened.

[0085] The polynucleotide encoding the gluconate repressor mutant may be any polynucleotide sequence that encodes the gluconate repressor mutant of the present application, for example, but not limited to, a polynucleotide sequence that encodes the amino acid sequence of the mutant of the present application.

[0086] 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: 32 to SEQ ID NO: 38, 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: 32 to SEQ ID NO: 38, 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.

[0087] 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.

[0088] 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.

[0089] 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 15 and 16). For example, the conditions include conditions under which polynucleotides having high homology or identity, such as 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 lower homology or identity 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, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS, which are washing conditions for normal Southern hybridization.

[0090] 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.

[0091] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application 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.

[0092] 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 15).

[0093] Specifically, the Corynebacterium microorganism of the present application capable of producing L-arginine is a microorganism lacking a polynucleotide encoding a gluconate repressor protein, but is not limited thereto. For example, the Corynebacterium microorganism of the present application capable of producing L-arginine is a microorganism lacking the nucleic acid base sequence of SEQ ID NO: 2, but is not limited thereto.

[0094] The Corynebacterium microorganism having L-arginine production ability of the present application contains an expression vector for expressing the gluconate repressor mutant in a host, thereby weakening the activity of the gluconate repressor protein compared to the endogenous activity, but is not limited thereto.

[0095] Alternatively, the Corynebacterium microorganism having L-arginine producing ability of the present application contains an expression vector for inactivating a gluconate repressor protein in a host cell, thereby inactivating the activity of the gluconate repressor protein, but is not limited thereto.

[0096] The vector of the present application means 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 a vector is transformed into a suitable host cell, it can replicate and function independently of the host genome, and is integrated into the genome itself.

[0097] 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, pSK, pSKH, pET, etc. may be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pSK, pSKH130, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. may be used.

[0098] 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 selective agent, only cells expressing the selection marker survive or show a different phenotype, so that transformed cells can be selected.

[0099] In the present application, "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or a microorganism, thereby expressing the polypeptide encoded by the polynucleotide in the host cell. The transformed polynucleotide may be any polynucleotide that is expressed in the host cell, regardless of whether it is inserted into the chromosome of the host cell 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 host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a 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 host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0100] 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.

[0101] In the microorganism of the present application, modification of a part or the whole of a polynucleotide can be induced by (a) homologous recombination using a vector for chromosomal introduction in the microorganism, or genome editing using engineered nuclease (e.g., CRISPR-Cas9), and / or (b) light such as ultraviolet light or radiation and / or chemical treatment, but is not limited thereto. Methods for modifying a part or the whole of the gene include methods using DNA recombination techniques. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene is introduced into the microorganism to cause homologous recombination, thereby deleting a part or the whole of the gene. The introduced nucleotide sequence or vector contains a dominant selection marker, but is not limited thereto.

[0102] Another aspect of the present application provides a method for producing L-arginine, comprising culturing a Corynebacterium microorganism of the present application in a medium.

[0103] In the method of the present application, any culture conditions and culture methods known in the art can be used for culturing the microorganisms. Such culture processes can be easily adjusted by those skilled in the art depending on the strain selected.

[0104] In the present application, "culturing" 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, or fed-batch culture, but is not limited thereto.

[0105] 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 that is used for culturing a normal microorganism, and the microorganism of the present application can be cultured in a normal 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] In the present application, 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 in a suitable manner during the cultivation of the microorganism. Also, during the cultivation, foam generation can be suppressed using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, in order to maintain the aerobic state of the culture, oxygen or an oxygen-containing gas can be injected into the culture, and in order to maintain the anaerobic and microaerobic state, no gas can be injected, and nitrogen, hydrogen, or carbon dioxide gas can be injected, but this is not limited thereto.

[0110] The temperature of the culture is 25° C. to 40° C., more specifically, but not limited to, 28° C. to 37° C. The culture period is continued until a desired amount of useful substance is produced, specifically, but not limited to, 1 hour to 100 hours.

[0111] The L-arginine produced by the culture of the present application is either secreted into the medium or remains intracellularly.

[0112] The L-arginine production method of the present application may further include a step of preparing a Corynebacterium microorganism of the present application, a step of preparing a medium for culturing the Corynebacterium microorganism, or a combination thereof (in any order), for example, prior to the culturing step.

[0113] 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) or the microorganism used in the culture. The recovery step may be further included after the culture step.

[0114] The recovery may be a method of collecting the target 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, or fed-batch culture. 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, and affinity chromatography, HPLC, or a combination thereof can be used, and the target L-arginine can be collected from the medium or the microorganism using a suitable method known in the art.

[0115] 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.

[0116] Yet another aspect of the present application provides a composition for producing L-arginine, comprising the Corynebacterium microorganism.

[0117] The composition of the present application may further comprise any suitable excipient commonly used in compositions for producing L-arginine, including, but not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, isotonicity agents, etc.

[0118] Yet another aspect of the present application provides use of the Corynebacterium microorganism for producing L-arginine. EXAMPLES

[0119] 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 a skilled engineer in the technical field of the present application or a similar technical field. EXAMPLES

[0120] Construction of a vector for attenuating gluconate repressor activity In order to attenuate the gluconate repressor activity, we attempted to insert the gntR1(I36N), gntR1(R59A), gntR1(T60L), gntR1(R63A), gntR1(R79A), and gntR1(A92L) mutants shown in SEQ ID NO:25 to SEQ ID NO:31, respectively, into Corynebacterium glutamicum KCCM10741P strain, and thus constructed vectors containing targeted mutations for attenuating the gluconate repressor activity. 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, 4 and 6, 3 and 7, 4 and 8, 3 and 9, 4 and 10, 3 and 11, 4 and 12, 3 and 13, 4 and 14, 3 and 15, 4 and 16), 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, R79A, and A92L mutation sequences (hereinafter referred to as "mutation-introduced fragment 1" to "mutation-introduced fragment 6", respectively). 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.

[0121] Next, the pDCM2 vector (Patent Document 2), which is incapable of replicating in Corynebacterium glutamicum, and the PCR-amplified fragment 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 6, respectively, were prepared: pDCM2-gntR1(I36N), pDCM2-gntR1(R59A), pDCM2-gntR1(T60L), pDCM2-gntR1(R63A), pDCM2-gntR1(R79A), and pDCM2-gntR1(A92L).

[0122] [Table 1] EXAMPLES

[0123] Construction of a strain with reduced gluconate repressor activity based on KCCM10741P and evaluation of its L-arginine productivity Example 2-1. Preparation of strains In order to attenuate the gluconate repressor protein activity of Corynebacterium glutamicum KCCM10741P (Patent Document 3), an L-arginine producing strain, the strain was transformed with the recombinant plasmids (pDCM2-gntR1(I36N), pDCM2-gntR1(R59A), pDCM2-gntR1(T60L), pDCM2-gntR1(R63A), pDCM2-gntR1(R79A), and pDCM2-gntR1(A92L)) prepared in Example 1 (Non-Patent Document 17). 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) on 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.

[0124] The transformed strains were named KCCM10741P-gntR1(I36N), KCCM10741P-gntR1(R59A), KCCM10741P-gntR1(T60L), KCCM10741P-gntR1(R63A), KCCM10741P-gntR1(R79A), and KCCM10741P-gntR1(A92L), respectively.

[0125] 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 with weakened gluconate repressor protein activity, KCCM10741P-gntR1(I36N), KCCM10741P-gntR1(R59A), KCCM10741P-gntR1(T60L), KCCM10741P-gntR1(R63A), KCCM10741P-gntR1(R79A), KCCM10741P-gntR1(A92L)), the L-arginine concentration in the culture medium was analyzed by culturing them in the following manner.

[0126] The parent strain Corynebacterium glutamicum KCCM10741P and the transformed strains (each gluconate repressor protein activity attenuated strain) prepared in Example 1-2 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)

[0127] After the cultivation, the L-arginine productivity (concentration) was analyzed by HPLC (Waters 2478). The analyzed L-arginine concentrations are shown in Table 2.

[0128] [Table 2]

[0129] 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 with weakened gluconate repressor protein activity, KCCM10741P-gntR1(I36N), KCCM10741P-gntR1(R59A), KCCM10741P-gntR1(T60L), KCCM10741P-gntR1(R63A), KCCM10741P-gntR1(R79A), KCCM10741P-gntR1(A92L)). EXAMPLES

[0130] Construction of a CJ1R-based strain with reduced gluconate repressor protein activity and evaluation of L-arginine production 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: 17 and 18, SEQ ID NOs: 19 and 20) in Table 3 and overlapping PCR using the primer pair of SEQ ID NOs: 17 and 20 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.

[0131] The constructed vector (recombinant plasmid) was named pDCM2-ΔargR.

[0132] [Table 3]

[0133] Next, the wild-type Corynebacterium glutamicum strain (ATCC13869) was transformed with the pDCM2-ΔargR prepared as described above (Non-Patent Document 17), 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.

[0134] Example 3-2. Construction of a strain with reduced gluconate repressor activity based on the CJ1R strain In order to weaken the gluconate repressor protein activity of 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(R79A), and CJ1R-gntR1(A92L), respectively.

[0135] 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 weakened gluconate repressor protein activity, CJ1R-gntR1(I36N), CJ1R-gntR1(R59A), CJ1R-gntR1(T60L), CJ1R-gntR1(R63A), 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.

[0136] 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.

[0137] [Table 4]

[0138] As a result, it was confirmed that all of the mutant strains (strains with weakened gluconate repressor protein activity, CJ1R-gntR1(I36N), CJ1R-gntR1(R59A), CJ1R-gntR1(T60L), CJ1R-gntR1(R63A), CJ1R-gntR1(R79A), CJ1R-gntR1(A92L)) had an average L-arginine production ability improved by approximately 19% compared to the parent strain. EXAMPLES

[0139] Construction of a strain with reduced gluconate repressor activity and evaluation of its L-arginine production ability Example 4-1. Preparation of strains To confirm the effect of attenuating gluconate repressor activity on L-arginine production, we constructed L-arginine-producing Corynebacterium glutamicum strains KCCM10741P and CJ1R by deleting the gntR1 gene.

[0140] Specifically, a recombinant vector for deleting the gntR1 gene was constructed in the same manner as in Example 3-1. The primers used in the vector construction are shown in Table 5. Specifically, a homologous recombination fragment having an argR deletion mutant sequence was obtained by performing PCR using the primer pairs (SEQ ID NOs: 21 and 22, SEQ ID NOs: 23 and 24) in Table 5 and overlapping PCR using the primer pair of SEQ ID NOs: 21 and 24, 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.

[0141] The constructed vector (recombinant plasmid) was named pDCM2-ΔgntR1.

[0142] [Table 5]

[0143] Next, the Corynebacterium glutamicum strains KCCM10741P and CJ1R capable of producing L-arginine were transformed with the pDCM2-△gntR1 prepared as described above (Non-Patent Document 17), and then secondary recombination was performed on a solid plate medium containing 4% sucrose. For the transformed strains in which secondary recombination was completed, PCR was performed using a primer pair (SEQ ID NOs: 21 and 24) to confirm that the gntR1 gene had been 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 strains were named KCCM10741P-△gntR1 and CJ1R-△gntR1, respectively.

[0144] Example 4-2. Evaluation of L-arginine production ability To compare the L-arginine production abilities of the L-arginine producing strains Corynebacterium glutamicum KCCM10741P and CJ1R and the gntR1 deletion strains (KCCM10741P-△gntR1, CJ1R-△gntR1) prepared in Example 4-1, the strains were cultured in the same manner as in Example 2-2 and the L-arginine concentration in the culture medium was analyzed.

[0145] 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 6.

[0146] [Table 6]

[0147] As a result, it was confirmed that the L-arginine producing ability of all the gntR1-deficient strains (KCCM10741P-ΔgntR1, CJ1R-ΔgntR1) was improved by an average of 13% to 19% compared to the parent strain.

[0148] 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 Corynebacterium microorganism in which gluconate repressor protein activity is weakened.

2. The microorganism of the genus Corynebacterium according to claim 1, which is capable of producing L-arginine.

3. The Corynebacterium microorganism according to claim 1 , wherein the gluconate repressor protein is a protein consisting of the amino acid sequence of SEQ ID NO:

1.

4. The microorganism according to claim 1 , wherein the Corynebacterium genus microorganism is Corynebacterium glutamicum.

5. The Corynebacterium microorganism according to any one of claims 1 to 4, wherein the microorganism has improved L-arginine producing ability.

6. A method for producing L-arginine, comprising the step of culturing a Corynebacterium microorganism in which gluconate repressor protein activity is attenuated in a medium.

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