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

Abstract

The present application relates to: a microorganism in which the activity of a gluconate repressor protein is weakened; a method for producing L-arginine using the microorganism of the present application; a composition for the production of L-arginine, comprising the microorganism of the present application; and the use of the microorganism of the present application for the production of L-arginine.

Description

Technical Field

[0001] The present disclosure relates to a microorganism in which the activity of a gluconate repressor protein is attenuated; a method for producing L-arginine using the microorganism of the present disclosure; a composition for producing L-arginine, comprising the microorganism of the present disclosure; and use of the microorganism of the present disclosure for producing L-arginine. Background Art

[0002] L-arginine is used in pharmaceuticals such as liver function improvers, brain function improvers, and complex amino acid preparations. It has recently attracted attention for use in foods such as fish cake additives, health drink additives, and salt substitutes for patients with hypertension. Research is ongoing on the use of microorganisms to produce high concentrations of industrially applicable arginine.

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

[0004] However, there is still an increasing demand for research on methods for efficiently producing L-arginine in high yield. Summary of the Invention

[0005] [Technical Issues]

[0006] The present inventors have developed a microorganism of the genus Corynebacterium in which the activity of the gluconate repressor protein is attenuated, a method for producing L-arginine using the microorganism, a composition for producing L-arginine comprising the microorganism, and use of the microorganism for producing L-arginine, thereby completing the present disclosure.

[0007] [Technical solution]

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

[0009] Another object of the present disclosure is to provide a method for producing L-arginine, which comprises culturing the microorganism of the genus Corynebacterium of the present disclosure in a culture medium.

[0010] Yet another object of the present disclosure is to provide a composition for producing L-arginine, comprising the microorganism of the genus Corynebacterium disclosed herein.

[0011] Yet another object of the present disclosure is to provide use of the microorganism of the genus Corynebacterium disclosed herein for producing L-arginine.

[0012] [Beneficial Effects]

[0013] The microorganism of the genus Corynebacterium of the present invention in which the activity of the gluconate repressor protein is attenuated can produce L-arginine at a high yield, compared to the microorganism of the genus Corynebacterium in which the activity of the gluconate repressor protein is not attenuated. DETAILED DESCRIPTION

[0014] The present disclosure will be described in detail below. At the same time, each description and embodiment disclosed herein can also be applied to other descriptions and embodiments. In other words, all combinations of the various elements disclosed herein 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 of the prior art and the description of the present disclosure.

[0015] One aspect of the present disclosure provides a microorganism of the genus Corynebacterium, wherein the activity of a gluconate repressor protein is attenuated.

[0016] The microorganism of the genus Corynebacterium of the present disclosure may have L-arginine production ability. In addition, compared with the microorganism of the genus Corynebacterium in which the activity of the gluconate repressor protein is not weakened, the microorganism of the genus Corynebacterium of the present disclosure may have increased L-arginine production ability.

[0017] As used herein, the term "gluconate repressor (GntR) protein" refers to a regulatory protein involved in gluconate metabolism, sugar uptake, and the like.

[0018] Specifically, the gluconate repressor protein is known in the art, and the protein and gene sequences of the gluconate repressor protein can be obtained from known databases, such as NCBI's GenBank, but are not limited thereto. More specifically, the gluconate repressor protein can be a protein consisting of the amino acid sequence of SEQ ID NO: 1. In addition, the gluconate repressor protein of the present disclosure can have and / or include the amino acid sequence shown in SEQ ID NO: 1, or can be essentially composed of the amino acid sequence.

[0019] In addition, the gluconate repressor protein of the present disclosure may include the amino acid sequence shown in SEQ ID NO: 1, and an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity with the amino acid sequence of SEQ ID NO: 1. In addition, it is obvious that if the amino acid sequence has such homology or identity and exhibits the same or corresponding biological activity as the gluconate repressor protein of the present disclosure, a protein having an amino acid sequence having a deletion, modification, substitution, conservative substitution or addition in a portion thereof may also be included in the scope of the present disclosure.

[0020] In addition, the gluconate repressor protein having the amino acid sequence of SEQ ID NO: 1 can be encoded by a polynucleotide having or including 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, or 98% or more, and less than 100% homology or identity to the sequence of SEQ ID NO: 2, or the polynucleotide consists of or essentially consists of the base sequence of SEQ ID NO: 2, or consists of or essentially consists of a base sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more, and less than 100% homology or identity to the sequence of SEQ ID NO: 2, but is not limited thereto.

[0021] As used herein, the term "L-arginine" refers to an amino acid with the chemical formula C6H 14 L-arginine is an L-amino acid that is present in all living organisms and is a conditionally essential amino acid. L-arginine is known to be produced by microorganisms (primarily by microorganisms of the genus Corynebacterium); however, it is also known that they are subject to feedback inhibition by intracellular arginine (Vehary Sakanyan, et al., Microbiology, 142:9-108, 1996). Therefore, the high-yield production of L-arginine is known to be limited.

[0022] As used herein, the term "microorganism (or strain)" includes all wild-type microorganisms and microorganisms with natural or artificial genetic modifications. The term refers to a microorganism in which a specific mechanism is weakened or enhanced due to the insertion of an exogenous gene, the enhancement or inactivation of an endogenous gene activity, etc., and the microorganism may be a microorganism comprising a genetic modification for producing a target polypeptide, protein or product.

[0023] As used herein, the term "microorganism of the genus Corynebacterium having L-arginine-producing ability" refers to a microorganism strain of the genus Corynebacterium that can produce L-arginine in itself, including microorganisms in which L-arginine-producing ability is imparted to a parent strain that does not have L-arginine-producing ability, or microorganisms with inherent L-arginine-producing ability. L-arginine-producing ability can be imparted or enhanced by species improvement.

[0024] As used herein, the term "unmodified microorganism" does not exclude the bacterial strain comprising the mutation that may occur naturally in the microorganism, and can refer to initial wild-type strain or natural strain, or the bacterial strain before changing its properties due to the genetic mutation caused by natural or artificial factors. For example, the unmodified microorganism can refer to a bacterial strain in which the activity of the gluconate repressor protein described in this specification sheets is not weakened compared with its intrinsic activity, or the bacterial strain before the activity weakens." unmodified microorganism" can be used interchangeably with "strain before modification", "microorganism before modification", "unmutated strain", "unmodified strain", "unmutated microorganism" or "reference microorganism".

[0025] 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.

[0026] The microorganism with L-arginine production ability disclosed herein may be a genetically modified microorganism or a recombinant microorganism, wherein the L-arginine production ability is enhanced due to the weakening of the gluconate repressor protein activity compared to the inherent activity, but is not limited thereto. Specifically, the recombinant strain with enhanced L-arginine production ability may be a microorganism with enhanced L-arginine production ability compared to a natural wild-type microorganism or an unmodified microorganism with inherent gluconate repressor protein activity, but is not limited thereto. In one embodiment, the strain with enhanced L-arginine production ability disclosed herein may be a microorganism, wherein the L-arginine production ability is enhanced compared to a microorganism comprising the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding the same, but is not limited thereto.

[0027] For example, the microorganism having L-arginine-producing ability may be a microorganism inherently containing a gluconate repressor protein.

[0028] For example, the microorganism having L-arginine-producing ability may be a microorganism inherently containing a polynucleotide sequence capable of encoding a gluconate repressor protein.

[0029] In one embodiment, the L-arginine-producing capacity of the microorganism with enhanced L-arginine-producing capacity can be increased by 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, compared to the parent strain before modification or the unmodified microorganism having the intrinsic activity of the gluconate repressor protein. , 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 and 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), but the L-arginine productivity is not limited thereto, as long as the value of the L-arginine productivity of the microorganism has a positive change compared to the L-arginine productivity of the parent strain before modification or an unmodified microorganism. In another example, compared to the parent strain or unmodified microorganism before modification, the recombinant strain with enhanced productivity can have an L-arginine productivity increased by 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 and 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), but the L-arginine productivity is not limited thereto. As used herein, the term "about" refers to a range including all ± 0.5, ± 0.4, ± 0.3, ± 0.2, ± 0.1, etc., including all values ​​within the range equal to or similar to the term "about", but not limited thereto.

[0030] As used herein, the term "microorganisms of the genus Corynebacterium" may include all microorganisms of the genus Corynebacterium. Specifically, the microorganism of the genus Corynebacterium disclosed herein may be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens, more specifically, Corynebacterium glutamicum.

[0031] Although it is known that microorganisms of the genus Corynebacterium can produce L-arginine, their production capacity is very low, and the genes involved in the production mechanism or the principle of this mechanism have not been fully elucidated. Therefore, the Corynebacterium microorganisms with L-arginine production capacity disclosed herein include all natural wild-type microorganisms, Corynebacterium microorganisms in which the L-arginine production capacity is improved by enhancing or weakening the activity of genes related to the L-arginine production mechanism, or Corynebacterium microorganisms in which the L-arginine production capacity is improved by introducing or enhancing the activity of exogenous genes.

[0032] As used herein, the term "attenuating" the activity of the gluconate repressor protein is intended to include all concepts including reduced or absent activity compared to intrinsic activity. The activity of the gluconate repressor protein can be used interchangeably with terms such as "polypeptide activity" and "protein activity." Attenuation can be used interchangeably with inactivation, deletion, downregulation, reduction, decrease, weakening, and the like.

[0033] Attenuation may be inactivation, but is not limited thereto. Inactivation may include, but is not limited to, all cases where the activity of the gluconate protein is 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 intrinsic activity.

[0034] Inactivation may refer to a case where the gluconate repressor protein is not expressed at all, or even if expressed, its activity is absent or reduced compared to an unmodified microorganism.

[0035] The attenuation may also include: due to mutations in the polynucleotide encoding the polypeptide, the polypeptide activity is reduced or disappears compared to the polypeptide activity originally possessed by the microorganism; due to inhibition of gene expression of the polynucleotide encoding the polypeptide or inhibition of translation into the polypeptide, the overall polypeptide activity and / or concentration (expression level) in the cell is lower than that in the natural strain; the polynucleotide is not expressed; and / or the protein has no activity despite the expression of the polynucleotide.

[0036] The term "intrinsic activity" refers to the activity of a specific polypeptide originally possessed by the parent strain or wild-type or unmodified microorganism before transformation, when the trait is altered due to genetic mutation caused by natural or artificial factors. This term is used interchangeably with "activity before modification." Compared to the intrinsic activity, "inactivation," "deletion," "reduction," "downregulation," "reduction," or "attenuation" of a polypeptide activity means that the activity of the polypeptide is reduced compared to the activity of the specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation.

[0037] Such attenuation of the polypeptide activity can be performed by any method known in the art, but is not limited thereto, and can be achieved by applying various methods well known in the art (e.g., Nakashima N et al., Bacterialcellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014; 15(2): 2773-2793; Sambrook et al., Molecular Cloning 2012, etc.).

[0038] Specifically, the method for reducing the activity of the polypeptide of the present disclosure may be:

[0039] 1) deleting all or part of the gene encoding the polypeptide;

[0040] 2) modifying the expression control region (or expression control sequence) to reduce the expression of the gene encoding the polypeptide;

[0041] 3) modifying the amino acid sequence constituting the polypeptide (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence) to eliminate or weaken the activity of the polypeptide;

[0042] 4) modifying the gene sequence encoding the polypeptide to eliminate or weaken the activity of the protein (for example, deleting / substituting / adding one or more nucleic acid bases in the nucleic acid base sequence of the polypeptide gene to encode a protein modified to eliminate or weaken the activity of the polypeptide);

[0043] 5) modifying the base sequence of the start codon or 5'-UTR region of the transcript encoding the gene, which gene encodes the polypeptide;

[0044] 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide;

[0045] 7) adding a sequence complementary to the Shine-Dalgarno sequence of the gene encoding the polypeptide to the upstream region of the Shine-Dalgarno sequence, thereby forming a secondary structure that makes it impossible for ribosomes to attach;

[0046] 8) adding a promoter to be reverse transcribed to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE); or

[0047] 9) A combination of two or more selected from the above items 1) to 8), but not particularly limited thereto.

[0048] For example, these methods could be described as follows:

[0049] Deleting all or part of the gene encoding the polypeptide in the above item 1) may include eliminating all polynucleotides encoding the endogenous target polypeptide in the chromosome, replacing the polynucleotide with a polynucleotide in which some nucleotides are deleted, or replacing the polynucleotide with a marker gene.

[0050] Modification of the expression control region (or expression control sequence) in the above item 2) may include inducing mutations in the expression control region (or expression control sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or replacing the expression control region with a sequence having weaker activity. The expression control region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence for controlling transcription and translation termination.

[0051] Modification of the amino acid sequence or polynucleotide sequence in items 3) and 4) above may include, but is not limited to, inducing sequence mutations 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, thereby reducing the activity of the polypeptide, or replacing them with modified amino acid sequences or polynucleotide sequences having weaker activity or modified amino acid sequences or polynucleotide sequences that are inactive. For example, gene expression can be inhibited or reduced by introducing a mutation into the polynucleotide sequence to form a stop codon, but is not limited to this.

[0052] The modification of the base sequence encoding the start codon or 5'-UTR region of the transcript encoding the polypeptide in the above item 5) may include, for example, replacing the base sequence with a base sequence encoding a different start codon having a lower polypeptide expression rate compared to the endogenous start codon, but is not limited thereto.

[0053] Regarding the introduction of an antisense oligonucleotide (e.g., antisense RNA) that complementarily binds to the transcript of the gene encoding the polypeptide in the above item 6), reference can be made to, for example, the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews-Trends in Genetics, Vol. 1(1) 1986].

[0054] In the above-mentioned item 7), a sequence complementary to the Shine-Dalgarno sequence of the gene encoding the polypeptide is added to the upstream region of the Shine-Dalgarno sequence, thereby forming a secondary structure that makes it impossible for ribosomes to attach, which may involve making it impossible to translate mRNA or reducing its translation rate.

[0055] The addition of a promoter to be reverse transcribed to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE) in the above item 8) may involve making the antisense nucleotide complementary to the transcript of the gene encoding the polypeptide, thereby attenuating the activity.

[0056] Specifically, the Corynebacterium microorganism having L-arginine-producing ability disclosed herein may include a gluconate repressor variant constituting the amino acid sequence of the gluconate repressor protein, which is modified to eliminate or weaken the activity of the gluconate repressor protein, a polynucleotide encoding the gluconate repressor variant, or a vector comprising the polynucleotide, but is not limited thereto.

[0057] More specifically, the gluconate repressor variant can 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 by other amino acids, but are not limited thereto.

[0058] As used herein, the term "variant" refers to a polypeptide having an amino acid sequence that is different from the amino acid sequence of the variant before modification by conservative substitution and / or modification of one or more amino acids, but retains its function or properties. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. In other words, the ability of the variant can be increased, unchanged, or decreased compared to the polypeptide before modification. In addition, some variants may include variants in which one or more parts, such as an N-terminal leader sequence or a transmembrane domain, have been removed. Other variants may include variants in which a portion of the sequence has been removed from the N- and / or C-terminus of the mature protein. The term "variant" can be used interchangeably with terms such as modification, modified polypeptide, modified protein, mutant, mutant protein, and isoform, and is not limited thereto, as long as the term is used to represent a variation.

[0059] In addition, the variants may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence that is co-translationally or post-translationally involved in protein translocation may be conjugated to the N-terminus of the variant. In addition, the variants may be conjugated to other sequences or linkers for identification, purification or synthesis.

[0060] The term "other amino acid" is not limited as long as the amino acid is different from the amino acid before substitution. At the same time, it is obvious that when the expression "replacing a specific amino acid" is used in the present disclosure, the amino acid is substituted with an amino acid different from the amino acid before substitution, even if there is no special description indicating that the amino acid is substituted with a different amino acid.

[0061] Amino acids can generally be classified based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues.

[0062] As an example of this classification, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; amino acids with non-polar side chains (non-polar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; amino acids with polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. As another example, amino acids can be classified as follows: arginine, lysine, histidine, glutamic acid, and aspartic acid, which are amino acids with charged side chains (charged amino acids); and glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, which are amino acids with uncharged side chains (uncharged amino acids; also known as neutral amino acids). As another example, phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. As another example, valine, leucine, and isoleucine can be classified as branched-chain amino acids. As another example, the 20 amino acids can be classified into five groups by size, starting with the relatively small amino acid group: glycine, alanine, and serine; cysteine, proline, threonine, aspartic acid, and asparagine; valine, histidine, glutamic acid, and glutamine; isoleucine, leucine, methionine, leucine, and arginine; and phenylalanine, tryptophan, and tyrosine. However, the amino acid classification is not limited thereto.

[0063] For example, the description "the amino acid corresponding to position 36 of SEQ ID NO: 1 is substituted with another amino acid" may mean that the amino acid corresponding to position 36 of SEQ ID NO: 1 is substituted with asparagine, phenylalanine, glycine, alanine, arginine, aspartic acid, cysteine, glutamic acid, leucine, glutamine, histidine, proline, serine, tyrosine, lysine, tryptophan, valine, methionine or threonine, excluding leucine, but is not limited thereto.

[0064] Obviously, when the present disclosure describes "a polypeptide or protein comprising an amino acid sequence shown in a specific SEQ ID NO," "a polypeptide or protein consisting of an amino acid sequence shown in a specific SEQ ID NO," or "a polypeptide or protein having an amino acid sequence shown in a specific SEQ ID NO," this means that any protein having a deletion, modification, substitution, conservative substitution, or addition in part of its amino acid sequence can be used in the present disclosure, as long as it has the same or corresponding activity as a polypeptide consisting of the amino acid sequence of the corresponding SEQ ID NO. For example, this includes the addition of sequences to the N-terminus and / or C-terminus of the amino acid sequence, naturally occurring mutations or silent mutations, or conservative substitutions thereof that do not alter the function of the protein.

[0065] For example, this includes additions or deletions to the N-terminus, C-terminus and / or internal regions of the amino acid sequence that do not alter the function of the gluconate repressor protein, naturally occurring mutations, silent mutations or conservative substitutions.

[0066] As used herein, the term "position N" may include position N and the amino acid position corresponding to position N. Specifically, it may include the amino acid position corresponding to any amino acid residue in the mature polypeptide disclosed in the specific amino acid sequence. The specific amino acid sequence may be the amino acid sequence of SEQ ID NO: 1.

[0067] 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 an amino acid residue at a listed position in the polypeptide. The amino acid at the corresponding position can be identified as a specific amino acid in a defined sequence, which refers to a specific sequence. As used herein, the term "corresponding region" generally refers to a similar or corresponding position in a related protein or a reference protein.

[0068] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, and based on this, each amino acid residue of any amino acid sequence can be numbered with reference to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, a sequence alignment algorithm as described in the present disclosure can determine the position of an amino acid or the position at which a modification, such as a substitution, insertion, or deletion, has occurred by comparison to a query sequence (also referred to as a "reference sequence").

[0069] As such an 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, as well as sequence alignment programs, pairwise sequence comparison algorithms, etc., which are all known in the art and can be used appropriately.

[0070] As used herein, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid of similar structure and / or chemical properties. Such amino acid substitutions typically 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; and 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. Amino acids with charged side chains include aspartic acid, glutamic acid, lysine, arginine, and histidine. Amino acids with uncharged side chains can be further divided into nonpolar amino acids and polar amino acids; nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, and 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 a protein or polypeptide.

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

[0072] The sequence homology or identity of conservative polynucleotide or polypeptide can be determined by standard comparison algorithm, and the acquiescence gap penalty set up by the used program can be applied together.Basically, homology or identical sequence can hybridize with each other in whole or in part usually under moderate or highly stringent condition.Obviously, said hybridization also comprises the hybridization of the polynucleotide containing common codon or the codon that takes into account the degeneracy of codon in the polynucleotide.

[0073] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, by known computer algorithms, such as the "FASTA" program, using default parameters as in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, they can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), 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, SF, [et al., J Molec Biol 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994 and [CARILLO et al. (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 Information.

[0074] Homology, similarity or identity between polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, for example Needleman et al., (1970), J Mol Biol. 48:443, as known in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar divided by the total number of symbols in the shorter of the two sequences. The default parameters for the GAP program may include:

[0075] (1) A binary comparison matrix (containing a value of 1 indicating identity and a value of 0 indicating non-identity) as disclosed in Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979) 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 penalty of 10 for gap opening and a penalty of 0.5 for gap extension); and (3) no penalty for terminal gaps.

[0076] In one embodiment, the variant may include one or more substitutions selected from the group consisting of: substitution of the amino acid at position 36 of the amino acid sequence corresponding to SEQ ID NO: 1 with asparagine, substitution of the amino acid at position 59 of the amino acid sequence corresponding to SEQ ID NO: 1 with alanine, substitution of the amino acid at position 60 of the amino acid sequence corresponding to SEQ ID NO: 1 with leucine, substitution of the amino acid at position 63 of the amino acid sequence corresponding to SEQ ID NO: 1 with alanine, substitution of the amino acid at position 79 of the amino acid sequence corresponding to SEQ ID NO: 1 with alanine, and substitution of the amino acid at position 92 of the amino acid sequence corresponding to SEQ ID NO: 1 with leucine, but are not limited thereto.

[0077] In one embodiment of the above embodiments, the variant provided in the present disclosure may include substitution of the amino acid corresponding to position 36 from the N-terminus of SEQ ID NO: 1 with a different amino acid.

[0078] In one embodiment of the above embodiment, the amino acid at position 36 from the N-terminus of SEQ ID NO: 1 in the variant 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 one in which the amino acid at position 36 from the N-terminus of SEQ ID NO: 1 is substituted with asparagine (N).

[0079] In one embodiment of the above embodiments, the variant provided in the present disclosure may include substitution of the amino acid corresponding to position 59 from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0080] In one embodiment of the above embodiment, the amino acid at position 59 from the N-terminus of SEQ ID NO: 1 in the variant may be substituted with a non-polar amino acid. For example, the amino acid may be selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. In one embodiment of the above embodiment, the variant may be a variant in which the amino acid at position 59 from the N-terminus of SEQ ID NO: 1 is substituted with asparagine (N).

[0081] In one embodiment of the above embodiments, the variant provided in the present disclosure may include substitution of the amino acid corresponding to position 60 from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0082] In one embodiment of the above embodiment, the amino acid at position 60 from the N-terminus of SEQ ID NO: 1 in the variant may be substituted with a non-polar amino acid. For example, the amino acid may be selected from glycine, alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. In one embodiment of the above embodiment, the variant may be a variant in which the amino acid at position 60 from the N-terminus of SEQ ID NO: 1 is substituted with asparagine (N).

[0083] In one embodiment of the above embodiments, the variant provided in the present disclosure may include substitution of the amino acid corresponding to position 63 from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0084] In one embodiment of the above embodiment, the amino acid at position 63 from the N-terminus of SEQ ID NO: 1 in the variant may be substituted with a non-polar amino acid. For example, the amino acid may be selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. In one embodiment of the above embodiment, the variant may be a variant in which the amino acid at position 63 from the N-terminus of SEQ ID NO: 1 is substituted with asparagine (N).

[0085] In one embodiment of the above embodiments, the variant provided in the present disclosure may include substitution of the amino acid corresponding to position 79 from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0086] In one embodiment of the above embodiment, the amino acid at position 79 from the N-terminus of SEQ ID NO: 1 in the variant may be substituted with a non-polar amino acid. For example, the amino acid may be selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. In one embodiment of the above embodiment, the variant may be a variant in which the amino acid at position 79 from the N-terminus of SEQ ID NO: 1 is substituted with asparagine (N).

[0087] In one embodiment of the above embodiments, the variant provided in the present disclosure may include substitution of the amino acid corresponding to position 92 from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0088] In one embodiment of the above embodiment, the amino acid at position 92 from the N-terminus of SEQ ID NO: 1 in the variant may be substituted with a non-polar amino acid. For example, the amino acid may be selected from glycine, alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. In one embodiment of the above embodiment, the variant may be a variant in which the amino acid at position 92 from the N-terminus of SEQ ID NO: 1 is substituted with asparagine (N).

[0089] At the same time, those skilled in the art will be able to identify in any amino acid sequence 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 of the present disclosure. In addition, when describing "an amino acid at a specific position of a specific SEQ ID NO", it is obvious that this includes reference to "amino acids at positions corresponding to the aforementioned positions" in any amino acid sequence.

[0090] In addition, the variants of the present disclosure 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% homology or identity with the amino acid sequence shown in SEQ ID NO: 1, wherein 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 are substituted with another amino acid. In addition, it is obvious that as long as the amino acid sequence has such homology or identity and exhibits an effect corresponding to the variants of the present disclosure, variants having partial deletion, modification, substitution, conservative composition or addition of the amino acid sequence are all within the scope of the present disclosure.

[0091] For example, the variants of the present disclosure may have or include an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 25-31, or may consist essentially of the above amino acid sequences. 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% homology or identity with an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 25-31.

[0092] Alternatively, the Corynebacterium microorganism having L-arginine-producing ability of the present disclosure may specifically have inactivated gluconate repressor activity, but is not limited thereto.

[0093] Specifically, the method for deleting all or part of the genes of coded proteins can be used for the activity of the gluconate repressor protein described in inactivation s. Specifically, it is possible to carry out by using the polynucleotides or marker genes that have wherein deleted a part of nucleotide sequence to replace the polynucleotides encoding endogenous target protein in the chromosome for the vector for chromosome insertion in microorganisms. As the example of the method for deleting all or part of this polynucleotide, it is possible to use the method for deleting polynucleotides by homologous recombination, but the method is not limited thereto. As another example, the method for deleting all or part of the gene can be by using light (such as ultraviolet rays) or chemical substances to induce mutations, and from the gained variant, select the bacterial strain of target gene defect to carry out.

[0094] Methods for gene deletion include methods based on genetic recombination technology. For example, homologous recombination can be induced by inserting a polynucleotide sequence or a vector containing a polynucleotide sequence having homology to the target gene into a microorganism. In addition, the inserted polynucleotide sequence or vector may contain a dominant selection marker, but is not limited thereto.

[0095] As used herein, the term "polynucleotide" refers to a polymer of nucleotides in which nucleotide monomers are extended longitudinally by covalent bonds, and it is a DNA or RNA chain of a certain length, more specifically, a polynucleotide fragment encoding the protein.

[0096] Specifically, the Corynebacterium microorganism having L-arginine-producing ability disclosed herein may include a polynucleotide encoding a modified gluconate repressor variant, wherein the amino acid sequence constituting the gluconate repressor protein is modified to eliminate or weaken the activity of the gluconate repressor protein, but is not limited thereto.

[0097] The polynucleotide encoding the gluconate repression variant may include any sequence without limitation, as long as the sequence is a polynucleotide sequence encoding the gluconate repression variant described in the present disclosure. For example, the polynucleotide encoding the gluconate repression 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.

[0098] For example, the polynucleotide encoding the gluconate repressor variant of the present disclosure can be a polynucleotide sequence selected from the group consisting of the polynucleotide sequences shown in SEQ ID NOs: 32-38, or a polynucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology or identity with a polynucleotide sequence selected from the group consisting of the polynucleotide sequences shown in SEQ ID NOs: 32-38, but is not limited thereto. Nucleotide sequences having homology or identity can exclude sequences having 100% identity from the scope, or can be sequences having less than 100% identity.

[0099] Taking into account the codon degeneracy or the preferred codons in the organism in which the variants of the present invention are to be expressed, the polynucleotides of the present invention may have various modifications in the coding region without changing the amino acid sequence of the variants of the present invention. Therefore, it is clear that polynucleotides that can be translated into a polypeptide consisting of the amino acid sequence of the variants of the present invention or a polypeptide having homology or identity thereto by codon degeneracy are also included.

[0100] Furthermore, the polynucleotides of the present disclosure may include any sequence without limitation, as long as the sequence can hybridize under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to all or part of the polynucleotide sequence of the present disclosure.

[0101] The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. These conditions are described in detail in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; and FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). Examples of stringent conditions may include conditions in which polynucleotides with higher homology or identity hybridize to each other, for example, polynucleotides with 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 with lower homology or identity do not hybridize to each other; or typical Southern hybridization washing conditions, i.e., washing once, twice, or three times under conditions corresponding to a salt concentration and temperature of 1×SSC, 0.1% SDS at 60° C., specifically 0.1×SSC, 0.1% SDS at 60° C., or more specifically 0.1×SSC, 0.1% SDS at 68° C.

[0102] Hybridization requires that the two nucleic acids have complementary sequences, although mismatches between bases are possible 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, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of the present disclosure may also include isolated nucleic acid fragments that are complementary to the entire sequence, as well as substantially similar nucleic acid sequences.

[0103] Specifically, polynucleotides having homology or identity to the polynucleotides of the present disclosure can be detected by using hybridization conditions that include a hybridization step at a Tm of 55°C and using the conditions described above. m The value may be 60° C., 63° C., or 65° C., but is not limited thereto, and may be appropriately adjusted by those skilled in the art according to the purpose.

[0104] 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).

[0105] Alternatively, the L-arginine-producing microorganism of the genus Corynebacterium disclosed herein may be a microorganism in which the polynucleotide encoding the gluconate repressor protein is deleted, but is not limited thereto. For example, the L-arginine-producing microorganism of the genus Corynebacterium disclosed herein may be a microorganism in which the nucleic acid base sequence of SEQ ID NO: 2 is deleted, but is not limited thereto.

[0106] The microorganism of the genus Corynebacterium having L-arginine-producing ability of the present disclosure may have attenuated gluconate repressor protein activity compared to inherent activity by including an expression vector for expressing a gluconate repressor variant in a host, but is not limited thereto.

[0107] Alternatively, the microorganism of the genus Corynebacterium having L-arginine-producing ability of the present disclosure may have its gluconate repressor activity inactivated by including an expression vector for inactivating the gluconate repressor protein in host cells, but is not limited thereto.

[0108] The vector of the present disclosure may include a DNA construct comprising a base sequence of a polynucleotide encoding a target polypeptide, operably linked to a suitable expression control region (or expression control sequence) for expressing the target polypeptide in a suitable host. The expression control region may include a promoter capable of initiating transcription, any operator sequence for controlling such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for controlling transcription and translation termination. After transformation into a suitable host cell, the vector may replicate or function independently of the host genome, or may be integrated into the genome itself.

[0109] The vector used in the present disclosure is not particularly limited, and any vector known in the art can be used. Common examples of vectors include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ-based, pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, and pET-based vectors can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0110] In one embodiment, the polynucleotide encoding the target polypeptide can be inserted into the chromosome by a vector for chromosome insertion in the cell. The polynucleotide can be inserted into the chromosome by using any method known in the art, such as homologous recombination, but is not limited thereto. The vector can further include a selection marker for confirming chromosome insertion. The selection marker is used to select cells transformed by the vector, i.e., to confirm the insertion of the target nucleic acid molecule, and can use markers that confer selectable phenotypes such as drug resistance, auxotrophy, cytotoxic drug resistance or surface polypeptide expression. In an environment treated with a selection agent, only cells expressing the selection marker can survive or show different phenotypic expressions, thereby enabling the selection of transformed cells.

[0111] As used herein, the term "transformation" refers to the introduction of a vector comprising a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The polynucleotide of transformation can include any polypeptide, as long as it can be expressed in the host cell, no matter whether it is inserted into and located in the chromosome of the host cell or located outside the chromosome. In addition, the polynucleotide includes DNA and / or RNA encoding the target polypeptide. The polynucleotide can be introduced in any form, as long as the polynucleotide can be introduced and expressed in the host cell. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a genetic construct comprising all factors 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 that are operably connected to the polynucleotide. The expression cassette can be in the form of an expression vector that can self-replicate. In addition, the polynucleotide can be introduced into the host cell as is and be operably connected to the sequence required for expression in the host cell, but is not limited thereto.

[0112] The term "operably linked" above refers to a functional connection between a promoter sequence and a polynucleotide sequence for initiating and mediating transcription of a polynucleotide encoding a target protein of the present disclosure.

[0113] In the microorganisms disclosed herein, all or part of the modification of the polynucleotides can be induced by (a) homologous recombination using a vector for chromosome insertion into the microorganism or genome editing using an engineered nuclease (e.g., CRISPR-Cas9) and / or (b) treatment with light, such as UV rays and radioactive rays, and / or chemicals, but not limited thereto. Methods for modifying all or part of a gene can include methods using DNA recombination technology. For example, by injecting a nucleotide sequence or a vector comprising a nucleotide sequence having homology to the target gene into the microorganism, thereby inducing homologous recombination, all or part of the gene can be deleted. The injected nucleotide sequence or vector can include a dominant selection marker, but not limited thereto.

[0114] Another aspect of the present disclosure provides a method for producing L-arginine, comprising culturing the microorganism of the genus Corynebacterium of the present disclosure in a culture medium.

[0115] In the method of the present disclosure, the culture of microorganisms can utilize any culture conditions and culture methods known in the art. Depending on the selected strain, those skilled in the art can easily change and apply this culture process.

[0116] As used herein, the term "cultivation" refers to culturing the microorganisms of the present disclosure under appropriately controlled environmental conditions. The cultivation process of the present disclosure can be carried out according to suitable culture media and culture conditions known in the art. Depending on the selected strain, those skilled in the art can easily modify and adapt such cultivation processes. Specifically, the cultivation can be carried out in batch, continuous and / or fed-batch mode, but is not limited thereto.

[0117] As used herein, the term "culture medium" refers to a mixed substance containing nutrients required for culturing the microorganisms of the present disclosure as main components, and the culture medium provides nutrients, growth factors, etc., including water that is indispensable for survival and development. Specifically, any culture medium and other culture conditions can be used to culture the microorganisms of the present disclosure without particular limitation, as long as the culture medium is commonly used for culturing microorganisms. The microorganisms described in the present disclosure can be cultured in a common culture medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and / or vitamin, etc., while controlling temperature, pH value and other conditions under aerobic conditions.

[0118] In the present disclosure, the carbon source includes 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 nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice bran, cassava, sugarcane residues, and corn steep liquor can be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugars) can be used, and appropriate amounts of other carbon sources can be used in various ways without limitation. These carbon sources can be used alone or in combination of two or more, but are not limited thereto.

[0119] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc., and organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition product, and defatted soybean cake or its decomposition product, etc. can be used alone or in combination of two or more, but are not limited thereto.

[0120] The phosphorus source may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or their corresponding sodium salts. As inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. may be used. These components or precursors may be added to the culture medium in batches or continuously, but the method of addition is not limited thereto.

[0121] In the process of cultivating the microorganism of the present disclosure, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, 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 anaerobic and microaerobic states, but is not limited thereto.

[0122] The temperature of the culture medium may be maintained at 25 to 40° C., more specifically, 28 to 37° C., but not limited thereto. Cultivation may be performed until a desired amount of useful substances is obtained, specifically for 1 to 100 hours, but not limited thereto.

[0123] L-arginine produced by the culture of the present disclosure may be released into the culture medium or may be retained in the cells.

[0124] The method for producing L-arginine of the present disclosure may further include, for example, preparing the microorganism of the genus Corynebacterium of the present disclosure, preparing a culture medium for culturing the microorganism of the genus Corynebacterium, or a combination thereof (in any order) before the culturing step.

[0125] The method for producing L-arginine of the present disclosure may further include recovering L-arginine from the culture medium according to the culture (the culture medium in which the culture is performed) or the microorganism. The method of the present disclosure may further include a recovery step after the culture step.

[0126] Recovery can be a method of culturing a microorganism according to the present disclosure, such as batch, continuous, fed-batch type, etc., using suitable methods known in the art to collect the desired L-arginine. The method may include, for example, centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, ultrasonication, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography and affinity chromatography), HPLC and combinations thereof, and the desired L-arginine can be recovered from the culture medium or the microorganism using suitable methods known in the art.

[0127] In addition, the method for producing L-arginine of the present disclosure may further include a purification step. Suitable methods known in the art can be used for purification. In one embodiment, when the method for producing L-arginine of the present disclosure includes a recovery step and a purification step, recovery and purification step can be carried out in a continuous or discontinuous manner, without considering the order, or can be carried out simultaneously by being integrated into a step, but the method is not limited thereto.

[0128] Yet another aspect of the present disclosure provides a composition for producing L-arginine, comprising the microorganism of the genus Corynebacterium disclosed herein.

[0129] The composition of the present disclosure may further include any suitable excipients commonly used in compositions for producing L-arginine, and the excipients may include, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, isotonic agents, etc., but are not limited thereto.

[0130] Yet another aspect of the present disclosure provides use of the microorganism of the genus Corynebacterium of the present disclosure for producing L-arginine.

[0131] [Modes for Carrying Out the Invention]

[0132] Hereinafter, the present disclosure will be described in detail with reference to examples. However, these examples are merely for illustrating preferred embodiments of the present disclosure, and therefore, these examples are not intended to limit the scope of the present disclosure. At the same time, those skilled in the art of the present disclosure or related fields can fully understand and easily implement technical matters not described herein.

[0133] Example 1 Preparation of a vector for reducing the activity of a gluconate repressor

[0134] In order to attenuate the activity of the gluconate repressor, gntR1(I36N), gntR1(R59A), gntR1(T60L), gntR1(R63A), gntR1(R79A) and gntR1(A92L) variants (represented by SEQ ID NOs: 25-31, respectively) were inserted into the Corynebacterium glutamicum KCCM10741P strain. Thus, a vector comprising a target mutation for attenuating the activity of the gluconate repressor was constructed. Specifically, using the genomic DNA of Corynebacterium glutamicum ATCC13869 as a template, PCR was performed using the primer pairs listed in Table 1 (SEQ ID NOs: 3 and 5, SEQ ID NOs: 4 and 6, SEQ ID NOs: 3 and 7, SEQ ID NOs: 4 and 8, SEQ ID NOs: 3 and 9, SEQ ID NOs: 4 and 10, SEQ ID NOs: 3 and 11, SEQ ID NOs: 4 and 12, SEQ ID NOs: 3 and 13, SEQ ID NOs: 4 and 14, SEQ ID NOs: 3 and 15, and SEQ ID NOs: 4 and 16), and overlapping PCR was performed using the primer pair SEQ ID NOs: 3 and 4 to obtain homologous recombination fragments with each of the gntR1-I36N, R59A, T60L, R63A, R79A, and A92L mutant sequences (hereinafter referred to as "mutation-introduced fragment 1" to "mutation-introduced fragment 6," respectively). PCR was performed by repeating 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute.

[0135] Subsequently, the pDCM2 vector (Korean Patent Application Publication No. 10-2020-0136813) and PCR amplified fragment that can not be replicated in Corynebacterium glutamicum are processed with restriction enzymes BamHI and XbaI for chromosomal integration. These fragments are then connected with DNA ligase to construct a plasmid having inserted each mutation introduction fragment. Each of these plasmids is transformed into Escherichia coli DH5α and then layered on LB solid medium containing kanamycin (25mg / L). Colonies transformed with the plasmid on LB solid medium were selected, and DNA was extracted using a DNA-spin plasmid DNA purification kit (iNtRON) to generate pDCM2-gntR1(I36N), pDCM2-gntR1(R59A), pDCM2-gntR1(T60L), pDCM2-gntR1(R63A), pDCM2-gntR1(R79A) and pDCM2-gntR1(A92L) vectors (recombinant plasmids) including mutation-introduced fragments 1 to 6, respectively.

[0136] [Table 1]

[0137]

[0138]

[0139] Example 2 Preparation of a strain with weakened gluconate repressor activity based on KCCM10741P strain and evaluation of L-arginine production capacity

[0140] Example 2-1 Preparation of strains

[0141] To attenuate the gluconate repressor activity in the L-arginine-producing strain Corynebacterium glutamicum KCCM10741P (US 8034602B2), each of the recombinant plasmids constructed in Example 1 (pDCM2-gntR1(I36N), pDCM2-gntR1(R59A), pDCM2-gntR1(T60L), pDCM2-gntR1(R63A), pDCM2-gntR1(R79A), and pDCM2-gntR1(A92L)) was transformed into the strain (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Subsequently, secondary recombination was performed on a solid plate medium containing 4% sucrose, and PCR was performed on the transgenic strain that had completed the secondary recombination using primer pairs (SEQ ID NOs: 3 and 4) to confirm that each mutation had been introduced into the chromosomal gntR1 gene. PCR was performed by repeating 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute.

[0142] Each transgenic strain was named KCCM10741P-gntR1(I36N), KCCM10741P-gntR1(R59A), KCCM10741P-gntR1(T60L), KCCM10741P-gntR1(R63A), KCCM10741P-gntR1(R79A) and KCCM10741P-gntR1(A92L).

[0143] Example 2-2 Evaluation of L-arginine production capacity

[0144] In order to compare the parent strain Corynebacterium glutamicum KCCM10741P and the transgenic strains constructed in Example 2-1 (strains with attenuated gluconate repressor activity, KCCM10741P-gntR1(I36N), KCCM10741P-gntR1(R59A), KCCM10741P-gntR1(T60L), KCCM10741P-gntR1(R63A), KCCM10741P-gntR1(R79A) and KCCM10741P-gntR1(A92L)), each strain was cultured by the following method, and the L-arginine concentration in the culture medium was analyzed.

[0145] Each parent strain Corynebacterium glutamicum KCCM10741P and the transgenic strain constructed in Example 1-2 (strain with weakened gluconate repressor activity) were inoculated into a 250 mL corner-baffled flask containing 25 mL of the following seed culture medium and cultured at 30° C. with shaking at 200 rpm for 20 hours. Then, 1 mL of the seed culture medium was inoculated into a 250 mL corner-baffled flask containing 24 mL of the production culture medium and cultured at 30° C. with shaking at 200 rpm for 72 hours. The compositions of the seed culture medium and the production culture medium used were as follows.

[0146] <Seed culture medium (pH 7.2)>

[0147] Glucose 20g, ammonium sulfate 45g, magnesium sulfate heptahydrate 2g, potassium dihydrogen phosphate 2g, ammonium chloride 10g, biotin 0.01mg, thiamine hydrochloride 0.1mg, calcium pantothenate 2mg, niacinamide 3mg, ferrous sulfate 10mg, manganese sulfate 10mg, zinc sulfate 0.02mg, copper sulfate 0.5mg (based on 1L of distilled water).

[0148] <Production Medium (pH 7.2)>

[0149] Glucose 60g, ammonium sulfate 45g, magnesium sulfate heptahydrate 2g, potassium dihydrogen phosphate 2g, ammonium chloride 10g, biotin 0.01mg, thiamine hydrochloride 0.1mg, calcium pantothenate 2mg, niacinamide 3mg, ferrous sulfate 10mg, manganese sulfate 10mg, zinc sulfate 0.02mg, copper sulfate 0.5mg, calcium carbonate 30g (based on 1 liter of distilled water).

[0150] After the cultivation was completed, the L-arginine productivity (concentration) was analyzed using HPLC (Waters 2478), and the obtained L-arginine concentrations are shown in Table 2 below.

[0151] [Table 2]

[0152]

[0153] As a result, it was confirmed that the L-arginine production capacity was increased by about 17% on average in all transgenic strains (strains with attenuated gluconate repressor activity; KCCM10741P-gntR1(I36N), KCCM10741P-gntR1(R59A), KCCM10741P-gntR1(T60L), KCCM10741P-gntR1(R63A), KCCM10741P-gntR1(R79A) and KCCM10741P-gntR1(A92L)) compared with the L-arginine production capacity of the parent strain.

[0154] Example 3 Preparation of a strain with weakened gluconate repressor activity based on the CJ1R strain and evaluation of L-arginine production capacity

[0155] Example 3-1 Preparation of CJ1R strain

[0156] In order to determine whether the effect of increasing L-arginine productivity is also observed in other Corynebacterium glutamicum strains with L-arginine productivity, a Corynebacterium glutamicum strain CJ1R with L-arginine productivity was constructed by introducing a mutation (ΔargR) into the wild-type strain (ATCC13869). Specifically, PCR was performed using the genomic DNA of Corynebacterium glutamicum ATCC13869 as a template, the primers in Table 3 (SEQ ID NO:17 and 18, and SEQ ID NO:19 and 20) were used to perform PCR, and the primers of SEQ ID NO:17 and 20 were used to perform overlapping PCR to obtain a homologous recombination fragment with the argR defective mutant sequence. PCR was performed by repeating the following cycle 30 times: 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute of polymerization.

[0157] The constructed vector (recombinant plasmid) was named pDCM2-ΔargR.

[0158] [Table 3]

[0159]

[0160] Subsequently, the constructed pDCM2-ΔargR vector was transformed into a wild-type Corynebacterium glutamicum strain (ATCC13869) (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999), and then, secondary recombination was carried out on a solid plate medium containing 4% sucrose. The transgenic strain that had completed secondary recombination was confirmed to have argR deleted on the chromosome by PCR using primers (SEQ ID NO: 19 and 22). PCR was performed by repeating the following cycle 30 times: 95°C denaturation for 30 seconds, 55°C annealing for 30 seconds, and 72°C polymerization for 1 minute. The transgenic strain was named CJ1R.

[0161] Example 3-2 Preparation of a strain with weakened gluconate repressor activity based on the CJ1R strain

[0162] In order to weaken the gluconate repressor activity of the CJ1R strain, mutant strains were constructed by introducing each vector produced 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.

[0163] Example 3-3 Evaluation of L-arginine production capacity

[0164] In order to compare the L-arginine production ability of the L-arginine-producing strain Corynebacterium glutamicum CJ1R and the mutant strains constructed in Example 3-2 (strains with attenuated gluconate repressor activity; CJ1R-gntR1(I36N), CJ1R-gntR1(R59A), CJ1R-gntR1(T60L), CJ1R-gntR1(R63A), CJ1R-gntR1(R79A) and CJ1R-gntR1(A92L)), each strain was cultured in the same manner as in Example 2-2, and the L-arginine concentration in the culture medium was determined.

[0165] After the cultivation was completed, the L-arginine productivity (concentration) was analyzed using HPLC (Waters 2478), and the obtained L-arginine concentrations are shown in Table 4 below.

[0166] [Table 4]

[0167]

[0168] The results confirmed that the L-arginine production capacity was increased by an average of about 19% in all mutant strains (strains with weakened gluconate repressor activity; CJ1R-gntR1(I36N), CJ1R-gntR1(R59A), CJ1R-gntR1(T60L), CJ1R-gntR1(R63A), CJ1R-gntR1(R79A) and CJ1R-gntR1(A92L)) compared with the parent strain.

[0169] Example 4 Preparation of strains with weakened gluconate repressor activity and evaluation of L-arginine production capacity

[0170] Example 4-1 Preparation of strains

[0171] In order to determine the effect of attenuating the gluconate repressor activity on L-arginine production, strains were constructed by deleting the gntR1 gene in Corynebacterium glutamicum strains KCCM10741P and CJ1R (both strains having L-arginine-producing ability).

[0172] Specifically, a recombinant vector for deleting the gntR1 gene was constructed in the same manner as in Example 3-1. The primers used to construct the vector are shown in Table 5. Specifically, genomic DNA of Corynebacterium glutamicum ATCC13869 was used as a template, PCR was performed using the primer pairs in Table 5 (SEQ ID NOs: 21 and 22, and SEQ ID NOs: 23 and 24), and overlapping PCR was performed using the primer pairs of SEQ ID NOs: 21 and 24 to obtain a homologous recombinant fragment having an argR-deficient mutant sequence. PCR was performed by repeating the following cycle 30 times: denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute.

[0173] The constructed vector (recombinant plasmid) was named pDCM2-ΔgntR1.

[0174] [Table 5]

[0175]

[0176] Subsequently, the constructed pDCM2-ΔgntR1 vector was transformed into the Corynebacterium glutamicum strains KCCM10741P and CJ1R (both strains have the ability to produce L-arginine (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999)), and then secondary recombination was carried out on a solid plate medium containing 4% sucrose. The strains with gntR1 deletion on the chromosome were confirmed by PCR using primer pairs (SEQ ID NO:21 and 24) on the transgenic strains that had completed the secondary recombination. PCR was performed by repeating the following cycle 30 times: denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute. The transgenic strains were named KCCM10741P-ΔgntR1 and CJ1R-ΔgntR1, respectively.

[0177] Example 4-2 Evaluation of L-arginine production capacity

[0178] In order to compare the L-arginine production capabilities of the L-arginine-producing Corynebacterium glutamicum strains KCCM10741P and CJ1R and the gntR1-deficient strains (KCCM10741P-ΔgntR1 and CJ1R-ΔgntR1) constructed in Example 4-1, each strain was cultured in the same manner as in Example 2-2, and the L-arginine concentration in the culture medium was determined.

[0179] After the cultivation was completed, the L-arginine productivity (concentration) was analyzed using HPLC (Waters 2478), and the obtained L-arginine concentrations are shown in Table 6 below.

[0180] [Table 6]

[0181]

[0182] As a result, it was confirmed that the L-arginine productivity of all gntR1-deficient strains (KCCM10741P-ΔgntR1 and CJ1R-ΔgntR1) was increased by 13% to 19% on average compared with the parent strain.

[0183] As described above, those skilled in the art will appreciate that the present disclosure may be implemented in other specific forms without departing from the technical essence or basic features of the present disclosure. In this regard, the above embodiments should be interpreted as illustrative rather than limiting the present disclosure. The scope of the present disclosure should be understood to include all changes or modifications derived from the definition and scope of the claims and their equivalents, rather than from the specific embodiments.

Claims

1. A microorganism of the genus Corynebacterium, wherein the activity of a gluconate repressor protein is attenuated. 2 . The microorganism of the genus Corynebacterium according to claim 1 , wherein the microorganism has an ability to produce L-arginine. 3 . The microorganism of the genus Corynebacterium according to claim 1 , wherein the gluconate repressor protein consists of the amino acid sequence of SEQ ID NO:

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

5. The microorganism according to any one of claims 1 to 4, wherein the microorganism has an increased L-arginine production capacity.

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

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