Corynebacterium genus microorganism producing L-arginine and method for producing L-arginine using the same

Abstract

The present application relates to an L-arginine-producing microorganism in which a protein comprising the amino acid sequence of SEQ ID NO:1 is attenuated, and a method for producing L-arginine using the same.

Description

[Technical Field]

[0001] The present application relates to an L-arginine-producing microorganism in which the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 is attenuated, and a method for producing L-arginine. [Background technology]

[0002] L-arginine is used in medicines such as liver function promoters, brain function improvers, and comprehensive amino acid preparations, and has recently been attracting attention for its use in foods such as kamaboko additives, health drink additives, and salt substitutes for hypertensive patients. Research into the use of microorganisms to produce industrially applicable high-concentration arginine has been ongoing, and methods using mutant strains derived from glutamate-producing microorganisms of the genus Brevibacterium or Corynebacterium, and methods using amino acid-producing strains with improved growth through cell fusion have been reported (Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] U.S. Patent No. 8,034,602 [Patent Document 2] U.S. Patent No. 7,662,943 [Patent Document 3] U.S. Patent No. 10,584,338 [Patent Document 4] U.S. Patent No. 10,273,491 [Patent Document 5] Korean Patent Publication No. 10-2020-0136813 [Non-patent literature]

[0004] [Non-Patent Document 1] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 [Non-licensed document 2] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-licensed document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

Non-licensed Document 17

Non-licensed Document 18

[0005] The problem to be solved by the present application is to provide a recombinant Corynebacterium microorganism capable of producing L-arginine, and a method for producing L-arginine using the same and uses thereof. [Means for solving the problem]

[0006] The present application aims to provide a recombinant microorganism of the genus Corynebacterium that produces L-arginine and has an attenuated activity of a protein comprising the amino acid sequence of SEQ ID NO: 1.

[0007] Another object of the present application is to provide a method for producing L-arginine, which includes a step of culturing in a medium a recombinant Corynebacterium microorganism in which the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 is attenuated. [Effects of the Invention]

[0008] A Corynebacterium microorganism in which the protein comprising the amino acid sequence of SEQ ID NO: 1 of the present application has been attenuated is capable of producing L-arginine at a high yield, and is therefore useful for industrial production. DETAILED DESCRIPTION OF THE INVENTION

[0009] These will be described in detail below. Note that each description and embodiment disclosed in this application also applies to other descriptions and embodiments. In other words, all combinations of various elements disclosed in this application are included in this application. Furthermore, this application is not limited to the specific descriptions below. Furthermore, many papers and patent documents are referenced throughout this specification, and citations thereof are provided. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety, thereby more clearly explaining the state of the art to which the present invention pertains and the content of the present invention.

[0010] One aspect of the present application provides a recombinant microorganism of the genus Corynebacterium in which the activity of a protein comprising the amino acid sequence of SEQ ID NO:1 is attenuated.

[0011] The protein of the present application may have the amino acid sequence shown in SEQ ID NO: 1, may contain the amino acid sequence, may consist of the amino acid sequence, or may essentially consist of the amino acid sequence.

[0012] In the present application, a protein comprising the amino acid sequence of SEQ ID NO: 1 is an endogenous protein of the microorganism of the present application, but is not limited thereto.

[0013] The amino acid sequence of SEQ ID NO: 1 can be obtained from the publicly known database, NIH GenBank. In the present application, the amino acid sequence of SEQ ID NO: 1 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 to the amino acid sequence represented by SEQ ID NO: 1. Needless to say, the present application also includes proteins having an amino acid sequence with such homology or identity, in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added, as long as the amino acid sequence has an effect equivalent to that of the protein of the present application.

[0014] For example, the amino acid sequence may have additions or deletions of sequences at the N-terminus, C-terminus and / or internally that do not change the function of the protein of the present application, naturally occurring mutations, silent mutations or conservative substitutions.

[0015] As used herein, the term "conservative substitution" refers to the substitution of an amino acid with another amino acid having similar structural and / or chemical properties. The protein may, for example, have at least one conservative substitution while still retaining at least one biological activity. Such amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues. For example, among amino acids having electrically charged side chains, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; among amino acids having uncharged side chains, nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; polar or hydrophilic amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and among the above amino acids, aromatic amino acids include phenylalanine, tryptophan, and tyrosine.

[0016] As used herein, "homology" or "identity" refers to the degree to which two given amino acid or nucleic acid base sequences are similar, expressed as a percentage. Homology and identity are often used interchangeably.

[0017] Sequence homology or identity of conserved polynucleotides or proteins can be determined by standard sequence algorithms, optionally 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 moderately or highly stringent conditions. Hybridization, of course, also includes hybridization to polynucleotides with common codons or codons that take codon degeneracy into account.

[0018] Whether any two polynucleotide or protein sequences have homology, similarity, or identity can be determined using known computer algorithms, such as the "FASTA" program, with default parameters, as described in, for example, Non-Patent Document 1. Alternatively, the Needleman-Wunsch algorithm (Non-Patent Document 3) can be used, as implemented in the Needleman program (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later) of the EMBOSS package (which includes the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, and FASTA (Non-Patent Documents 5, 6, and 7)). For example, BLAST or Clustal W from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0019] Homology, similarity, or identity of polynucleotides or proteins can be determined by comparing sequence information using a GAP computer program such as that disclosed in Non-Patent Document 3, as disclosed in Non-Patent Document 8. 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, non-identity a value of 0) and a weighted comparison matrix (or EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 9; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0020] In the present application, a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 1 is also referred to as an NCgl1469 gene.

[0021] 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 refers to a DNA or RNA chain longer than a certain length, and more specifically refers to a polynucleotide fragment that encodes the protein.

[0022] A polynucleotide encoding a protein of the present application may comprise a nucleic acid base sequence encoding the amino acid sequence represented by SEQ ID NO: 1. As an example of the present application, a polynucleotide of the present application may have or comprise the nucleic acid base sequence of SEQ ID NO: 2. Furthermore, a polynucleotide of the present application may consist of the nucleic acid base sequence of SEQ ID NO: 2, or may consist essentially of the nucleic acid base sequence of SEQ ID NO: 2.

[0023] The polynucleotide of the present application can be modified in various ways in the coding region thereof, taking into consideration codon degeneracy or preferred codons in the organism in which the protein of the present application is to be expressed, as long as the amino acid sequence of the protein of the present application is not changed. Specifically, the polynucleotide of the present application has a nucleic acid base sequence that is 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% identical to the nucleic acid base sequence of SEQ ID NO: 2, or comprises the nucleic acid base sequence, or consists of a nucleic acid base sequence that is 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% identical to the sequence of SEQ ID NO: 2, or is essentially composed of the nucleic acid base sequence, but is not limited thereto.

[0024] 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 sequence complementary to all or part of the polynucleotide sequence of the present application. 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 11 and 12). For example, conditions include those under which polynucleotides with high homology or identity, such as polynucleotides with a homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, hybridize with each other, but polynucleotides with lower homology or identity do not hybridize with each other; or conditions including washing once, specifically two to three times, at a salt concentration and temperature equivalent to those used in conventional Southern hybridization, namely, 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.

[0025] Hybridization requires that two nucleic acids have complementary sequences, even if 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 not only substantially similar nucleic acid base sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0026] 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 can be appropriately adjusted by those skilled in the art depending on the purpose.

[0027] 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 11).

[0028] The term "microorganism (or strain)" as used herein includes all wild-type microorganisms and naturally or artificially genetically modified microorganisms, and refers to microorganisms in which a specific mechanism has been weakened or strengthened by inserting an exogenous gene or by enhancing or inactivating the activity of an endogenous gene, and which have been genetically modified to produce a desired polypeptide, protein, or product.

[0029] The microorganism of the present application may be a microorganism in which a protein comprising the amino acid sequence of SEQ ID NO: 1 of the present application is attenuated, or a microorganism in which the polynucleotide encoding the protein is deleted, or a microorganism (e.g., a recombinant microorganism) that has been genetically modified with a vector so that the protein comprising the amino acid sequence of SEQ ID NO: 1 of the present application is attenuated or the polynucleotide encoding the protein is deleted, but is not limited to these.

[0030] The microorganism of the present application may be a microorganism capable of producing L-arginine.

[0031] The microorganism of the present application may be a microorganism having improved L-arginine-producing ability compared to the parent strain.

[0032] The microorganism of the present application is a microorganism to which L-arginine-producing ability has been imparted by attenuating a protein comprising the amino acid sequence of SEQ ID NO: 1 of the present application or by deleting a polynucleotide encoding the same from a parent strain (e.g., a parent strain that naturally has L-arginine-producing ability or does not have L-arginine-producing ability), but is not limited thereto.

[0033] As an example, the recombinant microorganism of the present application is a strain or microorganism that has been transformed with a vector so that a protein comprising the amino acid sequence of SEQ ID NO: 1 of the present application is attenuated or the polynucleotide encoding it is deleted, and thus the protein comprising the amino acid sequence of SEQ ID NO: 1 of the present application is attenuated or the polynucleotide encoding it is deleted, and is a naturally occurring wild-type microorganism or a microorganism that produces L-arginine, in which the protein comprising the amino acid sequence of SEQ ID NO: 1 of the present application is attenuated or the polynucleotide encoding it is deleted, thereby improving the L-arginine production ability compared to naturally occurring wild-type microorganisms or microorganisms in which the protein comprising the amino acid sequence of SEQ ID NO: 1 of the present application or the polynucleotide encoding it has not been modified, but is not limited to these.

[0034] For example, the unmodified microorganism or parent strain, which is the subject strain to be compared for whether the L-arginine production ability is improved, is the ATCC13869 strain, Corynebacterium glutamicum KCCM10741P (Non-Patent Document 13), or a strain in which the argR gene has been deleted in wild-type Corynebacterium glutamicum ATCC13869 (e.g., Corynebacterium glutamicum CJ1R), but is not limited to these.

[0035] For example, the recombinant strain with improved production ability has an L-arginine production ability that is improved 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, or about 14% or more (there is no particular upper limit, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 40% or less, about 30% or less, or about 25% or less), compared to the L-arginine production ability of the parent strain or unmodified microorganism before mutation. However, any strain with improved production ability may be used as long as it has an increase in the + value compared to the production ability of the parent strain or unmodified microorganism before mutation. In other examples, the recombinant strain with improved L-arginine production ability has an improved L-arginine production ability of at least about 1.1 times, at least about 1.12 times, at least about 1.13 times, or at least about 1.14 times (the upper limit is not particularly limited, for example, at most about 10 times, at most about 5 times, at most about 3 times, or at most about 2 times) compared to the parent strain or unmodified microorganism before mutation, but is not limited thereto. The term "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and may be any number in a range that is equal to or similar to the number following the term "about," but is not limited thereto.

[0036] In the present application, the term "unmodified microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, but refers to a wild-type strain or a naturally occurring strain itself, or a strain before its traits have been changed due to genetic mutation caused by natural or artificial factors. For example, the unmodified microorganism refers to a microorganism before a protein comprising the amino acid sequence of SEQ ID NO: 1 described herein has been attenuated, or before a polynucleotide encoding the protein has been deleted. The term "unmodified microorganism" is also used interchangeably with "strain before modification," "microorganism before modification," "non-mutated strain," "non-modified strain," "non-mutated microorganism," or "reference microorganism."

[0037] In other examples of the present application, the Corynebacterium microorganism of the present application may be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens.

[0038] Specifically, the recombinant microorganism of the present application may be a microorganism in which all or part of the polynucleotide encoding the protein consisting of the amino acid sequence of SEQ ID NO:1 is deleted.

[0039] Furthermore, the microorganism of the present application may be a microorganism in which the activity of arginine repressor (ArgR) has been further weakened. Specifically, the microorganism of the present application may be a microorganism in which the argR gene has been further deleted in whole or in part.

[0040] The ArgR may comprise a polypeptide represented by the amino acid sequence of SEQ ID NO: 13. The argR gene may comprise a polynucleotide represented by the nucleic acid base sequence of SEQ ID NO: 14, but is not limited thereto.

[0041] For example, the microorganism of the present application may be a strain of Corynebacterium glutamicum KCCM10741P (Non-Patent Document 13) in which the NCgl1469 gene has been deleted, or a strain of Corynebacterium glutamicum CJ1R in which the NCgl1469 gene has been deleted.

[0042] The microorganism of the present application may also contain ArgF (Ornithine carbamoyltransferase subunit F), a polynucleotide encoding the ArgF, or the argF gene. The ArgF of the present application may consist of the amino acid sequence shown in SEQ ID NO: 15. The argF gene of the present application may also consist of the nucleic acid base sequence shown in SEQ ID NO: 16.

[0043] In this application, the term "attenuation" of a protein refers to a reduction in activity compared to the endogenous activity or the absence of activity. The term "attenuation" is also used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation.

[0044] The attenuation includes at least one of the following: a reduction or elimination of the activity of the protein itself compared to the activity of the protein originally possessed by the microorganism due to, for example, a mutation in the polynucleotide encoding the protein; a reduction in the overall level and / or concentration (expression level) of the protein in cells compared to the native strain due to, for example, inhibition of gene expression of the encoding polynucleotide or inhibition of translation into protein; complete absence of expression of the polynucleotide; and absence of protein activity even if the polynucleotide is expressed. The term "endogenous activity" refers to the activity of a specific protein originally possessed by a parent strain, wild-type, or unmodified microorganism before the transformation, when a trait is changed due to genetic mutation caused by natural or artificial factors. This term is used interchangeably with "activity before transformation." "Inactivation," "deficiency," "reduction," "down-regulation," "reduction," or "attenuation" of a protein activity compared to the endogenous activity refers to a reduction in the activity of a specific protein compared to the activity originally possessed by a parent strain or unmodified microorganism before the transformation.

[0045] Such attenuation of protein activity can be achieved by applying various methods well known in the art, including, but not limited to, those described above (for example, Non-Patent Documents 14 and 15).

[0046] Specifically, the attenuation of a protein in the present application can be achieved by 1) deleting all or part of a gene encoding the protein, 2) modifying an expression regulatory region (or expression regulatory sequence) so as to reduce the expression of a gene encoding the protein, 3) modifying the amino acid sequence constituting the protein so as to delete or weaken the activity of the protein (for example, deleting / substituting / adding one or more amino acids in the amino acid sequence), or 4) modifying the gene sequence encoding the protein so as to delete or weaken the activity of the protein (for example, modifying the nucleic acid of the protein gene so as to encode a protein modified so as to delete or weaken the activity of the protein). The modification can be carried out by, but is not limited to, any of the following: (1) deleting / substituting / adding one or more nucleic acid bases in the nucleic acid sequence; (2) modifying the nucleic acid sequence encoding the start codon or 5'UTR region of the gene transcript encoding the protein; (3) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcript encoding the protein; (4) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding the protein so that a secondary structure that prevents ribosome attachment is formed; (5) adding a promoter to the 3' end of the ORF (open reading frame) of the gene sequence encoding the protein so that it can be reverse-transcribed (reverse transcription engineering, RTE); or (6) combining two or more of the above 1) to 8).

[0047] For example, 1) deleting a part or all of the gene encoding the protein may be carried out by deleting the entire polynucleotide encoding the endogenous target protein in the chromosome, or by substituting a polynucleotide with a partial deletion of nucleotides or a marker gene.

[0048] Furthermore, the modification of the expression regulatory region (or expression regulatory sequence) described above in 2) may be carried out 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 lower 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.

[0049] Furthermore, the nucleic acid sequence encoding the start codon or 5'UTR region of the gene transcript encoding the protein (3) can be modified, for example, by substituting it with a nucleic acid sequence encoding another start codon that has a lower protein expression rate than the endogenous start codon, but is not limited to this.

[0050] Furthermore, modifying the amino acid sequence or polynucleotide sequence of 4) and 5) 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 protein or the polynucleotide sequence encoding the protein so as to attenuate the activity of the protein, or by substituting an amino acid sequence or polynucleotide sequence that has been improved to have lower activity or to eliminate activity. For example, gene expression can be inhibited or attenuated by, but is not limited to, introducing a mutation into a polynucleotide sequence to form a stop codon.

[0051] 6) Introduction of an antisense oligonucleotide (for example, antisense RNA) that binds complementarily to the gene transcription product encoding the protein may be carried out, for example, as described in Non-Patent Document 16.

[0052] 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a protein so that a secondary structure that prevents ribosome attachment is formed may be achieved by disabling or slowing down mRNA translation.

[0053] 8) Adding a promoter to the 3' end of the ORF (open reading frame) of a gene sequence encoding a protein so as to reverse transcribe it (reverse transcription engineering, RTE) may be carried out by creating an antisense nucleotide complementary to the gene transcript encoding the protein, thereby attenuating its activity.

[0054] In the present application, "enhancing" a protein activity means improving the activity of the protein compared to its endogenous activity. The term "enhancing" is used interchangeably with "activation," "up-regulation," "overexpression," and "increase." Here, activation, enhancement, up-regulation, overexpression, and enhancement all encompass the development of an activity not originally present, as well as an improvement in activity compared to endogenous activity or activity before modification. The term "endogenous activity" refers to the activity of a specific protein originally possessed by a parent strain or unmodified microorganism prior to the phenotypic change, when a trait is altered through genetic mutation due to natural or artificial factors. This term is also used interchangeably with "activity before modification." "Enhancing," "up-regulating," "overexpressing," or "improving" a protein activity compared to its endogenous activity means an improvement in the activity and / or concentration (expression level) of a specific protein originally possessed by a parent strain or unmodified microorganism prior to the phenotypic change.

[0055] The enhancement may be achieved by introducing a foreign protein, or by enhancing the activity and / or increasing the concentration (expression level) of an endogenous protein. Whether the activity of the protein has been enhanced can be confirmed by an increase in the level of activity, expression level, or amount of a product produced from the protein.

[0056] Various methods well known in the art can be applied to enhance the activity of the protein, and any method can be used as long as it can enhance the activity of the target protein compared to the activity of the microorganism before modification. Specifically, these methods include, but are not limited to, conventional methods in molecular biology that use genetic engineering and / or protein engineering well known to those skilled in the art (e.g., Non-Patent Documents 15 and 17).

[0057] Specifically, the enhancement of a protein in the present application is carried out by, but is not limited to, 1) increasing the intracellular copy number of a polynucleotide encoding the protein, 2) replacing an expression regulatory region of a gene on a chromosome that encodes the protein with a sequence with stronger activity, 3) modifying the nucleic acid base sequence encoding the start codon or 5'UTR region of a transcript of a gene encoding the protein, 4) modifying the amino acid sequence of the protein so that the activity of the protein is enhanced, 5) modifying the polynucleotide sequence encoding the protein so that the activity of the protein is enhanced (for example, modifying the polynucleotide sequence of the protein gene so that the protein is modified to encode a protein whose activity is enhanced), 6) introducing a foreign protein that exhibits the activity of the protein or a foreign polynucleotide encoding it, 7) optimizing the codons of the polynucleotide encoding the protein, 8) analyzing the tertiary structure of the protein and selecting and modifying or chemically modifying exposed portions, or 9) a combination of two or more selected from 1) to 8) above.

[0058] More specifically, the 1) increase in the intracellular copy number of a polynucleotide encoding a protein may be achieved by introducing into a host cell a vector to which a polynucleotide encoding the protein is operably linked, the vector replicating and functioning independently of the host. Alternatively, the increase may be achieved by introducing one or more copies of the polynucleotide encoding the protein into a chromosome in the host cell. The introduction into a chromosome may be achieved by, but is not limited to, introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome in the host cell. The vector is as described above.

[0059] The 2) replacement of a gene expression regulatory region (or expression regulatory sequence) on a chromosome encoding a protein with a sequence with stronger activity can be achieved, for example, by generating a mutation in the sequence through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by replacing the expression regulatory region with a sequence with higher activity, so as to further enhance the activity of the expression regulatory region. The expression regulatory region includes, but is not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, sequences regulating the termination of transcription and translation, and the like. For example, this can be achieved by replacing the original promoter with a strong promoter, but is not limited to this.

[0060] Examples of known strong promoters include, but are not limited to, the CJ1 to CJ7 promoters (Patent Document 2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (Patent Document 3), the O2 promoter (Patent Document 4), the tkt promoter, and the yccA promoter.

[0061] The nucleic acid sequence encoding the start codon or 5'UTR region of the gene transcript encoding the protein (3) can be modified, for example, by substituting it with a nucleic acid sequence encoding another start codon that has a higher protein expression rate than the endogenous start codon, but is not limited to this.

[0062] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) can be achieved by, but is not limited to, generating a mutation in the amino acid sequence of the protein or the polynucleotide sequence encoding the protein through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to enhance the activity of the protein, or by substituting an amino acid sequence or polynucleotide sequence improved to have higher activity or improved activity. Specifically, the substitution can be achieved by, but is not limited to, inserting the polynucleotide into a chromosome through homologous recombination. The vector used here may further include a selection marker for confirming whether or not it has been inserted into the chromosome. The selection marker is as described above.

[0063] The introduction of a foreign polynucleotide that exhibits the activity of a protein (6) may be carried out by introducing into a host cell a foreign polynucleotide that encodes a protein that exhibits the same or similar activity as the protein. The foreign polynucleotide may be of any origin or sequence, as long as it exhibits the same or similar activity as the protein. The introduction can be carried out by a person skilled in the art using a known transformation method appropriately selected, and the introduced polynucleotide is expressed in the host cell as described above, thereby producing the protein and improving its activity.

[0064] 7) Optimizing the codons of a polynucleotide encoding a protein may be carried out by optimizing the codons of an endogenous polynucleotide so that transcription or translation is increased in the host cell, or by optimizing the codons of an exogenous polynucleotide so that optimized transcription and translation are achieved in the host cell.

[0065] 8) Analyzing the tertiary structure of a protein and selecting and altering or chemically modifying exposed portions may be carried out, for example, by comparing the sequence information of the protein to be analyzed with a database storing sequence information of known proteins, determining candidate template proteins based on the degree of sequence similarity, confirming the structure based on the candidate template proteins, and selecting and altering or modifying exposed portions to be altered or chemically modified.

[0066] Such enhancement of protein activity can be achieved by, but is not limited to, improving the activity, concentration, or expression level of the corresponding protein compared to the activity or concentration of the protein expressed in a wild-type or unmodified microbial strain, or by increasing the amount of a product produced from the protein.

[0067] In this application, the term "vector" refers to a DNA product comprising a nucleic acid base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable the target polypeptide to be expressed in a suitable host. The expression control region includes a promoter that initiates transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence that regulates the termination of transcription and translation. When transformed into a suitable host cell, the vector can replicate and function independently of the host genome and is integrated into the genome itself.

[0068] 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 naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage or cosmid vectors. Examples of plasmid vectors that may be used include pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors may be used.

[0069] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using a vector for intracellular chromosomal introduction. The insertion of the polynucleotide into a chromosome can be achieved by any method known in the art, including, but not limited to, homologous recombination. A selection marker for determining whether or not the polynucleotide has been inserted into the chromosome may be further included. The selection marker is used to select cells transformed with the vector, i.e., to determine whether or not the target nucleic acid molecule has been inserted. Markers that confer selectable phenotypes, such as drug resistance, auxotrophy, resistance to cytotoxic agents, and expression of surface polypeptides, are used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit a different phenotype, allowing the selection of transformed cells.

[0070] In the present application, "transformation" refers to introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism, thereby expressing the polypeptide encoded by the polynucleotide in the host cell. The transformed polynucleotide may be any polynucleotide that can be expressed in the host cell, regardless of whether it is located intrachromosomally or extrachromosomally. The polynucleotide may also include DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form that can be expressed in the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct 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 also be introduced into the host cell in its own form and operably linked to sequences necessary for expression in the host cell, but is not limited thereto.

[0071] Furthermore, 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 protein of the present application.

[0072] In the microorganisms of the present application, partial or complete modification of a polynucleotide can be induced by, but is not limited to, (a) homologous recombination using a vector for chromosomal introduction into the microorganism, or genome editing using engineered nucleases (e.g., CRISPR-Cas9), and / or (b) light and / or chemical treatment, such as ultraviolet light or radiation. Methods for partially or completely modifying the gene include methods using DNA recombination techniques. For example, partial or complete deletion of a gene can be achieved by introducing a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism and causing homologous recombination. The introduced nucleotide sequence or vector may contain, but is not limited to, a dominant selection marker.

[0073] Another aspect of the present application provides a method for producing L-arginine, comprising the step of culturing in a medium a recombinant Corynebacterium microorganism in which the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 is attenuated.

[0074] The proteins, attenuations, microorganisms, etc. containing the amino acid sequence of SEQ ID NO: 1 are as described above.

[0075] The Corynebacterium microorganism is Corynebacterium glutamicum, but is not limited thereto.

[0076] The microorganism may further have weakened arginine repressor (ArgR) activity or may further have a deleted argR gene, but is not limited thereto, as described above.

[0077] The term "culturing" in the present application means growing the Corynebacterium microorganism of the present application under appropriately adjusted environmental conditions. The culturing process of the present application can be carried out using a suitable medium and culture conditions known in the art. Those skilled in the art can easily adjust such a culturing process depending on the selected strain. Specifically, the culturing may be batch, continuous, and / or fed-batch culture, but is not limited thereto.

[0078] The term "culture medium" as used herein refers to a mixture of nutrients necessary for culturing the microorganisms of the genus Corynebacterium of the present application, primarily as ingredients, and provides nutrients such as water essential for survival and growth, growth factors, etc. Specifically, the medium and other culture conditions used for culturing the microorganisms of the genus Corynebacterium of the present application may be any medium used for culturing conventional microorganisms, and the microorganisms of the genus Corynebacterium of the present application can be cultured in a conventional medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins under aerobic conditions by adjusting the temperature, pH, etc.

[0079] Specifically, a culture medium for Corynebacterium microorganisms is disclosed in Non-Patent Document 18.

[0080] Examples of carbon sources used in the present application include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid can also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) can be used. Any other carbon source can also be used in an appropriate amount. These carbon sources can be used alone or in combination of two or more, but are not limited to these.

[0081] Examples of the nitrogen source that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate, and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its degradation products, and defatted soybean cake or its degradation products. These nitrogen sources can be used alone or in combination of two or more, but are not limited to these.

[0082] Examples of the phosphorus source include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and their corresponding sodium-containing salts. Examples of inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate. Other examples include amino acids, vitamins, and / or suitable precursors. These components or precursors can be added to the medium in a batch or continuous manner. However, the present invention is not limited to these.

[0083] Furthermore, during the cultivation of the Corynebacterium microorganism of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid may be added to the medium by a suitable method to adjust the pH of the medium. Furthermore, during cultivation, foam formation may be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, to maintain an aerobic state in the medium, oxygen or an oxygen-containing gas may be injected into the medium, and to maintain an anaerobic or microaerobic state, no gas may be injected, and nitrogen, hydrogen, or carbon dioxide gas may be injected, but this is not limited to these.

[0084] In the culture of the present application, the culture temperature is maintained at 20 to 45°C, specifically 25 to 40°C, and the culture is carried out for about 10 to 160 hours, but is not limited thereto.

[0085] The L-arginine produced by the culture of the present application is either secreted into the medium or remains within the cells.

[0086] The method for producing L-arginine of the present application may further include a step of preparing the Corynebacterium microorganism of the present application, a step of preparing a medium for culturing the microorganism, or a combination thereof (in any order), for example, before the culturing step.

[0087] The method for producing L-arginine of the present application may further include a step of recovering L-arginine from the culture medium (the medium in which the culture was carried out) or the Corynebacterium microorganism in the culture medium used for the culture. The recovery step may be further included after the culture step.

[0088] The recovery may involve 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, the target L-arginine can be collected from the medium or the microorganism using a suitable method known in the art, such as centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination thereof.

[0089] 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 may be performed continuously or discontinuously regardless of the order, or may be performed simultaneously or integrated into one step, but is not limited thereto.

[0090] Yet another aspect of the present application provides a composition for producing L-arginine, comprising a Corynebacterium microorganism in which the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 of the present application is attenuated, a culture medium in which the microorganism is cultured, or a combination thereof.

[0091] The composition of the present application may further contain any suitable excipient commonly used in compositions for producing amino acids, including, but not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, isotonicity agents, etc.

[0092] In the composition of the present application, the protein containing the amino acid sequence of SEQ ID NO: 1, the attenuation, the microorganism, the culture, the medium, etc. are as described above.

[0093] Yet another aspect of the present application provides a method for producing a Corynebacterium microorganism, the method comprising the step of attenuating the activity of a protein comprising the amino acid sequence of SEQ ID NO:1.

[0094] Yet another aspect of the present application provides use of a Corynebacterium microorganism having attenuated activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 for producing L-arginine.

[0095] The protein, attenuation, microorganism, etc. comprising the amino acid sequence of SEQ ID NO: 1 in the method for producing the Corynebacterium microorganism and use of the Corynebacterium microorganism for L-arginine production are as described above. [Example]

[0096] 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 well understood and easily implemented by skilled artisans in the technical field of the present application or a similar technical field. [Example]

[0097] Construction and screening of transposon-based random mutation libraries To obtain a strain with improved L-arginine production, Corynebacterium glutamicum KCCM10741P (Patent Document 1) was used as the parent strain, and EZ-Tn5 TM <R6Kγori / KAN-2> Tnp Transposome TM The plasmid obtained using a kit (Epicentre) was transformed by the electric pulse method (Non-patent Document 19), and then smeared on a complex plate containing kanamycin (25 mg / l) to obtain approximately 20,000 colonies. <Complex plate medium (pH 7.0)> Glucose 10g, peptone 10g, beef extract 5g, yeast extract 5g, Brain Heart Infusion 18.5g, NaCl 2.5g, urea 2g, sorbitol 91g, agar 20g (in 1 liter of distilled water)

[0098] Using the ninhydrin method (Non-patent Document 20), the top seven mutant strains with improved L-arginine productivity compared to the parent strain, Corynebacterium glutamicum KCCM10741P, were selected. [Example]

[0099] Analysis of L-arginine production ability of selected random mutant strains To finally select a strain that exhibits reproducible improvement in L-arginine production from the seven mutant strains selected in Example 1, flask culture was carried out using the following medium. After completion of the culture, the L-arginine concentration in the culture medium was analyzed using HPLC. The L-arginine production concentration of each mutant strain is shown in Table 1. <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)

[0100] [Table 1]

[0101] From the ten selected mutant strains, KCCM10741P / mt-7 was finally selected as a strain with significantly improved L-arginine productivity. [Example]

[0102] Elucidation of the cause of improved L-arginine productivity in the final selected strain In KCCM10741P / mt-7 of Example 2, the genes that were inactivated by random insertion of transposons were identified.

[0103] Specifically, the genomic DNA of KCCM10741P / mt-7 was extracted, digested, ligated, and transformed into E. coli DH5α, which was then spread onto LB solid medium containing kanamycin (25 mg / L). Twenty transformed colonies were selected, and a plasmid containing a portion of the unknown gene was obtained, which was then cloned into EZ-Tn5. TM <R6Kγori / KAN-2> Tnp Transposome TM The nucleic acid sequence was analyzed using primer 1 (SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4) of the kit. As a result, it was confirmed that the gene containing the nucleotide sequence of SEQ ID NO: 2 was inactivated based on the nucleic acid sequence reported in the National Institutes of Health Genbank.

[0104] [Table 2]

[0105] A blast search of SEQ ID NO: 2 in wild-type Corynebacterium glutamicum ATCC13869 confirmed that it was the NCgl1469 gene. [Example]

[0106] Construction of recombinant vectors to delete the NCgl1469 gene To reconfirm the effect of inactivation of the NCgl1469 gene on L-arginine production, a recombinant vector was constructed to delete the NCgl1469 gene on the chromosome of Corynebacterium sp. strains.

[0107] First, to prepare a fragment for deleting the gene, primers 3 to 6 were synthesized, which are shown in Table 3.

[0108] [Table 3]

[0109] To generate a vector with an ORF deletion based on SEQ ID NO:2, PCR was performed using the primer pair SEQ ID NO:5 and SEQ ID NO:6 and the primer pair SEQ ID NO:7 and SEQ ID NO:8 with wild-type Corynebacterium glutamicum ATCC 13869 gDNA as template. The PCR conditions were denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds, followed by polymerization at 72°C for 5 minutes. Using the mixture of the two fragments thus obtained as template, overlapping PCR was again performed using the primer pair SEQ ID NO:5 and SEQ ID NO:8 to obtain a fragment. The PCR was performed with denaturation at 94°C for 5 minutes, followed by 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, followed by polymerization at 72°C for 5 minutes. The pDCM2 vector (Patent Document 5) was treated with smaI, and the resulting PCR product was subjected to fusion cloning as described above. Fusion cloning was performed using the In-Fusion® HD Cloning Kit (Clontech). The resulting plasmid was designated pDCM2-ΔNCgl1469. [Example]

[0110] Construction of a strain lacking the NCgl1469 gene and evaluation of its L-arginine production ability - 1 pDCM2-△NCgl1469 was used to transform the L-arginine-producing strain Corynebacterium glutamicum KCCM10741P by homologous recombination on the chromosome (Non-patent Document 13).

[0111] After that, secondary recombination was carried out on a solid plate medium containing 4% sucrose. After secondary recombination, the transformed strain of Corynebacterium glutamicum was subjected to PCR using primers 3 and 6 to confirm that the gene of SEQ ID NO:2 had been deleted on the chromosome. The recombinant strain was named Corynebacterium glutamicum KCCM10741P-△NCgl1469.

[0112] To analyze the L-arginine producing ability of the Corynebacterium glutamicum KCCM10741P-△NCgl1469 strain prepared as described above, it was cultured together with the parent strain, Corynebacterium glutamicum KCCM10741P, in the following manner.

[0113] The parent strains Corynebacterium glutamicum KCCM10741P and KCCM10741P-△NCgl1469 were inoculated into 25 ml of the following seed medium in a 250 ml corner baffle flask 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 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)> 60g glucose, 45g ammonium sulfate, 2g magnesium sulfate heptahydrate, 2g potassium dihydrogen phosphate, 10g ammonium chloride, 0.01mg biotin, 0.1mg thiamine HCl, 2mg calcium pantothenate, 3mg nicotinamide, 10mg ferrous sulfate, 10mg manganese sulfate, 0.02mg zinc sulfate, 0.5mg copper sulfate, 30g calcium carbonate (in 1 liter of distilled water).

[0114] After the cultivation was completed, the L-arginine productivity was measured by HPLC (Waters 2478), and the results are shown in Table 4.

[0115] [Table 4]

[0116] As a result, it was confirmed that KCCM10741P-β had an average 19.8% improvement in L-arginine productivity compared to the parent strain. [Example]

[0117] Construction of a strain lacking the NCgl1469 gene and evaluation of its L-arginine production ability - 2 It was also confirmed whether the same effect as in Example 5 was observed in other Corynebacterium glutamicum strains that produce L-arginine.

[0118] Specifically, a single mutation (argR gene deletion, hereafter referred to as △argR) was introduced into a wild-type Corynebacterium glutamicum strain (ATCC13869) to create an L-arginine-producing strain.

[0119] The recombinant vector for ΔargR was prepared in the same manner as in Example 4. The primers used for vector preparation are shown in Table 5.

[0120] [Table 5]

[0121] A plasmid into which the target gene had been inserted was selected by PCR. This plasmid was designated pDCM2-ΔargR. Corynebacterium glutamicum wild-type strain ATCC13869 was transformed with pDCM2-ΔargR in the same manner as in Example 5. The transformed strain was subjected to PCR using primers 7 and 10 to identify a strain lacking argR on the chromosome, which was designated Corynebacterium glutamicum CJ1R. A strain lacking the NCgl1469 gene was constructed from Corynebacterium glutamicum CJ1R in the same manner as in Example 5, and designated CJ1R-ΔNCgl1469.

[0122] The CJ1R-ΔNCgl1469 strain was cultured in the same manner as in Example 5, and after the culture was completed, the L-arginine productivity was measured by HPLC (Waters 2478). The results are shown in Table 6.

[0123] [Table 6]

[0124] As a result, it was confirmed that CJ1R-β had an average 19.7% improvement in L-arginine productivity compared to the parent strain CJ1R.

[0125] From the above description, those 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 the technical idea or essential features thereof. It should be understood that the above examples are merely illustrative and not limiting. The present application should be construed as including all modifications and variations derived from the meaning and scope of the claims, rather than the specification, and their equivalent concepts.

Claims

1. A microorganism of the genus Corynebacterium that produces L-arginine, in which the activity of a protein consisting of the amino acid sequence of SEQ ID NO: 1 is attenuated, wherein the microorganism has improved L-arginine-producing ability compared to a parent strain that contains the protein consisting of the amino acid sequence of SEQ ID NO: 1 but in which the activity of the protein is not attenuated.

2. The microorganism of claim 1 , wherein the microorganism is Corynebacterium glutamicum.

3. The microorganism according to claim 1, further comprising an attenuated arginine repressor activity.

4. The microorganism according to claim 1, wherein the polynucleotide encoding the protein consisting of the amino acid sequence of SEQ ID NO: 1 is deleted.

5. A method for producing L-arginine, comprising the step of culturing a microorganism described in any one of claims 1 to 4 in a culture medium.

6. The method of claim 5, wherein the microorganism is Corynebacterium glutamicum.

7. The method according to claim 5 , wherein the Corynebacterium microorganism further has an attenuated arginine repressor.

8. The method according to claim 5, wherein the microorganism lacks a nucleic acid base sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO:

1.

9. Use of the microorganism according to any one of claims 1 to 4 for producing L-arginine.

Images