RU2837670C2 - Microorganism of genus corynebacterium, producing l-arginine, and method of producing l-arginine using said microorganism - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is use of a recombinant microorganism of the genus Corynebacterium, in which a polynucleotide coding a protein comprising the amino acid sequence SEQ ID NO: 1 is removed to obtain L-arginine. Also disclosed is a method of producing L-arginine, involving cultivation in a medium of a recombinant microorganism of the genus Corynebacterium, in which a polynucleotide coding a protein comprising the amino acid sequence SEQ ID NO: 1 is removed.

EFFECT: invention provides production of L-arginine with high output.

7 cl, 6 tbl, 6 ex

Description

Field of technology

The present invention relates to a microorganism producing L-arginine, in which the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 is weakened, and to a method for producing L-arginine.

Prior art

L-arginine is used in medicines such as liver function promoter, brain function promoter and amino acid complex preparation, and has recently attracted attention in foods such as fish pie additive, health drink additive and salt substitute for hypertension patients. Research on the use of microorganisms to produce industrially applicable arginine in high concentrations has been continuously carried out, and reports have been made on methods of using mutant strains obtained from microorganisms of the genus Brevibacterium or Corynebacterium, which are glutamate-producing strains, and methods of using amino acid-producing strains with improved growth by cell fusion, and the like (US 8034602 B2).

Technical task

In one aspect of the present invention, there is provided a recombinant microorganism of the genus Corynebacterium producing L-arginine, a method for producing L-arginine using it, and a use thereof.

Technical solution

According to one aspect of the present invention, there is provided a recombinant microorganism of the genus Corynebacterium producing L-arginine, in which the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 is weakened.

According to another aspect of the present invention, a method for producing L-arginine is provided, comprising culturing a recombinant microorganism of the genus Corynebacterium in a medium in which the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 is weakened.

Beneficial effects

A microorganism of the genus Corynebacterium in which the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 is weakened according to the present invention can produce L-arginine in high yield and thus can be advantageously used for industrial production.

Detailed description of the invention

The present invention will be specifically described as follows. Each description and embodiment disclosed in the present invention can also be used for other descriptions and embodiments. That is, all combinations of various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention is not limited to the specific description below. In addition, references to and citations from many articles and patent documents are provided throughout the entire description. The descriptions of the cited articles and patent documents are fully incorporated herein by reference, and the level of the art to which the present invention pertains and the details of the present invention are explained more clearly.

According to one aspect of the present invention, there is provided 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 weakened.

The protein of the present invention may have or include the amino acid sequence of SEQ ID NO: 1, or may consist of or consist essentially of that amino acid sequence.

In the present invention, the protein comprising the amino acid sequence of SEQ ID NO: 1 may be an endogenous protein of the microorganism of the present invention, but is not limited thereto.

The amino acid sequence of SEQ ID NO: 1 can be obtained from the known GeneBank database of the National Institutes of Health of the United States (NIH GenBank). In the present invention, the amino acid sequence of SEQ ID NO: 1 can 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 sequence indicated in SEQ ID NO: 1. It would be obvious that any protein that has an amino acid sequence with a deletion, modification, substitution, conservative substitution or addition in its region can also be included in the range of the present invention as long as the amino acid sequence has the same homology or identity and exhibits an efficiency corresponding to the protein of the present invention.

For example, an amino acid sequence may include an amino acid sequence having an addition or deletion, a naturally occurring mutation, a silent mutation, or a conservative substitution at the N-terminus, C-terminus, and/or within the amino acid sequence that does not alter the function of the protein of the present invention.

As used herein, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid that has similar structural and/or chemical properties. A protein may have, for example, one or more conservative substitutions while retaining one or more biological activities. Such an amino acid substitution can generally be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues. For example, among electrically charged amino acids, positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include glutamic acid and aspartic acid. Among uncharged amino acids, 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. Among the amino acids, aromatic amino acids include phenylalanine, tryptophan, and tyrosine.

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

Sequence homology or identity of conserved polynucleotides or proteins can be determined by standard alignment algorithms and the default gap penalties set by the program used can be shared. Essentially, all homologous or identical sequences or parts thereof can hybridize to each other as usual under moderate or very stringent conditions. It would be obvious that hybridization also includes hybridization to a polynucleotide containing conventional codons or codons taking into account codon degeneracy in the polynucleotide.

Homology, similarity or identity between any two polynucleotide or protein sequences can be determined using a known computer algorithm such as the FASTA program, using default parameters as specified in the literature (Pearson et al., (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444). Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) which is implemented in the Needleman program of the European Molecular Biology Open Source Software Suite (EMBOSS) (Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) (which includes the GCG (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)) program package, BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego,1994 and [CARILLO ETA/.] (1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using the BLAST information database of the National Center for Biotechnology Information or ClustalW.

Homology, similarity, or identity between polynucleotides or proteins can be determined by comparing sequence information using the computer program GAP, e.g., Needleman et al, (1970) J. Mol Biol. 48:443, as learned from Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program defines similarity as the number of aligned characters (i.e., nucleotides or amino acids) that are similar divided by the total number of characters in the shorter of the two sequences. Default parameters for the GAP program may include: (1) a binary comparison matrix (containing the value 1 for identity and 0 for non-identity) and a weighted comparison matrix Gribskov et al. (1986) Nucl. Acids Res. 14:6745, which are described in Schwartz and Dayhoff, eds., "Atlas of Protein Sequence and Structure," National Biomedical Research Foundation, pp.353-358 (1979) (or the EDNAFULL substitution matrix (EMBOSS version of NCBI NUC4.4); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each character in each gap (or a gap-opening penalty of 10 and a gap-extending penalty of 0.5); and (3) no penalty for trailing gaps.

In the present invention, a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 1 may be referred to as the NCgl1469 gene.

As used herein, the term "polynucleotide" refers to a polymer consisting of a nucleotide chain extended lengthwise by covalent bonds of nucleotide monomers, which represents a DNA or RNA chain of a specified length and, more particularly, a fragment of a polynucleotide encoding a protein.

A polynucleotide encoding a protein of the present invention may comprise a nucleic acid base sequence encoding an amino acid sequence set forth in SEQ ID NO: 1. As an example, a polynucleotide of the present invention may have or comprise a nucleic acid base sequence of SEQ ID NO: 2. Alternatively, a polynucleotide of the present invention may consist of or consist essentially of a nucleic acid base sequence of SEQ ID NO: 2.

In the polynucleotide of the present invention, various modifications can be made in the coding region as long as the amino acid sequence of the protein of the present invention does not change to take into account codon degeneracy or codons preferred in the organism that is intended to express the protein of the present invention. Specifically, a polynucleotide of the present invention may have or include a nucleic acid base sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, but less than 100% homology or identity to the nucleic acid sequence of SEQ ID NO: 2, or may consist of or consist essentially of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98%, but less than 100% homology or identity to the nucleic acid base sequence of SEQ ID NO: 2, but not limited to this.

In addition, a polynucleotide of the present invention may include, but is not limited to, a sequence that can hybridize, under stringent conditions, with a probe that can be derived from a known gene sequence, such as a sequence complementary to a portion or all of a polynucleotide sequence of the present invention. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Specifically, these conditions are described in detail in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; and F. M. 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 under which polynucleotides having higher homology or identity, such as polynucleotides having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity, hybridize with each other, but polynucleotides having lower homology or identity do not hybridize with each other; or typical Southern hybridization washing conditions, i.e., washing conditions of one, two, or three times, at a salt concentration and temperature corresponding to 1×SSC, 0.1% SDS (sodium dodecyl sulfate) at 60°C, specifically 0.1×SSC (sodium citrate and chloride solution), 0.1% SDS at 60°C, or, more specifically, 0.1×SSC, 0.1% SDS at 60°C. SSC, 0.1% SDS at 68°C.

Hybridization requires that two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Accordingly, a polynucleotide of the present invention may also include isolated nucleic acid fragments that are complementary to the entire sequence, as well as substantially similar nucleic acid base sequences.

Specifically, polynucleotides having homology or identity with the polynucleotide of the present invention can be detected by applying hybridization conditions including a step of hybridization at a temperature of Tm of 55°C and using the conditions described above. In addition, the value of Tm may be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by a person skilled in the art according to the purpose.

The appropriate degree of stringency for polynucleotide hybridization depends on the length of the polynucleotide and its degree of complementarity, and their variable parameters are well known in the art (e.g., Sambrook et al., supra).

As used herein, the term "microorganism (or strain)" includes all wild-type microorganisms or naturally or artificially genetically modified microorganisms, refers to a microorganism in which a specific mechanism is attenuated or enhanced by the insertion of an exogenous gene or the enhancement or inactivation of the activity of an endogenous gene, and may be a microorganism comprising a target protein or a genetic modification for the production of a protein or product.

The microorganism of the present invention may be a microorganism in which a protein comprising the amino acid sequence of SEQ ID NO: 1 of the present invention is attenuated or a polynucleotide encoding the protein is deleted; or a microorganism that has been genetically modified (e.g., a recombinant microorganism) by a vector to attenuate a protein comprising the amino acid sequence of SEQ ID NO: 1 of the present invention or to delete a polynucleotide encoding the protein, but is not limited thereto.

The microorganism of the present invention may be a microorganism having the ability to produce L-arginine.

The microorganism of the present invention may be a microorganism having an increased ability to produce L-arginine compared to the parent strain.

The microorganism of the present invention may be a microorganism obtained by imparting the ability to produce L-arginine to a parent strain (for example, a parent strain naturally having the ability to produce L-arginine or not having the ability to produce L-arginine) by attenuating a protein comprising the amino acid sequence of SEQ ID NO: 1 of the present invention or deleting a polynucleotide encoding the protein, but not limited thereto.

For example, the recombinant microorganism of the present invention may be a strain or a microorganism in which a protein comprising the amino acid sequence of SEQ ID NO: 1 of the present invention is attenuated or a polynucleotide encoding the protein is deleted, by transformation with a vector, in order to attenuate a protein comprising the amino acid sequence of SEQ ID NO: 1 of the present invention or delete a polynucleotide encoding the protein, wherein a protein comprising the amino acid sequence of SEQ ID NO: 1 is attenuated or a polynucleotide encoding the protein is deleted in a native wild-type microorganism or an L-arginine-producing microorganism, the ability of the microorganism to produce L-arginine is increased compared with a native wild-type microorganism or a microorganism in which a protein comprising the amino acid sequence of SEQ ID NO: 1 or a polynucleotide encoding the protein is not modified, but is not limited thereto.

For example, the unmodified microorganism or parent strain as a comparison strain for comparing the increase in the ability to produce L-arginine may be the strain ATCC13869 or Corynebacterium glutamicum KCCM10741P (van der Rest et al, Appl. MicrobiolBiotechnol 52:541-545, 1999) or a strain in which the argR gene has been deleted from the wild-type Corynebacterium glutamicum ATCC 13869 (e.g., Corynebacterium glutamicum CJ1R), but is not limited to these.

As an example, the recombinant strain with an increased production capacity may have an L-arginine producing capacity that is increased by at least about 1%, in particular by at least about 1%, at least about 2.5%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 10.5%, at least about 11%, at least about 11.5%, at least about 12.5%, at least about 13%, at least about 13.5%, or at least about 14% (the upper limit is not particularly limited, and the upper limit may be, for example, at most 200%, at most about 150%, at most about 100%, at most about 50%, not more than about 40%, not more than about 30%, or not more than about 25%), compared to that of the parent strain before modification or the unmodified microorganism, but the ability to produce L-arginine is not limited thereby as long as it has an increase of a positive value (+) compared to the producing ability of the parent strain before modification or the unmodified microorganism. As another example, a recombinant strain with an increased producing ability may have an ability to produce L-arginine that is increased by 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, and the upper limit may be, for example, no more than about 10 times, no more than about 5 times, no more than about 3 times, or no more than about 2 times) compared to that of the parent strain before modification or the unmodified microorganism, but the ability to produce L-arginine is not limited thereto. As used herein, the term “about” refers to a range encompassing all of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, and the like, and thus encompasses all values in the range equivalent or similar to those stated after that term, but is not limited thereto.

As used herein, the term "unmodified microorganism" may refer to a wild-type or native strain as such, or to a strain prior to transformation by genetic mutation caused by natural or artificial factors, without excluding strains comprising mutations that may occur naturally in microorganisms. For example, an unmodified microorganism may refer to a microorganism prior to attenuation of a protein comprising the amino acid sequence of SEQ ID NO: 1 herein, or deletion of a polynucleotide encoding the protein. "Unmodified microorganism" may be used interchangeably with "pre-modification strain", "pre-modification microorganism", "non-mutated strain", "non-modified strain", "non-mutated microorganism", or "reference microorganism".

As another example of the present invention, the microorganism of the genus Corynebacterium of the present invention 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.

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

In addition, the microorganism of the present invention may be a microorganism in which the activity of the arginine repressor (ArgR) is further weakened. Specifically, the microorganism of the present invention may be a microorganism in which the entire argR gene or a part thereof is further deleted.

ArgR may include a polypeptide specified in the amino acid sequence of SEQ ID NO: 13. The argR gene may include a polynucleotide specified in the nucleic acid base sequence of SEQ ID NO: 14, but is not limited to it.

As an example, the microorganism of the present invention may be a strain in which the NCgl1469 gene is deleted in Corynebacterium glutamicum KSCM10741P (van der Rest et al, Appl. Microbiol Biotechnol 52:541-545, 1999), or a strain in which the NCgl1469 gene is deleted in Corynebacterium glutamicum CJ1R.

In addition, the microorganism of the present invention may comprise a subunit F of ornithine carbamoyltransferase (ArgF), a polynucleotide encoding ArgF, or an argF gene. ArgF of the present invention may consist of the amino acid sequence indicated in SEQ ID NO: 15. The argF gene of the present invention may consist of the nucleic acid base sequence indicated in SEQ ID NO: 16.

As used herein, the term "attenuation" of a protein has a concept that encompasses all decreases in activity or absence of activity relative to the protein's intrinsic activity. Attenuation may be used interchangeably with inactivation, deficiency, down-regulation, reduction, reduction, or the like.

Attenuation may also include: a case where the activity of the protein itself is reduced or eliminated compared to the activity of the protein possessed by the original microorganism due to a mutation or the like in the polynucleotide encoding the protein; a case where the overall activity and/or concentration (expression level) of the protein in the cell is reduced compared to the native strain due to inhibition of the expression of the gene of the polynucleotide encoding the protein; or inhibition of translation into the protein; a case where expression of the polynucleotide is not achieved; and/or a case where the protein has no activity despite the expression of the polynucleotide. The term "intrinsic activity" refers to the activity of a particular protein originally possessed by the parent strain before modification, or to the activity of a wild-type or unmodified microorganism when the trait is changed due to a genetic mutation caused by a natural or artificial factor. This term may be used interchangeably with "activity before modification". "Inactivation," "deficiency," "reduction," "downregulation," "reduction," or "attenuation" of a protein's activity relative to its inherent activity means that the protein's activity is reduced relative to the activity of the particular protein originally possessed by the parent strain prior to transformation or by the unmodified microorganism.

The attenuation of the protein activity can be accomplished by any method known in the art, but is not limited thereto, and can be achieved by using various methods well known in the art (e.g. Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing, hit J Mol Sci. 2014; 15(2):2773-2793; and Sambrook et al. Molecular Cloning 2012).

Specifically, the attenuation of the protein of the present invention can be achieved by:

1) removal of all or part of the gene encoding the protein;

2) modifications of the expression control region (or expression control sequence) so as to reduce the expression of the gene encoding the protein;

3) modifications of the amino acid sequence (e.g. deletion/substitution/addition of at least one amino acid in the amino acid sequence) constituting the protein so as to eliminate or weaken the activity of the protein;

4) modifications of the sequence of a gene encoding a protein in order to eliminate or weaken the activity of the protein (e.g. deletion/replacement/addition of at least one nucleic acid base in the nucleic acid base sequence of a protein gene in order to encode a protein modified to eliminate or weaken the activity of the protein);

5) modifications of the base sequence of the nucleic acid encoding the initiation codon or 5'-UTR region of the transcript of the gene encoding the protein;

6) introduction of an antisense oligonucleotide (for example, antisense RNA) that complementarily binds to the transcript of the gene encoding the polypeptide;

7) adding a sequence complementary to the Shine-Dalgarno sequence of the protein-coding gene to the beginning of the Shine-Dalgarno sequence in such a way as to form a secondary structure that makes ribosome attachment impossible;

8) adding a promoter that is reverse transcribed to the 3' end of the open reading frame (ORF) of the protein-coding gene sequence (reverse transcription engineering, RTE); or

9) combinations of two or more of (1)-(8) above, but not limited to.

For example, they are described as follows.

The deletion of all or part of the gene encoding the protein referred to in paragraph (1) above may be the elimination of the entire polynucleotide encoding the endogenous target protein in the chromosome, replacement with a polynucleotide with deletions of some nucleotides, or replacement with a marker gene.

The modification of the expression control region (or expression control sequence) according to paragraph 2 above may be the induction of a mutation in the expression control region (or expression control sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or substitution with a sequence having a more weakened activity. The expression control region includes, but is not limited to, a promoter, an operator sequence, a sequence for encoding a ribosome binding site, and a sequence for controlling the termination of transcription and translation.

The modification of the nucleic acid base sequence encoding the initiation codon or the 5'-UTR region of the transcript of the gene encoding the protein according to paragraph 3 above may be, for example, a substitution with another initiation codon having a lower rate of expression of the protein encoding the nucleic acid base sequence, rather than with the endogenous initiation codon, but is not limited to this.

The modification of the amino acid sequence or polynucleotide sequence in paragraphs 4 and 5 above may be, but is not limited to, the induction of 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 weaken the activity of the protein, or substitution with an amino acid sequence or polynucleotide sequence modified to further weaken the activity, or an amino acid sequence or polynucleotide sequence modified to lack activity. For example, gene expression may be inhibited or weakened by introducing a mutation in the polynucleotide sequence to form a stop codon.

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

The addition of a sequence complementary to the Shine-Dalgarno sequence of a protein-coding gene upstream of the Shine-Dalgarno sequence to form a secondary structure that prevents ribosome attachment according to item 7 above may result in the failure of mRNA translation or a decrease in its rate.

The addition of a promoter that is reverse transcribable to the 3' end of the open reading frame (ORF) of the protein-coding gene sequence (reverse transcription engineering, RTE) of item 8 above may result in the creation of an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide, thereby attenuating the activity of the protein.

As used herein, the term "enhancement" of a protein activity refers to an increase in the activity of a protein compared to its own activity. The term "enhancement" may be used interchangeably with terms such as activation, up-regulation, overexpression, enhancement, and the like. Specifically, activation, enhancement, up-regulation, overexpression, and enhancement may include the expression of an activity that was originally possessed or the expression of an activity that has been improved compared to its own activity or the activity before modification. "Inherent activity" refers to the activity of a particular protein that was originally possessed by the parent strain before transformation or by an unmodified microorganism when the trait is altered by a genetic mutation caused by a natural or artificial factor. This term may be used interchangeably with "activity before modification." "Enhancement," "upregulation," "overexpression," or "increase" of the activity of a protein relative to its own activity refers to the activity and/or concentration (expression level) of a particular polypeptide that was originally possessed by the parent strain prior to transformation or the unmodified microorganism.

Enhancement may be achieved by introducing an exogenous protein or by increasing the activity and/or concentration (expression level) of an endogenous protein. Enhancement of protein activity may be confirmed by increasing the degree of activity or expression level of the corresponding protein or by increasing the amount of product released from the corresponding protein.

The enhancement of the protein activity can be achieved by using various methods well known in the art, and these methods are not limited as long as the methods can enhance the activity of the target protein compared to the activity of the microorganism before transformation. Specifically, the enhancement of the protein activity can be achieved by using genetic engineering and/or protein engineering well known to those skilled in the art, which are routine methods in molecular biology, but are not limited thereto (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16; and Sambrook et al. Molecular Cloning 2012).

Specifically, the increase in the efficiency of the protein of the present invention can be achieved by:

1) an increase in the number of intracellular copies of the polynucleotide encoding the protein;

2) replacement of the expression control region on the chromosome encoding the protein with a sequence with higher activity;

3) modifications of the base sequence of the nucleic acid encoding the initiation codon or 5'-UTR region of the transcript of the gene encoding the protein;

4) modifications of the amino acid sequence of a protein in such a way as to increase the activity of the protein;

5) modifications of a polynucleotide sequence encoding a protein to enhance the activity of the protein (e.g. modifications of a polynucleotide sequence of a protein gene to encode a protein modified to enhance the activity of the protein);

6) introduction of an exogenous protein that exhibits the activity of a given protein, or an exogenous polynucleotide that encodes it;

7) optimization of the codon of the polynucleotide encoding the protein;

8) modification or chemical modification of the exposed region selected by analysis of the tertiary structure of the protein; or

9) combinations of two or more options selected from paragraphs (1) to (8) above, but not specifically limited thereto.

A more specific description is provided below.

The increase in the number of intracellular copies of the polynucleotide encoding the protein according to claim 1 above can be achieved by introducing into the host cell a vector operably linked to the polynucleotide encoding the corresponding protein and capable of replicating and functioning independently of the host. Alternatively, the increase can be achieved by introducing at least one copy of the polynucleotide encoding the corresponding protein into the chromosome of the host cell. The introduction into the chromosome can be performed by introducing into the host cell a vector capable of inserting the polynucleotide into the chromosome in the host cell, but is not limited to this. The vector is as described above.

The replacement of the expression control region (or expression control sequence) on the chromosome encoding the protein with a sequence having a higher activity according to item 2 above may be, for example, inducing a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof to further enhance the activity of the expression control region, or replacement with a sequence having a stronger activity. The expression control region may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence controlling the termination of transcription and translation, and the like. For example, the replacement may be a replacement of the original promoter with a strong promoter, but is not limited thereto.

Known examples of a stronger promoter may include, but are not limited to, the CJ1 to CJ7 promoters (US 7662943 B2), 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 (US 10584338 B2), the O2 promoter (US 10273491 B2), the tkt promoter, the yccA promoter, and the like.

The modification of the nucleic acid base sequence encoding the initiation codon or the 5'-UTR region of the gene transcript encoding the protein according to paragraph 3 above may be, for example, a substitution with a nucleic acid base sequence encoding not its own initiation codon, but another initiation codon having a lower rate of expression from the protein, but without limitation thereto.

The modification of the amino acid sequence or polynucleotide sequence in paragraphs 4 and 5 above may be, but is not limited to, inducing 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 to enhance the activity of the protein, or substitution with an amino acid sequence or polynucleotide sequence modified to further enhance the activity, or an amino acid sequence or polynucleotide sequence modified to increase the activity of the protein. The substitution may be performed by, but is not limited to, integrating the polynucleotide into the chromosome by homologous recombination. The vector used herein may further comprise a selectable marker for identifying the integration into the chromosome. The selectable marker is as described above.

The introduction of an exogenous polypeptide exhibiting the activity of the polypeptide according to item 6 above may be the introduction into the host cell of an exogenous polynucleotide encoding a protein exhibiting the same/similar activity as this protein. The exogenous polynucleotide is not limited in origin or sequence as long as the exogenous polynucleotide exhibits the same activity as the protein. The introduction can be carried out by any known transformation method, which is appropriately selected by a person skilled in the art, and the introduced polynucleotide is expressed in the host cell, and thus the protein is produced and its activity can be increased.

Optimization of a codon of a polynucleotide encoding a protein according to claim 7 may be optimization of a codon of an endogenous polynucleotide to enhance its transcription or translation in a host cell, or optimization of a codon of an exogenous polynucleotide to perform optimized transcription or translation in a host cell.

The modification or chemical modification of the exposed region selected as a result of the analysis of the tertiary structure of the protein in paragraph 8 above may be, for example, a modification or chemical modification of the exposed region to be modified or chemically modified by comparing the sequence information of the protein to be analyzed with a database storing sequence information of existing proteins to determine a candidate template protein according to sequence similarity and identifying a structure based on the determined candidate.

Enhancement of protein activity may mean that the activity, concentration, or expression level of the corresponding protein is increased relative to the activity or concentration of the protein expressed in the wild-type microbial strain or the microbial strain before modification, or that the amount of product obtained from the corresponding protein is increased, but is not limited to this.

As used herein, the term "vector" refers to a DNA construct that includes a sequence of nucleotide bases of a polynucleotide encoding a target protein and operably linked to a suitable expression control region (or expression control sequence) such that the target polypeptide can be expressed 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 for encoding a suitable mRNA binding site for the ribosome, and a sequence for controlling the termination of transcription and translation. The vector, upon transformation into a suitable host cell, may replicate or function independently of the host genome or may be integrated into the genome itself.

The vector used in the present invention is not particularly limited, and any vector known in the art can be used. Examples of the vector that is usually used may include native 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, pBR, pUC, pBluescriptII, pGEM, and pTZ-based vectors, pCL, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pdc2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

As an example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome by a vector for chromosomal insertion into a chromosome. The insertion of a polynucleotide into a chromosome can be performed using any method known in the art, such as, but not limited to, homologous recombination. The vector can further comprise a selectable marker for examining insertion or non-insertion into a chromosome. The selectable marker is used to select cells transformed by the vector, i.e., to identify insertion or non-insertion of the target nucleic acid molecule, and markers that impart a selectable phenotype, such as drug resistance, auxotrophy, cytotoxic drug resistance, or expression of surface polypeptides, can be used. Under conditions of treatment with selective agents, only cells expressing selectable markers can survive or exhibit other phenotypic characteristics, which makes it possible to select transformed cells.

As used herein, the term "transformation" means that a vector containing a polynucleotide encoding a target polypeptide is introduced into a host cell or microorganism to allow expression of the polypeptide encoded by the polynucleotide in the host cell. Examples of a transformed polynucleotide may include any polypeptide as long as it can be expressed in the host cell, whether it is inserted and localized within a chromosome of the host cell or located outside the chromosome. In addition, a polynucleotide includes DNA and/or RNA encoding a target polypeptide. The polynucleotide may be introduced in any form as long as the polynucleotide can be introduced and expressed in the host cell. For example, a polynucleotide may be introduced as an expression cassette, which is a gene construct containing all the factors necessary for self-expression. An expression cassette may typically include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of a self-replicating expression vector. In addition, the polynucleotide may be introduced into the host cell as is for operative linkage to a sequence required for expression in the host cell, but is not limited thereto.

The above term "operably linked" refers to a functional link between a promoter sequence for initiating and mediating transcription of a polynucleotide encoding a target protein of the present invention and a polynucleotide sequence.

In the microorganism of the present invention, modification of all or part of a polynucleotide can be induced by (a) homologous recombination using a vector for inserting chromosomes into a chromosome or genome editing using a modified nuclease (e.g., CRISPR-Cas9) and/or (b) treatment with light, such as ultraviolet rays and radioactive rays and/or chemicals, but not limited to. Modification of all or part of a gene can include a method using recombinant DNA technology. For example, all or part of a gene can be deleted by introducing a nucleotide sequence or a vector that contains a nucleotide sequence homologous to a target gene into a microorganism to induce homologous recombination. The introduced nucleotide sequence or vector may contain a dominant selectable marker, but is not limited to this.

According to another aspect of the present invention, a method for producing L-arginine is provided, comprising culturing in a medium a recombinant microorganism of the genus Corynebacterium, in which the activity of a protein comprising the amino acid sequence SEQ ID NO: 1 is weakened.

The protein comprising the amino acid sequence of SEQ ID NO: 1, the attenuation, the microorganism and the like are as described in other aspects.

The microorganism of the genus Corynebacterium may be, but is not limited to, Corynebacterium glutamicum.

In a microorganism, the activity of the arginine repressor (ArgR) can be further attenuated, or the argR gene can be further deleted, but is not limited to this. Attenuation and deletion are described in other aspects.

As used herein, the term "cultivating" refers to growing the microorganism of the genus Corynebacterium of the present invention under appropriately selected environmental conditions. The culturing step of the present invention can be performed according to suitable media or culturing conditions known to those skilled in the art. The culturing step can be easily adjusted by a person skilled in the art according to the selected strain. Specifically, the culturing can be performed in a batch, continuous and/or fed-batch manner, but is not limited thereto.

As used herein, the term "medium" refers to a mixture containing nutrients necessary for culturing the microorganism of the present invention as main ingredients, wherein the medium provides nutrients, growth factors and the like, including water necessary for survival and growth. Specifically, the medium and other culture conditions for culturing the microorganism of the genus Corynebacterium of the present invention may be any medium that is used for conventional microorganism culturing, without particular limitations. However, the microorganism of the genus Corynebacterium of the present invention 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, while temperature, pH and the like are adjusted.

Specifically, culture media for microorganisms of the genus Corynebacterium can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D.C., USA, 1981].

In the present invention, examples of carbon sources may include: carbohydrates such as glucose, sucrose, 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. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, raw molasses, rice bran, cassava, bagasse and liquid corn steep liquor may be used, and in particular, carbohydrates such as glucose and sterile pre-treated molasses (i.e., molasses converted into reducing sugars) may be used, and appropriate amounts of other carbon sources may be used without limitation. These carbon sources may be used individually or in combination of two or more, but are not limited thereto.

Examples of nitrogen sources may include: inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; amino acids such as glutamic acid, methionine, and glutamine; and organic nitrogen sources such as peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn broth, casein hydrolysates, fish or fish degradation products, defatted soybean meal or fish degradation products. These nitrogen sources may be used individually or in combinations of two or more of them, but are not limited to them.

Examples of phosphorus sources may include monobasic potassium phosphate, dibasic potassium phosphate and their corresponding sodium salts. Examples of inorganic compounds may include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, calcium carbonate and the like, and may further include amino acids, vitamins and/or suitable precursors. These constituent ingredients or precursors may be added to the medium in a batch or continuous manner. However, examples of phosphorus sources are not limited thereto.

The pH of the medium can be adjusted during the cultivation of the microorganism of the genus Corynebacterium described in the present invention by appropriately adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the medium. In addition, an antifoam such as a fatty acid polyglycol ester can be added to suppress foaming during the cultivation. Additionally, oxygen or an oxygen-containing gas can be introduced into the medium to maintain an aerobic state of the medium, or nitrogen, hydrogen or carbon dioxide gas can be introduced without introducing a gas to maintain an anaerobic or non-aerobic state of the medium, but not limited thereto.

In the present description, the culturing temperature can be maintained at 20°C to 45°C, and specifically at 25°C to 40°C, and the culturing can be performed for about 10 to 160 hours, but not limited thereto.

L-arginine obtained by culturing according to the present invention may be released into the medium or may remain in the cells.

The method for producing L-arginine according to the present invention may further comprise obtaining a microorganism of the genus Corynebacterium according to the present invention, preparing a medium for culturing the microorganism, or a combination of these steps (irrespective of the order, in any order), for example, before the culturing step.

The method for producing L-arginine according to the present invention may further comprise extracting L-arginine from the medium obtained by culturing (the medium in which the culturing was performed) or from the microorganism of the genus Corynebacterium in the medium obtained by culturing. The method according to the present invention may further comprise an extraction step after the culturing step.

The recovery may be collecting the target L-arginine using an appropriate method known in the art according to the microorganism culturing method of the present invention, for example, batch, continuous or fed-batch type culturing. Examples of the method may include centrifugation, filtration, treatment with a crystallized protein precipitating agent (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 (high performance liquid chromatography) and a combination of these methods, and the target L-arginine can be recovered from the medium or the microorganism by using an appropriate method known in the art.

The method for producing L-arginine according to the present invention may further comprise a purification step. The purification may be performed using a suitable method known in the art. In one example, when the method for producing L-arginine according to the present invention comprises both an extraction step and a purification step, the extraction step and the purification step may be performed continuously or intermittently regardless of the order, or may be performed simultaneously or combined into one step, but are not limited to this.

According to another aspect of the present invention, a composition for producing L-arginine is provided, wherein the composition comprises: a microorganism of the genus Corynebacterium, in which the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 is weakened; a medium in which the microorganism was cultured, or a combination thereof.

The composition of the present invention may further contain any suitable excipient which is generally used in compositions for producing amino acids, and examples of the excipient may include, but are not limited to, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizing agent or an isotonic agent.

In the composition of the present invention, the protein comprising the amino acid sequence of SEQ ID NO: 1, the attenuation, the microorganism, the culturing, the medium and the like are as described in other aspects.

According to another aspect of the present invention, a method for producing a microorganism of the genus Corynebacterium is provided, wherein the method comprises reducing the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1.

According to another aspect of the present invention, there is provided the use of a microorganism of the genus Corynebacterium for producing L-arginine, in which the activity of a protein comprising the amino acid sequence SEQ ID NO: 1 is weakened.

In the method for producing a microorganism of the genus Corynebacterium and using the microorganism of the genus Corynebacterium to produce L-arginine, the protein comprising the amino acid sequence of SEQ ID NO: 1, the attenuator, the microorganism and the like are the same as those described in other aspects.

Embodiment of the invention

Hereinafter, the present invention will be described in detail with reference to Examples. However, these Examples are only preferred embodiments for illustrating the present invention and, therefore, are not intended to limit the scope of the rights of the present invention. However, technical issues not described herein can be sufficiently understood and easily implemented by a person skilled in the art or similar technical fields to the present invention.

Example 1: Generation of a library of random mutants using transposon and screening

To obtain strains having an increased ability to produce L-arginine, Corynebacterium glutamicum KSCM10741P (US 8034602 B2) as a parent strain was transformed with a plasmid obtained using the Transposome™ EZ-Tn5 <R6Kyori/KAN-2>Tnp kit (Epicentre) by the electropulse method (Appendix Microbial. Biotechnol. (1999) 52:541-545), and then the transformants were plated on a complex nutrient medium containing kanamycin (25 mg/L), resulting in approximately 20,000 colonies.

Complex medium for plates (pH 7.0)

Glucose 10 g, peptone 10 g, meat extract 5 g, yeast extract 5 g, brain heart extract 18.5 g, NaCl 2.5 g, urea 2 g, sorbitol 91 g, agar 20 g (based on 1 liter of distilled water)

The seven best mutant strains with improved ability to produce L-arginine compared to the Corynebacterium glutamicum strain KSCM10741P as the parent strain were selected using the ninhydrin method (Moore, S., Stein, W. H., Photometric ninhydrin method for use in the chromatography of amino acids. J. Biol. Chem. 1948, 176, 367-388).

Example 2: Analysis of L-arginine producing capacity in randomly selected mutant strains

For final selection, strains with reproducibly increased L-arginine production ability from the seven mutant strains selected in Example 1 were cultured in a flask using the following medium. After completion of the culture, the L-arginine concentration in the culture was analyzed by HPLC. The concentrations of L-arginine produced by each mutant strain are shown in Table 1 below.

Production medium (pH 7.2)

Glucose 60 g, ammonium sulfate 45 g, magnesium sulfate heptahydrate 2 g, monobasic potassium phosphate 2 g, ammonium chloride 10 g, biotin 0.01 mg, thiamine-HCl 0.1 mg, calcium pantothenate 2 mg, ricotinamide 3 mg, iron sulfate 10 mg, manganese sulfate 10 mg, zinc sulfate 0.02 mg, copper sulfate 0.5 mg, calcium carbonate 30 g (based on 1 liter of distilled water).

Among the ten selected mutant strains, KCCM10741P/mt-7 was finally selected as the strain with significantly improved L-arginine producing ability.

Example 3. Establishing the reasons for the increased ability to produce L-arginine in the finally selected strain

A gene inactivated by a random transposon insertion into KCCM10741P/mt-7 from Example 2 was identified.

Specifically, genomic DNA was isolated from KCCM10741P/mt-7, digested and then ligated, and the ligation product was transformed into E. coli DH5a. The transformants were plated on LB solid medium containing kanamycin (25 mg/L). After selecting 20 transformed colonies, plasmids containing the unknown part of the gene were obtained, and their nucleic acid sequences were analyzed using Primer 1 (SEQ ID NO: 3) and Primer 2 (SEQ ID NO: 4) of the Transposome™ EZ-Tn5 <R6Kγori/KAN-2> Tpr Kit. As a result, it was identified that the gene comprising the nucleotide sequence of SEQ ID NO: 2 was inactivated based on the nucleic acid sequence submitted to NIH GenBank U.S. National Institutes of Health.

A quick search of SEQ ID NO: 2 of Corynebacterium glutamicum wild type ATCC 13869 identified this sequence as TQuNCgl1469.

Example 4: Construction of a recombinant vector for deletion of the NCgl1469 gene

To re-identify the effect of NCgl1469 gene inactivation on L-arginine production, a recombinant vector was constructed to delete the NCgl1469 gene on the chromosome of a Corynebacterium strain.

To obtain fragments for gene deletion, primers 3 through 6 were synthesized and are shown in Table 3.

To construct a vector having a deletion of the ORF region based on SEQ ID NO: 2, PCR was performed using the wild-type Corynebacterium glutamicum ATCC 13869 gDNA as a template together with the primer pair of SEQ ID NOS: 5 and 6 and the primer pair of SEQ ID NOS: 7 and 8. PCR was performed under the conditions of denaturation at 95°C for 5 minutes, 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 and then polymerization at 72°C for 5 minutes. Overlapping PCR was again performed using the mixture of the two fragments obtained above as a template together with the primer pair of SEQ ID NOS: 5 and 8, thereby obtaining fragments. PCR was performed under conditions of denaturation at 94°C for 5 minutes, 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 (KR 10-2020-0136813) was digested with SmaI, and the resulting PCR products were subjected to fusion cloning. Fusion cloning was performed using the m-Fusion® HD Cloning Kit (Clontech). The resulting plasmid was named pDCM2-ΔNCgl1469.

Example 5: Obtaining a strain with a deleted NCgl1469 gene and assessing the ability to produce L-arginine - 1

Corynebacterium glutamicum KSCM10741P, which is an L-arginine-producing strain, was transformed with pDCM2-ΔNCgl1469 by homologous recombination on the chromosome (van der Rest et al., Appl. Microbiol Biotechnol 52:541–545, 1999).

After this, the transformants were subjected to secondary recombination on a Petri dish with solid medium containing 4% sucrose. The transformed Corynebacterium glutamicum strains after completion of secondary recombination were subjected to PCR using primers 3 and 6 to identify the strain in which the gene SEQ ID NO: 2 was deleted. The recombinant strain was named Corynebacterium glutamicum KССМ10741Р-ΔNCgl1469.

To analyze the L-arginine producing capacity of the obtained Corynebacterium glutamicum KCCM10741P-ΔNCgl1469 strain, it was cultured together with the parent strain Corynebacterium glutamicum KССМ10741Р in the following manner.

The parent strain Corynebacterium glutamicum KCCM10741P and Corynebacterium glutamicum KCCM10741P-ΔNCgl1469 were each inoculated into a 250-mL baffled flask containing 25 mL of the following seeding medium and cultured with shaking at 200 rpm at 30°C for 20 h. Then, 1 mL of the seed culture was inoculated into a 250-mL baffled flask containing 24 mL of production medium and cultured with shaking at 200 rpm at 30°C for 72 h. The compositions of the seeding medium and production medium were as follows.

Sowing medium (pH 7.2)

Glucose 20 g, ammonium sulfate 45 g, magnesium sulfate heptahydrate 2 g, monobasic potassium phosphate 2 g, ammonium chloride 10 g, biotin 0.01 mg, thiamine-HCl 0.1 mg, calcium pantothenate 2 mg, nicotinamide 3 mg, iron sulfate 10 mg, manganese sulfate 10 mg, zinc sulfate 0.02 mg, copper sulfate 0.5 mg (based on 1 liter of distilled water).

Production medium (pH 7.2)

Glucose 60 g, ammonium sulfate 45 g, magnesium sulfate heptahydrate 2 g, monobasic potassium phosphate 2 g, ammonium chloride 10 g, biotin 0.01 mg, thiamine-HCl 0.1 mg, calcium pantothenate 2 mg, nicotinamide 3 mg, iron sulfate 10 mg, manganese sulfate 10 mg, zinc sulfate 0.02 mg, copper sulfate 0.5 mg, calcium carbonate 30 g (based on 1 liter of distilled water).

After completion of the culture, the L-arginine producing ability was determined by HPLC (Waters 2478), and the results are shown in Table 4 below.

As a result, the L-arginine producing ability of KCCM10741P-ΔNCgl1469 increased by an average of 19.8% compared to the parental strain.

Example 6: Obtaining a strain with a deleted NCgl1469 gene and assessing the ability to produce L-arginine - 2

It was determined whether another strain of Corynebacterium glutamicum producing L-arginine exhibited the same effect as in Example 5.

Specifically, an L-arginine-producing strain was obtained by introducing one type of mutation (deletion of the argR gene, hereinafter referred to as ΔargR) into the wild-type Corynebacterium glutamicum strain (ATCC 13869).

The recombinant vector for ΔargR was constructed in the same manner as in the method for constructing the vector of Example 4, and the primers used for constructing the vector are shown in Table 5.

The plasmid into which the target gene was inserted was selected by PCR, and then the plasmid was named pDCM2-ΔargR. The wild-type Corynebacterium glutamicum strain ATCC13869 was transformed with pDCM2-ΔargR by the same method as in Example 5. The transformant strains were subjected to PCR using primers 7 and 10 to identify the strain in which argR was deleted in the chromosome, and the strain was named Corynebacterium glutamicum CJ1R. Corynebacterium glutamicum CJ1R was allowed to undergo the same method as in Example 5 to obtain a strain in which the NCgl1469 gene was deleted, and the strain was named CJ1R-ΔNCgl1469.

CJ1R-ΔNCgl1469 was cultured by the same method as in Example 5. After completion of the culture, the L-arginine producing ability was measured by HPLC (Waters 2478), and the results are shown in Table 6 below.

As a result, CJlR-ΔNCgl1469 showed an average increase in L-arginine producing capacity of 19.7% compared to the parent strain CJ1R.

As indicated above, a person skilled in the art to which this disclosure pertains will appreciate that the present invention may be embodied in other specific forms without departing from its technical character or essential characteristics. Accordingly, the embodiments described above should be considered as illustrative and not limiting of the present invention. The scope of the present invention should be understood so that all changes or modifications resulting from the definitions and scopes of the claims and their equivalents fall within the scope of the application.

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construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ctaccctgtggaacacctacatct</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="5">

<INSDSeq>

<INSDSeq_length>34</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q9">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer 3</INSDQualifier_value>

<NonEnglishQualifier_value>primer

3</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q10">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>artificial

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>tcgagctcggtaccctcagtgctccaactgcttg</INSDSeq_seque

nce>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="6">

<INSDSeq>

<INSDSeq_length>34</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q11">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer 4</INSDQualifier_value>

<NonEnglishQualifier_value>primer

4</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q12">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>artificial

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>cttcagggggataccagaatagcgaaatgaaaag</INSDSeq_seque

nce>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="7">

<INSDSeq>

<INSDSeq_length>34</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q13">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer 5</INSDQualifier_value>

<NonEnglishQualifier_value>primer

5</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q14">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>artificial

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ctggtatccccctgaaggcgatttaaggggtcga</INSDSeq_seque

nce>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="8">

<INSDSeq>

<INSDSeq_length>34</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q15">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer 6</INSDQualifier_value>

<NonEnglishQualifier_value>primer

6</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q16">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>artificial

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ctctagaggatccccgcaggggagtttgaaaaac</INSDSeq_seque

nce>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="9">

<INSDSeq>

<INSDSeq_length>34</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q17">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer 7</INSDQualifier_value>

<NonEnglishQualifier_value>primer

7</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q18">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>artificial

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>tcgagctcggtacccagcaggccttaagggtaag</INSDSeq_seque

nce>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="10">

<INSDSeq>

<INSDSeq_length>34</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q19">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer 8</INSDQualifier_value>

<NonEnglishQualifier_value>primer

8</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..34</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q20">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>artificial

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>aggggcgctgtcttacctcggctggtgggccagc</INSDSeq_seque

nce>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="11">

<INSDSeq>

<INSDSeq_length>36</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..36</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q21">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer 9</INSDQualifier_value>

<NonEnglishQualifier_value>primer

9</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..36</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q22">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>artificial

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ggtaagacagcgcccctagttcaaggcttgttaatc</INSDSeq_seq

uence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="12">

<INSDSeq>

<INSDSeq_length>35</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..35</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q23">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>primer 10</INSDQualifier_value>

<NonEnglishQualifier_value>primer

10</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..35</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q24">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

<NonEnglishQualifier_value>artificial

construction</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ctctagaggatccccaccgttgaactgcttgccag</INSDSeq_sequ

ence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="13">

<INSDSeq>

<INSDSeq_length>171</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..171</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q25">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>argR amino acid

sequence</INSDQualifier_value>

<NonEnglishQualifier_value>amino acid sequence

argR</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..171</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q41">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>Corynebacterium</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MSLGSTPSTPENLNPVTRTARQALILQILDKQKVTSQVQLSELLLDEGI

DITQATLSRDLDELGARKVRPDGGRAYYAVGPVDSIAREDLRGPSEKLRRMLDELLVSTDHSGNIAMLRT

PPGAAQYLASFIDRVGLKEVVGTIAGDDTVFVLARDPLTGKELGELLSGRTT</INSDSeq_sequence

>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="14">

<INSDSeq>

<INSDSeq_length>516</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..516</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q27">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>argR nucleotide

sequence</INSDQualifier_value>

<NonEnglishQualifier_value>nucleotide sequence

argR</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..516</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>genomic DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q38">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>Corynebacterium</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atgtcccttggctcaaccccgtcaacaccggaaaacttaaatcccgtga

ctcgcactgcacgccaagctctcattttgcagattttggacaaacaaaaagtcaccagccaggtacaact

gtctgaattgctgctggatgaaggcatcgatatcacccaggccaccttgtcccgagatctcgatgaactc

ggtgcacgcaaggttcgccccgatgggggacgcgcctactacgcggtcggcccagtagatagcatcgccc

gcgaagatctccggggtccgtcggagaagctgcgccgcatgcttgatgaactgctggtttctacagatca

ttccggcaacatcgcgatgctgcgcaccccgccgggagctgcccagtacctggcaagtttcatcgatagg

gtggggctgaaagaagtcgttggcaccatcgctggcgatgacaccgttttcgttctcgcccgtgatccgc

tcacaggtaaagaactaggtgaattactcagcgggcgcaccacttaa</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="15">

<INSDSeq>

<INSDSeq_length>319</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table> <INSDFeature>

<INSDFeature_key>REGION</INSDFeature_key>

<INSDFeature_location>1..319</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q29">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>argF amino acid

sequence</INSDQualifier_value>

<NonEnglishQualifier_value>amino acid sequence

argF</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..319</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q42">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>Corynebacterium</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MTSQPQVRHFLADDDLTPAEQAEVLTLAAKLKAAPFSERPLEGPKSVAV

LFDKTSTRTRFSFDAGIAHLGGHAIVVDSGSSQMGKGETLQDTAAVLSRYVEAIVWRTYAHSNFHAMAET

STVPLVNSLSDDLHPCQILADLQTIVENLSPEEGPAGLKGKKAVYLGDGDNNMANSYMIGFATAGMDISI

IAPEGFQPRAEFVERAEKRGQETGAKVVVTDSLDEVAGADVVITDTWVSMGMENDGIDRTTPFVPYQVND

EVMAKANDGAIFLHCLPAYRGKEVAASVIDGPASKVFDEAENRLHAQKALLVWLLAHQPR</INSDSeq_

sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="16">

<INSDSeq>

<INSDSeq_length>960</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>misc_feature</INSDFeature_key>

<INSDFeature_location>1..960</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier id="q31">

<INSDQualifier_name>note</INSDQualifier_name>

<INSDQualifier_value>argF nucleotide

sequence</INSDQualifier_value>

<NonEnglishQualifier_value>nucleotide sequence

argF</NonEnglishQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..960</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>genomic DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q43">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>Corynebacterium</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>atgacttcacaaccacaggttcgccatttcctggctgatgatgatctca

cccctgcagagcaggcagaggttttgaccctagccgcaaagctcaaggcagcgccgttttcggagcgtcc

actcgagggaccaaagtccgttgcagttctttttgataagacttcaactcgtactcgcttctccttcgac

gcgggcatcgctcatttgggtggacatgccatcgtcgtggattccggcagctcacagatgggtaagggcg

agaccctgcaggacaccgcagctgtattgtcccgctacgtggaagcaattgtgtggcgcacctacgcaca

cagcaatttccacgccatggcggagacgtccactgtgccactggtgaactccttgtccgatgatctgcac

ccatgccagattctggctgatctgcagaccatcgtggaaaacctcagccctgaagaaggcccagcaggcc

ttaagggtaagaaggctgtgtacctgggcgatggcgacaacaacatggccaactcctacatgattggctt

tgccaccgcgggcatggatatctccatcatcgctcctgaagggttccagcctcgtgcggaattcgtggag

cgcgcggaaaagcgtggccaggaaaccggcgcgaaggttgttgtcaccgacagcctcgacgaggttgccg

gcgccgatgttgtcatcaccgatacctgggtatccatgggtatggaaaacgacggcatcgatcgcaccac

acctttcgttccctaccaggtcaacgatgaggtcatggcgaaagctaacgacggcgccatcttcctgcac

tgccttcctgcctaccgcggcaaagaagtggcagcctccgtgattgatggaccagcgtccaaagttttcg

atgaagcagaaaaccgcctccacgctcagaaagcactgctggtgtggctgctggcccaccagccgaggta

a</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (7)

1. Use of a recombinant microorganism of the genus Corynebacterium, in which a polynucleotide encoding a protein comprising the amino acid sequence SEQ ID NO: 1 has been removed, for producing L-arginine. 2. The use according to claim 1, wherein the microorganism has an increased ability to produce L-arginine compared to the parent strain, in which the activity of the protein comprising the amino acid sequence SEQ ID NO:1 is not removed. 3. The use according to claim 1, wherein the microorganism is Corynebacterium glutamicum . 4. The use according to claim 1, wherein the activity of the arginine repressor is further weakened. 5. A method for producing L-arginine, comprising culturing in a medium a recombinant microorganism of the genus Corynebacterium , in which the polynucleotide encoding a protein comprising the amino acid sequence SEQ ID NO: 1 has been removed. 6. The method according to claim 5, wherein the microorganism is Corynebacterium glutamicum . 7. The method according to claim 5, wherein in the microorganism of the genus Corynebacterium the activity of the arginine repressor is further weakened.