Microorganisms producing L-amino acids with reduced carbamoyl phosphate synthetase activity and method for producing L-amino acids using the same

Abstract

The present application relates to an L-amino acid producing microorganism having reduced carbamoyl phosphate synthetase activity and a method for producing an L-amino acid using the same.

Description

[Technical field]

[0001] The present application relates to an L-amino acid producing microorganism having reduced carbamoyl phosphate synthetase activity and a method for producing an L-amino acid using the same. [Background technology]

[0002] In the process of producing L-amino acids using microorganisms, ATP is used as a cofactor for biosynthetic enzymes and is an important substance used in cell growth and various metabolic processes that maintain activity.

[0003] Carbamoyl-phosphate is an intermediate metabolite used in the pyrimidine and arginine biosynthetic pathways. Carbamoyl-phosphate synthetase is an enzyme that converts bicarbonate to carbamoyl-phosphate, using two molecules of ATP and one molecule of glutamine as a nitrogen donor during the synthesis of carbamoyl-phosphate. Glutamine is produced by glutamine synthetase using glutamate as a substrate, consuming one molecule of ATP in the process.

[0004] In this regard, it has been reported that the production capacity of L-arginine, citrulline, and pyrimidine derivatives is improved by introducing a mutant carbamoyl phosphate synthetase that is desensitized to feedback inhibition and maintains high activity into Escherichia coli (Patent Document 1).

[0005] However, it has been reported that excessive intracellular carbamoyl phosphate formation induces cytotoxicity (Non-Patent Document 1). Carbamoyl phosphate is a highly unstable substance, and cyanate, which is naturally converted from carbamoyl phosphate, can induce cytotoxicity.

[0006] Therefore, if the activity of carbamoyl phosphate synthetase in a microorganism can be regulated to maintain an appropriate level of carbamoyl phosphate in the cell and prevent unnecessary consumption of ATP, thereby regulating energy balance, the L-amino acid production ability can be improved. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2003-093083 A [Patent Document 2] U.S. Patent No. 7,662,943 [Patent Document 3] U.S. Pat. No. 1,058,438 [Patent Document 4] U.S. Pat. No. 10,273,491 [Patent Document 5] Korean Patent Publication No. 10-2020-0136813 [Non-patent literature]

[0008] [Non-Patent Document 1] Charlier et al., Amino Acids. 2018; 50(12): 1647-1661. [Non-Patent Document 2] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 [Non-Patent Document 3] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-Patent Document 4] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-Patent Document 5] Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)

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

[0009] The present inventors have developed a Corynebacterium microorganism having an L-amino acid producing ability and reduced activity of carbamoyl phosphate synthetase, a method for producing an L-amino acid using said microorganism, a composition for producing L-arginine containing said microorganism, and use of said microorganism for producing L-arginine, and have completed the present application. [Means for solving the problem]

[0010] An object of the present application is to provide a microorganism of the genus Corynebacterium that has an ability to produce L-amino acids and has weakened carbamoyl-phosphate synthetase activity.

[0011] Another object of the present application is to provide a method for producing an L-amino acid, comprising the step of culturing in a medium a Corynebacterium microorganism having an ability to produce an L-amino acid and an attenuated activity of carbamoyl phosphate synthetase.

[0012] A further object of the present application is to provide a composition for producing L-amino acids, comprising the Corynebacterium microorganism of the present application, a medium in which it has been cultured, or a combination thereof.

[0013] Another object of the present application is to provide a method for producing a Corynebacterium microorganism having an L-amino acid producing ability, which comprises a step of attenuating carbamoyl phosphate synthetase.

[0014] A further object of the present application is to provide use of a Corynebacterium microorganism with weakened carbamoyl phosphate synthetase activity in production of L-amino acids. Effect of the Invention

[0015] The microorganism of the present application has an improved L-amino acid producing ability compared to conventional unmodified microorganisms because the activity of carbamoyl-phosphate synthetase is weakened. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] These will be described in detail below. Each description and embodiment disclosed in this application is also applicable to other descriptions and embodiments. That is, all combinations of various elements disclosed in this application are included in this application. In addition, this application is not limited to the following specific description. Furthermore, many papers and patent documents are referenced throughout this specification, and citations are provided. The disclosure contents of the cited papers and patent documents are incorporated in their entirety into this specification by reference, thereby more clearly explaining the state of the art to which this application pertains and the contents of this application.

[0017] One aspect of the present application provides a Corynebacterium microorganism having an ability to produce an L-amino acid and reduced activity of carbamoyl-phosphate synthetase.

[0018] In this application, "carbamoyl-phosphate synthetase" refers to an enzyme that converts bicarbonate to carbamoyl-phosphate.

[0019] The carbamoyl phosphate synthase may be composed of a carbamoyl phosphate synthase small subunit (CarA) and a carbamoyl phosphate synthase large subunit (CarB), and the amino acid sequence of CarA or CarB can be obtained from a publicly known database such as NCBI Genebank.

[0020] As an example, CarA or CarB of the present application may be derived from a microorganism, specifically from a microorganism of the genus Corynebacterium, more specifically from Corynebacterium glutamicum, Corynebacterium deserti, Corynebacterium crudilactis, Corynebacterium efficiens, Corynebacterium callunae, etc., but is not limited thereto.

[0021] As another example, the amino acid sequence of CarA in the present application is ANU33813.1 derived from Corynebacterium glutamicum ATCC13869, and the amino acid sequence of CarB is ANU33812.1 derived from Corynebacterium glutamicum ATCC13869, but it goes without saying that proteins having CarA or CarB activity of various origins are included.

[0022] In the present application, CarA 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, and CarB may have the amino acid sequence shown in SEQ ID NO: 2, may contain the amino acid sequence, may consist of the amino acid sequence, or may essentially consist of the amino acid sequence.

[0023] In the present application, the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2 includes 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 and / or SEQ ID NO: 2. Needless to say, the present application also includes a protein having an amino acid sequence in which a part of the sequence has been deleted, modified, substituted, conservatively substituted or added, so long as the amino acid sequence has such homology or identity and exhibits efficacy equivalent to that of a protein comprising the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0024] For example, the amino acid sequence may include additions or deletions of sequences that do not change the function of the protein of the present application, naturally occurring mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or internally.

[0025] The term "conservative substitution" refers to the replacement of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0026] In this application, "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, expressed as a percentage. Homology and identity are often used interchangeably.

[0027] Sequence homology or identity of conserved polynucleotides or polypeptides may be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences will generally hybridize to all or part of the sequence under moderate or high stringent conditions. Hybridization, of course, also includes hybridization to polynucleotides having common codons or codons that take into account the degeneracy of codons in the polynucleotide.

[0028] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program with default parameters as in, for example, Non-Patent Document 2. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 4) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 3) (version 5.0.0 or later), which includes the GCG program package (Non-Patent Document 5), BLASTP, BLASTN, FASTA (Non-Patent Documents 6, 7 and 8). For example, BLAST or Clustal W from the National Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0029] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as that disclosed in, for example, J. Biol. 1999, 143:1311-1354 (2001). Briefly, the GAP program defines the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include (1) a binary comparison matrix (identity takes a value of 1 and non-identity takes a value of 0) and a weighted comparison matrix (or EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in, (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5), and (3) no penalty for terminal gaps.

[0030] CarA and CarB of the present application may be encoded by the carAB operon genes.

[0031] For example, the carA gene is a polynucleotide encoding ANU33813.1 derived from Corynebacterium glutamicum ATCC13869, and the carB gene is a polynucleotide encoding ANU33812.1 derived from Corynebacterium glutamicum ATCC13869, but is not limited thereto and naturally includes carA and / or carB genes of various origins that encode proteins having CarA and / or CarB activity.

[0032] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and means a DNA or RNA chain longer than a certain length, and more specifically, means a polynucleotide fragment that encodes the protein.

[0033] The polynucleotide encoding the carbamoyl phosphate synthetase of the present application may comprise a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1 and / or SEQ ID NO: 2. As an example of the present application, the polynucleotide of the present application may have the sequence represented by SEQ ID NO: 3 or may comprise said sequence. Furthermore, the polynucleotide of the present application may consist of the sequence represented by SEQ ID NO: 3 or may essentially consist of said sequence. Specifically, the carbamoyl phosphate synthetase may be encoded by a polynucleotide represented by the nucleotide sequence represented by SEQ ID NO: 3.

[0034] The polynucleotide of the present application may be modified in various ways in the coding region, without changing the amino acid sequence of the carbamoyl phosphate synthetase, by taking into consideration the codon degeneracy or the codons preferred in the organism in which the carbamoyl phosphate synthetase of the present application is to be expressed. Specifically, the polynucleotide of the present application has a base sequence having 70% or more, 75% or more, 76% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more homology or identity to the sequence of SEQ ID NO: 3, or includes the base sequence, or consists of a base sequence having 70% or more, 75% or more, 76% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, or 98% or more homology or identity to the sequence of SEQ ID NO: 3, or is substantially composed of the base sequence, but is not limited thereto.

[0035] Furthermore, the polynucleotide of the present application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a complementary sequence to all or a part of the polynucleotide sequence of the present application. 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 12 and 13). For example, the conditions include conditions under which polynucleotides having high homology or identity, such as polynucleotides having a homology or identity of 70% or more, 75% or more, 76% 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 having lower homology or identity than that do not hybridize with each other, or conditions under which washing is performed once, specifically two to three times, at a salt concentration and temperature equivalent to those used in normal 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.

[0036] Hybridization requires that two nucleic acids have complementary sequences, even though mismatches between bases are possible depending on the stringency of hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application may include isolated nucleic acid fragments that are complementary to the entire sequence, as well as substantially similar nucleic acid sequences.

[0037] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the hybridization conditions described above, in which the hybridization step is performed at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and may be appropriately adjusted by those skilled in the art depending on the purpose.

[0038] 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 12).

[0039] In the present application, the term "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have been genetically modified, either naturally or artificially, and is a microorganism in which a specific mechanism has been weakened or enhanced by, for example, inserting an exogenous gene or enhancing or weakening the activity of an endogenous gene, and is a microorganism that has been genetically modified to produce a desired polypeptide, protein, or product.

[0040] As an example of the present application, the microorganism of the present application may have an attenuated activity of carbamoyl phosphate synthetase. The microorganism may have an attenuated activity of at least one selected from the group consisting of CarA and CarB. For example, the microorganism of the present application may have an attenuated activity of CarA and CarB, or the carAB operon genes encoding them, compared to the endogenous activity, but is not limited thereto.

[0041] In the present application, the term "attenuation" of the activity of a polypeptide (including, for example, proteins specified by the name of each enzyme) is a concept that includes all of the following: a decrease in activity compared to the endogenous activity, or the absence of activity. The term "attenuation" is also used interchangeably with other terms such as inactivation, deficiency, down-regulation, decrease, reduction, and attenuation.

[0042] The weakening includes at least one of the following: the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide originally possessed by the microorganism due to a mutation in the polynucleotide encoding the polypeptide, the degree and / or concentration (expression amount) of the overall polypeptide activity in the cell is reduced compared to the natural strain due to inhibition of gene expression of the encoding polynucleotide or inhibition of translation into the polypeptide, the complete absence of expression of the polynucleotide, and the absence of polypeptide activity even if the polynucleotide is expressed. The "endogenous activity" refers to the activity of a specific polypeptide originally possessed by a parent strain, a wild type, or an unmodified microorganism before the trait change when the trait is changed by genetic mutation due to natural or artificial factors. This is used interchangeably with "activity before modification." The activity of a polypeptide is "weakened," "inactivated," "lacking," "reduced," "down-regulated," "decreased," or "attenuated" compared to the endogenous activity means that the activity of a specific polypeptide is reduced compared to the activity of a specific polypeptide originally possessed by a parent strain or an unmodified microorganism before the trait change.

[0043] For the purpose of this application, the microorganism of this application has improved L-amino acid production ability due to attenuated activity of carbamoyl phosphate synthetase, and the subject strains for comparison as to whether the L-amino acid production ability is improved or not include, but are not limited to, the wild-type Corynebacterium glutamicum ATCC13869 strain, the Corynebacterium glutamicum ATCC13869 strain with attenuated activity of ArgF protein, which is known as an L-ornithine producing strain, and the Corynebacterium glutamicum ATCC13869 strain with attenuated activity of ArgR protein.

[0044] Such attenuation of the activity of a polypeptide can be achieved by applying various methods well known in the art, including, but not limited to, those described in Non-Patent Documents 14 and 15.

[0045] Specifically, the activity of a polypeptide of the present application can be attenuated by 1) deleting all or a part of a gene encoding the polypeptide, 2) modifying an expression regulatory region (or an expression regulatory sequence) so as to reduce the expression of a gene encoding the polypeptide, 3) modifying an amino acid sequence constituting the polypeptide so as to delete or attenuate the activity of the polypeptide (e.g., deleting / substituting / adding one or more amino acids in the amino acid sequence), or 4) modifying a gene sequence encoding the polypeptide so as to delete or attenuate the activity of the polypeptide (e.g., deleting one or more nuclei of the nucleic acid base sequence of the polypeptide gene so as to encode a polypeptide modified so as to delete or attenuate the activity of the polypeptide). The modification can be carried out by, but is not limited to, the following: deleting / substituting / adding an acid or base; 5) modifying the base sequence encoding the start codon, Shine-Dalgarno sequence, or 5'UTR region of the gene transcript encoding the polypeptide; 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide; 7) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding the polypeptide so that a secondary structure that makes ribosome attachment impossible is formed; 8) adding a promoter to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide so that it is reverse transcribed (reverse transcription engineering, RTE); or 9) a combination of two or more selected from 1) to 8) above.

[0046] For example, 1) deleting a part or the whole of the gene encoding the polypeptide may be carried out by deleting the entire polynucleotide encoding the endogenous target polypeptide in a chromosome, or by replacing it with a polynucleotide lacking some nucleotides or a marker gene.

[0047] The expression regulatory region (or expression regulatory sequence) may be modified by generating a mutation in the expression regulatory region (or expression regulatory sequence) through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by substituting a sequence having a weaker activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0048] The modification of the amino acid sequence or polynucleotide sequence in 3) and 4) above can be performed by, but is not limited to, generating a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide so as to weaken the activity of the polypeptide, or by replacing the sequence with an amino acid sequence or polynucleotide sequence improved to have a weaker activity, or an amino acid sequence or polynucleotide sequence improved to have no activity. For example, gene expression can be inhibited or weakened by, but is not limited to, introducing a mutation into a polynucleotide sequence to form a stop codon.

[0049] The nucleotide sequence encoding the start codon or 5'UTR region of the gene transcription product encoding the polypeptide (5) can be modified, for example, by substituting it with a nucleotide sequence encoding another start codon that has a lower expression rate of the polypeptide compared to the endogenous start codon, but is not limited to this.

[0050] 6) Introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcription product encoding the polypeptide may be described, for example, by referring to Non-Patent Document 16.

[0051] 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide so that a secondary structure is formed that makes ribosome attachment impossible may be achieved by making mRNA translation impossible or slowing down the rate.

[0052] In addition, the above 8) adding a promoter to the 3' end of the ORF (open reading frame) of a gene sequence encoding a polypeptide so as to reverse transcribe the ORF (reverse transcription engineering, RTE) may be carried out by creating an antisense nucleotide complementary to the gene transcription product encoding the polypeptide to weaken the activity.

[0053] As an example, the activity of the carbamoyl phosphate synthetase of the present application may be weakened by modifying the start codon of the gene transcript encoding the carbamoyl phosphate synthetase, specifically, by replacing the start codon of the carAB operon gene transcript encoding the carbamoyl phosphate synthetase with another start codon with a low polypeptide expression rate. As another example, the activity of the carbamoyl phosphate synthetase of the present application may be weakened by modifying the promoter of the gene encoding the carbamoyl phosphate synthetase, or by replacing it with a weakened promoter, but is not limited thereto.

[0054] In the microorganism of the present application, modification of a part or the whole of a polynucleotide can be induced by (a) homologous recombination using a vector for chromosomal introduction in the microorganism, or genome editing using engineered nuclease (e.g., CRISPR-Cas9), and / or (b) light such as ultraviolet light or radiation and / or chemical treatment, but is not limited thereto. Methods for modifying a part or the whole of the gene include methods using DNA recombination techniques. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene is introduced into the microorganism to cause homologous recombination, thereby deleting a part or the whole of the gene. The introduced nucleotide sequence or vector contains a dominant selection marker, but is not limited thereto.

[0055] The vector of the present application means a DNA product comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host. The expression control region includes a promoter for initiating transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating the termination of transcription and translation. When transformed into a suitable host cell, the vector can replicate or function independently of the host genome, and is integrated into the genome itself.

[0056] The vector used in the present application is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. may be used as phage or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc. may be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. may be used.

[0057] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome by a vector for intracellular chromosome introduction. The polynucleotide can be inserted into a chromosome by any method known in the art, such as, but not limited to, homologous recombination. A selection marker for confirming whether or not it has been inserted into the chromosome may be further included. The selection marker is for selecting cells transformed with the vector, i.e., for confirming whether or not the target nucleic acid molecule has been inserted, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide is used. In an environment treated with a selection agent, only cells expressing the selection marker survive or show a different phenotype, so that transformed cells can be selected.

[0058] In the present application, "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or a microorganism, thereby expressing the polypeptide encoded by the polynucleotide in the host cell. The transformed polynucleotide may be any polynucleotide that is expressed in the host cell, regardless of whether it is inserted into the chromosome of the host cell or located extrachromosomally. The polynucleotide may include DNA and / or RNA that encodes the target polypeptide. The polynucleotide may be introduced in any form as long as it is introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic structure containing all elements necessary for its own expression. Typically, the expression cassette contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. The polynucleotide may be introduced into the host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0059] Additionally, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target polypeptide of the present application.

[0060] In another embodiment of the present application, the microorganism of the present application may be a microorganism capable of producing an L-amino acid.

[0061] The microorganism of the present application may be a microorganism with improved L-amino acid producing ability.

[0062] The microorganism of the present application may have an attenuated activity of carbamoyl phosphate synthetase. The microorganism may have an attenuated activity of at least one selected from the group consisting of CarA and CarB. Specifically, the microorganism of the present application may be a microorganism in which CarA and CarB, or the carAB operon genes encoding them, are attenuated, or may be a microorganism (e.g., a recombinant microorganism) genetically modified so that CarA and CarB, or the carAB operon genes encoding them, are attenuated, but is not limited thereto.

[0063] The microorganism of the present application may be a microorganism that naturally has L-amino acid production ability, or a microorganism in which L-amino acid production ability has been imparted to a parent strain that does not have L-amino acid production ability by weakening carbamoyl phosphate synthetase or a polynucleotide encoding the same, but is not limited thereto.

[0064] For the purposes of this application, a recombinant microorganism of the present application is a naturally occurring wild-type microorganism or a microorganism that produces L-amino acids containing a carbamoyl phosphate synthetase or a polynucleotide encoding the same, in which the carbamoyl phosphate synthetase or the polynucleotide encoding the same is weakened, and the microorganism has improved L-amino acid production ability compared to the naturally occurring wild-type microorganism or a microorganism that produces L-amino acids containing a carbamoyl phosphate synthetase or a polynucleotide encoding the same, but is not limited thereto. For example, the subject strains for comparison as to whether the L-amino acid producing ability is improved and which are unmodified microorganisms in which carbamoyl phosphate synthetase is not weakened include the wild-type Corynebacterium glutamicum ATCC13869 strain, the Corynebacterium glutamicum ATCC13869 strain with weakened ArgF protein activity, which is known as an L-ornithine producing strain, and the Corynebacterium glutamicum ATCC13869 strain with weakened ArgR protein activity, but are not limited to these.

[0065] As an example, the recombinant strain with improved production ability has an L-amino acid production ability of about 1% or more, specifically about 2% 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 13% or more, about 15% or more, about 16% or more, about 17% or more, about 18% or more, about 19% or more, about 20% or more, about 21% or more, about 24% or more, about 25% or more, about 27% or more, about 30% or more, about 31% or more, about 35% or more, about 40% or more, about 4 ... The improvement is 9% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 97% or more (there is no particular limitation on the upper limit, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, or about 15% or less), but any increase in the + value compared to the productivity of the parent strain or unmodified microorganism before the mutation is acceptable. In other examples, the microorganism with improved production ability has an L-amino acid producing ability that is about 1.01-fold or more, about 1.02-fold or more, about 1.05-fold or more, about 1.06-fold or more, about 1.07-fold or more, about 1.08-fold or more, about 1.09-fold or more, about 1.1-fold or more, about 1.13-fold or more, about 1.15-fold or more, about 1.16-fold or more, about 1.17-fold or more, about 1.18-fold or more, about 1.19-fold or more, about 1.20-fold or more, about 1.21-fold or more, about 1.24-fold or more, about 1.25-fold or more, about 1.27-fold or more, about 1.28-fold or more, about 1.29-fold or more, about 1.30-fold or more, about 1.31-fold or more, about 1.32-fold or more, about 1.33-fold or more, about 1.34-fold or more, about 1.35-fold or more, about 1.36-fold or more, about 1.37-fold or more, about 1.38-fold or more, about 1.39-fold or more, about 1.40-fold or more, about 1.41-fold or more, about 1.42-fold or more, about 1.43-fold or more, about 1.44-fold or more, about 1.45-fold or more, about 1.46-fold or more, about 1.47-fold or more, about 1.48-fold or more, about 1.49-fold or more, about 1.50-fold or more, about 1.51-fold or more, about 1.52-fold or more, about 1.53 0.30 times or more, about 1.31 times or more, about 1.35 times or more, about 1.40 times or more, about 1.45 times or more, about 1.49 times or more, about 1.50 times or more, about 1.55 times or more, about 1.60 times or more, about 1.65 times or more, about 1.70 times or more, about 1.75 times or more, about 1.80 times or more, about 1.85 times or more, about 1.90 times or more, about 1.95 times or more, or about 1.97 times or more (there is no particular limit to the upper limit, and it is, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less), but is not limited to these.

[0066] In the present application, the term "unmodified microorganism" does not exclude strains containing naturally occurring mutations in microorganisms, but refers to a wild-type or naturally occurring strain itself, or a strain before its characteristics are changed by genetic mutation due to natural or artificial factors. For example, the unmodified microorganism refers to a strain in which the carbamoyl phosphate synthetase or the polynucleotide encoding it described herein is not attenuated or has not been attenuated yet. The term "unmodified microorganism" is used interchangeably with "strain before modification", "microorganism before modification", "non-mutated strain", "non-modified strain", "non-mutated microorganism" or "reference microorganism".

[0067] In the present application, the L-amino acid may be at least one selected from the group consisting of L-ornithine, L-glutamic acid, L-aspartic acid, and L-phenylalanine.

[0068] As yet another example of the present application, the microorganism of the present application is Corynebacterium glutamicum, Corynebacterium stationis, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens, specifically but not limited to Corynebacterium glutamicum.

[0069] As yet another example of the present application, the recombinant microorganism of the present application may be a microorganism whose L-amino acid production ability is enhanced by further enhancing the activity of a part of a protein in an L-amino acid biosynthetic pathway or by further weakening the activity of a part of a protein in an L-amino acid degradation pathway.

[0070] Specifically, the microorganism of the present application may be a microorganism in which the activity of ornithine carbamoyltransferase subunit F (ArgF) is further weakened or the argF gene encoding it is further deleted, or the microorganism may be a microorganism in which the activity of arginine repressor (ArgR) is further weakened or the argR gene encoding it is further deleted.

[0071] The amino acid sequence of ArgF or ArgR can be obtained from a known database such as NCBI Genebank. For example, the amino acid sequence of ArgF in the present application includes ANU33618.1 (SEQ ID NO: 19) derived from Corynebacterium glutamicum ATCC13869 or an amino acid sequence having 90% or more homology thereto, and the amino acid sequence of ArgR includes ANU33619.1 (SEQ ID NO: 24) derived from Corynebacterium glutamicum ATCC13869 or an amino acid sequence having 90% or more homology thereto, but is not limited thereto, and it goes without saying that proteins having ArgF or ArgR activity of various origins are included.

[0072] However, the weakening of the ArgF and / or ArgR protein activity, or the deletion of the argF and / or argR genes are merely examples and are not intended to be limiting. The microorganism of the present application may be a microorganism in which the protein activity of various known L-amino acid biosynthetic pathways is enhanced, or a microorganism in which the protein activity of a degradation pathway is weakened.

[0073] In the present application, "enhancing" the activity of a polypeptide means improving the activity of the polypeptide compared to its endogenous activity. The term "enhancing" is used interchangeably with "activation," "up-regulation," "overexpression," "increase," and the like. Here, activation, enhancement, up-regulation, overexpression, and increase all include exhibiting an activity that was not originally present, and improving the activity compared to the endogenous activity or the activity before modification. The term "endogenous activity" refers to the activity of a specific polypeptide that was originally possessed by a parent strain or an unmodified microorganism before the trait change when the trait is changed due to genetic mutation caused by natural or artificial factors. This term is used interchangeably with "activity before modification." The term "enhancing," "up-regulating," "overexpressing," or "improving" the activity of a polypeptide compared to its endogenous activity means improving the activity and / or concentration (expression level) of a specific polypeptide that was originally possessed by a parent strain or an unmodified microorganism before the trait change.

[0074] The enhancement may be achieved by introducing a foreign polypeptide, or by enhancing the activity and / or increasing the concentration (expression level) of an endogenous polypeptide. Whether the activity of the polypeptide has been enhanced can be confirmed by an increase in the level of activity of the polypeptide, the expression level, or the amount of a product produced from the polypeptide.

[0075] To enhance the activity of the polypeptide, various methods well known in the art can be applied, and any method can be used as long as it can enhance the activity of the target polypeptide compared to the unmodified microorganism. Specifically, the method is a conventional method in molecular biology that uses genetic engineering and / or protein engineering well known to those skilled in the art, but is not limited to these (e.g., Non-Patent Documents 15, 17, etc.).

[0076] Specifically, the enhancement of the polypeptide activity of the present application is carried out by 1) increasing the intracellular copy number of a polynucleotide encoding the polypeptide, 2) replacing the expression regulatory region of a gene on a chromosome encoding the polypeptide with a sequence having a stronger activity, 3) modifying the base sequence encoding the start codon or 5'UTR region of a gene transcript encoding the polypeptide, 4) modifying the amino acid sequence of the polypeptide so as to enhance the polypeptide activity, 5) modifying the polynucleotide sequence encoding the polypeptide so as to enhance the polypeptide activity (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the polypeptide activity), 6) introducing a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same, 7) optimizing the codons of the polynucleotide encoding the polypeptide, 8) analyzing the tertiary structure of the polypeptide and selecting and modifying or chemically modifying exposed portions, or 9) a combination of two or more selected from 1) to 8) above, but is not particularly limited thereto.

[0077] More specifically, the above 1) increasing the intracellular copy number of the polynucleotide encoding the polypeptide may be achieved by introducing into the host cell a vector to which the polynucleotide encoding the polypeptide is operably linked, the vector replicating and functioning independently of the host. Alternatively, the above may be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide into a chromosome in the host cell. The introduction into the chromosome is achieved by introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome in the host cell, but is not limited thereto. The vector is as described above.

[0078] The expression regulatory region (or expression regulatory sequence) of the gene on the chromosome encoding the polypeptide (2) may be replaced with a sequence having a stronger activity, for example, by generating a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so that the activity of the expression regulatory region is further enhanced, or by replacing the expression regulatory region with a sequence having a higher activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating the termination of transcription and translation, and the like. For example, the expression regulatory region may be replaced with a strong promoter, but is not limited to this.

[0079] 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.

[0080] The nucleotide sequence encoding the start codon or 5'UTR region of the gene transcription product encoding the polypeptide (3) can be modified, for example, by substituting it with a nucleotide sequence encoding another start codon that has a higher expression rate of the polypeptide compared to the endogenous start codon, but is not limited to this.

[0081] The modification of the amino acid sequence or polynucleotide sequence in 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 polypeptide or the polynucleotide sequence encoding the polypeptide, so as to enhance the activity of the polypeptide, or by replacing the sequence with an improved amino acid sequence or polynucleotide sequence having a higher activity, or an improved amino acid sequence or polynucleotide sequence having an improved activity. Specifically, the replacement can be performed by, but is not limited to, inserting the polynucleotide into a chromosome by homologous recombination. Here, the vector used may further include a selection marker for checking whether it has been inserted into the chromosome.

[0082] The above 6) introduction of a foreign polynucleotide exhibiting the activity of the polypeptide may be carried out by introducing into a host cell a foreign polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide. The foreign polynucleotide may be of any origin or sequence as long as it exhibits the same / similar activity as the polypeptide. The introduction can be carried out by a person skilled in the art by appropriately selecting a known transformation method, and the introduced polynucleotide is expressed in the host cell as described above, thereby producing a polypeptide and improving its activity.

[0083] 7) Optimizing the codons of a polynucleotide encoding a polypeptide may be carried out by optimizing the codons of an endogenous polynucleotide so as to increase transcription or translation in a host cell, or by optimizing the codons of an exogenous polynucleotide so as to perform optimized transcription or translation in a host cell.

[0084] 8) Analyzing the tertiary structure of a polypeptide and selecting and altering or chemically modifying an exposed portion may be carried out, for example, by comparing sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored, determining candidates for template proteins depending on the degree of sequence similarity, confirming the structure based on the candidates, and selecting and altering or modifying the exposed portion to be altered or chemically modified.

[0085] Such enhancement of polypeptide activity can be achieved by, but is not limited to, improving the activity, concentration, or expression level of the corresponding polypeptide compared to the activity or concentration of the polypeptide expressed in a wild-type or unmodified microbial strain, or by increasing the amount of a product produced from the polypeptide.

[0086] Another aspect of the present application provides a method for producing an L-amino acid, comprising the step of culturing in a medium a Corynebacterium microorganism having an ability to produce an L-amino acid and an attenuated activity of carbamoyl phosphate synthetase.

[0087] The method for producing an L-amino acid of the present application may include a step of culturing in a medium a microorganism in which CarA and CarB, or the carAB operon genes encoding them, are attenuated, or a microorganism genetically modified to attenuate CarA and CarB, or the carAB operon genes encoding them. The microorganism is as described above.

[0088] "Culturing" in the present application means growing the microorganism of the present application under appropriately adjusted environmental conditions. The culturing process of the present application can be carried out in a suitable medium and under suitable culture conditions known in the art. Such a culturing process can be easily adjusted and used by a person skilled in the art depending on the microorganism selected. Specifically, the culturing can be batch, continuous and / or fed-batch culture, but is not limited thereto.

[0089] In the present application, the term "culture medium" refers to a substance that is a mixture of nutrients necessary for culturing the microorganism of the present application as the main components, and that supplies nutrients such as water essential for survival and growth, growth factors, etc. Specifically, the culture medium and other culture conditions used for culturing the microorganism of the present application may be any medium used for culturing ordinary microorganisms, and the microorganism of the present application can be cultured in an ordinary culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, etc., under aerobic conditions by adjusting the temperature, pH, etc.

[0090] Specifically, a culture medium for Corynebacterium microorganisms is disclosed in Non-Patent Document 18.

[0091] In the present application, the carbon source may be carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc., sugar alcohols such as mannitol, sorbitol, etc., organic acids such as pyruvic acid, lactic acid, citric acid, etc., amino acids such as glutamic acid, methionine, lysine, etc. Also, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, corn steeping liquid, etc. may be used, specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and any other suitable carbon source may be used. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0092] The nitrogen source may be an inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, or ammonium nitrate, or an organic nitrogen source such as an amino acid such as glutamic acid, methionine, or glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its decomposition product, or defatted soybean cake or its decomposition product. These nitrogen sources may be used alone or in combination of two or more kinds, but are not limited thereto.

[0093] The phosphorus source may be potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or a sodium-containing salt equivalent thereto. The inorganic compound may be sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, or other suitable compounds, including amino acids, vitamins, and / or suitable precursors. These components or precursors may be added to the medium in a batch or continuous manner. However, they are not limited thereto.

[0094] In addition, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the medium in a suitable manner during the cultivation of the microorganism of the present application. Furthermore, during the cultivation, foam formation can be suppressed using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, and in order to maintain the anaerobic and microaerobic state, no gas may be injected, and nitrogen, hydrogen, or carbon dioxide gas may be injected, but is not limited thereto.

[0095] 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.

[0096] The L-amino acids produced by the culture of the present application are either secreted into the medium or remain intracellularly.

[0097] The L-amino acid may be at least one selected from the group consisting of L-ornithine, L-glutamic acid, L-aspartic acid, and L-phenylalanine.

[0098] The L-amino acid production method of the present application may further comprise a step of preparing a microorganism of the present application, a step of preparing a medium for culturing said microorganism, or a combination thereof (in any order), e.g., prior to said culturing step.

[0099] The method for producing an L-amino acid of the present application may further include a step of recovering the L-amino acid from the medium used in the culture (the medium in which the culture was carried out) or from the cultured microorganism. The recovery step may be further included after the culture step.

[0100] The recovery may involve collecting the target L-amino acid using a suitable method known in the art depending on the culture method of the microorganism of the present application, such as batch, continuous, or fed-batch culture. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographies such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination thereof can be used, and the target L-amino acid can be collected from the medium or the microorganism using a suitable method known in the art.

[0101] In addition, the L-amino acid 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-amino acid 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.

[0102] The carbamoyl phosphate synthetase, polynucleotide, vector, microorganism, etc. in the method of the present application are as described above.

[0103] Yet another aspect of the present application provides a composition for producing an L-amino acid, comprising a Corynebacterium microorganism having attenuated carbamoyl phosphate synthetase, a medium in which the microorganism is cultured, or a combination thereof.

[0104] The composition of the present application may further contain any suitable excipient that is commonly used in compositions for producing L-amino acids, including, but not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, isotonicity agents, etc.

[0105] Yet another aspect of the present application provides a method for producing a Corynebacterium microorganism having an ability to produce an L-amino acid, the method comprising the step of attenuating carbamoyl phosphate synthetase.

[0106] Yet another aspect of the present application provides use of a Corynebacterium microorganism with an attenuated carbamoyl phosphate synthetase activity for producing an L-amino acid.

[0107] The carbamoyl phosphate synthetase, the weakening, the Corynebacterium microorganism, etc. are as described above. EXAMPLES

[0108] The present application will be described in more detail below with reference to examples. However, these examples are merely preferred embodiments illustrating the present application, and the present application is not limited thereto. Note that technical matters not described in this specification are fully understood and easily implemented by skilled engineers in the technical field of the present application or a similar technical field. EXAMPLES

[0109] Vector construction Example 1-1: Construction of a vector for carAB promoter replacement A vector was constructed to replace the promoter of the carA gene encoding the small subunit of carbamoyl phosphate synthase (CarA). Information on the carAB genes (SEQ ID NO: 3) encoding CarA (ANU33813.1, SEQ ID NO: 1) and the adjacent nucleic acid sequence (NZ_CP016335.1) was obtained from the National Institutes of Health GenBank, and using the information, a vector was constructed to replace the promoter site of carA with the promoter of betP (ANU33153.1) (SEQ ID NO: 4). Using the genome of wild-type C. glutamicum ATCC 13869 as a template, the betP promoter was generated using the primer pair of SEQ ID NOs: 5 and 6, the homologous recombinant A arm was generated using the primer pair of SEQ ID NOs: 7 and 8, and the homologous recombinant B arm was generated using the primer pair of SEQ ID NOs: 9 and 10 by PCR (Sol TM The DNA fragments were amplified using Pfu-X DNA polymerase.

[0110] As described above, the obtained promoter and recombinant arm gene fragments were cloned into the vector pDCM2 (Patent Document 5) cleaved with SalI and BamHI restriction enzymes by the Gibson assembly method (Non-Patent Document 19) to prepare a vector. The prepared vector was transformed into Escherichia coli DH5α and spread on a kanamycin (25 mg / l)-containing LB solid medium. To select colonies transformed with the vector, PCR was performed using the primer pair of SEQ ID NOs: 11 and 12, and a plasmid was obtained from the selected colonies by a conventional plasmid extraction method. As described above, the obtained plasmid was named pDCM2-PbetP-carA.

[0111] Example 1-2: Construction of a vector for carA start codon mutation A vector was constructed to replace the start codon gtg of gene carA with ttg. The genome of wild-type C. glutamicum ATCC 13869 was used as a template to amplify the homologous recombinant A arm using the primer pair of SEQ ID NO: 7 and 13, and the homologous recombinant B arm using the primer pair of SEQ ID NO: 10 and 14. Then, a plasmid was obtained as described above. It was named pDCM2-carA(g1t).

[0112] Example 1-3: Construction of a vector for carB start codon mutation A vector was constructed to replace the initiation codon atg of gene carB with ttg. The carAB gene (SEQ ID NO: 3) encoding the large subunit of carbamoyl phosphate synthase (SEQ ID NO: 2) and information on the adjacent nucleic acid sequence (NZ_CP016335.1) were obtained from the National Institutes of Health gene bank. Using the genome of wild-type C. glutamicum ATCC 13869 as a template, the homologous recombinant A arm was amplified using the primer pair of SEQ ID NO: 15 and 16, and the homologous recombinant B arm was amplified using the primer pair of SEQ ID NO: 17 and 18. Then, a plasmid was obtained as described above. It was named pDCM2-carB(a1t).

[0113] The primer sequences used in Examples 1-1 to 1-3 are shown in Table 1.

[0114] [Table 1] EXAMPLES

[0115] Construction of L-ornithine-producing strains Example 2-1: Construction of a vector for ArgF inactivation To generate an L-ornithine producing strain, a vector was constructed to replace the serine at position 55 of the protein sequence of ArgF (ANU33618.1, SEQ ID NO: 19) with a stop codon. Using the genome of wild-type C. glutamicum ATCC13869 as a template, the homologous recombinant A arm was amplified using the primer pair of SEQ ID NO: 20 and 21, and the homologous recombinant B arm was amplified using the primer pair of SEQ ID NO: 22 and 23. Then, a plasmid was obtained as described above. It was named pDCM2-argF(S55*).

[0116] Example 2-2: Construction of a vector for ArgR inactivation A vector was constructed to replace the glutamic acid at position 47 of the protein sequence of ArgR (ANU33619.1, SEQ ID NO: 24) with a stop codon. Using the genome of wild-type C. glutamicum ATCC 13869 as a template, the homologous recombinant A arm was amplified using the primer pair of SEQ ID NO: 25 and 26, and the homologous recombinant B arm was amplified using the primer pair of SEQ ID NO: 27 and 28. Then, a plasmid was obtained as described above. The plasmid was named pDCM2-argR(E47*).

[0117] The primer sequences used in Examples 2-1 and 2-2 are shown in Table 2.

[0118] [Table 2]

[0119] Example 2-3: Preparation of L-ornithine-producing strain To prepare an L-ornithine producing strain, the wild type C. glutamicum ATCC 13869 was transformed by electroporation (Non-Patent Document 20) using the pDCM2-argF(S55*) vector prepared in Example 2-1, and then a secondary crossover process was performed to obtain a strain in which the 164th base sequence of argF was replaced by adenine (a) instead of cytosine (c) and the 55th protein sequence was replaced by a stop codon. PCR and base sequence analysis were performed using a primer pair of SEQ ID NOs: 20 and 23, which amplify the adjacent site including the position where the gene was inserted, to confirm the genetic manipulation. The strain obtained in this manner was named C. gl::argF*.

[0120] In order to prepare a strain with improved ornithine production in C.gl::argF*, the pDCM2-argR(E47*) vector prepared in Example 2-2 was used to obtain a strain by the method described above. PCR and base sequence analysis were performed using the primer pair of SEQ ID NOs: 25 and 28, which amplify the adjacent site including the position where the gene was inserted, to confirm the genetic manipulation. The strain thus obtained was named C.gl::argF*_argR*. EXAMPLES

[0121] Generation of CarAB-attenuated strains The pDCM2-PbetP-carA and pDCM2-carA(g1t) vectors prepared in Examples 1-1 and 1-2 were transformed into wild-type C. glutamicum ATCC 13869, C. gl::argF* and C. gl::argF*_argR* prepared in Example 2-3 by electroporation, and then a secondary crossover process was performed to obtain strains. PCR and base sequence analysis were performed using the primer pair of SEQ ID NOs: 6 and 9 to confirm the genetic manipulation. The strains obtained in this manner were named C.gl::PbetP-carA, C.gl::carA(g1t), C.gl::argF*_PbetP-carA, C.gl::argF*_carA(g1t), C.gl::argF*_argR*_PbetP-carA, and C.gl::argF*_argR*_carA(g1t), respectively.

[0122] To prepare the carB promoter and start codon replacement strains, the plasmid pDCM2-carB(a1t) vector obtained in Example 1-3 was used to transform wild-type C. glutamicum ATCC 13869, C. gl::argF*, and C. gl::argF*_argR* by electroporation, and then a secondary crossover process was performed to obtain strains. PCR and base sequence analysis were performed using the primer pair of SEQ ID NOs: 16 and 19, which amplify the adjacent site including the position where the gene was inserted, to confirm the genetic manipulation. The strains obtained in this manner were named C. gl::carB(a1t), C. gl::argF*_carB(a1t), and C. gl::argF*_argR*_carB(a1t), respectively. EXAMPLES

[0123] Evaluation of L-amino acid production ability of recombinant strains The C.gl::PbetP-carA, C.gl::carA(g1t), and C.gl::carB(a1t) strains prepared in Example 3 and the wild-type C. glutamicum control strain were cultured, and the bacterial mass and L-amino acid productivity were compared.

[0124] First, each strain was inoculated into a 250-ml corner baffle flask containing 25 ml of seed medium, and cultured with shaking at 200 rpm at 30° C. for 16 hours. 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 200 rpm at 33° C. for 40 hours.

[0125] After the cultivation was completed, the production amounts of 20 L-amino acids were measured by HPLC. <Seed medium (pH 7.0)> Glucose 20g, peptone 10g, yeast extract 5g, urea 1.5g, KH2PO4 4g, K2HPO48g, MgSO4·7H2O 0.5g, biotin 0.1mg, thiamine HCl 1mg, calcium pantothenate 22mg, nicotinamide 2mg (in 1 liter of distilled water) <Production medium (pH 7.0)> Raw sugar 50g, (NH4)2SO4 25g, yeast extract 1g, KH2PO4 0.55g, MgSO4·7H2O 0.6g, biotin 0.9mg, thiamine hydrochloride 4.5mg, calcium pantothenate 4.5mg, nicotinamide 30mg, MnSO4 9mg, FeSO4 9mg, ZnSO4 0.45mg, CuSO4 0.45mg, CaCO3 30g (in 1 liter of distilled water)

[0126] The above experiment was repeated three times. The culture results (average values) are shown in Table 3. The concentration increase rate of each L-amino acid was calculated as (experimental group - control group) / control group.

[0127] [Table 3]

[0128] As shown in Table 3, the C.gl::PbetP-carA, C.gl::carA(g1t), and C.gl::carB(a1t) strains had improved L-glutamic acid, L-aspartic acid, and L-phenylalanine production abilities and yields compared to the control strain, wild-type C. glutamicum ATCC 13869. EXAMPLES

[0129] Evaluation of L-ornithine producing ability of recombinant strains The C.gl::argF*_PbetP-carA, C.gl::argF*_carA(g1t), C.gl::argF*_carB(a1t), C.gl::argF*_argR*_PbetP-carA, C.gl::argF*_argR*_carA(g1t), C.gl::argF*_argR*_carB(a1t) strains prepared in Example 3, and the control strains C.gl::argF* and C.gl::argF*_argR* were cultured using the following medium in the same manner as in Example 4, and the bacterial mass and L-ornithine production ability were compared. <Seed medium (pH 7.0)> Glucose 20g, peptone 10g, yeast extract 5g, urea 1.5g, KH2PO4 4g, K2HPO48g, MgSO4·7H2O 0.5g, biotin 0.1mg, thiamine HCl 1mg, calcium pantothenate 22mg, nicotinamide 2mg (in 1 liter of distilled water) <Production medium (pH 7.0)> Raw sugar 50g, (NH4)2SO4 25g, yeast extract 1g, KH2PO4 0.55g, MgSO4·7H2O 0.6g, L-arginine 0.2g, biotin 0.9mg, thiamine hydrochloride 4.5mg, calcium pantothenate 4.5mg, nicotinamide 30mg, MnSO4 9mg, FeSO4 9mg, ZnSO4 0.45mg, CuSO4 0.45mg, CaCO3 30g (in 1 liter of distilled water)

[0130] The above experiment was repeated three times. The culture results (average values) are shown in Table 4. The L-ornithine yield was calculated by ornithine concentration / consumed sugar, and the concentration increase rate was calculated by (L-ornithine concentration produced by the experimental group-control group (L-ornithine concentration produced by the wild-type C. glutamicum ATCC 13869 strain)) / control group (L-ornithine concentration produced by the wild-type C. glutamicum ATCC 13869 strain).

[0131] [Table 4]

[0132] As shown in Table 4, the C.gl::argF*_PbetP-carA, C.gl::argF*_carA(g1t), and C.gl::argF*_carB(a1t) strains showed improved productivity and yield compared to the control strain C.gl::argF*. Similarly, the C.gl::argF*_argR*_PbetP-carA, C.gl::argF*_argR*_carA(g1t), and C.gl::argF*_argR*_carB(a1t) strains showed improved productivity and yield compared to the control strain C.gl::argF*_argR*.

[0133] From the above description, a person skilled in the art to which the present application pertains will understand that the present application can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. This application should be interpreted as including all modifications or alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

[0134] [Sequence table] JPEG2025509684000005.jpg228170JPEG2025509684000006.jpg241170JPEG2025509684 000007.jpg241170JPEG2025509684000008.jpg241170JPEG2025509684000009.jpg24117 0JPEG2025509684000010.jpg241170JPEG2025509684000011.jpg241170JPEG2025509684 000012.jpg241170JPEG2025509684000013.jpg241170JPEG2025509684000014.jpg41170

Claims

1. A microorganism of the genus Corynebacterium having an ability to produce an L-amino acid and in which the activity of carbamoyl phosphate synthetase is weakened.

2. The microorganism according to claim 1, wherein the L-amino acid is at least one selected from the group consisting of L-ornithine, L-glutamic acid, L-aspartic acid, and L-phenylalanine.

3. The microorganism according to claim 1, wherein the microorganism has attenuated activity of at least one selected from the group consisting of carbamoyl phosphate synthase small subunit (CarA) and carbamoyl phosphate synthase large subunit (CarB).

4. The microorganism of claim 1 , wherein the carbamoyl phosphate synthetase is encoded by a carAB operon gene.

5. The microorganism according to claim 3 , wherein CarA and CarB have the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively, or amino acid sequences having 90% or more homology thereto.

6. The microorganism according to claim 1, further comprising an attenuated activity of ornithine carbamoyltransferase subunit F (ArgF).

7. The microorganism according to claim 1 , further comprising an attenuated activity of an arginine repressor (ArgR).

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

9. A method for producing an L-amino acid, comprising the step of culturing in a medium a Corynebacterium microorganism having an ability to produce an L-amino acid and an attenuated activity of carbamoyl phosphate synthetase.

10. 10. The method according to claim 9, wherein the L-amino acid is at least one selected from the group consisting of L-ornithine, L-glutamic acid, L-aspartic acid, and L-phenylalanine.

11. The method according to claim 9, wherein the microorganism further has attenuated activity of ornithine carbamoyltransferase subunit F (ArgF).

12. The method according to claim 9 , wherein the microorganism further has attenuated activity of arginine repressor (ArgR).

13. The present invention relates to a composition for producing an L-amino acid, the composition comprising a microorganism of the genus Corynebacterium in which carbamoyl phosphate synthetase activity is attenuated, a medium in which the microorganism is cultured, or a combination thereof.

14. Use of a Corynebacterium sp. microorganism with weakened carbamoyl phosphate synthetase activity for producing an L-amino acid.

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