Carbamoyl phosphate synthetase large subunit mutant and method for producing L-ornithine using the same

Abstract

The present application relates to carbamoyl phosphate synthetase large subunit mutants, polynucleotides encoding the mutants, microorganisms containing the mutants or polynucleotides, and methods for producing L-ornithine using the same.

Description

[Technical field]

[0001] The present application relates to a carbamoyl phosphate synthetase large subunit mutant and a method for producing L-ornithine 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 glutamic acid as a substrate, and one molecule of ATP is consumed 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. Patent No. 10,273,491 [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-Patent Document 6] Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990)

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 novel carbamoyl phosphate synthetase large subunit mutant that increases L-ornithine production, a polynucleotide encoding the mutant, a Corynebacterium microorganism containing the mutant or the polynucleotide, and a method for producing L-ornithine using the microorganism, and have completed the present application. [Means for solving the problem]

[0010] The present application aims to provide a carbamoyl phosphate synthase large subunit (CarB) mutant derived from the genus Corynebacterium, which comprises a substitution of the amino acid corresponding to position 799 in the amino acid sequence of SEQ ID NO:1 with isoleucine, leucine or valine, a substitution of the amino acid corresponding to position 918 with leucine or valine, a substitution of the amino acid corresponding to position 1079 with isoleucine, leucine or valine, or a combination thereof.

[0011] The present application also aims to provide polynucleotides encoding the variants of the present application.

[0012] A further object of the present application is to provide a Corynebacterium microorganism comprising the mutant of the present application or a polynucleotide encoding the mutant.

[0013] Furthermore, the present application aims to provide a method for producing L-ornithine, comprising a step of culturing in a medium a Corynebacterium microorganism containing the mutant of the present application or a polynucleotide encoding the mutant.

[0014] Furthermore, the present application aims to provide a composition for producing L-ornithine, comprising any one selected from the group consisting of the mutant of the present application, a polynucleotide encoding the mutant, a vector comprising the polynucleotide, a microorganism comprising the mutant, a polynucleotide encoding the mutant, or a vector comprising the polynucleotide, a culture of the microorganism, and a combination of at least two thereof.

[0015] Furthermore, the present application aims to provide use of the microorganism of the present application for producing L-ornithine.

[0016] Furthermore, the present application aims to provide use of a carbamoyl phosphate synthase large subunit (CarB) mutant derived from the genus Corynebacterium, which comprises a substitution of the amino acid corresponding to position 799 in the amino acid sequence of SEQ ID NO:1 with isoleucine, leucine or valine, a substitution of the amino acid corresponding to position 918 with leucine or valine, a substitution of the amino acid corresponding to position 1079 with isoleucine, leucine or valine, or a combination thereof, for L-ornithine production. Effect of the Invention

[0017] A microorganism into which the mutant of the present application has been introduced has improved L-ornithine producing ability compared to a conventional unmodified microorganism.

[0018] 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.

[0019] One aspect of the present application provides a carbamoyl phosphate synthase large subunit (CarB) mutant derived from the genus Corynebacterium, which comprises, in the amino acid sequence of SEQ ID NO:1, a substitution of the amino acid corresponding to position 799 with another amino acid, a substitution of the amino acid corresponding to position 918 with another amino acid, a substitution of the amino acid corresponding to position 1079 with another amino acid, or a combination thereof.

[0020] The variant of the present application may be one in which at least one amino acid at the position corresponding to 799, 918, or 1079 in the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid different from the amino acid before substitution. Alternatively, the variant may be one having an uncharged amino acid side chain and substituted with an amino acid different from the amino acid before substitution, but is not limited thereto.

[0021] Specifically, the mutant has, in the amino acid sequence of SEQ ID NO: 1, i) a substitution of the amino acid corresponding to position 799 with at least one amino acid selected from the group consisting of arginine, lysine, histidine, aspartic acid, glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; or ii) a substitution of the amino acid corresponding to position 918 with at least one amino acid selected from the group consisting of arginine, lysine, histidine, aspartic acid, glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. or iii) the amino acid at position 1079 has been substituted with at least one amino acid selected from the group consisting of arginine, lysine, histidine, glutamic acid, aspartic acid, glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, cysteine, tyrosine, asparagine, and glutamine, but is not limited thereto.

[0022] More specifically, the mutant has, in the amino acid sequence of SEQ ID NO:1, i) a substitution of the amino acid corresponding to position 799 with at least one amino acid selected from the group consisting of alanine, isoleucine, leucine, and valine, ii) a substitution of the amino acid corresponding to position 918 with at least one amino acid selected from the group consisting of alanine, isoleucine, leucine, and valine, or iii) a substitution of the amino acid corresponding to position 1079 with at least one amino acid selected from the group consisting of alanine, isoleucine, leucine, and valine, but is not limited thereto.

[0023] More specifically, the variant of the present application may have an amino acid sequence represented by SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23 or SEQ ID NO:25, may contain the amino acid sequence, may consist of the amino acid sequence, or may essentially consist of the amino acid sequence.

[0024] The variant of the present application is an amino acid sequence represented by SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:25, in which the amino acid corresponding to the 799th position of the amino acid sequence of SEQ ID NO:1 is at least one selected from the group consisting of alanine, isoleucine, leucine, and valine, and the amino acid corresponding to the 918th position of the amino acid sequence is at least one selected from the group consisting of alanine, isoleucine, leucine, and valine. and the amino acid corresponding to position 1079 of the amino acid sequence is at least one selected from the group consisting of alanine, isoleucine, leucine, and valine, and the amino acid sequence has 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 shown in SEQ ID NO: 1. It goes without saying that the present application also includes amino acid sequences having such homology or identity and exhibiting efficacy equivalent to the variants of the present application, in which a part of the sequence has been deleted, modified, substituted, conservatively substituted, or added.

[0025] For example, the variants may have additions or deletions of sequences at the N-terminus, C-terminus and / or internally of the amino acid sequence that do not alter the function of the variants of the present application, naturally occurring mutations, silent mutations or conservative substitutions.

[0026] 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.

[0027] For example, among amino acids having electrically charged side chains, positively charged (basic) amino acids include arginine, lysine, and histidine, negatively charged (acidic) amino acids include glutamic acid and aspartic acid, and amino acids having uncharged side chains include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.

[0028] In the present application, the term "variant" refers to a polypeptide that differs from the amino acid sequence of the variant by conservative substitution and / or modification of at least one amino acid, but maintains its functions or properties. Such variants can generally be identified by modifying at least one amino acid in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant is improved, unchanged, or decreased compared to the polypeptide before the mutation. Some variants also include variants in which at least one portion, such as an N-terminal leader sequence or a transmembrane domain, has been removed. Other variants also include variants in which a portion has been removed from the N- and / or C-terminus of a mature protein. The term "variant" is used interchangeably with mutated, modified, mutant polypeptide, mutated protein, mutation, etc. (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), but may be any term that means a mutation. For the purposes of this application, the variant may be a polypeptide comprising an amino acid sequence represented by SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23 or SEQ ID NO:25, in which at least one of the following has been performed: substitution of glutamic acid, which is the amino acid corresponding to position 799 of the amino acid sequence of SEQ ID NO:1, with at least one amino acid selected from the group consisting of alanine, isoleucine, leucine and valine; substitution of glutamic acid, which is the amino acid corresponding to position 918, with at least one amino acid selected from the group consisting of alanine, isoleucine, leucine and valine; and substitution of threonine, which is the amino acid corresponding to position 1079, with at least one amino acid selected from the group consisting of alanine, isoleucine, leucine and valine.

[0029] The variant may also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the N-terminus of the variant may be linked to a signal (or leader) sequence involved in co-translationally or post-translationally protein translocation. The variant may also be linked to other sequences or linkers to allow identification, purification, or synthesis.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] As an example of the present application, the mutant of the present application may have improved L-ornithine production ability. Specifically, the mutant of the present application may have an activity that improves L-ornithine production ability compared to a wild-type polypeptide having carbamoyl phosphate synthetase activity.

[0035] In this application, "carbamoyl-phosphate synthetase" refers to an enzyme that converts bicarbonate to carbamoyl-phosphate.

[0036] 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.

[0037] As an example, the 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. For the purposes of the present application, a mutant of CarB from Corynebacterium glutamicum is provided.

[0038] As another example, the CarB amino acid sequence of the present application is ANU33812.1 derived from Corynebacterium glutamicum ATCC13869, but it goes without saying that proteins having CarB activity of various origins are included.

[0039] For purposes of this application, the mutants of this application may be carbamoyl phosphate synthetase large subunit mutants, i.e. CarB mutants.

[0040] The CarB 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.

[0041] The amino acid sequence of SEQ ID NO: 1 can be obtained from the publicly known database, NIH GenBank. In the present application, the amino acid sequence of SEQ ID NO: 1 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. It goes without saying that 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 an efficacy equivalent to that of a protein containing the amino acid sequence of SEQ ID NO: 1.

[0042] 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.

[0043] "Corresponding to" in this application means the amino acid residue at the recited position in the polypeptide, or an amino acid residue similar, identical or equivalent to the recited residue in the polypeptide. Identifying the amino acid at the corresponding position will determine the particular amino acid of the sequence to which the particular sequence is referenced. "Corresponding region" in this application generally refers to a similar or corresponding position in a related or reference protein.

[0044] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, and based thereon, each amino acid residue in the amino acid sequence can be numbered with reference to the number and position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, the sequence alignment algorithm in the present application can identify the positions of amino acids or positions where modifications such as substitutions, insertions, deletions, etc. occur when compared to a query sequence (referred to as a "reference sequence").

[0045] For such alignment, for example, the Needleman-Wunsch algorithm (Non-Patent Document 4) or the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 3) can be used, but is not limited to these. Sequence alignment programs and pairwise sequence comparison algorithms known in the technical field can be used as appropriate.

[0046] Another aspect of the present application provides polynucleotides encoding the variants of the present application.

[0047] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and means a DNA or RNA chain longer than a certain length, and more specifically, means a polynucleotide fragment encoding the mutant.

[0048] The polynucleotide encoding the variant of the present application may comprise a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:25. As an example of the present application, the polynucleotide of the present application may have or comprise the sequence represented by SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26. The polynucleotide of the present application may consist of the sequence represented by SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26, or may essentially consist of the sequence represented by SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26.

[0049] The polynucleotide of the present application may be modified in various ways in the coding region, taking into account codon degeneracy or preferred codons in an organism in which the variant of the present application is to be expressed, without changing the amino acid sequence of the variant of the present application. Specifically, the polynucleotide of the present application has or includes a base sequence that has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or less than 100% homology or identity to the sequence of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26, or is a base sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or less than 100% homology or identity to the sequence of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:26, or is substantially composed of the above base sequence, but is not limited thereto. Here, in the sequence having homology or identity, the codon encoding the amino acid corresponding to position 799 of SEQ ID NO: 1 may be one of the codons encoding at least one amino acid selected from the group consisting of alanine, isoleucine, leucine and valine, the codon encoding the amino acid corresponding to position 918 of SEQ ID NO: 1 may be one of the codons encoding at least one amino acid selected from the group consisting of alanine, isoleucine, leucine and valine, and the codon encoding the amino acid corresponding to position 1079 of SEQ ID NO: 1 may be one of the codons encoding at least one amino acid selected from the group consisting of alanine, isoleucine, leucine and valine.

[0050] 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, 6% 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 normal Southern hybridization washing conditions of 60°C, 1 x SSC, 0.1% SDS, specifically 60°C, 0.1 x SSC, 0.1% SDS, more specifically 68°C, 0.1 x SSC, 0.1% SDS.

[0051] 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.

[0052] 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.

[0053] 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).

[0054] Yet another aspect of the present application provides a vector comprising a polynucleotide of the present application, the vector being, but not limited to, an expression vector for expressing the polynucleotide in a host cell.

[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 variant of the present application.

[0060] Yet another aspect of the present application provides a microorganism comprising the variant of the present application or the polynucleotide of the present application.

[0061] 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 reduced or enhanced by, for example, inserting an exogenous gene or enhancing or inactivating the activity of an endogenous gene, and is a microorganism that has been genetically modified to produce a desired polypeptide, protein, or product.

[0062] A strain of the present application is, but is not limited to, a strain that comprises at least one of a variant of the present application, a polynucleotide of the present application, and a vector comprising a polynucleotide of the present application, a strain that has been modified to express a variant of the present application or a polynucleotide of the present application, a strain that expresses a variant of the present application or a polynucleotide of the present application (e.g., a recombinant strain), or a strain that has an activity of a variant of the present application (e.g., a recombinant strain).

[0063] As an example of the present application, the strain of the present application may be a strain capable of producing L-ornithine.

[0064] The microorganism of the present application may have improved L-ornithine producing ability.

[0065] The strain of the present application may be a microorganism that naturally has carbamoyl phosphate synthetase or L-ornithine producing ability, or may be a microorganism into which a mutant of the present application or a polynucleotide encoding the mutant (or a vector containing the polynucleotide) has been introduced into a parent strain and / or which has been imparted with L-ornithine producing ability, but is not limited thereto.

[0066] For example, the strain of the present application is a cell or microorganism transformed with a vector containing the polynucleotide of the present application or a polynucleotide encoding the mutant of the present application and expressing the mutant of the present application, and for the purposes of the present application, the strain of the present application may be any microorganism that produces L-ornithine, including the mutant of the present application. For example, the strain of the present application may be a recombinant strain in which a polynucleotide encoding the mutant of the present application is introduced into a natural wild-type microorganism or a microorganism that produces L-ornithine, thereby expressing a mutant of the large subunit of carbamoyl phosphate synthetase, thereby improving L-ornithine production ability. The recombinant strain with improved L-ornithine producing ability is a microorganism with improved L-ornithine producing ability compared to a natural wild-type microorganism or a microorganism with unmodified carbamoyl phosphate synthetase (i.e., a microorganism expressing a wild-type carbamoyl phosphate synthetase (SEQ ID NO: 1), or a microorganism not expressing a mutant (SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, or SEQ ID NO: 25) protein), but is not limited thereto. For example, the carbamoyl phosphate synthetase unmodified microorganisms, which are the subject strains for comparing whether the L-ornithine production ability is improved, include, but are not limited to, 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.

[0067] As an example, the recombinant strain with improved production ability has an L-ornithine production ability improved by about 1% or more, specifically about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 15% or more, about 16% or more, about 17% or more, about 18% or more, about 19% or more, or about 20% or more (there is no particular limit to 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) compared to the parent strain or unmodified microorganism before mutation, but may be any that shows an increase in + value compared to the production ability of the parent strain or unmodified microorganism before mutation. In other examples, the recombinant strain with improved production ability has an L-ornithine production ability improved to about 1.01 times or more, about 1.02 times or more, about 1.03 times or more, about 1.05 times or more, about 1.06 times or more, about 1.07 times or more, about 1.08 times or more, about 1.09 times or more, about 1.10 times or more, about 1.15 times or more, about 1.16 times or more, about 1.17 times or more, about 1.18 times or more, about 1.19 times or more, or about 1.20 times or more (there is no particular limit to the upper limit, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less) compared to the parent strain or unmodified microorganism before the mutation, but is not limited thereto.

[0068] 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 into which the carbamoyl phosphate synthetase large subunit mutant described herein has not been introduced or has not been introduced. 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".

[0069] In other examples of the present application, the microorganism of the present application is Corynebacterium stationis, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium The bacterium may be Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens.

[0070] 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.

[0071] 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.

[0072] 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: 48) 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: 53) 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.

[0073] 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.

[0074] In the present application, the term "attenuation" of the activity of a polypeptide 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.

[0075] 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 cells 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 "inactivated," "lack," "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.

[0076] 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.

[0077] 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., modifying the gene sequence encoding the polypeptide so as to encode a polypeptide modified so as to delete or attenuate the activity of the polypeptide). a nucleic acid sequence encoding a polypeptide; 5) modifying a nucleic acid sequence encoding the start codon or 5'UTR region of a gene transcript encoding a polypeptide; 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding a polypeptide; 7) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding a polypeptide so that a secondary structure that makes ribosome attachment impossible is formed; 8) adding a promoter to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide so that it is reverse transcribed (reverse transcription engineering, RTE); or 9) a combination of two or more selected from 1) to 8) above, but is not particularly limited thereto.

[0078] 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.

[0079] 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.

[0080] Furthermore, 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 lose 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.

[0081] 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.

[0082] 6) The introduction of 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.).

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] The mutants, polynucleotides, L-ornithine, and the like in the microorganisms of the present application are as described above.

[0100] Yet another aspect of the present application provides a method for producing L-ornithine, comprising culturing a microorganism comprising a variant of the present application or a polynucleotide of the present application in a medium.

[0101] The method for producing L-ornithine of the present application may include a step of culturing a microorganism comprising a variant of the present application, a polynucleotide of the present application, or a vector of the present application in a medium.

[0102] "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.

[0103] 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.

[0104] Specifically, a culture medium for Corynebacterium microorganisms is disclosed in Non-Patent Document 18.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] L-ornithine produced by the culture of the present application is secreted into the medium or remains intracellularly.

[0111] The L-ornithine production method of the present application may further include a step of preparing a microorganism of the present application, a step of preparing a medium for culturing the microorganism, or a combination thereof (in any order), for example before the culturing step.

[0112] The method for producing L-ornithine of the present application may further include a step of recovering L-ornithine from the medium used in the culture (the medium in which the culture was carried out) or the Corynebacterium glutamicum strain. The recovery step may be further included after the culture step.

[0113] The recovery may be a method of recovering the target L-ornithine using a suitable method known in the art according to the culture method of the microorganism of the present application, such as batch, continuous, or fed-batch culture. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographies such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination thereof can be used, and the target L-ornithine can be recovered from the medium or the microorganism using a suitable method known in the art.

[0114] In addition, the L-ornithine production method of the present application may further comprise a purification step. Said purification can be carried out by any suitable method known in the art.For example, when the L-ornithine production method of the present application comprises both a recovery step and a purification step, said recovery step and said purification step can be carried out continuously or discontinuously regardless of the order, and can be carried out simultaneously or integrated into one step, but is not limited thereto.

[0115] The mutants, polynucleotides, vectors, strains, etc. in the methods of the present application are as described above.

[0116] Yet another aspect of the present application provides a composition for producing L-ornithine, comprising any one selected from the group consisting of a mutant of the present application, a polynucleotide encoding the mutant, a vector comprising the polynucleotide, a microorganism comprising the mutant, a polynucleotide encoding the mutant, or a vector comprising the polynucleotide, a culture of the microorganism, and a combination of at least two thereof.

[0117] The composition of the present application may further contain any suitable excipient that is commonly used in compositions for producing L-ornithine. Such excipients include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, isotonicity agents, etc.

[0118] Yet another aspect of the present application provides use of a variant of the present application, a polynucleotide encoding the variant, a vector comprising the polynucleotide, or a microorganism comprising the polynucleotide of the present application for producing L-ornithine.

[0119] The carbamoyl phosphate synthetase, mutant, polynucleotide, vector, strain, medium, L-ornithine, etc. are as described above.

[0120] Yet another aspect of the present application provides use of a carbamoyl phosphate synthase large subunit (CarB) mutant derived from the genus Corynebacterium, which comprises a substitution of the amino acid corresponding to position 799 in the amino acid sequence of SEQ ID NO:1 with isoleucine, leucine or valine, a substitution of the amino acid corresponding to position 918 with leucine or valine, a substitution of the amino acid corresponding to position 1079 with isoleucine, leucine or valine, or a combination thereof, for L-ornithine production. EXAMPLES

[0121] 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. Example 1

[0122] Construction of carB-effective mutation vector In order to improve L-ornithine producing ability, CarB mutants were prepared in which amino acids at specific positions (positions 799, 918, or 1079 in SEQ ID NO: 1) in the amino acid sequence of CarB were substituted with other amino acids.

[0123] First, in order to introduce an effective mutation into the carB gene, we created a vector to replace the 799th glutamic acid in the protein sequence of the carB gene with four amino acids (alanine, isoleucine, leucine, and valine) that have similar size and chemical properties to valine.

[0124] To prepare a vector for substitution with alanine, the genome of wild-type C. glutamicum ATCC 13869 was used as a template, and the homologous recombinant A arm was amplified using the primer pair of SEQ ID NOs: 27 and 28, and the homologous recombinant B arm was amplified using the primer pair of SEQ ID NOs: 29 and 30. Similarly, to prepare an isoleucine-substituted vector, the homologous recombinant A arm and B arm were amplified using the primer pair of SEQ ID NOs: 27 and 28, and the primer pair of SEQ ID NOs: 31 and 30, to prepare a leucine-substituted vector, the homologous recombinant A arm and B arm were amplified using the primer pair of SEQ ID NOs: 27 and 28, and the primer pair of SEQ ID NOs: 32 and 31, and to prepare a valine-substituted vector, the homologous recombinant A arm and B arm were amplified using the primer pair of SEQ ID NOs: 27 and 28, and the primer pair of SEQ ID NOs: 33 and 30. Then, the plasmid was obtained using a conventional plasmid extraction method. They were named pDCM2-carB(E799A), pDCM2-carB(E799I), pDCM2-carB(E799L), and pDCM2-carB(E799V).

[0125] A vector was constructed to replace glutamic acid at position 918 in the protein sequence of the carB gene with four amino acids (alanine, isoleucine, leucine, and valine).

[0126] To prepare a vector for substitution with alanine, the genome of wild-type C. glutamicum ATCC 13869 was used as a template, and the homologous recombinant A arm was amplified using the primer pair of SEQ ID NO: 34 and 35, and the homologous recombinant B arm was amplified using the primer pair of SEQ ID NO: 36 and 37. Similarly, to prepare an isoleucine-substituted vector, the homologous recombinant A arm and B arm were amplified using the primer pair of SEQ ID NO: 34 and 35, and the primer pair of SEQ ID NO: 38 and 37, to prepare a leucine-substituted vector, the homologous recombinant A arm and B arm were amplified using the primer pair of SEQ ID NO: 34 and 35, and the primer pair of SEQ ID NO: 39 and 37, and to prepare a valine-substituted vector, the homologous recombinant A arm and B arm were amplified using the primer pair of SEQ ID NO: 34 and 35, and the primer pair of SEQ ID NO: 40 and 37. Then, the plasmid was obtained using a conventional plasmid extraction method. They were named pDCM2-carB(E918A), pDCM2-carB(E918I), pDCM2-carB(E918L), and pDCM2-carB(E918V), respectively.

[0127] A vector was constructed to replace threonine at position 1079 of the protein sequence of gene carB with four amino acids (alanine, isoleucine, leucine, and valine).

[0128] To prepare a vector for substitution with alanine, the genome of wild-type C. glutamicum ATCC 13869 was used as a template, and the homologous recombinant A arm was amplified using the primer pair of SEQ ID NO: 41 and 42, and the homologous recombinant B arm was amplified using the primer pair of SEQ ID NO: 43 and 44. Similarly, to prepare an isoleucine-substituted vector, the homologous recombinant A arm and B arm were amplified using the primer pair of SEQ ID NO: 41 and 42, and the primer pair of SEQ ID NO: 45 and 44, to prepare a leucine-substituted vector, the homologous recombinant A arm and B arm were amplified using the primer pair of SEQ ID NO: 41 and 42, and the primer pair of SEQ ID NO: 46 and 44, to prepare a valine-substituted vector, the homologous recombinant A arm and B arm were amplified using the primer pair of SEQ ID NO: 41 and 42, and the primer pair of SEQ ID NO: 47 and 44. Then, the plasmid was obtained using a conventional plasmid extraction method. They were named pDCM2-carB(T1079A), pDCM2-carB(T1079I), pDCM2-carB(T1079L), and pDCM2-carB(T1079V), respectively.

[0129] The primer sequences used in Example 1 are shown in Table 1.

[0130] [Table 1] Example 2

[0131] Creation of L-ornithine-producing microorganisms To generate an L-ornithine-producing microorganism, a vector was constructed to replace the serine at position 55 of the protein sequence of ArgF (ANU33618.1, SEQ ID NO: 48) 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: 49 and 50, and the homologous recombinant B arm was amplified using the primer pair of SEQ ID NO: 51 and 52. Then, a plasmid was obtained as described above. It was named pDCM2-argF(S55*).

[0132] To generate a microorganism with improved L-ornithine production, a vector was constructed to replace the glutamic acid at position 47 of the protein sequence of ArgR (ANU33619.1, SEQ ID NO: 53) 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: 54 and 55, and the homologous recombinant B arm was amplified using the primer pair of SEQ ID NO: 56 and 57. Then, a plasmid was obtained as described above. The plasmid was named pDCM2-argR(E47*).

[0133] The primer sequences used in Example 2 are shown in Table 2.

[0134] [Table 2]

[0135] The wild-type C. glutamicum ATCC 13869 was transformed by electroporation (Non-Patent Document 19) using the pDCM2-argF(S55*) vector prepared as described above, and then a secondary crossover process was performed to obtain a microorganism 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: 49 and 52, which amplify the adjacent site including the position where the gene was inserted, to confirm the genetic manipulation. The microorganism obtained in this manner was named C. gl::argF*.

[0136] In order to prepare a microorganism with improved ornithine production in C.gl::argF*, the pDCM2-argR(E47*) vector was used to obtain a microorganism by the method described above. PCR and sequence analysis were performed using the primer pair of SEQ ID NOs: 54 and 57, which amplify the adjacent site including the position where the gene was inserted, to confirm the genetic manipulation. The microorganism thus obtained was named C.gl::argF*_argR*. Example 3

[0137] Construction of an L-ornithine-producing strain-based carB mutant To prepare mutant strains in glutamic acid at position 799 of the protein sequence of carB based on the L-ornithine producing strain, the pDCM2-carB(E799A), pDCM2-carB(E799I), pDCM2-carB(E799L), and pDCM2-carB(E799V) vectors prepared in Example 1 were transformed into C.gl::argF*_argR* prepared in Example 2 by electroporation, and then a secondary crossover process was performed to obtain microorganisms in which glutamic acid was replaced with alanine, isoleucine, leucine, and valine, respectively. PCR and base sequence analysis were performed using the primer pair of SEQ ID NO: 27 and SEQ ID NO: 30, which amplifies the adjacent site including the position where the gene was inserted, to confirm the genetic manipulation. The obtained microorganisms were named C.gl::argF*_argR*_carB(E799A), C.gl::argF*_argR*_carB(E799I), C.gl::argF*_argR*_carB(E799L), and C.gl::argF*_argR*_carB(E799V), respectively.

[0138] To prepare mutant strains in glutamic acid at position 918 of the protein sequence of carB based on the L-ornithine producing strain, the pDCM2-carB(E918A), pDCM2-carB(E918I), pDCM2-carB(E918L), and pDCM2-carB(E918V) vectors prepared in Example 5 were transformed into C.gl::argF*_argR* prepared in Example 2 by electroporation, and then a secondary crossover process was performed to obtain microorganisms in which glutamic acid was replaced with alanine, isoleucine, leucine, and valine, respectively. PCR and base sequence analysis were performed using the primer pair of SEQ ID NO: 34 and SEQ ID NO: 37, which amplifies the adjacent site including the position where the gene was inserted, to confirm the genetic manipulation. The obtained microorganisms were named C.gl::argF*_argR*_carB(E918A), C.gl::argF*_argR*_carB(E918I), C.gl::argF*_argR*_carB(E918L), and C.gl::argF*_argR*_carB(E918V), respectively.

[0139] To prepare a mutant strain at threonine at position 1079 of the protein sequence of carB based on the L-ornithine producing strain, the pDCM2-carB(T1079A), pDCM2-carB(T1079I), pDCM2-carB(T1079L), and pDCM2-carB(T1079V) vectors prepared in Example 5 were transformed into C.gl::argF*_argR* prepared in Example 2 by electroporation, and then a secondary crossover process was performed to obtain a microorganism in which glutamic acid was replaced with alanine, isoleucine, leucine, and valine, respectively. PCR and base sequence analysis were performed using the primer pair of SEQ ID NO: 41 and SEQ ID NO: 44, which amplifies the adjacent site including the position where the gene was inserted, to confirm the genetic manipulation. The obtained microorganisms were named C.gl::argF*_argR*_carB(T1079A), C.gl::argF*_argR*_carB(T1079I), C.gl::argF*_argR*_carB(T1079L), and C.gl::argF*_argR*_carB(T1079V), respectively. Example 4

[0140] Comparison of L-ornithine productivity in carB mutants C.gl::argF*_argR*_carB(E799A), C.gl::argF*_argR*_carB(E799I), C.gl::argF*_argR*_carB(E799L), C.gl::argF*_argR*_carB(E799V), C.gl::argF*_argR*_carB(E918A), C.gl::argF*_argR*_carB(E918I), C.gl::argF The *_argR*_carB(E918L), C.gl::argF*_argR*_carB(E918V), C.gl::argF*_argR*_carB(T1079A), C.gl::argF*_argR*_carB(T1079I), C.gl::argF*_argR*_carB(T1079L), C.gl::argF*_argR*_carB(T1079V) microorganisms, wild-type C. glutamicum ATCC 13869 as control microorganisms, and C.gl::argF*_argR* microorganisms were cultured in the following production medium, and the bacterial mass and L-ornithine productivity were compared.

[0141] First, each microorganism 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. <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)

[0142] The above experiment was repeated three times. The culture results (average values) are shown in Table 3. 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).

[0143] [Table 3]

[0144] As shown in Table 3, C.gl::argF*_argR*_carB(E799I), C.gl::argF*_argR*_carB(E799L), C.gl::argF*_argR*_carB(E799V), C.gl::argF*_argR*_carB(E918L), C.gl::argF*_argR*_carB(E918V), C.gl::argF*_argR*_carB(T1079I), C.gl::argF*_argR*_carB(T1079L), and C.gl::argF*_argR*_carB(T1079V) had improved productivity and yield compared to the control microorganism C.gl::argF*_argR*.

[0145] 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.

[0146] [Sequence table] JPEG2025509686000004.jpg194170JPEG2025509686000005.jpg242170JPEG2025509686000006.jpg242170JPEG2025509686000007.jpg242170JPEG2025509686000008.jpg242170JPEG2025509686000009.jpg242170JPEG2025509686000010.jpg242170JPEG2025509686000011.jpg242170JPEG2025509686000012.jpg242170JPEG2025509686000013.jpg242170JPEG2025509686000014.jpg242170JPEG2025509686000015.jpg242170JPEG2025509686000016.jpg242170JPEG2025509686000017.jpg242170JPEG2025509686000018.jpg242170JPEG2025509686000019.jpg242170JPEG2025509686000020.jpg242170JPEG2025509686000021.jpg242170JPEG2025509686000022.jpg242170JPEG2025509686000023.jpg242170JPEG2025509686000024.jpg242170JPEG2025509686000025.jpg242170JPEG2025509686000026.jpg242170JPEG2025509686000027.jpg242170JPEG2025509686000028.jpg242170JPEG2025509686000029.jpg242170JPEG2025509686000030.jpg242170JPEG2025509686000031.jpg242170JPEG2025509686000032.jpg242170JPEG2025509686000033.jpg242170JPEG2025509686000034.jpg242170JPEG2025509686000035.jpg242170JPEG2025509686000036.jpg242170JPEG2025509686000037.jpg242170JPEG2025509686000038.jpg242170JPEG2025509686000039.jpg242170JPEG2025509686000040.jpg242170JPEG2025509686000041.jpg242170JPEG2025509686000042.jpg242170JPEG2025509686000043.jpg242170JPEG2025509686000044.jpg242170JPEG2025509686000045.jpg242170JPEG2025509686000046.jpg242170JPEG2025509686000047.jpg242170JPEG2025509686000048.jpg242170JPEG2025509686000049.jpg242170JPEG2025509686000050.jpg242170JPEG2025509686000051.jpg242170JPEG2025509686000052.jpg242170JPEG2025509686000053.jpg242170JPEG2025509686000054.jpg242170JPEG2025509686000055.jpg242170JPEG2025509686000056.jpg242170JPEG2025509686000057.jpg242170JPEG2025509686000058.jpg242170JPEG2025509686000059.jpg242170JPEG2025509686000060.jpg242170JPEG2025509686000061.jpg242170JPEG2025509686000062.jpg242170JPEG2025509686000063.jpg242170JPEG2025509686000064.jpg242170JPEG2025509686000065.jpg242170JPEG2025509686000066.jpg242170JPEG2025509686000067.jpg242170JPEG2025509686000068.jpg168170.

Claims

1. A mutant of the large subunit of carbamoyl phosphate synthetase derived from the genus Corynebacterium, comprising a substitution of the amino acid corresponding to position 799 in the amino acid sequence of SEQ ID NO:1 with isoleucine, leucine or valine, a substitution of the amino acid corresponding to position 918 with leucine or valine, a substitution of the amino acid corresponding to position 1079 with isoleucine, leucine or valine, or a combination thereof.

2. A polynucleotide encoding the variant of claim 1.

3. The carbamoyl phosphate synthetase large subunit mutant of claim 1 , which has a sequence identity of 80% or more with SEQ ID NO:

1.

4. A Corynebacterium microorganism comprising any one selected from the group consisting of carbamoyl phosphate synthetase large subunit mutants, which include, in the amino acid sequence of SEQ ID NO:1, a substitution of the amino acid corresponding to position 799 with isoleucine, leucine or valine, a substitution of the amino acid corresponding to position 918 with leucine or valine, a substitution of the amino acid corresponding to position 1079 with isoleucine, leucine or valine, or a combination thereof, and a polynucleotide encoding the mutant.

5. The microorganism according to claim 4, which has an ability to produce L-ornithine.

6. The microorganism according to claim 4, further comprising an attenuated ornithine carbamoyltransferase (ArgF) activity.

7. The microorganism according to claim 4 , further comprising an attenuated activity of arginine repressor (ArgR).

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

9. A method for producing L-ornithine, comprising the step of culturing in a medium a Corynebacterium microorganism comprising any one selected from the group consisting of carbamoyl phosphate synthetase large subunit mutants which contain, in the amino acid sequence of SEQ ID NO:1, a substitution of the amino acid corresponding to position 799 with isoleucine, leucine or valine, a substitution of the amino acid corresponding to position 918 with leucine or valine, a substitution of the amino acid corresponding to position 1079 with isoleucine, leucine or valine, or a combination thereof, and a polynucleotide encoding said mutant.

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

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

12. A composition for producing L-ornithine, comprising any one selected from the group consisting of a carbamoyl phosphate synthetase large subunit mutant derived from the genus Corynebacterium, which mutant contains an amino acid corresponding to position 799 in the amino acid sequence of SEQ ID NO:1, a substitution of the amino acid corresponding to position 918 with leucine or valine, a substitution of the amino acid corresponding to position 1079 with isoleucine, leucine or valine, or a combination thereof; a polynucleotide encoding the mutant; a vector comprising the polynucleotide; a microorganism comprising the mutant, a polynucleotide encoding the mutant, or a vector comprising the polynucleotide; a culture of the microorganism; and a combination of at least two of these.

13. Use of a Corynebacterium microorganism for L-ornithine production comprising any one selected from the group consisting of carbamoyl phosphate synthetase large subunit mutants comprising, in the amino acid sequence of SEQ ID NO:1, a substitution of the amino acid corresponding to position 799 with isoleucine, leucine or valine, a substitution of the amino acid corresponding to position 918 with leucine or valine, a substitution of the amino acid corresponding to position 1079 with isoleucine, leucine or valine, or a combination thereof, and a polynucleotide encoding the mutant.

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