Carbamyl phosphate synthase large subunit variant and method for producing L-ornithine using same

Abstract

The present application relates to a large subunit variant of carbamoyl phosphate synthetase, a polynucleotide encoding the variant, a microorganism comprising the variant or the polynucleotide, and a method for producing L-ornithine using the same.

Description

Technical Field

[0001] The present disclosure relates to a carbamoyl phosphate synthetase large subunit variant and a method for producing L-ornithine using the same. Background Art

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

[0003] Carbamoyl phosphate is an intermediate metabolite used in the pyrimidine biosynthetic pathway and the arginine biosynthetic pathway. Carbamoyl phosphate synthetase is an enzyme that converts bicarbonate into carbamoyl phosphate, using two molecules of ATP and one molecule of glutamine as nitrogen donors in the production 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 when a variant carbamoyl phosphate synthetase in which feedback inhibition is desensitized and high activity is maintained is introduced into Escherichia coli (E. coli), the production of L-arginine, citrulline, and pyrimidine derivatives increases (Patent Document 1).

[0005] However, it has been reported that excessive formation of intracellular carbamoyl phosphate leads to cytotoxicity (Non-Patent Document 1). Carbamoyl phosphate is a very unstable substance, and cyanate naturally converted from carbamoyl phosphate may cause cytotoxicity.

[0006] Therefore, if the activity of carbamoyl phosphate synthetase in microorganisms is controlled to maintain an appropriate intracellular level of carbamoyl phosphate, prevent unnecessary waste of ATP, and control energy balance, it is possible to increase the production of L-amino acids.

[0007] [Prior art literature]

[0008] [Patent Literature]

[0009] (Patent Document 1) JP 2003-093083 A

[0010]

Non-patent documents

[0011] (Non-patent document 1) Charlier et al., Amino Acids. 2018; 50(12): 1647-1661. Summary of the invention

[0012]

Technical issues

[0013] The present inventors have developed a novel carbamoyl phosphate synthetase large subunit variant that increases L-ornithine production, a polynucleotide encoding the variant, a microorganism of the genus Corynebacterium comprising the variant or the polynucleotide, and a method for producing L-ornithine using the microorganism, thereby completing the present disclosure.

[0014]

Technical solution

[0015] The object of the present disclosure is to provide a variant of the large subunit (CarB) of carbamoyl phosphate synthetase derived from the genus Corynebacterium, which variant includes in the amino acid sequence of SEQ ID NO:1, the amino acid corresponding to position 799 is replaced by isoleucine, leucine or valine; the amino acid corresponding to position 918 is replaced by leucine or valine; the amino acid corresponding to position 1079 is replaced by isoleucine, leucine or valine; or a combination of the above.

[0016] Another object of the present disclosure is to provide polynucleotides encoding the variants of the present disclosure.

[0017] Another object of the present disclosure is to provide a microorganism of the genus Corynebacterium comprising the variant of the present disclosure; or a polynucleotide encoding the variant.

[0018] Another object of the present disclosure is to provide a method for producing L-ornithine, which comprises the step of culturing a microorganism of the genus Corynebacterium comprising the variant of the present disclosure; or a polynucleotide encoding the variant in a culture medium.

[0019] Another object of the present disclosure is to provide a composition for producing L-ornithine, the composition comprising: a variant of the present disclosure; a polynucleotide encoding the variant; a vector comprising the polynucleotide; a microorganism comprising the variant, the polynucleotide encoding the variant, or the vector comprising the polynucleotide; a culture of a microorganism; or a combination of two or more of the foregoing.

[0020] Another object of the present disclosure is to provide use of the microorganism of the present disclosure in producing L-ornithine.

[0021] Another object of the present disclosure is to provide a use of a variant of the large subunit (CarB) of carbamoyl phosphate synthetase derived from the genus Corynebacterium in producing L-ornithine, wherein the variant includes in the amino acid sequence of SEQ ID NO: 1, the amino acid corresponding to position 799 is replaced by isoleucine, leucine or valine; the amino acid corresponding to position 918 is replaced by leucine or valine; the amino acid corresponding to position 1079 is replaced by isoleucine, leucine or valine; or a combination of the above.

[0022]

Beneficial Effects

[0023] Compared to existing unmodified microorganisms, a microorganism into which the variant of the present disclosure is introduced may have enhanced L-ornithine-producing ability. DETAILED DESCRIPTION

[0024] The present disclosure will be described in detail below. At the same time, each description and embodiment disclosed in the present disclosure can also be applied to other descriptions and embodiments. That is to say, all combinations of the various elements disclosed in the present disclosure fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the specific description described below. In addition, many papers and patent documents are referenced and cited throughout the specification. The disclosures of the cited papers and patent documents are incorporated herein by reference as a whole to further illustrate the level and scope of the subject matter to which the present disclosure belongs.

[0025] One aspect of the present disclosure provides a variant of a large subunit (CarB) of carbamoyl phosphate synthetase derived from the genus Corynebacterium, wherein in the amino acid sequence of SEQ ID NO:1, the amino acid corresponding to position 799 is replaced by a different amino acid; the amino acid corresponding to position 918 is replaced by a different amino acid; the amino acid corresponding to position 1079 is replaced by a different amino acid; or a combination of the above.

[0026] The variant disclosed herein may be a variant in which any one or more amino acids at positions 799, 918 or 1079 in the amino acid sequence corresponding to SEQ ID NO: 1 are substituted with an amino acid different from the amino acid before substitution. Alternatively, the variant may be a variant having uncharged amino acids substituted with an amino acid different from the amino acid before substitution, but is not limited thereto.

[0027] Specifically, the variant can be a variant wherein in the amino acid sequence of SEQ ID NO: 1, i) the amino acid corresponding to position 799 is replaced by any one or more amino acids 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; ii) the amino acid corresponding to position 918 is replaced by any one or more amino acids 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 corresponding to position 1079 is replaced by any one or more amino acids 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.

[0028] More specifically, the variant may be a variant in which, in the amino acid sequence of SEQ ID NO:1, i) the amino acid corresponding to position 799 is replaced by any one or more amino acids selected from the group consisting of alanine, isoleucine, leucine and valine, ii) the amino acid corresponding to position 918 is replaced by any one or more amino acids selected from the group consisting of alanine, isoleucine, leucine and valine, or iii) the amino acid corresponding to position 1079 is replaced by any one or more amino acids selected from the group consisting of alanine, isoleucine, leucine and valine, but is not limited thereto.

[0029] Even more specifically, the variant may have, comprise or consist of the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or SEQ ID NO:9 or SEQ ID NO:11 or SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17 or SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:23 or SEQ ID NO:25, or may essentially consist of the amino acid sequence.

[0030] The variant disclosed herein may be a variant wherein in the amino acid sequence represented by SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or SEQ ID NO:9 or SEQ ID NO:11 or SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17 or SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:23 or SEQ ID NO:25, the amino acid corresponding to position 799 based on the amino acid of SEQ ID NO:1 is any one or more amino acids selected from the group consisting of alanine, isoleucine, leucine and valine, the amino acid corresponding to position 918 is any one or more amino acids selected from the group consisting of alanine, isoleucine, leucine and valine, and / or the amino acid corresponding to position 1079 is any one or more amino acids selected from the group consisting of alanine, isoleucine, leucine and valine, and may contain the amino acid sequence represented by SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or SEQ ID NO:11 or SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17 or SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:23 or SEQ ID NO:25, the amino acid corresponding to position 799 based on the amino acid of SEQ ID NO:1 is any one or more amino acids selected from the group consisting of alanine, isoleucine, leucine and valine, and The amino acid sequence shown in NO: 1 has an amino acid sequence with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity. In addition, it is clear that any variant of the amino acid sequence with deletion, modification, substitution, conservative substitution or addition in the partial sequence can also fall within the scope of the present disclosure, as long as the amino acid sequence has such homology or identity and shows the effect corresponding to the variant of the present disclosure.

[0031] Examples include sequence additions or deletions, naturally occurring mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or within the amino acid sequence that do not alter the function of the variants of the disclosure.

[0032] "Conservative substitutions" refer to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions generally occur based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues. Typically, conservative substitutions may have little or no effect on the activity of a protein or polypeptide.

[0033] For example, among the amino acids having charged side chains (charged amino acids), the positively charged (basic) amino acids include arginine, lysine and histidine, and the negatively charged (acidic) amino acids include glutamic acid and aspartic acid; and the amino acids having uncharged side chains include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine and glutamine.

[0034] As used herein, the term "variant" refers to a polypeptide in which one or more amino acids are conservatively replaced and / or modified to be different from the amino acid sequence of the variant before modification, while retaining its function or property. Such variants can generally be identified by modifying one or more amino acids in the polypeptide amino acid sequence and evaluating the properties of the modified polypeptide. In other words, the ability of the variant can be enhanced, unchanged or reduced compared to the polypeptide before modification. In addition, some variants may include variants in which one or more parts, such as N-terminal leader sequences or transmembrane domains have been removed. Other variants may include variants in which a part has been removed from the N- and / or C-terminal of the mature protein. The term "variant" may also be used to refer to a modified body, a modified polypeptide, a modified protein, a mutant, a mutant protein, a divergent mutation, etc., and any term is not limited as long as it is used in the sense of being mutated. For the purposes of the present disclosure, a variant may be a polypeptide comprising the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or SEQ ID NO:9 or SEQ ID NO:11 or SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17 or SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:23 or SEQ ID NO:25, wherein, in the amino acid sequence of SEQ ID NO:1, the glutamic acid corresponding to the amino acid at position 799 is replaced by any one or more amino acids selected from the group consisting of alanine, isoleucine, leucine and valine, the glutamic acid corresponding to the amino acid at position 918 is replaced by any one or more amino acids selected from the group consisting of alanine, isoleucine, leucine and valine, and / or the threonine corresponding to the amino acid at position 1079 is replaced by any one or more amino acids selected from the group consisting of alanine, isoleucine, leucine and valine.

[0035] The variants may also include deletions or additions of amino acids that have minimal effects on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated to a signal (or leader) sequence at the N-terminus of the variant, which co-translationally or post-translationally directs the transfer of the protein. The variants may also be conjugated to other sequences or linkers for identification, purification or synthesis.

[0036] As used herein, the term "homology" or "identity" refers to the degree of similarity between two given amino acid sequences or nucleotide sequences, and can be expressed as a percentage. The terms "homology" and "identity" are often used interchangeably.

[0037] The sequence homology or identity of conservative polynucleotides or polypeptides is determined by standard comparison algorithms, and the default gap penalty set up by the used program can be used together. Basically, homologous or identical sequences are usually able to hybridize with all or part of the sequence under medium or high stringency conditions. Obviously, hybridization also includes hybridization with polynucleotides containing universal codons or codons that take into account codon degeneracy.

[0038] Known computer algorithms (such as the "FASTA" program) can be used to determine whether any two polynucleotide or polypeptide sequences have homology, similarity or identity, for example using the default parameters in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) can be used, which is implemented as follows: the Needleman program (version 5.0.0 or later) in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (including the GCG program package (Devereux, J. et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [et al., J MOLEC BIOL 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego Diego, 1994, and [CARILLO et al. / .] (1988) SIAM J Applied Math 48: 1073) to determine homology, similarity or identity. For example, BLAST of the National Center for Biotechnology Information, or ClustalW can be used to determine homology, similarity or identity.

[0039] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using, for example, the GAP computer program, e.g., Needleman et al. (1970), J Mol Biol. 48:443, as described in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In summary, the GAP program can be defined as the value obtained by dividing the number of similar aligned symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program may include (1) a binary comparison matrix (including a value of 1 for identity and a value of 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Acids Res. 14:6745 (or the EDNAFULL (EMBOSS version of NCBINUC 4.4) substitution matrix), as disclosed in Schwartz and Dayhoff, ed., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (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 end gaps.

[0040] In one embodiment of the present disclosure, the variant of the present disclosure may have an enhanced L-ornithine production capacity. Specifically, compared with a wild-type polypeptide having carbamoyl phosphate synthetase activity, the variant of the present disclosure may have an activity that enhances L-ornithine production capacity.

[0041] As used herein, the term "carbamoyl-phosphate synthetase" refers to an enzyme that converts bicarbonate into carbamoyl-phosphate.

[0042] Carbamoyl phosphate synthetase may be composed of a carbamoyl phosphate synthetase small subunit (CarA) and a carbamoyl phosphate synthetase large subunit (CarB), and the amino acid sequence of CarA or CarB may be obtained from a known database such as Genbank of NCBI.

[0043] For example, CarB of the present disclosure can be derived from a microorganism. The microorganism can be specifically derived from a microorganism of the genus Corynebacterium, more specifically, Corynebacterium glutamicum, Corynebacterium deserti, Corynebacterium crudilactis, Corynebacterium efficiens, Corynebacterium callunae, etc., but are not limited thereto. For the purpose of the present disclosure, the present disclosure can provide a CarB variant derived from Corynebacterium glutamicum.

[0044] For another example, the amino acid of CarB of the present disclosure may be ANU33812.1 derived from Corynebacterium glutamicum ATCC 13869, but it is apparent that proteins having CarB activity derived from various sources are included.

[0045] For the purposes of the present disclosure, the variants of the present disclosure may be carbamoyl phosphate synthetase large subunit variants, ie, CarB variants.

[0046] CarB may have, include, or consist of the amino acid sequence shown in SEQ ID NO: 1, or may consist essentially of the amino acid sequence.

[0047] The amino acid sequence of SEQ ID NO: 1 can be obtained from known databases such as NIH Genbank. In the present disclosure, the amino acid sequence of SEQ ID NO: 1 may include an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity with the amino acid sequence shown in SEQ ID NO: 1. In addition, it is obvious that any protein having an amino acid sequence with deletion, modification, substitution, conservative substitution or addition in part of the sequence may also fall within the scope of the present disclosure, as long as the amino acid sequence has such homology or identity and exhibits an efficacy corresponding to the protein comprising the amino acid sequence SEQ ID NO: 1.

[0048] Examples thereof include sequence additions or deletions, naturally occurring mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or within the amino acid sequence that do not alter the function of the protein of the present disclosure.

[0049] As used herein, the term "corresponding to" refers to an amino acid residue at a listed position in a polypeptide, or an amino acid residue that is similar, identical, or homologous to an amino acid residue listed in a polypeptide. Identifying amino acids at corresponding positions can include determining specific amino acids in a sequence that correspond to a particular sequence. As used herein, a "corresponding region" generally refers to a similar or corresponding position in a related protein or a reference protein.

[0050] For example, an arbitrary amino acid sequence is aligned with SEQ ID NO: 1, and on this basis, each amino acid residue in the amino acid sequence can be numbered with reference to the amino acid residues and the numerical positions of the corresponding amino acid residues in SEQ ID NO: 1. For example, the sequence alignment algorithm described in the present disclosure can determine the position of an amino acid or the position of a modification such as a substitution, insertion or deletion by comparing it with the position of an amino acid in a query sequence (also referred to as a "reference sequence").

[0051] For this alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Biol. 48: 443-453), the Needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), etc. can be used, but are not limited to these, and sequence alignment programs, pairwise sequence alignment algorithms, etc. known in the art can be appropriately used.

[0052] Another aspect of the present disclosure provides a polynucleotide encoding a variant of the present disclosure.

[0053] As used herein, the term "polynucleotide" is a polymer of nucleotides in which nucleotide monomers are linked by covalent bonds into a long chain, and refers to a DNA or RNA chain of predetermined or longer length, and more specifically, refers to a polynucleotide fragment encoding a variant.

[0054] The polynucleotide encoding the variant of the present disclosure may include a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 or SEQ ID NO: 11 or SEQ ID NO: 13 or SEQ ID NO: 15 or SEQ ID NO: 17 or SEQ ID NO: 19 or SEQ ID NO: 21 or SEQ ID NO: 23 or SEQ ID NO: 25. In one embodiment of the present disclosure, the polynucleotide of the present disclosure may have or include a sequence of or SEQ ID NO: 4 or SEQ ID NO: 6 or SEQ ID NO: 8 or SEQ ID NO: 10 or SEQ ID NO: 12 or SEQ ID NO: 14 or SEQ ID NO: 16 or SEQ ID NO: 18 or SEQ ID NO: 20 or SEQ ID NO: 22 or SEQ ID NO: 24 or SEQ ID NO: 26. In addition, the polynucleotides of the present disclosure may consist of or consist essentially of the sequence of SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16 or SEQ ID NO:18 or SEQ ID NO:20 or SEQ ID NO:22 or SEQ ID NO:24 or SEQ ID NO:26.

[0055] In the polynucleotides of the present disclosure, various modifications may be made in the coding region, taking into account the degeneracy of codons or codons preferred in an organism in which the variants of the present disclosure are intended to be expressed, as long as the amino acid sequence of the variants of the present disclosure is not changed. Specifically, the polynucleotides of the present disclosure may have or include a nucleotide sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the sequence of SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16 or SEQ ID NO:18 or SEQ ID NO:20 or SEQ ID NO:22 or SEQ ID NO:24 or SEQ ID NO:26, or may consist of or consist essentially of a nucleotide sequence having 100% or more homology or identity with the sequence of SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16 or SEQ ID NO:18 or SEQ ID NO:20 or SEQ ID NO:22 or SEQ ID NO:24 or SEQ ID NO: The sequence of IDNO:26 has a nucleotide sequence composition of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity. In this regard, in the sequence having such homology or identity, the codon encoding the amino acid corresponding to position 799 of SEQ IDNO:1 may be one of the codons encoding any one or more amino acids 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 any one or more amino acids 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 any one or more amino acids selected from the group consisting of alanine, isoleucine, leucine and valine.

[0056] In addition, the polynucleotides of the present disclosure may include probes prepared from known gene sequences, for example, any sequence, as long as it is a sequence that can hybridize with all or part of the complementary sequence of the polynucleotide sequence of the present disclosure under stringent conditions, is not limited. The term "stringent conditions" refers to conditions that enable specific hybridization between polynucleotides. These conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). Examples include conditions in which polynucleotides with higher homology or identity, i.e., polynucleotides with 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, whereas polynucleotides with lower homology or identity do not hybridize with each other, or conditions in which washing is performed once, specifically 2 to 3 times, at a salt concentration and temperature equivalent to 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, which are general washing conditions for Southern hybridization.

[0057] Hybridization requires that the two nucleic acids have complementary sequences, although mismatches between bases are allowed depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, for DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of the present disclosure may also include substantially similar nucleic acid sequences and isolated nucleic acid fragments that are complementary to the entire sequence.

[0058] Specifically, the hybridization conditions including a hybridization step with a Tm value of 55° C. and the above conditions can be used to detect polynucleotides having homology or identity with the polynucleotides disclosed herein. The Tm value can be 60° C., 63° C. or 65° C., but is not limited thereto, and those skilled in the art can make appropriate adjustments according to the purpose.

[0059] The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementation, and these variables are well known in the art (eg, J. Sambrook et al., supra).

[0060] Another aspect of the present disclosure provides a vector comprising a polynucleotide of the present disclosure. The vector may be an expression vector for expressing the polynucleotide in a host cell, but is not limited thereto.

[0061] The vector of the present disclosure may comprise a DNA construct comprising a nucleotide sequence of a polynucleotide encoding a desired polypeptide, which is operably linked to a suitable expression regulatory region (or expression control sequence) so that the desired polypeptide can be expressed in a suitable host. The expression regulatory region may comprise a promoter capable of initiating transcription, any operon sequence controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling transcription and translation termination. The vector may be transformed into a suitable host cell and then replicated or functioned independently of the host genome, or may be integrated into the genome itself.

[0062] The vector used in the present disclosure is not particularly limited, but any vector known in the art can be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as phage vectors or cosmid vectors; pDZ system, pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, pET system, etc. can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

[0063] For example, the polynucleotide encoding the desired polypeptide can be inserted into the chromosome by the vector for intracellular chromosome insertion. The polynucleotide can be inserted into the chromosome by any method known in the art, such as homologous recombination, but is not limited thereto. The carrier can also include a selection marker for confirming chromosome insertion. The selection marker is used to select cells transformed with the carrier, i.e., for confirming the insertion of the desired nucleic acid molecule, and can use a marker that grants a selectable phenotype (such as drug resistance, auxotrophy, cytotoxic agent resistance or surface polypeptide expression). In the environment of processing with a selection agent, only the cells expressing the selection marker survive or show other phenotypic traits, and therefore the cells transformed can be selected.

[0064] As used herein, the term "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. As long as the converted polynucleotide can be expressed in the host cell, it can be positioned, regardless of the position by being inserted into the chromosome of the host cell or being located outside the chromosome. In addition, the polynucleotide comprises DNA and / or RNA encoding the desired polypeptide. The polynucleotide can be introduced in any form, as long as it can be introduced into the host cell and expressed. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a gene construct comprising all the elements required for autonomous expression. The expression cassette can generally include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably connected to the polynucleotide. The expression cassette can be in the form of an expression vector capable of self-replication. In addition, the polynucleotide can be introduced into the host cell in its own form and operably connected to the sequence required for expression in the host cell, but is not limited thereto.

[0065] Furthermore, the term "operably linked" means that a polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates the transcription of a polynucleotide encoding a desired variant of the present disclosure.

[0066] Another aspect of the present disclosure provides a microorganism comprising a variant of the present disclosure or a microorganism comprising a polynucleotide of the present disclosure.

[0067] As used herein, the term "microorganism (or strain)" includes all wild-type microorganisms or naturally or artificially genetically modified microorganisms, and it can be a microorganism in which a specific mechanism is weakened or enhanced due to the insertion of exogenous genes or the enhancement or inactivation of the activity of endogenous genes, and it can be a microorganism comprising a genetic modification for producing a target polypeptide, protein or product.

[0068] The strain disclosed herein may be a strain comprising any one or more of the variant disclosed herein, the polynucleotide disclosed herein, and a vector comprising the polynucleotide disclosed herein; a strain modified to express the variant disclosed herein or the polynucleotide disclosed herein; a strain expressing the variant disclosed herein or the polynucleotide disclosed herein (e.g., a recombinant strain); or a strain having the activity of the variant disclosed herein (e.g., a recombinant strain), but is not limited thereto.

[0069] In one embodiment of the present disclosure, the strain of the present disclosure may be a strain having the ability to produce L-ornithine.

[0070] The microorganism of the present disclosure may be a microorganism having improved L-ornithine-producing ability.

[0071] The strain disclosed herein may be a microorganism naturally having carbamoyl phosphate synthetase or L-amino acid production ability, or a microorganism prepared by introducing a variant disclosed herein or a polynucleotide encoding the same (or a vector comprising the polynucleotide) into a parent strain and / or by providing L-ornithine production ability, but is not limited thereto.

[0072] For example, the strain of the present invention is a cell or microorganism transformed with a polynucleotide of the present invention or a vector comprising a polynucleotide encoding a variant of the present invention to express a variant of the present invention. For the purpose of the present invention, the strain of the present invention may include all microorganisms capable of producing L-ornithine by comprising a variant of the present invention. For example, the strain of the present invention may be a recombinant strain with enhanced L-ornithine production capacity, wherein the large subunit variant of carbamoyl phosphate synthetase is expressed by introducing a polynucleotide encoding a variant of the present invention into a natural wild-type microorganism or a microorganism producing L-ornithine. The recombinant strain with enhanced L-ornithine production ability may be a microorganism with enhanced L-ornithine production ability compared to a natural wild-type microorganism or a microorganism in which carbamoyl phosphate synthetase is not modified (e.g., a microorganism expressing a wild-type carbamoyl phosphate synthetase (SEQ ID NO: 1) or a microorganism not expressing a variant (SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 or SEQ ID NO: 11 or SEQ ID NO: 13 or SEQ ID NO: 15 or SEQ ID NO: 17 or SEQ ID NO: 19 or SEQ ID NO: 21 or SEQ ID NO: 23 or SEQ ID NO: 25) protein), but is not limited thereto. For example, the microorganism in which carbamoyl phosphate synthetase is not modified (which is a target strain for comparing whether the L-ornithine production ability is enhanced) may be a wild-type Corynebacterium glutamicum ATCC 13869 strain, a Corynebacterium glutamicum ATCC 13869 strain with attenuated ArgF protein activity known as an L-ornithine producing strain, or a Corynebacterium glutamicum ATCC 13869 strain with attenuated ArgR protein activity, but is not limited thereto.

[0073] For example, compared with the parent strain before modification or unmodified microorganism, the recombinant strain with enhanced productivity can have an increase of 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, about 20% or more (the upper limit is not particularly limited, but can be, 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) L-ornithine productivity, but the increase is not limited thereto, as long as the productivity has a + value increase compared to the productivity of the parent strain before modification or unmodified microorganism. In another example, the recombinant strain with enhanced production capacity can have an increased L-ornithine production capacity of 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, about 1.20 times or more (the upper limit is not particularly limited, but can be, 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 before modification or an unmodified microorganism, but is not limited thereto.

[0074] As used herein, the term "unmodified microorganism" does not exclude bacterial strains comprising mutations that may occur naturally in microorganisms, and may be wild-type strains or natural strains themselves, or may be strains before the genetic variation caused by natural or artificial factors changes the character. For example, an unmodified microorganism may refer to a strain into which the carbamoyl phosphate synthetase large subunit variant described herein is not imported or has not yet been imported." unmodified microorganism" may be used interchangeably with "strain before modification", "microorganism before modification", "unchanged strain", "unmodified strain", "unchanged microorganism" or "reference microorganism".

[0075] In another embodiment of the present disclosure, the microorganism of the present disclosure may be Corynebacterium stationis, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens.

[0076] In another embodiment of the present disclosure, the recombinant microorganism of the present disclosure may be a microorganism having enhanced L-amino acid production capacity by additionally enhancing the activity of some proteins in the L-amino acid biosynthesis pathway or by additionally weakening the activity of some proteins in the L-amino acid degradation pathway.

[0077] Specifically, the microorganism of the present disclosure may be a microorganism in which the activity of ornithine carbamoyltransferase subunit F (ArgF) is additionally attenuated, or the argF gene encoding ArgF is additionally deleted, and / or the activity of arginine repressor protein (ArgR) is additionally attenuated, or the argR gene encoding ArgR is additionally deleted.

[0078] The amino acid sequence of ArgF or ArgR can be obtained from known databases such as Genbank of NCBI. For example, the amino acid sequence of ArgF of the present disclosure may include ANU33618.1 (SEQ ID NO: 48) derived from Corynebacterium glutamicum ATCC 13869 or an amino acid sequence having 90% or higher homology thereto, and the amino acid sequence of ArgR may include ANU33619.1 (SEQ ID NO: 53) derived from Corynebacterium glutamicum ATCC 13869 or an amino acid sequence having 90% or higher homology thereto, but is not limited thereto, and obviously includes proteins having ArgF or ArgR activity derived from various sources.

[0079] However, the reduction of the activity of ArgF and / or ArgR proteins or the deletion of argF and / or argR genes is only an example and is not limited thereto. The microorganism disclosed in the present invention can be a microorganism with enhanced activity of proteins in various known L-amino acid biosynthesis pathways or reduced activity of proteins in degradation pathways.

[0080] As used herein, the term "reduction" of polypeptide activity has all concepts covering reduced activity or loss of activity compared to endogenous activity. Reduction can be used interchangeably with terms such as inactivation, lack, downregulation, reduction, decrease, weakening, etc.

[0081] Attenuation can also include: the situation where the activity of the polypeptide itself is weakened or disappears compared with the original polypeptide activity of the microorganism due to the variation of the polynucleotide encoding the polypeptide; the situation where the overall polypeptide activity level and / or concentration (expression level) in the cell is lower than that of the natural strain due to the inhibition of gene expression of the polynucleotide encoding the polypeptide or the inhibition of translation into polypeptide; the situation where the polynucleotide is not expressed at all; and / or the situation where there is no polypeptide activity even when the polynucleotide is expressed. "Endogenous activity" refers to the activity of a specific polypeptide originally present in the parent strain or wild-type or unmodified microorganism before the trait is changed when the trait is changed due to genetic variation caused by natural or artificial factors. This can be used interchangeably with "activity before modification". "Inactivation, lack, reduction, downregulation, reduction or attenuation" of polypeptide activity compared with its endogenous activity means that the activity of the polypeptide is reduced compared with the activity of the original specific polypeptide of the parent strain or unmodified microorganism before the trait is changed.

[0082] The reduction of the activity of the polypeptide can be carried out by any method known in the art, but is not limited thereto, and can be achieved by applying various methods well known in the art (for example, Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014; 15(2): 2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).

[0083] Specifically, attenuating the activity of the polypeptide of the present disclosure can be achieved by:

[0084] 1) Deletion of all or part of the gene encoding the polypeptide;

[0085] 2) modifying the expression control region (or expression control sequence) to reduce the expression of the gene encoding the polypeptide;

[0086] 3) Modifying the amino acid sequence constituting the polypeptide to eliminate or weaken the activity of the polypeptide (for example, deleting / replacing / adding one or more amino acids in the amino acid sequence);

[0087] 4) Modifying a gene sequence encoding a polypeptide to eliminate or weaken the activity of the polypeptide (for example, deleting / replacing / adding one or more nucleotides in the nucleotide sequence of the polypeptide gene to encode the modified polypeptide, thereby eliminating or weakening the activity of the polypeptide);

[0088] 5) modifying the nucleotide sequence encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide;

[0089] 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to a gene transcript encoding a polypeptide;

[0090] 7) adding a sequence complementary to the Shine-Dalgarno sequence of the gene encoding the polypeptide upstream of the Shine-Dalgarno sequence to form a secondary structure that prevents ribosomes from attaching;

[0091] 8) adding a promoter to be reverse transcribed to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE); or

[0092] 9) A combination of two or more selected from 1) to 8), but not particularly limited thereto.

[0093] For example,

[0094] 1) Deleting part or all of a gene encoding a polypeptide may be by deleting all of the polynucleotide encoding the endogenous target polypeptide in the chromosome, replacing it with a polynucleotide in which some nucleotides are deleted, or replacing it with a marker gene.

[0095] 2) Modification of the expression control region (or expression control sequence) can be mutation of the expression control region (or expression control sequence) by deletion, insertion, non-conservative substitution or conservative substitution or a combination thereof, or replacement with a sequence having weaker activity. The expression control region includes a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence controlling transcription and translation termination, but is not limited thereto.

[0096] In addition, 3) and 4) modification of the amino acid sequence or polynucleotide sequence may be a mutation in the sequence through deletion, insertion, non-conservative substitution or conservative substitution or a combination thereof in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, or replacement with an amino acid sequence or polynucleotide sequence improved to have weaker activity, or replacement with an amino acid sequence or polynucleotide sequence improved to have no activity, thereby weakening the activity of the polypeptide, but not limited thereto. For example, the expression of a gene may be inhibited or weakened by introducing a mutation in the polynucleotide sequence to form a stop codon, but not limited thereto.

[0097] 5) Modification of the nucleotide sequence encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, replaced with a nucleotide sequence encoding another start codon having a lower polypeptide expression rate instead of the endogenous start codon, but is not limited thereto.

[0098] 6) Introduction of antisense oligonucleotides (e.g., antisense RNA) that complementarily bind to gene transcripts encoding polypeptides can be found in, for example, the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews-Trends in Genetics, Vol. 1 (1) 1986].

[0099] 7) A sequence complementary to the Shine-Dalgarno sequence of the gene encoding the polypeptide is added upstream of the Shine-Dalgarno sequence to form a secondary structure that prevents ribosomes from attaching, which may make the mRNA unable to be translated or reduce its translation rate.

[0100] In addition, 8) adding a promoter to be reverse transcribed to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE) may make the antisense nucleotide complementary to the gene transcript encoding the polypeptide, thereby weakening the activity.

[0101] As used herein, the term "enhancement" of polypeptide activity refers to the enhancement of the activity of the polypeptide compared to its endogenous activity. Enhancement can be used interchangeably with terms such as activation, upregulation, overexpression, increase, etc. Here, activation, enhancement, upregulation, overexpression and increase can include two situations in which the activity that was not originally present or the activity enhancement is shown compared to the endogenous activity or the activity before modification." Endogenous activity" refers to the activity of the original specific polypeptide of the parent strain or unmodified microorganism before the property change when the property changes due to the genetic variation caused by natural or artificial factors. This can be used interchangeably with "activity before modification". Compared to the endogenous activity, the fact that the activity of the polypeptide is "enhanced", "upregulated", "overexpressed" or "increased" means that the activity of the polypeptide is improved compared to the activity and / or concentration (expression level) of the original specific polypeptide of the parent strain or unmodified microorganism before the property change.

[0102] Enhancement can be achieved by introducing exogenous polypeptides or enhancing the activity and / or concentration (expression level) of endogenous polypeptides. The enhancement of polypeptide activity can be confirmed by an increase in the activity degree and expression level of the corresponding polypeptide or an increase in the amount of the corresponding polypeptide released product.

[0103] In order to enhance the activity of polypeptide, various methods well known in the art can be applied, as long as the activity of the desired polypeptide can be enhanced compared with the microorganism before modification, the method is not limited. Specifically, genetic engineering and / or protein engineering well known to those skilled in the art can be used, which is a conventional method of molecular biology, but the method is not limited thereto (e.g., Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2, 1-16, Sambrook et al., Molecular Cloning 2012, etc.).

[0104] Specifically, the polypeptides that enhance the present disclosure may be:

[0105] 1) Increasing the copy number of the polynucleotide encoding the polypeptide in the cell;

[0106] 2) Replace the gene expression regulatory region on the chromosome encoding the polypeptide with a more active sequence;

[0107] 3) modifying the nucleotide sequence encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide;

[0108] 4) Modify the amino acid sequence of a polypeptide to enhance the activity of the polypeptide;

[0109] 5) modifying a polynucleotide sequence encoding a polypeptide to enhance the activity of the polypeptide (e.g., modifying a polynucleotide sequence of a polypeptide gene to encode a modified polypeptide to enhance the activity of the polypeptide);

[0110] 6) introducing an exogenous polypeptide expressing the polypeptide activity or an exogenous polynucleotide encoding the polypeptide;

[0111] 7) Codon optimization of polynucleotides encoding polypeptides;

[0112] 8) analyzing the tertiary structure of the polypeptide to select and modify or chemically modify exposed sites; or

[0113] 9) A combination of two or more selected from 1) to 8), but not particularly limited thereto.

[0114] More specifically, 1) increasing the number of copies of a polynucleotide encoding a polypeptide in a cell can be achieved by introducing a vector into a host cell, the vector can replicate and function independently of the host, and the polynucleotide encoding the corresponding polypeptide is operably linked to the vector. Alternatively, this increase can be achieved by introducing one copy or two or more copies of the polynucleotide encoding the corresponding polypeptide into the chromosome of the host cell. The introduction into the chromosome can be carried out by introducing a vector capable of inserting the polynucleotide into the chromosome of the host cell, but is not limited thereto. The vector is as described above.

[0115] 2) Replacing the gene expression regulatory region (or expression control sequence) on the chromosome encoding the polypeptide with a sequence showing strong activity may be, for example, a variation in the sequence due to deletion, insertion, non-conservative or conservative substitution or a combination thereof, or replacing it with a sequence showing stronger activity, thereby further enhancing the activity of the expression regulatory region. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence controlling transcription and translation termination, etc. For example, the replacement may be replacing the original promoter with a strong promoter, but is not limited thereto.

[0116] Examples of known strong promoters include CJ1 to CJ7 promoters (U.S. 7662943B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (U.S. 10584338B2), O2 promoter (U.S. 10273491B2), tkt promoter, yccA promoter, etc., but are not limited to these.

[0117] 3) Modification of the nucleotide sequence encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, replaced with a nucleotide sequence encoding another start codon having a higher polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0118] 4) and 5) modification of the amino acid sequence or polynucleotide sequence may be due to a deletion, insertion, non-conservative substitution or conservative substitution or a combination thereof of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, or substitution with an amino acid sequence or polynucleotide sequence that is improved to exhibit stronger activity, or substitution with an amino acid sequence or polynucleotide sequence that is improved to be more active, thereby enhancing the activity of the polypeptide, but is not limited thereto. The replacement may be specifically performed by inserting the polynucleotide into the chromosome by homologous recombination, but is not limited thereto. The vector used herein may also contain a selection marker for confirming chromosomal insertion.

[0119] 6) The introduction of an exogenous polynucleotide that exhibits polypeptide activity can be to introduce an exogenous polynucleotide into a host cell, and the polypeptide encoded by the exogenous polynucleotide exhibits the same or similar activity as the polypeptide. There is no limitation on its source or sequence, as long as the exogenous polynucleotide exhibits the same or similar activity as the polypeptide. The method used in the introduction can be carried out by appropriately selecting a known transformation method by a person skilled in the art. As the introduced polynucleotide is expressed in the host cell, a polypeptide can be produced, and its activity can be enhanced.

[0120] 7) Codon optimization of a polynucleotide encoding a polypeptide can be codon optimization of an endogenous polynucleotide to enhance transcription or translation in a host cell, or codon optimization of an exogenous polynucleotide to optimize transcription and translation in a host cell.

[0121] 8) Analyzing the tertiary structure of a polypeptide to select and modify or chemically modify an exposed site can be, for example, by comparing the sequence information of the polypeptide to be analyzed with a database storing known protein sequence information, determining a template protein candidate according to the degree of sequence similarity, confirming the structure based on this, and modifying or chemically modifying the exposed site to be modified or chemically modified.

[0122] Based on the activity or concentration of the polypeptide expressed in the wild type or microbial strain before modification, this enhancement of polypeptide activity can be an increase in the activity or concentration / expression level of the corresponding polypeptide, or an increase in the amount of the product produced by the corresponding polypeptide, but is not limited thereto.

[0123] The modification of some or all of the polynucleotides in the microorganisms disclosed herein can be induced by: (a) homologous recombination using a vector for chromosome insertion in the microorganism, or genome editing using an engineered nuclease (e.g., CRISPR-Cas9), and / or (b) treatment with light (e.g., ultraviolet light and radiation) and / or chemicals, but not limited thereto. Methods for modifying some or all of the genes may include methods by DNA recombination technology. For example, a nucleotide sequence or vector comprising a nucleotide sequence homologous to the target gene can be introduced into the microorganism to cause homologous recombination, resulting in partial or complete deletion of the gene. The nucleotide sequence or vector to be introduced may include a dominant selection marker, but is not limited thereto.

[0124] Regarding the microorganisms, variants, polynucleotides, L-ornithine, etc. of the present disclosure are as described in other aspects.

[0125] Another aspect of the present disclosure provides a method for producing L-ornithine, the method comprising the step of culturing a microorganism comprising the variant of the present disclosure or the polynucleotide of the present disclosure in a culture medium.

[0126] The method for producing L-ornithine of the present disclosure may include the step of culturing a microorganism comprising the variant of the present disclosure, the polynucleotide of the present disclosure, or the vector of the present disclosure in a culture medium.

[0127] As used herein, the term "culture" refers to growing the microorganisms of the present disclosure under appropriately adjusted environmental conditions. The culture procedure of the present disclosure can be carried out according to suitable culture media or culture conditions known in the art. Those skilled in the art can easily adjust this culture procedure according to the selected strain. Specifically, the culture can be batch type, continuous type and / or fed-batch type, but is not limited thereto.

[0128] As used herein, "culture medium" refers to a mixture containing nutrients required for culturing the microorganisms of the present disclosure as the main components, wherein the culture medium provides nutrients including water, growth factors, etc. necessary for survival and growth. Specifically, for the culture medium and other culture conditions for culturing the microorganisms of the present disclosure, any culture medium used for conventional culture of microorganisms can be used without particular limitation. However, the microorganisms of the present disclosure can be cultured in a general culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and / or vitamin, etc. under aerobic conditions, while controlling temperature, pH, etc.

[0129] Specifically, a culture medium of a microorganism of the genus Corynebacterium can be found in the document ["Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington, DC, USA, 1981)].

[0130] In the present disclosure, the carbon source may include carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrient sources such as starch hydrolysate, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steep liquor may be used, and in particular, carbohydrates such as glucose and aseptically pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and appropriate amounts of various other carbon sources may be used without restriction. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0131] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc., amino acids such as glutamic acid, methionine, glutamine, etc., and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition product, defatted soybean cake or its degradation product, etc. can be used. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0132] The phosphorus source may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate and their corresponding sodium salts. The inorganic compound may include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc., and may also include amino acids, vitamins and / or suitable precursors. These components or precursors may be added to the culture medium in batches or continuously. However, the present disclosure is not limited thereto.

[0133] In addition, in the process of cultivating the microorganism of the present disclosure, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the culture medium in an appropriate manner. In addition, defoamers such as fatty acid polyethylene glycol esters can be used to suppress foam formation during cultivation. In addition, oxygen or oxygen-containing gas can be injected into the culture medium to maintain the aerobic state of the culture medium, or gas can not be injected, or nitrogen, hydrogen or carbon dioxide gas can be injected to maintain the anaerobic or non-aerobic state of the culture medium, but it is not limited thereto.

[0134] In the culture of the present disclosure, the culture temperature may be maintained at 20° C. to 45° C., specifically, at 25° C. to 40° C., and the culture may be performed for about 10 hours to about 160 hours, but is not limited thereto.

[0135] L-ornithine produced by the culture of the present disclosure may be released into the medium, or may be retained in the cells.

[0136] The method for producing L-ornithine of the present disclosure may further include, for example, before the culturing step, the step of preparing the microorganism of the present disclosure, preparing a culture medium for culturing the microorganism, or a combination thereof (regardless of the order, in any order).

[0137] The method for producing L-ornithine of the present disclosure may further include a step of recovering L-ornithine from the culture medium (cultured culture medium) or the Corynebacterium glutamicum strain produced by the culture. A recovery step may also be included after the culture step.

[0138] Recovery can include: according to the method of culturing microorganisms disclosed herein, such as batch, continuous or fed-batch type culture, collecting the desired L-ornithine by using suitable methods known in the art. For example, centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, ultrasonic treatment, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography and affinity chromatography, etc.), HPLC and combinations of these methods can be used, and the desired L-ornithine can be recovered from the culture medium or microorganism by using suitable methods known in the art.

[0139] In addition, the method for producing L-ornithine disclosed herein may further include a purification step. Purification may be performed by using a suitable method known in the art. In an exemplary embodiment, when the method for producing L-ornithine disclosed herein includes a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously without regard to the order, or may be performed simultaneously or integrated into one step, but is not limited thereto.

[0140] With regard to the methods, variants, polynucleotides, vectors, strains, etc. of the present disclosure are as described in other aspects.

[0141] Another aspect of the present disclosure provides a composition for producing L-ornithine, comprising: a variant of the present disclosure; a polynucleotide encoding the variant; a vector comprising the polynucleotide; a microorganism comprising the variant, the polynucleotide encoding the variant, or the vector comprising the polynucleotide; a culture of a microorganism; or a combination of two or more of the foregoing.

[0142] The composition of the present disclosure may also include any suitable excipients commonly used in compositions for producing L-ornithine, and such excipients may include, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, isotonic agents, etc., but are not limited thereto.

[0143] Another aspect of the present disclosure provides use of a variant of the present disclosure, a polynucleotide encoding the variant, a vector comprising the polynucleotide, or a microorganism comprising the polynucleotide of the present disclosure in producing L-ornithine.

[0144] Carbamoyl phosphate synthetase, variants, polynucleotides, vectors, strains, culture media, and L-ornithine are as described in other aspects.

[0145] Another aspect of the present disclosure provides the use of a variant of the large subunit (CarB) of carbamoyl phosphate synthetase derived from Corynebacterium in the production of L-ornithine, wherein the variant includes in the amino acid sequence of SEQ ID NO: 1, the amino acid corresponding to position 799 is replaced by isoleucine, leucine or valine; the amino acid corresponding to position 918 is replaced by leucine or valine; the amino acid corresponding to position 1079 is replaced by isoleucine, leucine or valine; or a combination of the above.

[0146] [Mode for carrying out the invention]

[0147] The present disclosure will be described in more detail below by way of exemplary embodiments. However, the following exemplary embodiments are only for illustrating preferred embodiments of the present disclosure, and therefore are not intended to limit the scope of the present disclosure thereto. At the same time, those skilled in the art or similar technical fields to which the present disclosure belongs can fully understand and easily implement technical matters not described in this specification.

[0148] Example 1: Construction of a vector for introducing effective mutations in carB

[0149] In order to increase the production of L-ornithine, CarB variants were prepared in which an amino acid at a specific position (SEQ ID NOS: 1 to 799, 918 or 1079) in the amino acid sequence of CarB was replaced with another amino acid.

[0150] First, in order to introduce an effective mutation into the carB gene, a vector was constructed to replace glutamic acid at position 799 in the protein sequence of the carB gene with four amino acids (alanine, isoleucine, leucine, and valine) by considering the similar size and chemical properties to valine.

[0151] To construct a vector for alanine replacement, the wild-type Corynebacterium glutamicum ATCC 13869 genome was used as a template, and the homologous recombination A arm and the homologous recombination B arm were amplified using primer pairs SEQ ID NOS: 27 and 28, primer pairs SEQ ID NOS: 29 and 30, respectively. Similarly, to construct a vector for isoleucine replacement, the homologous recombination A arm and B arm were amplified using primer pairs SEQ ID NOS: 27 and 28, primer pairs SEQ ID NOS: 31 and 30, respectively. To construct a vector for leucine replacement, the homologous recombination A arm and B arm were amplified using primer pairs SEQ ID NOS: 27 and 28, primer pairs SEQ ID NOS: 32 and 31, respectively. To construct a vector for valine replacement, the homologous recombination A arm and B arm were amplified using primer pairs SEQ ID NOS: 27 and 28, primer pairs SEQ ID NOS: 33 and 30, respectively. Thereafter, plasmids were obtained using a known plasmid extraction method and named pDCM2-carB(E799A), pDCM2-carB(E799I), pDCM2-carB(E799L), and pDCM2-carB(E799V).

[0152] A vector was constructed in which four amino acids (alanine, isoleucine, leucine and valine) were used to replace the glutamic acid at position 918 in the protein sequence of the carB gene.

[0153] To construct a vector for alanine replacement, the wild-type Corynebacterium glutamicum ATCC 13869 genome was used as a template, and the homologous recombination A arm and the homologous recombination B arm were amplified using primer pairs SEQ ID NOS: 34 and 35 and primer pairs SEQ ID NOS: 36 and 37, respectively. Similarly, to construct a vector for isoleucine replacement, the homologous recombination A arm and the B arm were amplified using primer pairs SEQ ID NOS: 34 and 35 and primer pairs SEQ ID NOS: 38 and 37, respectively. To construct a vector for leucine replacement, the homologous recombination A arm and the B arm were amplified using primer pairs SEQ ID NOS: 34 and 35 and primer pairs SEQ ID NOS: 39 and 37, respectively. To construct a vector for valine replacement, the homologous recombination A arm and the B arm were amplified using primer pairs SEQ ID NOS: 34 and 35 and primer pairs SEQ ID NOS: 40 and 37, respectively. Thereafter, plasmids were obtained using a known plasmid extraction method and named pDCM2-carB(E918A), pDCM2-carB(E918I), pDCM2-carB(E918L), and pDCM2-carB(E918V) in this order.

[0154] A vector was constructed in which four amino acids (alanine, isoleucine, leucine and valine) were used to replace the threonine at position 1079 in the protein sequence of the carB gene.

[0155] To construct a vector for alanine replacement, the wild-type Corynebacterium glutamicum ATCC 13869 genome was used as a template, and the homologous recombination A arm and the homologous recombination B arm were amplified using primer pairs SEQ ID NOS: 41 and 42 and primer pairs SEQ ID NOS: 43 and 44, respectively. Similarly, to construct a vector for isoleucine replacement, the homologous recombination A arm and the B arm were amplified using primer pairs SEQ ID NOS: 41 and 42 and primer pairs SEQ ID NOS: 45 and 44, respectively. To construct a vector for leucine replacement, the homologous recombination A arm and the B arm were amplified using primer pairs SEQ ID NOS: 41 and 42 and primer pairs SEQ ID NOS: 46 and 44, respectively. To construct a vector for valine replacement, the homologous recombination A arm and the B arm were amplified using primer pairs SEQ ID NOS: 41 and 42 and primer pairs SEQ ID NOS: 47 and 44, respectively. Thereafter, plasmids were obtained using a known plasmid extraction method and named pDCM2-carB(T1079A), pDCM2-carB(T1079I), pDCM2-carB(T1079L), and pDCM2-carB(T1079V) in this order.

[0156] The sequences of the primers used in Example 1 are shown in Table 1 below.

[0157] [Table 1]

[0158]

[0159]

[0160] Example 2: Preparation of microorganisms for producing L-ornithine

[0161] In order to prepare a microorganism producing L-ornithine, a vector was constructed in which the serine at position 55 of the ArgF (ANU33618.1, SEQ ID NO: 48) protein sequence was replaced with a stop codon. The wild-type Corynebacterium glutamicum ATCC1 3869 genome was used as a template, and the homologous recombination A arm and the homologous recombination B arm were amplified using primer pairs SEQ ID NOS: 49 and 50 and primer pairs SEQ ID NOS: 51 and 52, respectively. Thereafter, a plasmid was obtained in the same manner as above and named pDCM2-argF (S55*).

[0162] In order to prepare a microorganism with further improved L-ornithine production capacity, a vector was constructed in which the glutamic acid at position 47 of the ArgR (ANU33619.1, SEQ ID NO: 53) protein sequence was replaced with a stop codon. The wild-type Corynebacterium glutamicum ATCC1 3869 genome was used as a template, and the homologous recombination A arm and the homologous recombination B arm were amplified using primer pairs SEQ ID NOS: 54 and 55 and primer pairs SEQ ID NOS: 56 and 57, respectively. Thereafter, a plasmid was obtained in the same manner as above and named pDCM2-argR (E47*).

[0163] The sequences of the primers used in Example 2 are shown in Table 2 below.

[0164] [Table 2]

[0165]

[0166] The pDCM2-argF (S55 *) vector constructed above was transformed into wild-type Corynebacterium glutamicum ATCC 13869 by electroporation (Appl. Microbiol. Biotechnol. (1999) 52: 541-545), and then a second crossover process was performed to obtain a microorganism in which the nucleotide sequence at position 164 of argF was replaced by adenine (a) from cytosine (c), and thus the protein sequence at position 55 was replaced by a stop codon. PCR and sequencing analysis were performed using primers SEQ ID NOS: 49 and 52, which were able to amplify the adjacent region containing the corresponding gene insertion position and confirmed the corresponding genetic manipulation. The microorganism thus obtained was named C.gl::argF *.

[0167] In order to prepare a microorganism with further improved ornithine production in C.gl::argF*, a microorganism was obtained in the same manner as above using the pDCM2-argR(E47*) vector. PCR and sequencing analysis were performed using primer pairs SEQ ID NOS:54 and 57, which are capable of amplifying the adjacent region containing the corresponding gene insertion position, and the corresponding genetic manipulation was confirmed. The microorganism thus obtained was named C.gl::argF*_argR*.

[0168] Example 3: Preparation of carB mutant strain based on L-ornithine producing strain

[0169] In order to prepare a mutant strain of glutamic acid at position 799 of the carB protein sequence, the L-ornithine-producing strain C.gl::argF*_argR* prepared in Example 2 was transformed by electroporation with the pDCM2-carB(E799A), pDCM2-carB(E799I), pDCM2-carB(E799L), and pDCM2-carB(E799V) vectors constructed in Example 1, respectively, and then a second exchange process was performed to obtain a microorganism in which glutamic acid was replaced by alanine, isoleucine, leucine, or valine. PCR and sequencing analysis were performed using primers SEQ ID NOS: 27 and 30, which were able to amplify the adjacent region containing the corresponding gene insertion position, and the corresponding genetic manipulation was confirmed. The microorganisms thus obtained 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.

[0170] In order to prepare a mutant strain of glutamic acid at position 918 of the carB protein sequence, the L-ornithine-producing strain C.gl::argF*_argR* prepared in Example 2 was transformed by electroporation with the pDCM2-carB(E918A), pDCM2-carB(E918I), pDCM2-carB(E918L), and pDCM2-carB(E918V) vectors constructed in Example 5, respectively, and then a second exchange process was performed to obtain a microorganism in which glutamic acid was replaced by alanine, isoleucine, leucine, or valine. PCR and sequencing analysis were performed using primer pairs SEQ ID NOS: 34 and 37, which were able to amplify the adjacent region containing the corresponding gene insertion position, and the corresponding genetic manipulation was confirmed. The microorganisms thus obtained 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.

[0171] In order to prepare a mutant strain of threonine at position 1079 of the carB protein sequence, the L-ornithine-producing strain C.gl::argF*_argR* prepared in Example 2 was transformed by electroporation with the pDCM2-carB(T1079A), pDCM2-carB(T1079I), pDCM2-carB(T1079L), and pDCM2-carB(T1079V) vectors constructed in Example 5, respectively, and then a second exchange process was performed to obtain a microorganism in which glutamic acid was replaced by alanine, isoleucine, leucine, or valine. PCR and sequencing analysis were performed using primers SEQ ID NOS: 41 and 44, which were able to amplify the adjacent region containing the corresponding gene insertion position, and the corresponding genetic manipulation was confirmed. The microorganisms thus obtained 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.

[0172] Example 4: Comparison of L-ornithine production capacity between carB mutant strains

[0173] 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 prepared in Example 3 were cultured using the following production medium. The cell mass and L-ornithine production capacity of C.gl::argF*_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), as well as the control microorganisms wild-type Corynebacterium glutamicum ATCC 13869 and C.gl::argF*_argR* microorganism were compared.

[0174] First, each microorganism was inoculated into a 250 ml corner-baffled flask containing 25 ml of seed culture medium and cultured at 30° C. with shaking at 200 rpm for 16 hours. 1 ml of the seed culture was inoculated into a 250 ml corner-baffled flask containing 24 ml of production culture medium and cultured at 33° C. with shaking at 200 rpm for 40 hours.

[0175] <Seed culture medium (pH 7.0)>

[0176] 20g glucose, 10g peptone, 5g yeast extract, 1.5g urea, 4g KH 2 PO 4 8g K 2 HPO 4 、0.5gMgSO 4 7H 2 O, 0.1 mg biotin, 1 mg thiamine HCl, 22 mg calcium pantothenate, 2 mg niacinamide (based on 1 liter of distilled water)

[0177] <Production medium (pH 7.0)>

[0178] 50g raw sugar, 25g (NH 4 ) 2 SO 4 , 1g yeast extract, 0.55g KH 2 PO4, 0.6 g MgSO 4 7H 2 O, 0.2g L-arginine, 0.9mg biotin, 4.5mg thiamine HCl, 4.5mg calcium pantothenate, 30mg niacinamide, 9mg MnSO 4 , 9mg FeSO 4 , 0.45mg ZnSO 4 , 0.45 mg CuSO 4 、30g CaCO 3 (Based on 1 liter of distilled water)

[0179] The above experiment was repeated three times, and the culture results (average values) are shown in Table 3. The L-ornithine yield was calculated as ornithine concentration / sugar consumption, the L-ornithine yield was calculated as ornithine concentration / sugar consumption, and the concentration increase rate was calculated as (L-ornithine concentration produced in the experimental group-L-ornithine concentration produced in the control group (wild-type Corynebacterium glutamicum ATCC 13869 strain)) / L-ornithine concentration produced in the control group (wild-type Corynebacterium glutamicum ATCC 13869 strain).

[0180] [Table 3]

[0181]

[0182]

[0183] As shown in Table 3, compared with the control microorganism C.gl::argF*_argR*, 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) showed increased production capacity and yield.

[0184] Based on the above description, it will be appreciated by those skilled in the art that the present disclosure may be implemented in other specific forms without departing from its technical spirit or essential features. In this regard, the above embodiments should be understood to be illustrative rather than restrictive in all respects. The scope of the present disclosure should be understood as the meaning and scope of the appended claims rather than detailed description, and all changes or modifications derived from equivalent substitutions fall within the scope of the present disclosure.

Claims

1. A large subunit variant of a carbamoyl phosphate synthetase derived from the genus Corynebacterium, wherein the variant comprises in the amino acid sequence of SEQ ID NO: 1, the amino acid corresponding to position 799 is replaced by isoleucine, leucine or valine; the amino acid corresponding to position 918 is replaced by leucine or valine; the amino acid corresponding to position 1079 is replaced by isoleucine, leucine or valine; or a combination of the above.

2. A polynucleotide encoding the variant according to claim 1. 3 . The carbamoyl phosphate synthetase large subunit variant according to claim 1 , wherein the carbamoyl phosphate synthetase large subunit variant has 80% or more sequence identity with SEQ ID NO:

1. 4 .

4. A Corynebacterium microorganism comprising a carbamoyl phosphate synthetase large subunit variant or a polynucleotide encoding the variant, wherein the variant is included in the amino acid sequence of SEQ ID NO:1, the amino acid corresponding to position 799 is replaced by isoleucine, leucine or valine; the amino acid corresponding to position 918 is replaced by leucine or valine; the amino acid corresponding to position 1079 is replaced by isoleucine, leucine or valine; or a combination of the above. The microorganism according to claim 4 , wherein the microorganism has L-ornithine-producing ability. The microorganism according to claim 4 , wherein the activity of ornithine carbamoyltransferase (ArgF) is further attenuated.

7. The microorganism according to claim 4, wherein the activity of arginine repressor (ArgR) is further attenuated.

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

9. A method for producing L-ornithine, comprising culturing a Corynebacterium microorganism comprising a carbamoyl-phosphate synthetase large subunit variant or a polynucleotide encoding the variant in a culture medium, wherein the variant comprises in the amino acid sequence of SEQ ID NO: 1, the amino acid corresponding to position 799 is substituted by isoleucine, leucine or valine; the amino acid corresponding to position 918 is substituted by leucine or valine; the amino acid corresponding to position 1079 is substituted by isoleucine, leucine or valine; or a combination thereof.

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

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

12. A composition for producing L-ornithine, comprising a large subunit variant of carbamoyl phosphate synthetase derived from the genus Corynebacterium, wherein the variant comprises, in the amino acid sequence of SEQ ID NO: 1, an amino acid corresponding to position 799 is substituted by isoleucine, leucine or valine, an amino acid corresponding to position 918 is substituted by leucine or valine, and an amino acid corresponding to position 1079 is substituted by isoleucine, leucine or valine; or a combination thereof; a polynucleotide encoding the variant; a vector comprising the polynucleotide; a microorganism comprising the variant, the polynucleotide encoding the variant or the vector comprising the polynucleotide; a culture of the microorganism; or a combination of two or more thereof.

13. Use of a microorganism of the genus Corynebacterium in producing L-ornithine, the microorganism comprising a large subunit variant of carbamoyl phosphate synthetase, wherein the variant comprises, in the amino acid sequence of SEQ ID NO: 1, the amino acid corresponding to position 799 is substituted by isoleucine, leucine or valine, the amino acid corresponding to position 918 is substituted by leucine or valine, and the amino acid corresponding to position 1079 is substituted by isoleucine, leucine or valine; or a combination of the above; or a polynucleotide encoding the variant.

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