Novel BBD29RS14450 variant and method for producing l-arginine using same

Abstract

The present application relates to: a novel BBD29RS14450 variant polypeptide; a polynucleotide encoding the variant polypeptide; a microorganism comprising any one or more of the variant polypeptide, a polynucleotide encoding the variant polypeptide, and a vector comprising the polynucleotide; and a method for producing L-arginine, the method comprising a step of culturing the microorganism in a culture medium.

Description

Technical Field

[0001] The present disclosure relates to novel BBD29_RS14450 variant polypeptides; polynucleotides encoding the variant polypeptides; microorganisms comprising any one or more of the variant polypeptides, the polynucleotides encoding the variant polypeptides, and vectors comprising the polynucleotides; and methods for producing L-arginine, comprising the step of culturing the microorganisms in a culture medium. Background Art

[0002] L-arginine is used for medical purposes, such as as a liver function enhancer, brain function enhancer, and polyamino acid preparation. It has also recently attracted attention for food applications, such as as a fish cake additive, a health drink additive, and a salt substitute for patients with hypertension. Therefore, research into the use of microorganisms is ongoing to produce high concentrations of arginine for industrial applications.

[0003] In order to produce L-amino acids and other useful substances, various studies have been conducted to develop efficient production microorganisms. For example, for the production of L-arginine, target substance-specific methods are mainly used, such as methods for increasing the expression of genes encoding enzymes mainly involved in L-arginine biosynthesis in Corynebacterium strains, or methods for deleting genes not required for L-arginine biosynthesis (Korean Patent No. 10-1102263).

[0004] However, there is a growing demand for research on methods that can efficiently produce L-arginine in high yields. Summary of the Invention

[0005] [Technical Issues]

[0006] The problem to be solved by the present disclosure is to provide a novel BBD29_RS14450 variant polypeptide; a polynucleotide encoding the variant polypeptide; a microorganism comprising the variant polypeptide, the polynucleotide encoding the variant polypeptide or a vector comprising the polynucleotide; and a method for producing L-arginine, the method comprising the step of culturing the microorganism in a culture medium.

[0007] [Technical solution]

[0008] One aspect of the present disclosure is to provide a BBD29_RS14450 variant polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, wherein the amino acid leucine at position 49 corresponding to SEQ ID NO: 3 is substituted with proline.

[0009] Another aspect of the present disclosure is to provide polynucleotides encoding the BBD29_RS14450 variant polypeptides of the present disclosure.

[0010] Another aspect of the present disclosure is to provide a microorganism comprising any one or more of the BBD29_RS14450 variant polypeptide of the present disclosure, a polynucleotide encoding the variant polypeptide, and a vector comprising the polynucleotide.

[0011] Another aspect of the present disclosure is to provide a method for producing L-arginine, comprising the step of culturing a microorganism in a culture medium, wherein the microorganism comprises any one or more of the BBD29_RS14450 variant polypeptide of the present disclosure, a polynucleotide encoding the BBD29_RS14450 variant polypeptide, and a vector comprising the polynucleotide.

[0012] Another aspect of the present disclosure is to provide a composition for producing L-arginine, the composition comprising the BBD29_RS14450 variant polypeptide of the present disclosure; a microorganism comprising any one or more of the variant polypeptide, a polynucleotide encoding the variant polypeptide, and a vector comprising the polynucleotide; or a combination thereof.

[0013] Another aspect of the present disclosure is to provide the BBD29_RS14450 variant polypeptide of the present disclosure and the use of a microorganism in producing L-arginine, wherein the microorganism comprises any one or more of the variant polypeptide, a polynucleotide encoding the variant polypeptide, and a vector comprising the polynucleotide.

[0014] [Beneficial Effects]

[0015] When a microorganism comprising the variant polypeptide of the present disclosure is cultured, L-arginine can be produced at a high yield compared to a microorganism having the existing unmodified polypeptide. DETAILED DESCRIPTION

[0016] 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. In other words, 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 in their entirety to further illustrate the level and scope of the subject matter to which the present disclosure belongs.

[0017] One aspect of the present disclosure provides a BBD29_RS14450 variant polypeptide, wherein the amino acid corresponding to position 49 of SEQ ID NO: 3 is substituted with a different amino acid.

[0018] In the present disclosure, the term “substituted with a different amino acid” is not limited as long as it is a substitution with an amino acid different from the amino acid before substitution. Meanwhile, in the present disclosure, when it is stated that “a specific amino acid has been substituted”, it is obvious that the amino acid has been substituted with an amino acid different from the amino acid before substitution, even if it is not specifically stated that the amino acid has been substituted with a different amino acid.

[0019] In one embodiment of the aforementioned aspects, there is provided a BBD29_RS14450 variant polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, wherein the amino acid leucine at position 49 corresponding to SEQ ID NO: 3 is substituted with proline.

[0020] As used herein, the term "BBD29_RS14450 variant polypeptide" refers to a variant of a BBD29_RS14450 polypeptide comprising one or more amino acid substitutions in the amino acid sequence of a BBD29_RS14450 polypeptide; or a variant of a BBD29_RS14450 polypeptide comprising one or more amino acid substitutions in a parent sequence, wherein the parent sequence is the amino acid sequence of a BBD29_RS14450 polypeptide.

[0021] The gene encoding BBD29_RS14450 may be derived from a microorganism of the genus Corynebacterium, specifically, Corynebacterium glutamicum, but is not limited thereto.

[0022] The BBD29_RS14450 variant polypeptide of the present disclosure may have the amino acid sequence shown in SEQ ID NO: 1, or may essentially consist of the amino acid sequence.

[0023] In addition, the BBD29_RS14450 variant polypeptide of the present disclosure may comprise 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 higher homology or identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid at position 49 corresponding to the amino acid sequence based on SEQ ID NO: 3 is proline. It is also obvious that variant polypeptides having amino acid sequences with deletions, modifications, substitutions, conservative substitutions or additions of certain sequences also fall within the scope of the present disclosure, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the variant polypeptide of the present disclosure.

[0024] For example, there are cases involving additions or deletions to the N-terminus, C-terminus and / or interior of the amino acid sequence that do not alter the function of the variants of the present disclosure, naturally occurring mutations, silent mutations or conservative substitutions thereof.

[0025] A "conservative substitution" refers to the replacement of one amino acid with another having similar structural and / or chemical properties. Such amino acid substitutions generally occur based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues. Typically, conservative substitutions may have little or no effect on the activity of a protein or polypeptide.

[0026] In addition, the BBD29_RS14450 protein having the amino acid sequence of SEQ ID NO: 1 can be encoded by a polynucleotide having or comprising the nucleotide sequence of SEQ ID NO: 2, or 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, or 99% or more, and less than 100% homology or identity to the sequence of SEQ ID NO: 2, or consisting of or essentially consisting of the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having 60% or more, 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 98% or more, and less than 100% homology or identity to the sequence of SEQ ID NO: 2, but is not limited thereto.

[0027] As used herein, the term "variant" refers to a polypeptide in which one or more amino acids are conservatively substituted 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 amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. In other words, the ability of the variant can be increased, 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 portion has been removed from the N- and / or C-terminus of the mature protein. The term "variant" may also be used interchangeably with modifications, modified polypeptides, modified proteins, mutants, mutant proteins, divergents, etc., and any term is not limited as long as it is used in the sense of being mutated.

[0028] For the purposes of the present disclosure, the BBD29_RS14450 variant may be a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid leucine at position 49 corresponding to the amino acid sequence of SEQ ID NO: 3 is substituted with proline.

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

[0030] As used herein, the term "homology" or "identity" refers to the degree of similarity between two given amino acid sequences or nucleotide sequences, which can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0031] The sequence homology or the identity of conservative polynucleotide or polypeptide are determined by standard comparison algorithms, and can be used together with the default gap penalty set up by the program to be used. Basically, homology or identical sequence can hybridize with whole or part sequence usually under moderate or highly stringent conditions. Obviously, hybridization also includes hybridization with the polynucleotide that comprises universal codons or takes into account the codon degeneracy.

[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, for example, using the default parameters as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as performed in the Needleman program of the EMBOSS software package (EMBOSS: The European Molecular Biology OpenSoftware Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or higher) (comprising 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, CA 901125. Diego, 1994 and [CARILLO et al.] (1988) SIAM J Applied Math 48:1073). For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0033] Homology, similarity or identity between polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, such as that described by Needleman et al., (1970), J Mol Biol. 48: 443, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2: 482. Briefly, the GAP program defines identity as the value obtained by dividing the number of similarly arranged 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 (comprising a value of 1 for identity and a value of 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 as disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353–358 (1979) (or the EDNAFULL (EMBOSS version of NCBINUC 4.4) substitution matrix); (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 opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0034] In one embodiment of the present disclosure, variant of the present disclosure can have GntR family transcriptional regulatory activity. In addition, compared with wild-type BBD29_RS14450 polypeptide, variant of the present disclosure can have the activity of increasing L-arginine production capacity. In the present disclosure, the sequence of wild-type BBD29_RS14450 polypeptide can be obtained from known databases (Genbank of NCBI etc.).

[0035] As used herein, "position N" may include position N and the amino acid position corresponding to position N. Specifically, it may include the amino acid position corresponding to any amino acid residue in the mature polypeptide disclosed in the specific amino acid sequence. The specific amino acid sequence may be the amino acid sequence of SEQ ID NO: 3.

[0036] 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 a listed residue in a polypeptide. Identifying an amino acid at a corresponding position can be a specific amino acid in a defined sequence, which sequence is referred to as a specific sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related protein or a reference protein.

[0037] For example, any amino acid sequence is aligned with SEQ ID NO: 3, based on which each amino acid residue of the amino acid sequence can be numbered by reference to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 3. For example, sequence alignment algorithms as described herein can identify the position of an amino acid, or the position at which a modification, such as a substitution, insertion, or deletion occurs, compared to a query sequence (also referred to as a "reference sequence").

[0038] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needleman program of the EMBOSS software 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 thereto, and sequence alignment programs, pairwise sequence comparison algorithms, etc. known in the art can be appropriately used.

[0039] Another aspect of the present disclosure provides a BBD29_RS01115 variant polypeptide consisting of the amino acid sequence of SEQ ID NO: 5, wherein the amino acid threonine at position 11 corresponding to SEQ ID NO: 7 is substituted with isoleucine. The BBD29_RS01115 variant polypeptide of the present disclosure may have the amino acid sequence shown in SEQ ID NO: 5, or may consist essentially of the amino acid sequence.

[0040] In addition, the BBD29_RS01115 variant polypeptide of the present disclosure may comprise 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 set forth in SEQ ID NO:5, wherein the amino acid corresponding to position 11 of the amino acid sequence based on SEQ ID NO:7 is isoleucine.

[0041] In addition, the BBD29_RS01115 protein having the amino acid sequence of SEQ ID NO:5 can be encoded by a polynucleotide having or comprising the nucleotide sequence of SEQ ID NO:6, or 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, or 99% or more and less than 100% homology or identity to the sequence of SEQ ID NO:6, or the polynucleotide consists of or consists essentially of the nucleotide sequence of SEQ ID NO:6, or 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, or 99% or more and less than 100% homology or identity to the sequence of SEQ ID NO:6, but is not limited thereto.

[0042] Another aspect of the present disclosure provides a BBD29_RS02580 variant polypeptide consisting of the amino acid sequence of SEQ ID NO: 9, wherein the amino acid phenylalanine at position 280 corresponding to SEQ ID NO: 11 is substituted with valine. The BBD29_RS02580 variant polypeptide of the present disclosure may have the amino acid sequence shown in SEQ ID NO: 9, or may consist essentially of the amino acid sequence.

[0043] In addition, the BBD29_RS02580 variant polypeptide of the present disclosure may comprise 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 to the amino acid sequence set forth in SEQ ID NO:9, wherein the amino acid corresponding to position 280 based on the amino acid sequence of SEQ ID NO:11 is valine.

[0044] In addition, the BBD29_RS02580 protein having the amino acid sequence of SEQ ID NO: 9 can be encoded by a polynucleotide having or comprising the nucleotide sequence of SEQ ID NO: 10, or 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, or 99% or more and less than 100% homology or identity to the sequence of SEQ ID NO: 10, or the polynucleotide consists of or consists essentially of the nucleotide sequence of SEQ ID NO: 10, or 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, or 99% or more and less than 100% homology or identity to the sequence of SEQ ID NO: 10, but is not limited thereto.

[0045] Another aspect of the present disclosure provides a BBD29_RS07530 variant polypeptide consisting of the amino acid sequence of SEQ ID NO: 13, wherein the amino acid alanine at position 2 corresponding to SEQ ID NO: 17 is substituted with valine. The BBD29_RS07530 variant polypeptide of the present disclosure may have the amino acid sequence shown in SEQ ID NO: 13, or may consist essentially of the amino acid sequence.

[0046] In addition, the BBD29_RS07530 variant polypeptide of the present disclosure may comprise 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 to the amino acid sequence set forth in SEQ ID NO: 13, wherein the amino acid corresponding to position 2 of the amino acid sequence based on SEQ ID NO: 17 is valine.

[0047] In addition, the BBD29_RS07530 protein having the amino acid sequence of SEQ ID NO: 13 can be encoded by a polynucleotide having or comprising the nucleotide sequence of SEQ ID NO: 14, or 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, or 99% or more and less than 100% homology or identity to the sequence of SEQ ID NO: 14, or the polynucleotide consists of or consists essentially of the nucleotide sequence of SEQ ID NO: 14, or 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, or 99% or more and less than 100% homology or identity to the sequence of SEQ ID NO: 14, but is not limited thereto.

[0048] Another aspect of the present disclosure provides a BBD29_RS07530 variant polypeptide consisting of the amino acid sequence of SEQ ID NO: 15, wherein the amino acid alanine corresponding to position 2 of SEQ ID NO: 17 is substituted with valine, and the amino acids corresponding to positions 79 to 82 are deleted. The BBD29_RS07530 variant polypeptide of the present disclosure may have the amino acid sequence shown in SEQ ID NO: 15, or may consist essentially of the amino acid sequence.

[0049] In addition, the BBD29_RS07530 variant polypeptide of the present disclosure may comprise 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 set forth in SEQ ID NO: 15, wherein the amino acid corresponding to position 2 of the amino acid sequence based on SEQ ID NO: 17 is valine, and the amino acids corresponding to positions 79 to 82 of the amino acid sequence based on SEQ ID NO: 17 are deleted.

[0050] In addition, the BBD29_RS07530 protein having the amino acid sequence of SEQ ID NO: 15 can be encoded by a polynucleotide having or comprising the nucleotide sequence of SEQ ID NO: 16, or 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, or 99% or more and less than 100% homology or identity to the sequence of SEQ ID NO: 16, or the polynucleotide consists of or consists essentially of the nucleotide sequence of SEQ ID NO: 16, or 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, or 99% or more and less than 100% homology or identity to the sequence of SEQ ID NO: 16, but is not limited thereto.

[0051] Another aspect of the present disclosure provides a BBD29_RS07535 variant polypeptide consisting of the amino acid sequence of SEQ ID NO: 19, wherein the amino acid alanine at position 203 corresponding to SEQ ID NO: 21 is substituted with threonine. The BBD29_RS07535 variant polypeptide of the present disclosure may have the amino acid sequence set forth in SEQ ID NO: 19, or may consist essentially of the amino acid sequence.

[0052] In addition, the BBD29_RS07535 variant polypeptide of the present disclosure may comprise 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 to the amino acid sequence set forth in SEQ ID NO: 19, wherein the amino acid corresponding to position 203 based on the amino acid sequence of SEQ ID NO: 21 is threonine.

[0053] In addition, the BBD29_RS07535 protein having the amino acid sequence of SEQ ID NO: 19 can be encoded by a polynucleotide having or comprising the nucleotide sequence of SEQ ID NO: 20, or 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, or 99% or more and less than 100% homology or identity to the sequence of SEQ ID NO: 20, or the polynucleotide consists of or consists essentially of the nucleotide sequence of SEQ ID NO: 20, or 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, or 99% or more and less than 100% homology or identity to the sequence of SEQ ID NO: 20, but is not limited thereto.

[0054] Another aspect of the present disclosure provides a BBD29_RS02010 variant polypeptide consisting of the amino acid sequence of SEQ ID NO: 97, wherein the amino acid arginine at position 349 corresponding to SEQ ID NO: 99 is substituted with glutamine. The BBD29_RS02010 variant polypeptide of the present disclosure may have the amino acid sequence shown in SEQ ID NO: 97, or may consist essentially of the amino acid sequence.

[0055] In addition, the BBD29_RS02010 variant polypeptide of the present disclosure may comprise 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 to the amino acid sequence set forth in SEQ ID NO:97, wherein the amino acid corresponding to position 349 based on the amino acid sequence of SEQ ID NO:99 is glutamine.

[0056] In addition, the BBD29_RS02010 protein having the amino acid sequence of SEQ ID NO: 97 can be encoded by a polynucleotide having or comprising the nucleotide sequence of SEQ ID NO: 98, or 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, or 99% or more and less than 100% homology or identity to the sequence of SEQ ID NO: 98, or the polynucleotide consists of or consists essentially of the nucleotide sequence of SEQ ID NO: 98, or 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, or 99% or more and less than 100% homology or identity to the sequence of SEQ ID NO: 98, but is not limited thereto.

[0057] Another aspect of the present disclosure provides a polynucleotide encoding the BBD29_RS14450 polypeptide variant of the present disclosure.

[0058] As used herein, the term "polynucleotide" refers to a DNA or RNA chain of a polymer of nucleotides of a certain length or longer, wherein nucleotide monomers are linked into a long chain by covalent bonds, and more specifically refers to a polynucleotide fragment encoding the variant polypeptide.

[0059] The polynucleotide encoding the BBD29_RS14450 variant polypeptide of the present disclosure may comprise a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1. For example, the polynucleotide of the present disclosure may have or comprise the nucleotide sequence of SEQ ID NO: 2. Furthermore, the polynucleotide of the present disclosure may consist of or consist essentially of the nucleotide sequence of SEQ ID NO: 2.

[0060] Taking into account codon degeneracy or codon preference in organisms expressing the disclosed variants, the polynucleotides of the present disclosure may be modified in various ways in the coding region without changing the amino acid sequence of the disclosed variants. Specifically, the polynucleotides of the present disclosure may have or comprise a nucleotide sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more and less than 100% homologous or identical to the sequence of SEQ ID NO: 2, or may consist of or consist essentially of a nucleotide sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more and less than 100% homologous or identical to the sequence of SEQ ID NO: 2, but are not limited thereto. In this regard, in a sequence having such homology or identity, the codon encoding the amino acid corresponding to position 49 of SEQ ID NO: 1 may be one of the codons encoding proline.

[0061] In addition, the polynucleotides of the present disclosure may include probes that can be prepared from known gene sequences, for example, any sequence without limitation, as long as it is a sequence that can hybridize under stringent conditions with a complementary sequence of all or part of the polynucleotide sequence of the present disclosure.

[0062] "Stringent conditions" refer to conditions that allow specific hybridization between polynucleotides. These conditions are described in detail in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, conditions under which polynucleotides having high homology or identity, i.e., polynucleotides 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 99% or more homology or identity, hybridize to each other, while polynucleotides having lower homology or identity than the above do not hybridize to each other, or washing conditions for ordinary Southern hybridization, i.e., washing once, specifically, twice or three times at a salt concentration and temperature corresponding to 60°C, 1хSSC, 0.1% SDS, specifically 60°C, 0.1хSSC, 0.1% SDS, more specifically 68°C, 0.1хSSC, 0.1% SDS.

[0063] Hybridization requires that the two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to each other. For example, with respect to 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, as well as isolated nucleic acid fragments that are complementary to the complete sequence.

[0064] Specifically, polynucleotides having homology or identity with the polynucleotides disclosed herein can be detected using hybridization conditions comprising a Tm value of 55° C. and a hybridization step under the above conditions. In addition, the Tm value may be 60° C., 63° C., or 65° C., but is not limited thereto and can be appropriately adjusted by those skilled in the art according to the purpose.

[0065] The appropriate stringency for hybridization of polynucleotides depends on the length of the polynucleotides and the degree of complementation, and variables are well known in the art (eg, J. Sambrook et al., supra).

[0066] Another aspect of the present disclosure provides a vector comprising the polynucleotide of the present disclosure. The vector may be an expression vector for expressing the polynucleotide in a host cell (eg, a microorganism), but is not limited thereto.

[0067] The carrier of the present disclosure can comprise a DNA construct comprising a nucleotide sequence of a polynucleotide encoding a desired polypeptide, wherein the polynucleotide is operably linked to a suitable expression control region (or expression control sequence) such that the desired polypeptide can be expressed in a suitable host. The expression control region can comprise a promoter capable of initiating transcription, any operon sequence for controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for controlling transcription and translation termination. The carrier can be transformed into a suitable host cell and then replicated or functioned independently of the host genome, or can be integrated into the genome itself.

[0068] 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 phages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as phage vectors or cosmid vectors, and pDZ system, pBR system, pUC system, pBluescriptII system, pGEM system, pTZ system, pCL system, pET system, etc. can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2 (Korean Patent Publication No. 10-2020-0136813, WO2021-187781 A1), pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

[0069] For example, the polynucleotide encoding the desired polypeptide can be inserted into the chromosome by the vector for the 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 vector can also include a selection marker for confirming chromosome insertion. The selection marker is used to select cells transformed with the vector, i.e., for confirming the insertion of the desired nucleic acid molecule, and can use a marker that gives selectable phenotypes such as drug resistance, auxotrophy, cytotoxic agent resistance or surface polypeptide expression. In the environment of processing with a selection agent, only cells expressing the selection marker survive or show other phenotypic traits, so the cells that are transformed can be selected.

[0070] 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. Regardless of the location, the transformed polynucleotide can be located by inserting into the chromosome of the host cell or outside the chromosome, as long as it can be expressed in the host cell. 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 genetic construct containing all the elements required for self-expression. The expression cassette can generally contain a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal that are operably linked 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 be operably linked to the sequence required for expression in the host cell, but is not limited thereto.

[0071] Furthermore, as used herein, the term "operably linked" means that a polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the desired variant of the present disclosure.

[0072] Yet another aspect of the present disclosure provides a strain comprising the BBD29_RS14450 polypeptide variant of the present disclosure or the polynucleotide of the present disclosure.

[0073] The strain of the present disclosure may comprise any one or more of the BBD29_RS14450 variant polypeptide of the present disclosure, a polynucleotide encoding the polypeptide, and a vector comprising the polynucleotide of the present disclosure.

[0074] As used herein, the term "strain (or microorganism)" includes all wild-type microorganisms or natural or artificial genetically modified microorganisms, and it can be a microorganism in which a specific mechanism is weakened or enhanced due to the insertion of an exogenous gene or the enhanced or inactivated activity of an endogenous gene, and it can be a microorganism comprising a genetic modification for producing a desired polypeptide, protein or product. In the present disclosure, "strain" and "microorganism" can be used interchangeably without restriction and have the same meaning.

[0075] The strain disclosed herein may be a strain comprising any one or more of the BBD29_RS14450 variant polypeptide disclosed herein, the polynucleotide disclosed herein, and a vector comprising the polynucleotide disclosed herein; a strain modified to express the BBD29_RS14450 variant polypeptide disclosed herein or the polynucleotide disclosed herein; a strain expressing the BBD29_RS14450 variant polypeptide disclosed herein or the polynucleotide disclosed herein (e.g., a recombinant strain); or a strain having the BBD29_RS14450 variant activity disclosed herein (e.g., a recombinant strain), but is not limited thereto.

[0076] The strain disclosed herein may be a strain having the ability to produce L-arginine.

[0077] The strain disclosed herein may be a microorganism that naturally has the ability to produce BBD29_RS14450 or L-arginine, or a microorganism prepared by introducing any one or more of the variant polypeptide disclosed herein, the polynucleotide encoding the variant polypeptide, and the vector comprising the polynucleotide into a parent strain that does not have the ability to produce BBD29_RS14450 or L-arginine, and / or by providing it with the ability to produce L-arginine, but is not limited thereto.

[0078] For example, bacterial strain of the present invention can be transformed with the cell or microorganism of variant polypeptide of the present invention with polynucleotide of the present invention or the vector comprising the polynucleotide of encoding variant polypeptide of the present invention to express variant polypeptide of the present invention.About purpose of the present disclosure, bacterial strain of the present invention can include all microorganisms capable of producing L-arginine by comprising variant polypeptide of the present invention.For example, bacterial strain of the present invention can be the recombinant bacterial strain with increased L-arginine productivity, wherein by importing the polynucleotide of encoding variant polypeptide of the present invention into natural wild-type microorganism or the microorganism producing L-arginine to express BBD29_RS14450 variant.Compared with the microorganism of natural wild-type microorganism or unmodified BBD29_RS14450 (for example, the microorganism expressing wild-type BBD29_RS14450 (SEQ ID NO:3) or the microorganism not expressing variant (SEQ ID NO:1) protein of the present invention), the recombinant bacterial strain with increased L-arginine productivity can be the microorganism whose L-arginine productivity increases, but is not limited thereto. For example, the unmodified BBD29_RS14450 microorganism used as the strain for comparison of whether the L-arginine productivity is increased may be strain KCCM10741P (KR 10-0791659B1, EP 2041265 B1) or CJ1R strain (KR 10-2022-0139085A, WO 2022-216088 A1), but is not limited thereto.

[0079] For example, the recombinant strain with increased productivity can have an L-arginine productivity of about 1% or more, about 2.5% or more, about 5% or more, about 7% or more, about 10% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 17% or more, about 19% or more, or 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) compared to the parental strain before the modification or the unmodified microorganism, but the amount increased is not limited thereto, as long as the productivity has a + value of increase compared to the productivity of the parental strain before the modification or the unmodified microorganism. In another example, compared with the parental strain before modification or unmodified microorganism, the recombinant bacterial strain with increase production capacity can have the L-arginine production capacity of about 1.01 times or more, about 1.02 times or more, about 1.05 times or more, about 1.07 times or more, about 1.1 times or more, about 1.12 times or more, about 1.14 times or more, about 1.15 times or more, about 1.17 times or more, about 1.19 times or more or 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, about 2 times or less or about 1.5 times or less) increase, but is not limited to this.Term " about " refers to the scope comprising whole ± 0.5, ± 0.4, ± 0.3, ± 0.2, ± 0.1 etc., and comprises all values that are equal to or similar to the value after these values, but this scope is not limited to this.

[0080] As used herein, term " unmodified microorganism " does not get rid of the bacterial strain that comprises the mutation that may naturally occur in microorganism, and can refer to wild-type strain or natural strain itself, or because natural or artificial factors change the bacterial strain before proterties by genetic variation.For example, unmodified microorganism can refer to the bacterial strain that wherein does not import BBD29_RS14450 variant polypeptide as herein described or the bacterial strain before importing." unmodified microorganism " can be used interchangeably with " bacterial strain before modification ", " microorganism before modification ", " unvaried bacterial strain ", " unmodified bacterial strain ", " unvaried microorganism " or " reference microorganism ".

[0081] Regarding the microorganism according to any of the aforementioned specific embodiments, the strain or microorganism of the present disclosure can be a microorganism of the genus Corynebacterium sp., Escherichia sp., Erwinia sp., Serratia sp., Providencia sp., Pseudomonas sp., Leptospira sp., Salmonella sp., Brevibacteria sp., Hypomononas sp., Chromobacterium sp. or Nocardia sp., or a microorganism of fungus or yeast, specifically, a microorganism of the genus Corynebacterium, but not limited thereto.

[0082] For another example of the present disclosure, the strain or microorganism of the present disclosure can be a microorganism of the genus Corynebacterium, specifically, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testis. testudinoris) or Corynebacterium flavescens.

[0083] As used herein, the term "reduction" of polypeptide activity is a concept that includes two situations in which the activity is reduced or absent compared to the endogenous activity. Reduction can be used interchangeably with terms such as inactivation, lack, downregulation, reduction, lowering, and weakening.

[0084] Attenuation can also include situations where the activity of the polypeptide itself is reduced or eliminated due to variations in the polynucleotide encoding the polypeptide, etc., compared to the polypeptide activity originally possessed by the microorganism; situations where the total activity level and / or concentration (expression level) of the polypeptide in the cell is lower than that of the natural strain due to inhibition of the expression of the gene encoding the polynucleotide or by inhibition of translation into the polypeptide; situations where the polynucleotide is not expressed at all; and / or situations where the polypeptide activity does not exist even when the polynucleotide is expressed. "Endogenous activity" refers to the activity of a specific polypeptide originally possessed by the parent strain, or the 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". The fact that the activity of a polypeptide is "inactivated, lacking, reduced, downregulated, lowered or weakened" compared to the endogenous activity means that the activity of the polypeptide is reduced compared to the activity of the specific polypeptide originally possessed by the parent strain or the unmodified microorganism before the trait is changed.

[0085] Such attenuation of polypeptide activity can be performed by any method known in the art, but the method is not limited thereto, and attenuation can be achieved by applying various methods well known in the art (e.g., 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.).

[0086] Specifically, the reduction of polypeptide activity in the present disclosure may be:

[0087] 1) Deletion of all or part of the gene encoding the polypeptide;

[0088] 2) modifying the expression regulatory region (or expression regulatory sequence) to reduce the expression of the gene encoding the polypeptide;

[0089] 3) modifying the amino acid sequence constituting the polypeptide to eliminate or weaken the activity of the polypeptide (e.g., deleting / substituting / adding one or more amino acids in the amino acid sequence);

[0090] 4) modifying a gene sequence encoding a polypeptide to eliminate or reduce the activity of the polypeptide (e.g., deleting / substituting / adding one or more nucleic acid bases in the nucleic acid sequence of the polypeptide gene to encode a polypeptide that has been modified to eliminate or reduce the activity of the polypeptide);

[0091] 5) modifying the nucleotide sequence of the start codon or 5'-UTR region of the gene transcript encoding the polypeptide;

[0092] 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to the transcript of the gene encoding the polypeptide;

[0093] 7) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding the polypeptide to form a secondary structure to which the ribosome cannot attach;

[0094] 8) adding a promoter that transcribes in the opposite direction to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE); or

[0095] 9) A combination of two or more selected from 1) to 8), but not particularly limited thereto.

[0096] For example,

[0097] 1) Deletion of part or all of the gene encoding the polypeptide can be achieved by removing the complete polynucleotide encoding the endogenous target polypeptide in the chromosome, replacing it with a polynucleotide lacking some nucleotides, or replacing it with a marker gene.

[0098] In addition, 2) modification of the expression regulatory region (or expression regulatory sequence) can be a variation in the expression regulatory region (or expression regulatory sequence) due to deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or replacement with a sequence exhibiting 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 sequences regulating transcription and translation termination.

[0099] Furthermore, 3) modification of the nucleotide sequence of the start codon or 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, replacement with a nucleotide sequence encoding another start codon having a lower polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0100] Furthermore, modifications of the amino acid sequence or polynucleotide sequence in 4) and 5) may be variations in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide due to deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or substitutions resulting in an amino acid sequence or polynucleotide sequence modified to have weaker activity or an amino acid sequence or polynucleotide sequence modified to be inactive, thereby reducing the activity of the polypeptide, but are not limited thereto. For example, gene expression can be inhibited or reduced by introducing a mutation into the polynucleotide sequence and forming a stop codon, but are not limited thereto.

[0101] 6) For the introduction of antisense oligonucleotides (e.g., antisense RNA) that bind complementarily to the transcript of the gene encoding the polypeptide, reference can be made to literature, for example [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews-Trends in Genetics, Vol. 1(1) 1986].

[0102] 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide to form a secondary structure to which ribosomes cannot attach, which may make mRNA translation impossible or slow down the mRNA translation rate.

[0103] 8) Adding a promoter that transcribes in the opposite direction to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE) may reduce activity by making the antisense nucleotide complementary to the transcript of the gene encoding the polypeptide.

[0104] As used herein, the term "enhancing" of a polypeptide activity refers to that the activity of the polypeptide increases compared to endogenous activity. Enhancing can be used interchangeably with terms such as activating, raising, over-expressing and increasing. Here, activating, enhancing, raising, over-expressing and increasing can include showing an activity that was not initially present and showing an activity that was improved compared to the activity before endogenous activity or modification. "Endogenous activity" refers to that when proterties change because of the genetic variation caused by natural or artificial factors, the activity of the specific polypeptide that the parental strain or unmodified microorganism originally had before the proterties change. This can be used interchangeably with "activity before modification". Compared to endogenous activity, the fact that the activity of a polypeptide is "enhanced", "raised", "over-expressed" or "increased" refers to that the activity and / or concentration (expression level) of the specific polypeptide that the parental strain or unmodified microorganism originally had before the activity and proterties change of the polypeptide are compared to improve.

[0105] Enhancement can be achieved by introducing an exogenous polypeptide or enhancing the endogenous activity and / or concentration (expression level) of the polypeptide. The activity enhancement of a polypeptide 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 product produced from the corresponding polypeptide.

[0106] For the enhancement of polypeptide activity, various methods well known in the art can be applied, and the method is not limited, as long as the activity of the target polypeptide can be enhanced compared to the microorganism before modification. Specifically, genetic engineering and / or protein engineering well known to those skilled in the art can be used, which are conventional methods of molecular biology, but the method is not limited thereto (for example, Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2.1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0107] Specifically, the enhancement of the polypeptide in the present disclosure can be:

[0108] 1) increasing the intracellular copy number of a polynucleotide encoding a polypeptide;

[0109] 2) replacing the gene expression regulatory region on the chromosome encoding the polypeptide with a sequence showing strong activity;

[0110] 3) modifying the nucleotide sequence of the start codon or 5'-UTR region of the gene transcript encoding the polypeptide;

[0111] 4) Modifying the amino acid sequence of a polypeptide to enhance its activity;

[0112] 5) modifying a polynucleotide sequence encoding a polypeptide to enhance the activity of the polypeptide (e.g., modifying the polynucleotide sequence of a polypeptide gene to encode a polypeptide that has been modified to enhance the activity of the polypeptide);

[0113] 6) introducing an exogenous polypeptide exhibiting polypeptide activity or an exogenous polynucleotide encoding the polypeptide;

[0114] 7) codon optimization of the polynucleotide encoding the polypeptide;

[0115] 8) analyzing the tertiary structure of the polypeptide to select exposed sites and modifying or chemically modifying the exposed sites; or

[0116] 9) A combination of two or more selected from 1) to 8), but not particularly limited thereto.

[0117] More specifically,

[0118] 1) Increasing the intracellular copy number of a polynucleotide encoding a polypeptide can be achieved by introducing a vector into the host cell, wherein the vector can replicate and function independently of the host, and the polynucleotide encoding the corresponding polypeptide is operably linked to the vector. Alternatively, such 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. Introduction into the chromosome can be achieved by introducing a vector capable of inserting the polynucleotide into the host cell chromosome, but is not limited thereto. The vector is as described above.

[0119] 2) Replacing the gene expression regulatory region (or expression control sequence) on the chromosome encoding the polypeptide with a sequence exhibiting strong activity can be, for example, a variation in sequence due to deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or replacing it with a sequence exhibiting stronger activity to further enhance the activity of the expression regulatory region. The expression regulatory region may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence controlling transcription and translation termination, and the like. For example, the replacement may be replacing the original promoter with a strong promoter, but is not limited thereto.

[0120] Examples of known strong promoters include CJ1 to CJ7 promoters (US Patent No. 7662943B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Patent No. 10584338B2), O2 promoter (US Patent No. 10273491B2), tkt promoter, yccA promoter, etc., but are not limited thereto.

[0121] 3) Modification of the start codon or 5'-UTR nucleotide sequence of the gene transcript encoding the polypeptide may be, for example, replacement with a nucleotide sequence encoding another start codon having a higher polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0122] 4) and 5) modification of the amino acid sequence or polynucleotide sequence may be due to deletion, insertion, non-conservative 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 improved to exhibit stronger activity, or with an amino acid sequence or polynucleotide sequence improved to be more active, thereby enhancing the activity of the polypeptide, but is not limited thereto. The polynucleotide may be specifically replaced by insertion into a chromosome by homologous recombination, but is not limited thereto. The vector used herein may further comprise a selection marker for confirming chromosomal insertion. The selection marker is the same as described above.

[0123] 6) Introduction of an exogenous polynucleotide exhibiting polypeptide activity can involve introducing into host cells an exogenous polynucleotide encoding a polypeptide exhibiting the same or similar activity as the polypeptide. The exogenous polynucleotide is not limited in source or sequence, as long as it exhibits the same or similar activity as the polypeptide. The introduction process can be performed by appropriately selecting a known transformation method used by those skilled in the art. When the introduced polynucleotide is expressed in the host cell, the polypeptide can be produced and its activity can be increased.

[0124] 7) Codon optimization of a polypeptide-encoding polynucleotide may be performed by codon optimization of an endogenous polynucleotide to increase transcription or translation in a host cell, or by codon optimization of an exogenous polynucleotide to optimize transcription and translation in a host cell.

[0125] 8) Analyzing the tertiary structure of a polypeptide to select and modify or chemically modify exposed sites can be, for example, performed by comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, determining template protein candidates based on the degree of sequence similarity, confirming the structure based on the sequence, and modifying or chemically modifying the exposed sites to be modified or chemically modified.

[0126] Such enhancement of polypeptide activity can be an increase in the activity or concentration expression level of the corresponding polypeptide based on the activity or concentration of the polypeptide expressed in the wild-type or pre-modified microbial strain, or an increase in the amount of the product produced by the corresponding polypeptide, but is not limited thereto.

[0127] In the microorganisms disclosed herein, some or all of the modifications of the polynucleotides can be induced by: (a) homologous recombination using a vector for chromosomal 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 can include methods using DNA recombination technology. For example, by introducing a nucleotide sequence or a vector comprising a nucleotide sequence homologous to the target gene into the microorganism to cause homologous recombination, some or all of the genes can be deleted. The nucleotide sequence or vector to be introduced may comprise a dominant selection marker, but is not limited thereto.

[0128] The strains and microorganisms disclosed herein may comprise any one or more of the BBD29_RS14450 variant polypeptide disclosed herein, a polynucleotide encoding the variant polypeptide, and a vector comprising the polynucleotide, and may also comprise any one or more of the following: any one or more of the BBD29_RS01115 variant polypeptide, the BBD29_RS02580 variant polypeptide, two BBD29_RS07530 variant polypeptides (e.g., SEQ ID NO: 13, SEQ ID NO: 15), the BBD29_RS07535 variant polypeptide, and the BBD29_RS02010 variant polypeptide, a polynucleotide encoding the variant polypeptide, and a vector comprising the polynucleotide.

[0129] In the microorganism disclosed herein, the variant polypeptide, polynucleotide, L-arginine, etc. are as described in other aspects.

[0130] Yet another aspect of the present disclosure provides a method for producing an L-amino acid, comprising the step of culturing a Corynebacterium glutamicum strain comprising the BBD29_RS14450 variant polypeptide of the present disclosure or the polynucleotide of the present disclosure in a culture medium.

[0131] The method for producing an L-amino acid of the present disclosure may include the step of culturing a strain comprising the variant of the present disclosure, the polynucleotide of the present disclosure, or the vector of the present disclosure in a culture medium.

[0132] Furthermore, the L-amino acid of the present disclosure may be L-arginine.

[0133] As used herein, the term "culture" refers to culturing the strains of the present invention under appropriately adjusted environmental conditions. The culture procedures disclosed herein can be carried out according to suitable culture media or culture conditions known in the art. Depending on the selected strain, those skilled in the art can easily adjust this culture process for use. Specifically, the culture can be batch, continuous and / or fed-batch, but is not limited thereto.

[0134] As used herein, "culture medium" refers to a mixture containing nutrients required for culturing the strains of the present invention as 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 strains of the present invention, any culture medium conventionally used for culturing microorganisms can be used without particular limitation. However, the strains of the present invention can be cultured in a common culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and / or vitamin under aerobic conditions, while controlling temperature, pH, etc.

[0135] Specifically, the culture medium of the strains disclosed herein (particularly coryneform bacteria) can be found in the literature ["Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington DC, USA, 1981)].

[0136] In the present disclosure, the carbon source may include 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.; 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 to reducing sugars) may be used, and various other carbon sources may be used in appropriate amounts without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0137] 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 products, defatted soybean cake or its degradation products, etc. can be used. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0138] The phosphorus source may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and corresponding sodium salts. As inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. may be used. In addition, amino acids, vitamins, and / or suitable precursors may also be included. These components or precursors may be added to the culture medium in batches or continuously. However, the present disclosure is not limited thereto.

[0139] In addition, during the cultivation of the bacterial strain of the present disclosure, the pH of the culture medium can be regulated by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid in a suitable manner to the culture medium. In addition, defoamers such as fatty acid polyethylene glycol esters can be used to suppress the formation of foam during the cultivation process. 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 be not injected or nitrogen, hydrogen or carbon dioxide gas can be injected to maintain the anaerobic or non-aerobic state of the culture medium, but is not limited thereto.

[0140] In the culture of the present disclosure, the culture temperature may be maintained at 20° C. to 45° C., specifically, 25° C. to 40° C., and the culture may be performed for about 10 to 160 hours, but is not limited thereto.

[0141] The L-amino acid produced by the culture of the present disclosure may be released into the culture medium or may be retained in the cells.

[0142] The method for producing L-amino acids disclosed herein may further include the steps of preparing the strain disclosed herein, preparing a culture medium for culturing the strain, or a combination of these steps (regardless of the order, in any order), for example, before the culturing step.

[0143] The method for producing L-amino acids of the present disclosure may further include a step of recovering the L-amino acids from the culture medium (culture medium) or the strain of the present disclosure. A recovery step may be further included after the culture step.

[0144] Recovery can be a method of culturing a microorganism according to the present disclosure, such as batch, continuous or fed-batch type culture, by collecting the desired L-amino acid using suitable methods known in the art. For example, centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, ultrasonication, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography and affinity chromatography), HPLC and combinations of these methods can be used, and the desired L-amino acid can be recovered from the culture medium or microorganism by using suitable methods known in the art.

[0145] In addition, the method for producing L-amino acids disclosed herein may further include a purification step. Purification may be performed using suitable methods known in the art. In exemplary embodiments, when the method for producing L-amino acids disclosed herein includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously without regard to order, or may be performed simultaneously or integrated into one step, but is not limited thereto.

[0146] In the methods disclosed herein, the variants, polynucleotides, vectors, strains, etc. are as described in other aspects.

[0147] Another aspect of the present disclosure provides a composition for producing L-amino acids, the composition comprising the BBD29_RS14450 variant polypeptide of the present disclosure; a polynucleotide encoding the variant polypeptide; a vector comprising the polynucleotide; a strain comprising any one or more of the BBD29_RS14450 variant polypeptide of the present disclosure, the polynucleotide of the present disclosure, and the vector comprising the polynucleotide; a culture medium for culturing the strain; or a combination of two or more thereof.

[0148] The composition of the present disclosure may further comprise any suitable excipient conventionally used in compositions for producing amino acids, and such excipients may include, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, isotonic agents, etc., but are not limited thereto.

[0149] In the compositions of the present disclosure, the variants, polynucleotides, vectors, strains, culture media and L-amino acids are as described in other aspects.

[0150] Another aspect of the present disclosure provides the use of the BBD29_RS14450 variant polypeptide and a microorganism of the present disclosure in producing L-arginine, wherein the microorganism comprises any one or more of the BBD29_RS14450 variant polypeptide, a polynucleotide encoding the BBD29_RS14450 variant polypeptide, and a vector comprising the polynucleotide.

[0151] The variant polypeptides, polynucleotides, vectors, microorganisms, and L-arginine disclosed herein are as described in other aspects.

[0152] [Modes for Carrying Out the Invention]

[0153] Hereinafter, the present disclosure will be described in more detail by way of exemplary embodiments. However, the following exemplary embodiments are merely for illustrating preferred embodiments of the present disclosure and are therefore not intended to limit the scope of the present disclosure thereto. Furthermore, technical matters not described in this specification may be fully understood and easily implemented by those skilled in the art of the present disclosure or similar technical fields.

[0154] Example 1. Preparation of a mutant strain with increased arginine production capacity by artificial mutagenesis

[0155] In order to select mutant strains with increased arginine production capacity, random mutations were induced on the genome using ultraviolet irradiation (UV mutagenesis). The arginine-producing strain KCCM10741P (Korean Patent No. 10-0791659, EP 2041265B1) was diluted and spread on a composite plate medium containing agar, irradiated with UV, and cultured at 30°C for 48 hours to obtain mutant colonies. Approximately 20,000 colonies thus obtained were screened and evaluated under 96 deep-well plate conditions. The composition of the composite plate medium is as follows.

[0156] <Compound Plate Medium (pH 7.0)>

[0157] 10g glucose, 10g peptone, 5g beef extract, 5g yeast extract, 18.5g brain heart infusion, 2.5g NaCl, 2g urea, 91g sorbitol, 20g agar (based on 1L distilled water)

[0158] Example 2. Screening and evaluation of mutant strains with increased arginine production capacity

[0159] About 20,000 colonies obtained in Example 1 were inoculated into 300 μl of the following selection medium and cultured in a 96-deep-well plate at 30° C. and 1,000 rpm for about 24 hours.

[0160] <Selection medium (pH 8.0)>

[0161] 10g glucose, 5.5g ammonium sulfate, 1.2g MgSO47H2O, 0.8g KH2PO4, 16.4g K2HPO4, 100μg biotin, 1mg thiamine hydrochloride, 2mg calcium pantothenate, 2mg niacinamide (based on 1L distilled water)

[0162] The ninhydrin method was used to analyze the production of L-arginine produced in the culture medium (Moore, S., Stein, WH, Photometric ninhydrin method for use in the chromatography of aminoacids. J. Biol. Chem. 1948, 176, 367-388).

[0163] To this end, after completing cultivation of each mutant strain, 10 μl of the culture supernatant and 190 μl of the ninhydrin reaction solution were reacted at 65°C for 30 minutes, and the absorbance of the reaction solution was measured at a wavelength of 570 nm using a spectrophotometer. Approximately 30 mutant colonies exhibiting high absorbance compared to the control strain, Corynebacterium glutamicum KCCM10741P, were selected. The remaining colonies were confirmed to exhibit similar or decreased absorbance compared to the control strain, Corynebacterium glutamicum KCCM10741P.

[0164] The 30 types of strains selected above were re-cultured using the same method, and the ninhydrin reaction was repeated to select the top 10 mutant strains having the most improved L-arginine-producing ability compared with the parent strain, Corynebacterium glutamicum KCCM10741P strain.

[0165] Example 3: Analysis of L-arginine production capacity of selected random mutant strains

[0166] To finally select a strain with reproducibly increased L-arginine production capacity from the 10 types of mutant strains selected in Example 2, shake flask cultures were performed using the following medium. After completion of the cultures, the L-arginine concentration in the culture medium was analyzed using HPLC. The concentration of L-arginine produced in each mutant strain is shown in Table 1 below.

[0167] <Production Medium (pH 7.0)>

[0168] 6% glucose, 3% ammonium sulfate, 0.1% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, 1.5% corn steep liquor (CSL), 1% NaCl, 0.5% yeast extract, 100 mg / l biotin, 3% CaCO3 (based on 1 liter of distilled water).

[0169] [Table 1]

[0170]

[0171] From the 10 selected mutant strains, KCCM10741P / UV-7 was ultimately selected as showing the most significant improvement in L-arginine production. Results confirmed that the KCCM10741P / UV-7 strain exhibited a 20% increase in arginine production compared to the control KCCM10741P strain, indicating improved arginine yield.

[0172] Example 4: Identification of the cause of increased L-arginine production capacity in the finally selected strain

[0173] NGS sequence analysis was performed on the mutant strain KCCM10741P / UV-7, which was ultimately selected from Example 3, and compared with the parent strain KCCM10741P and the wild-type Corynebacterium glutamicum ATCC13869. Genes whose protein sequences were altered due to nucleotide sequence mutations were identified, and the types of gene substitutions are shown in Table 2 below.

[0174] Among the mutation types, it was confirmed that the most common mutation type was amino acid mutation introduced due to nucleotide sequence substitution, of which there were 6 cases. BBD29_RS07530 was confirmed to be a variant protein in which, in addition to the nucleotide sequence substitutions shown in Table 2 below, 4 amino acids were deleted by an additional deletion of 12 bp of nucleotide sequence from positions 233 to 245.

[0175] [Table 2]

[0176]

[0177]

[0178] Example 5: Construction of recombinant vectors to detect the arginine production ability of identified gene mutations

[0179] In order to detect the effects of the protein variants of the KCCM10741P / UV-7 strain identified in Example 4 on the arginine production capacity of the microorganism, a recombinant vector capable of inducing mutations (substitution mutations or deletions) in each protein was constructed. The recombinant vector was constructed based on the pDCM2 (Korean Patent Publication No. 10-2020-0136813, WO 2021-187781A1) plasmid and used to replace the gene in the chromosome of the Corynebacterium strain.

[0180] The gDNA (genomic DNA) of KCCM10741P / UV-7 selected in Example 3 was used as a template, and upstream and downstream gene fragments based on the mutation site were obtained by PCR to introduce various mutant sequences. The names of the constructed vectors and the sequence information of the primers used are listed in Table 3 below. For each gene, PCR was performed using the F1 and R2 primer pairs to obtain the upstream gene fragment based on the mutation site, and the F3 and R4 primer pairs were used to obtain the downstream fragment. PCR was performed as follows: denaturation at 94°C for 5 minutes; followed by 30 cycles of: 30 seconds at 94°C, 30 seconds at 55°C, and 1 minute 30 seconds at 72°C; then 5 minutes at 72°C. After DNA purification, the DNA was purified using In-Fusion The two fragments thus obtained were connected (fusion cloned) to the pDCM2 plasmid treated with the SmaI restriction enzyme using a cloning kit (Clontech). The resulting vectors were named pDCM2-BBD29_RS01115(C32T), pDCM2-BBD29_RS02580(T838G), pDCM2-BBD29_RS07530(C5T), pDCM2-BBD29_RS07530(Δ233-245), pDCM2-BBD29_RS07535(G607A), pDCM2-BBD29_RS14450(T146C), and pDCM2-BBD29_RS02010(G1046A).

[0181] [Table 3]

[0182]

[0183]

[0184]

[0185] Example 6: Preparation of microorganisms expressing protein variants and evaluation of L-arginine production capacity

[0186] The seven types of vectors constructed in Example 5 were each transformed into Corynebacterium glutamicum CJ1R (Korean Patent Application Publication No. 10-2022-0139085, WO 2022-216088 A1). The corresponding vectors and host strains were transformed by electroporation (Appl. Microbiol. Biotechnol. (1999) 52: 541-545), and strains in which gene mutations were inserted were obtained by secondary exchange. The mutant strains obtained in this way were named CJ1R-BBD29_RS01115(C32T), CJ1R-BBD29_RS02580(T838G), CJ1R-BBD29_RS07530(C5T), CJ1R-BBD29_RS07530(△233-245), CJ1R-BBD29_RS07535(G607A), CJ1R-BBD29_RS14450(T146C) and CJ1R-BBD29_RS02010(G1046A), respectively.

[0187] In order to compare the L-arginine production capabilities of the strains into which the gene mutations were introduced, these strains were cultured using the following method, and the L-arginine concentration in the culture medium was analyzed.

[0188] The parent strain, Corynebacterium glutamicum CJ1R strain, and a total of 8 types of transformed strains were each inoculated into a 250 ml baffled flask containing 25 ml of the following seed culture medium and cultured with shaking at 200 rpm at 30°C for 20 hours. Thereafter, 1 ml of the seed culture medium was inoculated into a 250 ml baffled flask containing 24 ml of the production culture medium and cultured with shaking at 200 rpm at 30°C for 72 hours. The compositions of the seed culture medium and the production culture medium are as follows.

[0189] <Seed culture medium (pH 7.2)>

[0190] 20g glucose, 45g ammonium sulfate, 2g magnesium sulfate heptahydrate, 2g potassium dihydrogen phosphate, 10g ammonium chloride, 0.01mg biotin, 0.1mg thiamine hydrochloride, 2mg calcium pantothenate, 3mg niacinamide, 10mg iron sulfate, 10mg manganese sulfate, 0.02mg zinc sulfate, 0.5mg copper sulfate (based on 1 liter of distilled water)

[0191] <Production Medium (pH 7.2)>

[0192] 60g glucose, 45g ammonium sulfate, 2g magnesium sulfate heptahydrate, 2g potassium dihydrogen phosphate, 10g ammonium chloride, 0.01mg biotin, 0.1mg thiamine hydrochloride, 2mg calcium pantothenate, 3mg niacinamide, 10mg iron sulfate, 10mg manganese sulfate, 0.02mg zinc sulfate, 0.5mg copper sulfate, 30g calcium carbonate (based on 1 liter of distilled water)

[0193] After the cultivation was completed, the L-arginine production capacity (concentration) was analyzed using HPLC (Waters 2478). The analyzed L-arginine concentrations are shown in Table 4 below.

[0194] [Table 4]

[0195]

[0196] As a result, it was confirmed that the L-arginine productivity of the strain CJ1R-BBD29_RS14450(T146C) into which the variant was introduced increased by 14% compared to the parent strain CJ1R, and showed an L-arginine productivity of 114%.

[0197] The above results confirmed that the BBD29_RS14450(T146C) mutation of the present disclosure increased the L-arginine production capacity of the microorganism. In addition, the 20% increase in the production capacity of the KCCM10741P / UV-7 mutant strain in Example 3 was attributed to the combined effects of the above mutations.

[0198] Based on the above description, it will be understood by those skilled in the art that the present disclosure can be implemented in different specific forms without changing its technical spirit or basic characteristics. In this regard, it should be understood that the above embodiments are not restrictive, but illustrative in all aspects. The scope of the present disclosure is defined by the appended claims rather than the description thereafter, and all changes and modifications that fall within the boundaries and scope of the claims, or the equivalents of these boundaries and scopes, are intended to be included in the claims.

Claims

1. A BBD29_RS14450 variant polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, wherein the amino acid leucine at position 49 corresponding to SEQ ID NO: 3 is substituted by proline. A polynucleotide encoding the variant polypeptide of claim 1 .

3. The polynucleotide according to claim 2, wherein the polynucleotide consists of the nucleotide sequence of SEQ ID NO:

2. 4 . A Corynebacterium glutamicum strain comprising any one or more of the variant polypeptide according to claim 1 and a polynucleotide encoding the variant polypeptide.

5. The Corynebacterium glutamicum strain according to claim 4, wherein the strain further comprises any one or more variant polypeptides of the following (a) to (f) and any one or more polynucleotides encoding the variant polypeptides: (a) a BBD29_RS01115 variant polypeptide consisting of the amino acid sequence of SEQ ID NO: 5, wherein the amino acid threonine at position 11 corresponding to SEQ ID NO: 7 is substituted with isoleucine; (b) a BBD29_RS02580 variant polypeptide consisting of the amino acid sequence of SEQ ID NO: 9, wherein the amino acid phenylalanine at position 280 corresponding to SEQ ID NO: 11 is substituted with valine; (c) a BBD29_RS07530 variant polypeptide consisting of the amino acid sequence of SEQ ID NO: 13, wherein the amino acid alanine at position 2 corresponding to SEQ ID NO: 17 is substituted with valine; (d) a BBD29_RS07530 variant polypeptide consisting of the amino acid sequence of SEQ ID NO: 15, wherein the amino acid alanine corresponding to position 2 of SEQ ID NO: 17 is substituted with valine, and the amino acids corresponding to positions 79 to 82 are deleted; (e) a BBD29_RS07535 variant polypeptide consisting of the amino acid sequence of SEQ ID NO: 19, wherein the amino acid alanine at position 203 corresponding to SEQ ID NO: 21 is substituted with threonine; and (f) A BBD29_RS02010 variant polypeptide consisting of the amino acid sequence of SEQ ID NO: 97, wherein the amino acid arginine at position 349 corresponding to SEQ ID NO: 99 is substituted with glutamine. 6 . The strain according to claim 4 , wherein the strain has increased L-arginine-producing ability compared to Corynebacterium glutamicum comprising the polypeptide of SEQ ID NO: 3 or a polynucleotide encoding the polypeptide of SEQ ID NO:

3.

7. A method for producing L-arginine, comprising the step of culturing a Corynebacterium glutamicum strain in a culture medium, wherein the Corynebacterium glutamicum strain comprises any one or more of the variant polypeptide according to claim 1 and a polynucleotide encoding the variant polypeptide.

8. A composition for the production of L-arginine, comprising any one or more of the following: a variant polypeptide according to claim 1; and a microorganism comprising the variant polypeptide, a polynucleotide encoding the variant polypeptide, or a combination thereof.

9. Use of a microorganism comprising the variant polypeptide according to claim 1, a polynucleotide encoding the variant polypeptide, or a combination thereof in producing L-arginine.

Images