Protein variant and L-arginine production method using same

Abstract

The present disclosure relates to: a protein variant; a polynucleotide encoding the protein variant; a microorganism of the genus Corynebacterium comprising at least one selected from the group consisting of the protein variant and the polynucleotide; and a method for producing L-arginine using the microorganism.

Description

Technical Field

[0001] Cross-reference to related applications

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10-2024-0071881 filed on May 31, 2024 and Korean Patent Application No. 10-2024-0135802 filed on October 7, 2024, and all disclosures in the documents of the corresponding Korean patent applications are incorporated herein by reference.

[0003] This disclosure relates to novel protein variants, Corynebacterium microorganisms containing said protein variants, and methods for producing L-arginine using said microorganisms. Background Technology

[0004] L-arginine is an amino acid that is a fundamental building block of proteins and is used as an animal feed additive, food additive, nutritional supplement, and pharmaceutical ingredient. Furthermore, L-arginine is an essential amino acid for animal growth and reproduction and is widely used in poultry and fish feed. It also promotes muscle formation and waste excretion, leading to increased use as a food-grade amino acid.

[0005] Based on these trends, various attempts have been made to improve the production capacity of methods for producing L-arginine using various microorganisms, including those of the genus Corynebacterium (US 2016-0145661 A1).

[0006] Despite these efforts, technologies to increase L-arginine production capacity still need to be developed. Summary of the Invention

[0007] [Technical Issues]

[0008] The purpose of this disclosure is to provide a novel protein variant.

[0009] Another object of this disclosure is to provide a polynucleotide encoding the protein variant.

[0010] Another object of this disclosure is to provide a Corynebacterium microorganism comprising at least one group selected from the protein variants and the polynucleotides encoding them.

[0011] Another object of this disclosure is to provide a method for producing L-arginine, which includes the step of culturing the microorganism in a culture medium.

[0012] Another object of this disclosure is to provide the use of the microorganism for the production of L-arginine.

[0013] [Technical Solution]

[0014] Its detailed description is as follows. Furthermore, each description and embodiment disclosed in this disclosure can also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this disclosure fall within the scope of this disclosure. Moreover, the scope of this disclosure should not be considered limited to the specific descriptions described below. Furthermore, those skilled in the art will recognize, or be able to determine, many equivalents of the specific aspects of this disclosure described herein using only conventional experiments. Furthermore, these equivalents are intended to be included in this disclosure.

[0015] One aspect of this disclosure provides a protein variant comprising an amino acid sequence in which the amino acid at position 922 from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 74 is replaced by an amino acid different from the original amino acid.

[0016] In this disclosure, the sequence of SEQ ID NO: 74 is shown in Table 1 below.

[0017] Table 1

[0018]

[0019] In one embodiment, the protein variant of the present disclosure described above may consist substantially of an amino acid sequence in which the amino acid at position 922 from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 74 is replaced by an amino acid different from the original amino acid. In another embodiment, the protein variant of the present disclosure described above may consist of an amino acid sequence in which the amino acid at position 922 from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 74 is replaced by an amino acid different from the original amino acid.

[0020] In this disclosure, the amino acid sequence of SEQ ID NO: 74 may be the sequence of a protein (excised nuclease ABC subunit A) encoded by the BBD29_07445 gene.

[0021] In one embodiment, the protein variant of the present disclosure described above may be a protein variant comprising an amino acid sequence in which the amino acid at position 922 from the N-terminus corresponding to the amino acid sequence of SEQ ID NO: 74 is replaced by glutamic acid (Glu, E).

[0022] In one embodiment, the amino acid at position 922 from the N-terminus in the amino acid sequence corresponding to SEQ ID NO: 74 may be glycine (Gly, G), but is not limited thereto.

[0023] The protein variant disclosed herein may have activity that increases L-arginine production capacity compared to the wild-type protein (peptide).

[0024] Protein variants of this disclosure may comprise an amino acid sequence in which the amino acid corresponding to the amino acid sequence of SEQ ID NO: 74, starting from the N-terminus at position 922, is replaced by an amino acid different from the original amino acid, or 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 that amino acid sequence. Furthermore, it is apparent that variants having partially deleted, modified, substituted, conservedly substituted, or inserted amino acid sequences are also included within the scope of this disclosure, provided they have this homology or identity and exhibit the same efficacy (increased L-arginine production activity) as variants of this disclosure.

[0025] For example, there may be additions or deletions, naturally occurring mutations, silent mutations, or conserved substitutions at the N-terminus, C-terminus, and / or internal portions of the amino acid sequence that do not alter the function of the variants disclosed herein.

[0026] The term "conservative substitution" refers to the replacement of one amino acid with another amino acid that has similar structure and / or chemical properties. Such amino acid substitutions typically occur based on the similarity of the residues' polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties. Typically, conservative substitutions have little or no effect on the activity of the protein or peptide.

[0027] In this disclosure, the term "variant" refers to a polypeptide in which one or more amino acids have been conservedly substituted and / or modified to form an amino acid sequence different from that of the polypeptide before the mutation, but which retains its function or properties. Such variants are typically identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the polypeptide before the mutation. Furthermore, some variants may include variants with one or more portions such as the N-terminal leader sequence or transmembrane domain missing. Other variants may include variants in which a portion is removed from the N- and / or C-terminus of the mature protein. The term "variant" may be used interchangeably with, and is not limited to, terms such as variant, modification, variant polypeptide, mutant protein, mutation, and variant (e.g., English expression modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), provided that the term is used in the sense of being mutated.

[0028] Furthermore, the variants may contain the deletion or addition of amino acids that have minimal impact on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence involved in protein co-translation or post-translational translocation may be linked to the N-terminus of the variant. Additionally, the variant may be linked to another sequence or adapter for identification, purification, or synthesis.

[0029] In one embodiment, the protein variant of the present disclosure described above may have 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher and less than 100% sequence homology or identity with the amino acid sequence of SEQ ID NO: 74.

[0030] Another aspect of this disclosure provides a protein variant comprising any amino acid sequence selected from the group consisting of amino acid sequences selected from SEQ ID NO: 15 to SEQ ID NO: 20. In one embodiment, the protein variant may be a protein variant comprising the amino acid sequence of SEQ ID NO: 20. In another embodiment, the variant of this disclosure may consist substantially of the amino acid sequence of SEQ ID NO: 20. In yet another embodiment, the variant of this disclosure may consist of the amino acid sequence of SEQ ID NO: 20.

[0031] In this disclosure, the amino acid sequence of SEQ ID NO: 20 may be the sequence of a protein variant in which the 922nd amino acid of the protein encoded by the BBD29_07445 gene (which excises nuclease ABC subunit A) is replaced with glutamic acid (Glu, E) instead of glycine (Gly, G).

[0032] Protein variants of this disclosure may comprise any amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NO: 15 to SEQ ID NO: 20 (e.g., the amino acid sequence of SEQ ID NO: 20), or 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 such amino acid sequence. Furthermore, it is apparent that variants having partially deleted, modified, substituted, conservedly substituted, or added amino acid sequences are also included within the scope of this disclosure, provided they possess such homology or identity and exhibit the efficacy (increased L-arginine production activity) corresponding to the variants of this disclosure.

[0033] For example, there may be sequence additions or deletions, naturally occurring mutations, silent mutations, or conserved substitutions at the N-terminus, C-terminus, and / or interior of the amino acid sequence that do not alter the function of the variants disclosed herein.

[0034] In the protein variants, conserved substitutions, variants, etc., are as described above.

[0035] In this disclosure, the terms “homology” or “identity” refer to the degree of similarity between two given amino acid sequences or nucleotide sequences, and may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0036] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms and can be combined with a default gap penalty established by the procedure used. Essentially, homologous or identical sequences can typically hybridize with the entire sequence or a portion thereof under moderately or highly stringent conditions. Obviously, hybridization also includes hybridization with polynucleotides containing universal codons or codons that take into account codon degeneracy within the polynucleotide.

[0037] Whether any two polynucleotide or polypeptide sequences are homologous or identical can be determined, for example, by using a known computer algorithm, such as the “FASTA” procedure with default parameters as described 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) executed in the Needleman program (version 5.0.0 or later) of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) can be used to determine (including the GCG 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, SanDiego, 1994 and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073). For example, homology or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information (NCBI) database.

[0038] Homology or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program known, for example, Smith and Waterman, Adv. Appl. Math (1981) 2:482 (e.g. Needleman et al. (1970), J MolBiol. 48:443). In summary, the GAP program can be defined as a value obtained by dividing the number of similarly aligned symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter sequence of the two sequences. The default parameters of the GAP procedure may include (1) a binary comparison matrix (containing a value of 1 for identity and a value of 0 for non-identity) and a weighted comparison matrix as disclosed in Schwartz and Dayhoff, Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353–358 (1979) by Gribskov et al. (1986) Nucl. Acids Res. 14: 6745 (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) replacement matrix); (2) a penalty of 3.0 for each vacancy and an additional penalty of 0.10 for each symbol in each vacancy (or a penalty of 10 for vacancy opening and 0.5 for vacancy extension); and (3) no penalty for terminal vacancy.

[0039] As an example of this disclosure, the variants of this disclosure described above may have activity that increases L-arginine production compared to wild-type proteins (peptides).

[0040] In this disclosure, the term "corresponding to" refers to an amino acid residue at a position listed in the polypeptide, or an amino acid residue that is similar to, identical to, or homologous to a residue listed in the polypeptide. Identifying the amino acid at the corresponding position may be done by referring to a specific sequence to determine a specific amino acid in the sequence. As used in this disclosure, "corresponding region" generally refers to a similar or corresponding position in a related protein or a reference protein.

[0041] For example, any amino acid sequence can be aligned with an amino acid sequence in which the amino acid corresponding to the amino acid sequence of SEQ ID NO: 74, at position 922 from the N-terminus, is replaced by an amino acid different from the original amino acid (e.g., the amino acid sequence of SEQ ID NO: 20). Based on this, each amino acid residue in the amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue in the amino acid sequence in which the amino acid corresponding to the amino acid sequence of SEQ ID NO: 74, at position 922 from the N-terminus, is replaced by an amino acid different from the original amino acid (e.g., the amino acid sequence of SEQ ID NO: 20). For example, the sequence alignment algorithm described in this disclosure can identify the position of an amino acid or the position where a modification such as substitution, insertion, or deletion occurs by comparing it with a query sequence (also referred to as a "reference sequence").

[0042] For such alignments, one can use, 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: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), but is not limited thereto; one can also use sequence alignment programs, pairwise sequence comparison algorithms, etc., known in the art as appropriate.

[0043] Another aspect of this disclosure is providing polynucleotides that encode protein variants of this disclosure.

[0044] In this disclosure, the term "polynucleotide" refers to a polymer of nucleotides in which nucleotide monomers are covalently linked together to form a long chain shape, which is a DNA or RNA chain of a certain length or longer, and more specifically refers to a polynucleotide fragment encoding the variant.

[0045] In one instance, the polynucleotide can be a polynucleotide encoding a protein variant containing an amino acid sequence in which the amino acid corresponding to the amino acid sequence of SEQ ID NO: 74 at position 922 from the N-terminus is replaced by an amino acid different from the original amino acid.

[0046] In one instance, the polynucleotide can be any nucleotide sequence comprising the group consisting of nucleotide sequences selected from SEQ ID NO: 21 to SEQ ID NO: 26, such as a polynucleotide of the nucleotide sequence of SEQ ID NO: 26.

[0047] The polynucleotide encoding a protein variant of this disclosure may have or comprise any nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NO: 21 to SEQ ID NO: 26, for example, a polynucleotide with the nucleotide sequence of SEQ ID NO: 26. In another example, the polynucleotide may comprise a nucleotide 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 nucleotide sequence. Furthermore, it is apparent that polynucleotides having nucleotide sequences with partial sequence deletions, modifications, substitutions, conserved substitutions, or insertions are also included within the scope of this disclosure, provided that they are sequences encoding polypeptides or proteins having such homology or identity and exhibiting activity (increased L-arginine production) corresponding to a variant of this disclosure.

[0048] In one instance, the nucleotide or amino acid sequences provided in this disclosure may include those sequences modified by conventional mutagenesis methods (e.g., directed evolution and / or site-directed mutagenesis) to achieve their original function or maintain the desired function to a certain extent. In one instance, the polynucleotide or polypeptide “comprising a specific nucleotide or amino acid sequence” may mean that the polynucleotide or polypeptide (i) consists of or substantially comprises a specific nucleotide or amino acid sequence, or (ii) consists of or substantially comprises an amino acid sequence having 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology to the specific nucleotide or amino acid sequence, and maintains the original function and / or the desired function.

[0049] Considering the degeneracy of codons or the preferred codons in organisms in which variants of this disclosure are to be expressed, the polynucleotides of this disclosure can be modified in various ways in the coding region without altering the amino acid sequence of the variants of this disclosure. Specifically, the polynucleotides of this disclosure may have or include any nucleotide sequence (e.g., the nucleotide sequence of SEQ ID NO: 26) having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher and less than 100% homology or identity with the nucleotide sequences selected from SEQ ID NO: 21 to SEQ ID NO: 26, or may consist of or substantially consist of a nucleotide sequence having 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher and less than 100% homology or identity with the nucleotide sequences, but are not limited thereto.

[0050] Furthermore, the polynucleotides disclosed herein may include, but are not limited to, probes that can be prepared from known gene sequences (e.g., sequences capable of hybridizing with all or part of the polynucleotide sequence of this disclosure under stringent conditions). “Stringent conditions” refers to conditions under which polynucleotides can specifically hybridize. For example, conditions in which polynucleotides having high homology or identity hybridize with each other, particularly those with 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology or identity, while polynucleotides with lower homology or identity do not hybridize with each other, or conditions involving washing once, specifically two to three times, at salt concentrations and temperatures corresponding 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, these are the washing conditions for conventional Southern hybridization.

[0051] Hybridization requires two nucleic acids to have complementary sequences, although mismatches between bases are possible depending on the strictness of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of this disclosure may also contain not only substantially similar nucleotide sequences, but also separate nucleic acid fragments complementary to the whole sequence.

[0052] Specifically, polynucleotides homologous to or identical with the polynucleotides disclosed herein can be detected using the following hybridization conditions, which include a Tm value of 55°C and a hybridization step using the conditions described above. Furthermore, the Tm value can be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by those skilled in the art for their purposes.

[0053] The appropriate stringency of hybrid polynucleotides depends on the length of the polynucleotide and the degree of complementarity of the polynucleotides, and the variables are well known in the art.

[0054] Other aspects of this disclosure provide vectors comprising the polynucleotides of this disclosure. The vectors may be expression vectors that express the polynucleotides in host cells, but are not limited thereto.

[0055] The vector disclosed herein may comprise a DNA construct containing the base sequence of a polynucleotide encoding a target polypeptide, the polynucleotide being operatively linked to a suitable expression control region (or expression control sequence) such that the target polypeptide can be expressed in a suitable host. The expression control region may include a promoter capable of initiating transcription, any operon sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a termination sequence for regulating transcription and translation. Upon transformation into a suitable host cell, the vector may replicate or function independently of the host genome, or it may integrate into the genome itself.

[0056] There are no particular limitations on the vectors used in this disclosure, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, granules, viruses, and bacteriophages in their natural or recombinant states. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc., can be used as phage vectors or granule vectors; and pDZ-based, pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, pET-based vectors, etc., can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, or pDC24 vectors, etc., can be used.

[0057] As an example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using a 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. A selection marker may be further included to confirm whether chromosome insertion has occurred. The selection marker is used to select cells transformed with the vector, i.e., to confirm whether the target nucleic acid molecule has been inserted, and may use a marker conferring a selectable phenotype (such as drug resistance, auxotrophic phenotype, resistance to cytotoxic agents, or expression of a surface polypeptide). In an environment treated with a selection agent, only cells expressing the selection marker survive or exhibit different phenotypic characteristics, thus allowing for the selection of transformed cells.

[0058] In this disclosure, the term "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism, such that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide can include all polynucleotides, whether located by insertion into the chromosome of the host cell or extrachromosomally, as long as they can be expressed in the host cell. Furthermore, the polynucleotide includes DNA and / or RNA encoding the target polypeptide. The polynucleotide can be introduced in any form, as long as it can be introduced into and expressed in a host cell. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a gene construct containing all the elements necessary for its own expression. The expression cassette typically includes a promoter, transcription termination signal, ribosome binding site, and translation termination signal operatively linked to the polynucleotide. The expression cassette can be in the form of a self-replicating expression vector. Furthermore, the polynucleotide is introduced into the host cell in its own form and operatively linked to the sequence required for expression in the host cell, but is not limited thereto.

[0059] Furthermore, the term "operably linked" as used above refers to the functional linking of a promoter sequence with a polynucleotide sequence to initiate and mediate the transcription of a polynucleotide encoding a target variant of this disclosure.

[0060] Another aspect of this disclosure provides microorganisms of the genus Corynebacterium, comprising at least one selected from the group consisting of protein variants and polynucleotides of this disclosure.

[0061] The microorganisms disclosed herein may contain protein variants of the present disclosure, polynucleotides encoding said protein variants, or vectors containing polynucleotides of the present disclosure.

[0062] In this disclosure, the term "microorganism (or strain)" includes all wild-type microorganisms or microorganisms in which natural or artificial genetic modifications have been made, and can be microorganisms whose specific mechanisms are weakened or enhanced due to reasons such as the insertion of exogenous genes or the enhancement or inactivation of endogenous gene activity, and which contain genetic modifications for the production of target peptides, proteins or products.

[0063] The microorganisms disclosed herein may be any one or more of the protein variants, polynucleotides, and vectors containing the polynucleotides of the present disclosure; microorganisms modified to express variants or polynucleotides of the present disclosure; microorganisms expressing variants or polynucleotides of the present disclosure (e.g., recombinant microorganisms); or microorganisms having the activity of variants of the present disclosure (e.g., recombinant microorganisms), but are not limited thereto.

[0064] The microorganisms disclosed herein may be microorganisms capable of producing L-arginine.

[0065] Compared to Corynebacterium microorganisms that do not contain at least one of the protein variants selected from the group consisting of the protein variants of the present disclosure and the polynucleotides encoding the protein variants (i.e., protein variants containing an amino acid sequence in which the amino acid corresponding to the 922nd amino acid from the N-terminus in the amino acid sequence of SEQ ID NO: 74 is replaced by glutamic acid; or microorganisms that do not express protein variants containing any amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 15 to SEQ ID NO: 20 (e.g., the amino acid sequence of SEQ ID NO: 20), or microorganisms that express wild-type proteins corresponding to the protein variants), the microorganisms of the present disclosure may have increased L-arginine production capacity.

[0066] The microorganisms disclosed herein may be microorganisms naturally capable of producing L-arginine, or microorganisms wherein a variant of the present disclosure or a polynucleotide encoding thereof (or a vector containing said polynucleotide) is introduced into a parent strain that does not have the ability to produce L-arginine, and / or microorganisms that are endowed with the ability to produce L-arginine, but are not limited thereto.

[0067] As an example, the microorganisms of this disclosure are cells or microorganisms that express protein variants of this disclosure by transformation with the polynucleotides of this disclosure or a vector containing a polynucleotide encoding a variant of this disclosure, and for the purposes of this disclosure, the strains of this disclosure may include all microorganisms including variants of this disclosure capable of producing L-arginine. For example, the microorganisms of this disclosure may be recombinant microorganisms with increased L-arginine production capacity by introducing a polynucleotide encoding a variant of this disclosure into a naturally occurring wild-type microorganism or an L-arginine-producing microorganism.

[0068] In one specific embodiment, the microorganisms of this disclosure may have increased L-arginine production capacity compared to Corynebacterium microorganisms that do not contain at least one of the protein variants selected from the group consisting of the polynucleotides encoding said protein variants. The Corynebacterium microorganisms that do not contain at least one of the protein variants selected from the group consisting of the protein variants selected from the group consisting of the polynucleotides encoding said protein variants may be naturally occurring wild-type microorganisms or unmodified microorganisms, or may be referred to as parental strains.

[0069] In this disclosure, "unmodified microorganism" does not exclude naturally occurring mutant microorganisms and may refer to wild-type microorganisms or naturally occurring strains themselves, or microorganisms whose characteristics have been altered by genetic variation due to natural or artificial factors. For example, unmodified microorganisms may refer to microorganisms in which the protein variants described in this disclosure have not been introduced, or strains in which such introduction occurred. "Unmodified microorganism" may be used interchangeably with "pre-modification strain," "pre-modification microorganism," "unmutated strain," "unmodified strain," "unmutated microorganism," or "reference microorganism."

[0070] In one embodiment, the unmodified microorganism may be, but is not limited to, a microorganism in which the biosynthetic pathway of L-arginine is additionally enhanced to increase L-arginine production.

[0071] In one embodiment, to enhance the biosynthetic pathway of L-arginine, the unmodified microorganism may be, for example, a microorganism in which the argR gene encoding the argine repressor (ArgR) is deleted, a genetic mutation (M54V) is introduced into the argB gene encoding acetylglutamate kinase (ArgB), and / or the activity of N-acetyl-γ-glutamyl-phosphoreductase (ArgC) is enhanced by promoter substitution, but is not limited thereto.

[0072] In one embodiment, the microorganism that increases L-arginine production according to this disclosure may be a microorganism whose L-arginine production capacity is increased by about 1% or more, about 1.5% or more, about 2% or more, about 2.5% or more, about 3% or more, about 3.5% or more, about 4% or more, about 4.5% or more, about 5% or more, about 5.5% or more, about 6% or more, or about 6.1% or more (no particular upper limit is specified, and it may be, for example, about 200% or less, about 150% or less, about 100% or less, or about 50% or less), but is not limited thereto. In another embodiment, the microorganism that increases L-arginine production according to this disclosure may be a microorganism whose L-arginine production capacity is increased by about 1.01 times or more, about 1.015 times or more, about 1.02 times or more, about 1.025 times or more, about 1.03 times or more, about 1.035 times or more, about 1.04 times or more, about 1.045 times or more, about 1.05 times or more, about 1.055 times or more, about 1.06 times or more, or about 1.061 times or more (there is no particular upper limit, and it may be, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less), but is not limited thereto.

[0073] The term “about” encompasses the entire range including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes, but is not limited to, all values ​​that are the same as or similar to the values ​​that appear after the term “about”.

[0074] In one embodiment, the microorganism disclosed herein may be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens, and more specifically, may be Corynebacterium glutamicum.

[0075] In this disclosure, the term "attenuation" of a polypeptide includes the concept of reduced or absent activity compared to endogenous activity. Attenuation may be used interchangeably with terms such as inactivation, lack, downregulation, reduction, decrease, and weakening.

[0076] The attenuation may also include situations where the activity of the polypeptide itself is reduced or eliminated compared to the polypeptide activity possessed by the original microorganism due to mutations in the polynucleotide encoding the polypeptide; situations where the overall polypeptide activity level and / or concentration (expression level) in the cell is lower than that of the natural strain due to inhibition of the expression of the gene encoding the polynucleotide or inhibition of its translation into a polypeptide; situations where the polynucleotide is not expressed at all; and / or situations where the polypeptide has no activity even if the polynucleotide is expressed. "Endogenous activity" refers to the activity of a specific polypeptide originally possessed by the parental strain or unmodified microorganism before the trait change, when the trait is altered by gene mutations caused by natural or human factors. This can be used interchangeably with "activity before modification." The phrase "inactivated, lacking, reduced, downregulated, decreased, or weakened" of polypeptide activity compared to endogenous activity means that its activity is lower than that of the specific polypeptide originally possessed by the parental strain or unmodified microorganism before the trait change.

[0077] The reduction of peptide activity can be achieved by any method known in the art, but not limited to it, and can be achieved by applying a variety of 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.).

[0078] Specifically, the peptides disclosed herein can be weakened by:

[0079] 1) The gene encoding all or part of the polypeptide is missing;

[0080] 2) Modify the expression control region (or expression control sequence) to reduce the expression of the gene encoding the polypeptide;

[0081] 3) Modify the amino acid sequence that makes up the polypeptide to eliminate or reduce the activity of the polypeptide (e.g., by deleting / replacing / adding one or more amino acids in the amino acid sequence).

[0082] 4) Modify the gene sequence encoding the polypeptide to eliminate or weaken the activity of the polypeptide (e.g., delete / replace / add 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).

[0083] 5) Modify the nucleotide sequence of the start codon or 5'-UTR region of the gene transcript (which encodes the polypeptide);

[0084] 6) Introduce an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to the transcript of the gene encoding the polypeptide.

[0085] 7) Add a sequence complementary to the Shine-Dalgarno sequence of the gene encoding the polypeptide to the front of the Shine-Dalgarno sequence to form a secondary structure to which ribosomes cannot attach.

[0086] 8) Add a promoter for reverse transcription (reverse transcription engineering, RTE) to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide; or

[0087] 9) Regulating the cellular localization of the protein (peptide); or

[0088] 10) Selected from two or more of the above 1) to 9), but not particularly limited thereto.

[0089] For example,

[0090] 1) The gene encoding the polypeptide that is missing part or all of the polypeptide may be a complete polynucleotide that has been removed from the chromosome that encodes the endogenous target polypeptide, replaced with a polynucleotide that has missing some nucleotides, or replaced with a selectable marker gene.

[0091] Furthermore, 2) modifications to the expression control region (or expression control sequence) can be mutations occurring in the expression control region (or expression control sequence) through deletion, insertion, non-conserved or conserved substitution, or a combination thereof; or substitution with a less active sequence. The expression control region includes, but is not limited to, promoter, operon sequences, sequences encoding ribosome binding sites, and sequences regulating transcription and translation termination.

[0092] In addition, 3) the nucleotide sequence that modifies the start codon or 5'-UTR region of the gene transcript (which encodes the polypeptide) may, for example, be replaced with a nucleotide sequence that encodes another start codon that has a lower polypeptide expression rate compared to the endogenous start codon, but is not limited thereto.

[0093] Furthermore, the modifications to the amino acid sequence or polynucleotide sequence described in 4) and 5) above can be achieved by mutations in the sequence through deletion, insertion, non-conservative or conserved substitutions, or combinations thereof, or by substitutions with amino acid sequences or polynucleotide sequences that are modified to have weaker activity or to be modified to have no activity, thereby weakening the activity of the polypeptide, but not limited thereto. For example, gene expression can be suppressed or weakened by introducing mutations into the polynucleotide sequence to form a stop codon, but not limited thereto.

[0094] For 6), antisense oligonucleotides (e.g., antisense RNA) that are complementary to the transcript of the gene encoding the polypeptide can be referenced, 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].

[0095] 7) Adding a sequence complementary to the Shine-Dalgarno sequence of the gene encoding the polypeptide to the front of the Shine-Dalgarno sequence to form a secondary structure in which ribosomes cannot attach, which may prevent mRNA from being translated or reduce its rate.

[0096] 8) Adding a promoter for reverse transcription (reverse transcription engineering, RTE) to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide can reduce activity by preparing an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide.

[0097] 9) Regulating the cellular localization of the protein (peptide) can be achieved by targeting the protein (peptide) to specific intracellular organelles or specific intracellular spaces. For example, it can be achieved by adding or removing a leader sequence for targeting the protein (peptide) to target the periplasm or cytoplasm, but is not limited thereto.

[0098] This reduction in peptide activity can be based on the activity or concentration of the peptide expressed in the unmodified wild-type or microbial strain, corresponding to a decrease in the activity or concentration (expression level) of the peptide, or a reduction in the amount of product produced by the peptide, but is not limited to these.

[0099] In this disclosure, the term "enhanced" peptide activity refers to an increase in the activity of a peptide compared to its endogenous activity. Enhancement may be used interchangeably with terms such as activation, upregulation, overexpression, and increase. In this document, activation, upregulation, overexpression, and increase may include exhibiting activity not originally present, or exhibiting increased activity compared to endogenous activity or activity before modification. "Endogenous activity" refers to the activity of a specific peptide originally possessed by the parental strain or unmodified microorganism before the trait change, when the trait is altered due to a gene mutation caused by natural or artificial factors. This may be used interchangeably with "activity before modification." The term "enhanced," "upregulated," "overexpressed," or "increased" peptide activity compared to endogenous activity means an increase in the activity and / or concentration (expression level) of a specific peptide originally possessed by the parental strain or unmodified microorganism before the trait change.

[0100] This enhancement can be achieved by introducing exogenous peptides or by increasing the activity and / or concentration (expression level) of endogenous peptides. Whether the peptide activity is enhanced can be confirmed by the degree of activity of the corresponding peptide, its expression level, or the increase in the amount of product released from the peptide.

[0101] Various methods well-known in the art can be used to enhance the activity of peptides, and there are no limitations as long as the activity of the target peptide can be enhanced more than that of the unmodified microorganism. Specifically, this can be achieved by using genetic engineering and / or protein engineering methods well-known to those skilled in the art, which are routine methods in molecular biology, but are not limited thereto (e.g., Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al., Molecular Cloning 2012, etc.).

[0102] Specifically, the enhancement of the peptides disclosed herein can be achieved through:

[0103] 1) Increase the intracellular copy number of the polynucleotide encoding the polypeptide;

[0104] 2) Replace the gene expression regulatory region on the chromosome encoding the polypeptide with a highly active sequence;

[0105] 3) Modify the nucleotide sequence of the start codon or 5'-UTR region of the gene transcript (which encodes the polypeptide);

[0106] 4) Modify the amino acid sequence of the polypeptide to enhance its activity;

[0107] 5) Modify the polynucleotide sequence encoding the polypeptide to enhance polypeptide activity (e.g., modify the polynucleotide sequence of a polypeptide gene to encode the modified polypeptide to enhance polypeptide activity).

[0108] 6) Import an exogenous polypeptide that exhibits the activity of the polypeptide or an exogenous polynucleotide encoding the polypeptide;

[0109] 7) Codon optimization of the polynucleotide encoding the polypeptide;

[0110] 8) By analyzing the tertiary structure of the peptide, sites are selected and modified or chemically modified to expose the peptide; or

[0111] 9) Regulating the cellular localization of the protein (peptide); or

[0112] 10) Selected from two or more of the above 1) to 9), but not particularly limited thereto.

[0113] 1) Increasing the intracellular copy number of the polynucleotide encoding the polypeptide can be achieved by introducing a recombinant vector into a host cell, the recombinant vector being operatively linked to the polynucleotide encoding the polypeptide and capable of replicating and functioning independently of the host. Alternatively, this can be achieved by introducing one or two or more copies of the polynucleotide encoding the polypeptide into the chromosome of the host cell. Chromosomal insertion can be performed 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.

[0114] 2) Replacing the gene expression control region (or expression control sequence) encoding the polypeptide on the chromosome with a sequence of high activity can be, for example, by mutating the sequence through deletion, insertion, non-conserved or conserved substitution, or a combination thereof, to further enhance the activity of the expression control region, or by replacing it with a sequence of even stronger activity. The expression control region is not particularly limited thereto, but may include promoters, operon sequences, sequences encoding ribosome binding sites, and sequences regulating transcription and translation termination. As an example, a strong promoter can replace the original promoter, but this is not limited to this.

[0115] Examples of known strong promoters include, but are not limited to, the CJ1 to CJ7 promoters (US Patent No. 7662943 B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the λ phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13(sm3) promoter (US Patent No. 10584338 B2), the O2 promoter (US Patent No. 10273491 B2), the tkt promoter, and the yccA promoter.

[0116] 3) The nucleotide sequence that modifies the start codon or 5'-UTR region of the gene transcript (which encodes the polypeptide) may, for example, be replaced with a nucleotide sequence that encodes another start codon that has a higher polypeptide expression rate compared to the endogenous start codon, but is not limited thereto.

[0117] The modifications to the amino acid or polynucleotide sequence described in 4) and 5) above can be achieved by mutations in the sequence through deletion, insertion, non-conservative or conserved substitution, or a combination thereof, or by substitution with an amino acid or polynucleotide sequence that has been modified to have stronger activity or to have increased activity, thereby enhancing the activity of the polypeptide, but are not limited thereto. Specifically, substitution can be achieved by inserting a polynucleotide into the genome through homologous recombination, but is not limited thereto. In this case, the vector used may also include a selection marker for confirming whether it has been inserted into the chromosome. The selection marker is as described above.

[0118] 6) The introduction of a foreign polynucleotide exhibiting the activity of the polypeptide can be achieved by introducing a foreign polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide into a host cell. The foreign polynucleotide is not limited by its source or sequence, as long as it exhibits the same / similar activity as the polypeptide. The method for introduction can be carried out by a known transformation method appropriately selected by those skilled in the art, and the polypeptide's activity is increased by producing it through expression of the introduced polynucleotide in the host cell.

[0119] 7) Codon optimization of the polynucleotide encoding the polypeptide may be performed on endogenous polynucleotides to increase transcription or translation in the host cell, or on exogenous polynucleotides to achieve optimized transcription and translation in the host cell.

[0120] 8) Selecting and modifying or chemically modifying exposure sites by analyzing the tertiary structure of the polypeptide can be, for example, by comparing the sequence information of the polypeptide to be analyzed with a database that stores the sequence information of known proteins, determining template protein candidates based on sequence similarity, identifying the structure based on this, and selecting and modifying or chemically modifying the exposure sites to be modified or chemically modified.

[0121] 9) Regulating the cellular localization of the protein (peptide) can be achieved by targeting the protein (peptide) to specific intracellular organelles or specific intracellular spaces. For example, it can be achieved by adding or removing a leader sequence for targeting the protein (peptide) to target the periplasm or cytoplasm, but is not limited thereto.

[0122] This enhancement of peptide activity may be due to an increase in the activity or concentration of the corresponding peptide based on the activity or concentration of the peptide expressed in the wild-type or unmodified microbial strain, or an increase in the amount of product produced by the peptide, but is not limited thereto.

[0123] Modification of some or all polynucleotides in the microorganisms disclosed herein (e.g., modifications encoding the protein variants described above) can be induced by: (a) genome editing using homologous recombination or engineered nucleases (e.g., CRISPR-Cas9) with a vector for chromosome insertion in the microorganism, and / or (b) treatment with light such as ultraviolet light and radiation and / or chemicals, but not limited thereto. Methods for modifying some or all genes can include methods using DNA recombination techniques. For example, deletion of some or all genes can be achieved by injecting a nucleotide sequence or a vector containing a nucleotide sequence homologous to the target gene into the microorganism to induce homologous recombination. The injected nucleotide sequence or vector may include, but is not limited to, a dominant selection marker.

[0124] In the microorganisms disclosed herein, the protein variants, polynucleotides, and L-arginine, etc., are as described in other aspects above.

[0125] Another aspect of this disclosure provides a method for producing L-arginine, comprising the step of culturing a Corynebacterium microorganism containing a protein variant or a polynucleotide of this disclosure in a culture medium.

[0126] The method for producing L-arginine disclosed herein may include the step of culturing a Corynebacterium microorganism containing a protein variant of the present disclosure, a polynucleotide of the present disclosure, or a vector of the present disclosure in a culture medium.

[0127] The term "culture" in this disclosure refers to the growth of Corynebacterium spp. microorganisms under appropriately controlled and artificially controlled environmental conditions. The culture process of this disclosure can be carried out using suitable culture media and conditions known in the art. Those skilled in the art can readily adapt and use this culture method according to the selected strain. Specifically, the culture can be batch, continuous, and / or fed-batch culture, but is not limited thereto.

[0128] In this disclosure, the term "culture medium" refers to a substance prepared by mixing nutrients required for culturing the Corynebacterium spp. microorganisms of this disclosure as the main component, and providing the water, nutrients, and growth factors necessary for survival and growth. Specifically, any culture medium used for culturing conventional microorganisms can be used as the culture medium and other culture conditions for culturing the Corynebacterium spp. microorganisms of this disclosure without particular limitation. However, the Corynebacterium spp. microorganisms of this disclosure can be cultured under aerobic conditions in a conventional culture medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, while controlling temperature, pH, etc.

[0129] Specifically, culture media for Corynebacterium species can be found in literature such as [“Manual of Methods for General Bacteriology”, American Society for Bacteriology (Washington, D.C., USA, 1981)].

[0130] In this disclosure, carbon sources 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 hydrolysates, molasses, molasses, rice bran, cassava, sugarcane residue, and corn steep liquor may be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) may be used, and various other suitable carbon sources may be used without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited thereto.

[0131] Nitrogen sources can include inorganic nitrogen sources, such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources, such as amino acids (e.g., glutamic acid, methionine, and glutamine), peptone, NZ-amines, meat extracts, yeast extracts, malt extracts, corn steep liquor, casein hydrolysate, fish or its decomposition products, and defatted soybean meal or its decomposition products. These nitrogen sources can be used alone or in combination of two or more, and are not limited thereto.

[0132] Phosphorus sources may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or their corresponding sodium-containing salts. As for 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 be included. These components or precursors may be added to the culture medium in batches or continuously. However, this is not the only possible approach.

[0133] Furthermore, during the cultivation of the Corynebacterium spp. disclosed herein, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the culture medium in an appropriate manner to adjust the pH of the medium. Additionally, antifoaming agents such as polyethylene glycol fatty acids can be used to suppress foam formation during cultivation. Furthermore, to maintain an aerobic state in the culture medium, oxygen or oxygen-containing gas can be injected into the medium; or to maintain an anaerobic or microaerobic state, no gas may be injected, or nitrogen, hydrogen, or carbon dioxide gas may be injected, but these are not limited to these methods.

[0134] In the cultivation process of this disclosure, the cultivation temperature can be from 20°C to 45°C, specifically 25°C to 40°C, 25°C to 40°C, 25°C to 37°C, 25°C to 35°C, 27°C to 40°C, 27°C to 37°C, 27°C to 35°C, 30°C to 40°C, 30°C to 37°C, or 30°C to 35°C, but is not limited thereto. In the cultivation process of this disclosure, the cultivation time can last from approximately 10 to 160 hours, but is not limited thereto.

[0135] L-arginine produced by the culture method disclosed herein can be secreted into the culture medium or retained inside the cell.

[0136] The method for producing L-arginine disclosed herein may further include, for example, the step of preparing the Corynebacterium genus of the present disclosure before the culturing step, the step of preparing a culture medium for culturing the strain, or a combination thereof (in any order).

[0137] The method for producing L-arginine disclosed herein may further include a step of recovering L-arginine from a culture medium (in which the culture is carried out) or a Corynebacterium microorganism. A recovery step may also be included after the culture step.

[0138] Recovery can be performed by collecting the target L-arginine product from the culture medium or microorganisms using appropriate methods known in the relevant field, depending on the culture method, such as batch, continuous, or fed-batch culture. For example, various types of chromatography can be used, such as centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography and affinity chromatography, HPLC, or combinations of these methods, but not limited to these. Appropriate methods known in the relevant field can be used to recover the desired L-arginine product from the culture medium or microorganisms.

[0139] Furthermore, the method for producing L-arginine disclosed herein may additionally include a purification step. Purification can be performed using suitable methods known in the relevant art. In one example, when the method for producing L-arginine disclosed herein includes a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the order, or may be performed simultaneously or integrated into a single step, but are not limited thereto.

[0140] In the methods disclosed herein, the variants, polynucleotides, vectors, and strains, etc., are as described in other aspects above.

[0141] Another aspect of this disclosure is to provide a composition for the production of L-arginine comprising at least one selected from the group consisting of: a protein variant of this disclosure, a polynucleotide encoding said protein variant and a carrier containing said polynucleotide, a Corynebacterium microorganism containing said polynucleotide; a culture medium for culturing said microorganism; or a combination of two or more of these.

[0142] The compositions disclosed herein may also include any suitable excipients commonly used in the production of amino acids (e.g., L-arginine), and such excipients may be, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents, but are not limited thereto.

[0143] In the compositions disclosed herein, the variants, polynucleotides, vectors, strains, culture media, and L-arginine, etc., are as described in other aspects above.

[0144] According to another aspect of this disclosure, this disclosure may provide a method for increasing the L-arginine production capacity of microorganisms, a method for conferring L-arginine production capacity on microorganisms, or a method for preparing microorganisms with increased L-arginine production capacity, comprising the steps of introducing (e.g., transforming) the novel protein variants of this disclosure, the polynucleotides encoding the variants, and / or the recombinant vectors containing the polynucleotides into the microorganisms.

[0145] In the method for preparing microorganisms disclosed herein, the polynucleotides, recombinant vectors, and microorganisms are as described above.

[0146] According to another aspect of this disclosure, this disclosure provides one or more uses selected from the group consisting of: the protein variants of the present disclosure described above; polynucleotides encoding the protein variants; recombinant vectors containing the polynucleotides; and microorganisms containing the protein variants, the polynucleotides encoding the protein variants, and / or recombinant vectors containing the polynucleotides, for the production of L-arginine, and / or the production of L-arginine-producing microorganisms, and / or the conferral and / or enhancement of the L-arginine production capacity of microorganisms.

[0147] In the use of the protein variants, polynucleotides, recombinant vectors, and microorganisms for the production of L-arginine, and / or for the production of L-arginine-producing microorganisms, and / or for the conferring and / or increasing of the L-arginine production capacity of microorganisms, the protein variants, polynucleotides, recombinant vectors, and microorganisms are as described above.

[0148] According to other aspects of this disclosure, this disclosure provides compositions, methods, products, processes, or uses characterized by one or more elements disclosed herein.

[0149] [Beneficial Effects]

[0150] When culturing Corynebacterium microorganisms containing novel protein variants of this disclosure, L-arginine can be produced in higher yields compared to microorganisms with existing unmodified peptides. Detailed Implementation

[0151] The present disclosure will be described in more detail below by way of embodiments. These embodiments are intended only to describe the present disclosure in more detail, and it will be apparent to those skilled in the art that the scope of the present disclosure is not limited to these embodiments based on the key points of the disclosure.

[0152] Example

[0153] (Throughout this disclosure, unless otherwise stated, the "%" used to express the concentration of a particular substance is (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid.)

[0154] Example 1. Construction of L-arginine production microorganisms

[0155] Example 1-1. Construction of Corynebacterium glutamicum CJR2 strain

[0156] To evaluate L-arginine production capacity, *Corynebacterium glutamicum* CJR2 was prepared, in which the ΔargR and argB(M54V) mutations were introduced into wild-type *Corynebacterium glutamicum* ATCC13869 (Ikeda, Masato et al., *Applied and environmental microbiology* 75(6)1635-41, 2009).

[0157] First, vectors for introducing argR deletion and argB(M54V) mutations were prepared. PCR was performed using primer pairs SEQ ID NO: 1 and 2 and SEQ ID NO: 3 and 4, with overlapping PCR using primer pairs SEQ ID NO: 1 and 4, using genomic DNA of *Corynebacterium glutamicum* ATCC13869 as a template to obtain homologous recombination fragments with the argR deletion mutation sequence. To prepare homologous recombination fragments with the argB(M54V) mutation in the same manner, PCR was performed using primer pairs SEQ ID NO: 5 and 6 and SEQ ID NO: 7 and 8, with overlapping PCR using SEQ ID NO: 5 and 8. PCR reactions were performed by denaturation at 95°C for 5 minutes; repeated 27 times: denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 1 minute; followed by polymerization at 72°C for 5 minutes. Following the purification process described above, the fragments were fused and cloned using the In-Fusion® HD Cloning Kit (Clontech) with the pDC24 vector (SEQ ID NO:81) treated with SmaI restriction enzyme, according to the manual, to obtain plasmids. The prepared vectors were named pDC24-ΔargR and pDC24-argB (M54V), respectively.

[0158] Subsequently, the argR deletion mutation was introduced into wild-type Corynebacterium glutamicum ATCC13869. Transformation was performed by electroporation using the prepared pDC24-ΔargR plasmid (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Secondary recombination was then performed on solid agar plates containing 4% sucrose, and PCR was conducted on the transformants that had completed the secondary recombination using primers of SEQ ID NO: 1 and 4 to confirm that the deletion mutation had been introduced into the argR gene on the chromosome. PCR was then performed under the same conditions as described above, and the resulting transformant was named CJR1.

[0159] Solid agar plate culture medium (pH 7.0)

[0160] 10 g glucose, 10 g peptone, 5 g beef extract, 5 g yeast extract, 18.5 g brain and heart extract, 2.5 g NaCl, 2 g urea, 91 g sorbitol, 20 g agar (based on 1 liter of distilled water)

[0161] The argB(M54V) mutation was introduced into Corynebacterium glutamicum CJR1 in the same manner as described above. Using the prepared pDC24-argB(M54V) plasmid, PCR was performed on the transformants that had undergone secondary recombination using primer pairs of SEQ ID NO: 5 and 8 to confirm that the M54V mutation had been introduced into the argB gene on the chromosome. The transformants were named CJR2.

[0162] Table 2 below describes the sequence information of the primers used in Examples 1-1.

[0163] Table 2

[0164]

[0165] Examples 1-2. Construction of Corynebacterium glutamicum strain CJR100

[0166] Based on the CJR2 strain prepared in Example 1-1, a CJR100 strain was prepared, in which the N-acetyl-γ-glutamyl-phosphoreductase (hereinafter referred to as argC) gene was enhanced.

[0167] To enhance the activity of argC (NCBI accession number BBD29_RS07530), N-acetyl-γ-glutamyl-phosphoreductase, a plasmid was prepared by replacing the wild-type promoter of the argC gene with the Po2 promoter (US 10273491 B2), a known strong promoter. The upstream and downstream regions of the argC gene were obtained. Specifically, to prepare a strain containing argC with the Po2 promoter, chromosomal DNA from *Corynebacterium glutamicum* ATCC13869 was used as a template for PCR. Primers SEQ ID NO: 9 and SEQ ID NO: 10 were used to amplify the upstream region of the argC gene, and primers SEQ ID NO: 11 and SEQ ID NO: 12 were used to amplify the downstream region of the argC gene. Furthermore, using the synthesized Po2 promoter as a template, Po2 promoter fragments were obtained using SEQ ID NO: 13 and SEQ ID NO: 14. Pfu Ultra™ high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction, and PCR was performed in the same manner as in Example 1-1. As a result, an 86 bp DNA fragment of the Po2 promoter region, a 610 bp DNA fragment upstream of Corynebacterium glutamicum ATCC13869 argC, and a 1086 bp DNA fragment downstream of argC were obtained. Using the amplified promoter and DNA fragments as templates, PCR was performed in the same manner as in Example 1-1 using primers of SEQ ID NO: 9 and SEQ ID NO: 12. After DNA purification, the two fragments obtained above were fused and cloned by ligating them to the pDC24 plasmid treated with SmaI restriction enzyme using the In-Fusion® HD Cloning Kit (Clontech). The resulting vector was named pDC24-Po2-argC.

[0168] Subsequently, the CJR2 strain prepared in Example 1-1 (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999) was transformed by electroporation with the pDC24-Po2-argC plasmid. Then, a second recombination was performed on solid agar plates containing 4% sucrose, and PCR was performed on the transformed strains with the primers of SEQ ID NO: 9 and 14 to confirm that the chromosomal argC gene was enhanced by the Po2 promoter. PCR was then performed under the same conditions as described above, and the resulting transformed strain was named CJR100.

[0169] Solid agar plate culture medium (pH 7.0)

[0170] 10 g glucose, 10 g peptone, 5 g beef extract, 5 g yeast extract, 18.5 g brain and heart extract, 2.5 g NaCl, 2 g urea, 91 g sorbitol, and 20 g agar (based on 1 liter of distilled water).

[0171] Table 3 below describes the sequence information of the primers used in Examples 1-2.

[0172] Table 3

[0173]

[0174] Example 2: Selecting mutant strains with increased arginine production capacity through artificial mutagenesis

[0175] Example 2-1: Random Mutation Induced by UV Radiation

[0176] To select mutant strains with increased arginine production capacity, the arginine-producing strain CJR100 prepared in Example 2-1 was plated on agar-containing nutrient medium and incubated at 30°C for 16 hours. Hundreds of colonies obtained therefrom were then irradiated with UV light at room temperature (UV mutagenesis) to induce random mutations in the strain's genome.

[0177] <Nutritional medium (pH 7.2)>

[0178] 10 g glucose, 5 g meat extract, 10 g peptone, 2.5 g sodium chloride, 5 g yeast extract, 20 g agar, 2 g urea (per liter of distilled water)

[0179] Example 2-2: Selection of strains with increased L-arginine production capacity

[0180] In order to select mutant strains with increased arginine production capacity compared with parent strain CJR100, CJR100 strain and mutant strains with induced random mutations in Example 2-1 were cultured by the following methods.

[0181] Each of the aforementioned strains was inoculated into 96-DeepWell Plate-Dome (Bioneer) plates containing 400 µl of seed medium and cultured in a plate shaking incubator (TAITEC) at 32°C and 1200 rpm for approximately 48 hours. Arginine concentrations in approximately 3000 cultured strains were examined using near-infrared (NIR) spectroscopy, and the top four mutant strains exhibiting increased arginine production compared to the parent strain CJR100 were selected.

[0182] Seed culture medium (pH 7.0)

[0183] 20 g glucose, 10 g peptone, 5 g yeast extract, 1.5 g urea, 4 g KH2PO4, 8 g K2HPO4, 0.5 g MgSO4•7H2O, 100 μg biotin, 1000 μg thiamine hydrochloride, 2000 μg calcium pantothenate, and 2000 μg nicotinamide (based on 1 liter of distilled water).

[0184] To ultimately select strains with reproducibly increased L-arginine production capacity from four chosen mutant strains, they were cultured and the L-arginine concentration produced was evaluated using the following methods.

[0185] The control strain and the four mutant strains were inoculated into 250 mL corner-baffle flasks containing 25 mL of arginine production medium, and then cultured at 32 °C with shaking at 200 rpm for 20 hours. Then, 1 mL of seed culture was inoculated into a 250 mL corner-baffle flask containing 24 mL of production medium and cultured at 30 °C with shaking at 200 rpm for 54 hours.

[0186] Seed culture medium (pH 7.0)

[0187] Glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH2PO4 4 g, K2HPO4 8 g, MgSO4•7H2O 0.5 g, biotin 100 ug, thiamine hydrochloride 1000 ug, calcium pantothenate 2000 ug, and nicotinamide 2000 ug (based on 1 liter of distilled water)

[0188] <Production medium (pH 7.2)>

[0189] 5% glucose, 3% ammonium sulfate, 0.1% potassium dihydrogen phosphate, 0.2% magnesium sulfate heptahydrate, 1.5% CSL (corn steep liquor), 1% NaCl, 0.5% yeast extract, biotin 100 mg / L

[0190] After cultivation, the concentration of L-arginine in the culture medium was analyzed by high performance liquid chromatography (HPLC). The L-arginine production concentration of each mutant strain is shown in Table 4 below.

[0191] Table 4

[0192]

[0193] As shown in Table 4 above, among the four selected mutant strains, CJR100_mt3 was ultimately selected as the mutant strain with the greatest increase in L-arginine production.

[0194] Example 3. Identification of mutations by whole-genome sequencing (WGS)

[0195] By performing whole-genome sequencing (WGS) on the CJR100_mt3 strain selected in Examples 2-2 above and comparing it with the parent strain CJR100, genes with protein sequence changes due to nucleotide sequence mutations were identified. The amino acid sequences of the protein variants and the nucleotide sequences of the genes are described in Tables 5 and 6 below.

[0196] Table 5

[0197] Mutations in the CDS (coding sequence) region

[0198]

[0199] *Deletion: Through nucleotide sequence substitution in a gene, the codon encoding the original amino acid is mutated into a stop codon, thus deleting the corresponding amino acid sequence.

[0200] Table 6

[0201] RNA region mutation

[0202]

[0203] Table 7

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215] Example 4: Construction of L-arginine-producing strains with introduced mutant promoters

[0216] Example 4-1: Construction of a recombinant vector introducing a mutant promoter

[0217] To evaluate the effectiveness of the protein variants identified in Example 3 above, a vector capable of introducing them into chromosomes was prepared.

[0218] Specifically, in order to introduce each of the BBD29_00030, BBD29_05930, BBD29_08260, BBD29_02985, BBD29_06380, BBD29_07445, BBD29_12405, BBD29_07610 and BBD29_04520 gene mutations into the CJR100 strain, a vector containing the target mutation was prepared.

[0219] Specifically, genomic DNA was extracted from strain CJR100_mt3 using the G-spin Total DNA Extraction MiniKit (Intron, catalog number 17045) according to the protocol provided in the kit, and the genomic DNA was used as a template for PCR. Solg™ Pfu-X DNA polymerase was used as the polymerase, and the PCR conditions were as follows: denaturation at 95°C for 4 minutes; 27 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 50 seconds; followed by polymerization at 72°C for 5 minutes. The sequences of the primer pairs used in the experiment are shown in Table 8 below.

[0220] The mutant insertion fragment obtained above and the pDC24 vector (SEQ ID NO: ) treated with the restriction enzyme SmaI were cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY2009, NEBuilder HiFi DNA Assembly Master Mix). 81), to obtain recombinant plasmids, and the vectors containing each introduced mutation fragment were named pDC24-BBD29_00030*; pDC24-BBD29_05930*; pDC24-BBD29_08260*; pDC24-BBD29_02985*; pDC24-BBD29_06380*; pDC24-BBD29_07445*; pDC24-BBD29_12405*; pDC24-BBD29_07610*; and pDC24-BBD29_04520*.

[0221] Table 8

[0222]

[0223] Example 4-2: Construction of L-arginine-producing strains with introduced mutant genes

[0224] Nine vectors prepared in Example 4-1 were transformed into the arginine-producing strain CJR100 using electroporation. Strains that inserted the vector into the chromosome via homologous sequence recombination were selected using kanamycin medium. Subsequently, for transformants that completed a second recombination, the strains that had introduced the variant promoter were confirmed by PCR using the primer pairs listed in Table 9.

[0225] Table 9

[0226]

[0227] The recombinant strains were named CJR100△BBD29_00030::BBD29_00030*; CJR100△BBD29_05930::BBD29_05930*; CJR100△BBD29_08260::BBD29_08260*; CJR100△BBD29_02985::BBD29_02985*; CJR100△BBD29 _06380::BBD29_06380*;CJR100△BBD29_07445::BBD29_07445*;CJR100△BBD29_12405::BBD 29_12405*; CJR100△BBD29_07610::BBD29_07610*; and CJR100△BBD29_04520::BBD29_04520*.

[0228] Example 5. Evaluation of L-arginine production capacity of L-arginine-producing strains with introduced mutant promoters

[0229] To evaluate the L-arginine production capacity of the recombinant strains constructed in Example 4-2, they were cultured and evaluated using the following methods.

[0230] Each strain was inoculated into a 250-ml baffled Erlenmeyer flask containing 25 ml of seed culture medium and incubated at 30°C with shaking at 200 rpm for 20 hours. 1 ml of seed culture was inoculated into a 250-ml baffled Erlenmeyer flask containing 24 ml of production culture medium and incubated at 30°C with shaking at 200 rpm for 54 hours. The culture medium composition was the same as in Examples 2-2, and the experiment was repeated three times.

[0231] After cultivation, the amount of L-arginine produced was measured using high performance liquid chromatography (HPLC), and the average values ​​of the analytical results are shown in Table 10 below.

[0232] Table 10

[0233]

[0234] The results, as shown in Table 10, confirmed that most arginine-producing strains expressing protein variants had arginine production capacity equal to or better than that of the parental strains. In particular, the arginine production capacity of the CJR100ΔBBD29_07445::BBD29_07445* strain was significantly increased compared to that of the parental strain CJR100.

[0235] This confirms that L-arginine can be produced more efficiently by introducing a protein variant in which the 922nd amino acid of the protein encoded by the BBD29_07445 gene (which removes the nuclease ABC subunit A) is replaced with glutamic acid (Glu, E) instead of glycine (Gly, G).

[0236] Based on the foregoing description, those skilled in the art to which this disclosure pertains will understand that this disclosure can be practiced in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the above embodiments are illustrative in all respects and not restrictive. The scope of this disclosure should be interpreted to include all variations or modifications derived from the meaning and scope of the appended claims and their equivalents (and not the detailed description above).

Claims

1. A protein variant comprising an amino acid sequence in which the amino acid at position 922 from the N-terminus of the amino acid sequence corresponding to SEQ ID NO: 74 is replaced by glutamic acid.

2. The protein variant according to claim 1, wherein the protein variant has 90% or more and less than 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO:

74.

3. The protein variant of claim 1, wherein the protein variant comprises the amino acid sequence of SEQ ID NO:

20.

4. A polynucleotide encoding a protein variant according to claim 1.

5. A Corynebacterium microorganism comprising at least one selected from the group consisting of a protein variant according to claim 1 and a polynucleotide encoding the protein variant.

6. The microorganism according to claim 5, wherein the Corynebacterium genus microorganism is Corynebacterium glutamicum.

7. The microorganism of claim 5, wherein the microorganism has an increased L-arginine production capacity compared to a Corynebacterium microorganism that does not contain at least one of the groups consisting of the protein variant and the polynucleotide encoding the protein variant.

8. A method for producing L-arginine, comprising culturing the microorganism according to claim 5 in a culture medium.

9. The method for producing L-arginine according to claim 8, wherein the method further comprises recovering L-arginine from a culture medium or cultured microorganisms.

10. Use of the microorganism according to any one of claims 5 to 7 for the production of L-arginine.

11. Compositions, methods, products, processes, or uses characterized by one or more elements disclosed in this disclosure.

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