Novel promoter and method for producing L-arginine using same

Abstract

The present disclosure relates to a novel polynucleotide having promoter activity and a method for producing L-arginine using the same. A microorganism into which the novel polynucleotide of the present disclosure, which has a mutation at a specific position in the promoter region of the BBD2914250 gene, has been introduced, shows a significantly increased ability to produce L-arginine, and thus the novel polynucleotide can be effectively used for efficient production of L-arginine.

Description

Technical Field

[0001] Cross-reference to related applications

[0002] This disclosure claims the benefit of priority based on Korean Patent Application No. 10-2024-0071881 filed on May 31, 2024 and Korean Patent Application No. 10-2024-0135801 filed on October 7, 2024, and the entire contents disclosed in the documents of the corresponding Korean patent applications are incorporated herein as part of this disclosure.

[0003] This disclosure relates to novel promoters and methods for producing L-arginine using them, and more particularly, to novel polynucleotides having promoter activity, vectors containing said polynucleotides, host cells transformed with said vectors, and methods for producing L-arginine using said host cells. Background Technology

[0004] To utilize microorganisms to produce target substances such as amino acids or useful compounds at high titers for various purposes, including feed, pharmaceuticals, and food, researchers are continuously working on methods such as genetic manipulation and / or the introduction of exogenous genes into biosynthetic pathways. One such method involves inducing overexpression of target genes in microorganisms, requiring efficient gene expression systems. Since promoters are among the factors that significantly influence gene expression levels and regulation, developing useful promoters is crucial for developing expression systems.

[0005] Corynebacterium microorganisms are those that produce amino acids including L-arginine. Unlike other industrial microorganisms such as Escherichia coli or Bacillus subtilis, the general structure of promoter sequences used for gene expression in Corynebacterium microorganisms is unknown. Therefore, promoters were developed by removing the promoter portion of antibiotic resistance genes such as chloramphenicol, inserting chromosomal DNA isolated from Corynebacterium microorganisms after cleavage with a suitable restriction endonuclease, and then measuring the antibiotic resistance of strains obtained by transforming Corynebacterium microorganisms with these promoters. Various strengths of the Pcj1–7 promoters derived from Corynebacterium microorganisms are known (US 7662943 B2).

[0006] With the increasing demand for L-arginine, there remains a need to develop strong promoters for overexpressing L-arginine-related genes and to use this as a method for producing L-arginine in high yields in microorganisms belonging to the genus Corynebacterium. Summary of the Invention

[0007] [Technical Issues]

[0008] One object of this disclosure is to provide a novel polynucleotide.

[0009] Another aspect of this disclosure is to provide an expression cassette comprising the polynucleotide and the target gene.

[0010] Another aspect of this disclosure is to provide a microorganism comprising the polynucleotide; or the polynucleotide and a target gene operatively linked thereto.

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

[0012] Another aspect 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 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] Furthermore, those skilled in the art can identify or confirm numerous equivalents of the specific aspects of this application disclosed herein using only ordinary experiments. Moreover, these equivalents are intended to be included in this application.

[0016] One aspect of this disclosure provides a polynucleotide comprising any nucleotide sequence selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO: 21. In one embodiment, the polynucleotide may be a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 21.

[0017] In this disclosure, the phrase "a polynucleotide or polypeptide comprising a specific nucleotide sequence (nucleic acid sequence, base sequence) or amino acid sequence" can mean that a polynucleotide or polypeptide is composed of or substantially comprises a specific nucleotide sequence (nucleic acid sequence, base sequence) or amino acid sequence.

[0018] In this disclosure, the term "polynucleotide" can refer to a polynucleotide comprising 2 or more, 5 or more, 10 or more, 13 or more, 20 or more, or 30 or more nucleotide monomers covalently linked to form a chain.

[0019] The polynucleotides disclosed herein may have promoter activity and / or can be used as universal promoters.

[0020] In one embodiment, the polynucleotide may have promoter activity expressed in Corynebacterium microorganisms.

[0021] In this disclosure, the term "promoter-active polynucleotide" may be used interchangeably with "mutant promoter," and all of the foregoing terms may be used in this disclosure.

[0022] According to one embodiment of this disclosure, a polynucleotide can be used as a synthetic promoter with strong inducible expression activity.

[0023] In this disclosure, the term "promoter" can refer to a DNA region containing a polymerase binding site and initiating transcription of a target gene downstream therefrom. The promoter can be located at the 5' end of the transcription start site. The promoter can be operatively and / or controllably (enhancing or weakening expression) linked upstream (5' end) of the target gene. For example, the promoter can be linked forward to the upstream side of the target gene to enhance (increase) gene expression, or it can be linked backward to the downstream side of the target gene to weaken (decrease) gene expression. When the promoter is introduced backward downstream of the target gene, for example, downstream of a stop codon, preferably between the stop codon and the tip of a transcription terminator, it can weaken gene expression by inducing the corresponding gene to transcribe in the opposite direction to the normal transcription direction, thereby causing the RNA polymerase complex to conflict with the normally oriented RNA polymerase complex during transcription.

[0024] The polymerase, also known as RNA polymerase or DNA-dependent RNA polymerase, can refer to an enzyme that synthesizes primary transcribed RNA from DNA. The polymerase can be a prokaryotic RNA polymerase or a eukaryotic RNA polymerase (e.g., RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, or RNA polymerase V, etc.).

[0025] According to one implementation scheme, the polynucleotide can be natural or non-natural, for example, it can be a non-natural polynucleotide synthesized by chemical synthesis or recombinant synthesis.

[0026] In this disclosure, the phrase "polynucleotide or polypeptide comprising a specific nucleotide sequence (nucleic acid sequence, base sequence) or amino acid sequence" can mean that the polynucleotide or polypeptide is composed of or substantially composed of a specific nucleotide sequence (nucleic acid sequence, base sequence) or amino acid sequence, and can be interpreted as including (or not excluding) sequences in which mutations (deletions, substitutions, modifications, and / or additions) have been added to the specific nucleic acid sequence (base sequence) or amino acid sequence to maintain the original function and / or desired function of the polynucleotide or polypeptide. Furthermore, the expression "composed of a nucleotide sequence" does not exclude the possibility of nucleotide additions, and / or deletions, and / or mutations occurring during the linking of the target gene (e.g., using a restriction enzyme) when the polynucleotide is used as a promoter by linking a target gene.

[0027] In one instance, a polynucleotide or polypeptide “containing a specific nucleotide sequence (nucleic acid sequence, base sequence) or amino acid sequence” can mean that the polynucleotide or polypeptide (i) consists of or is substantially composed of a specific nucleotide sequence (nucleic acid sequence, base sequence) or amino acid sequence, or (ii) consists of or is substantially composed of a nucleotide sequence or amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98%, 99.5%, or 99.9% homology with the specific nucleotide sequence (nucleic acid sequence, base sequence) or amino acid sequence, and retains its original function and / or desired function.

[0028] In one embodiment, the polynucleotide of this disclosure may comprise the nucleotide sequence of SEQ ID NO: 15, and the nucleotide sequence of SEQ ID NO: 15 may be a sequence in which a portion of the promoter sequence of the PBBD29_12015 gene is mutated.

[0029] In one embodiment, the polynucleotide of this disclosure may comprise the nucleotide sequence of SEQ ID NO: 16, and the nucleotide sequence of SEQ ID NO: 16 may be a sequence in which a portion of the promoter sequence of the PBBD29_07560 gene is mutated.

[0030] In one embodiment, the polynucleotide of this disclosure may comprise the nucleotide sequence of SEQ ID NO: 17, and the nucleotide sequence of SEQ ID NO: 17 may be a sequence in which a portion of the promoter sequence of the PBBD29_12275 gene is mutated.

[0031] In one embodiment, the polynucleotide of this disclosure may comprise the nucleotide sequence of SEQ ID NO: 18, and the nucleotide sequence of SEQ ID NO: 18 may be a sequence in which a portion of the promoter sequence of the PBBD29_01740 gene is mutated.

[0032] In one embodiment, the polynucleotide of this disclosure may comprise the nucleotide sequence of SEQ ID NO: 19, and the nucleotide sequence of SEQ ID NO: 19 may be a sequence in which a portion of the promoter sequence of the PBBD29_04965 gene is mutated.

[0033] In one embodiment, the polynucleotide of this disclosure may comprise the nucleotide sequence of SEQ ID NO: 20, and the nucleotide sequence of SEQ ID NO: 20 may be a sequence in which a portion of the promoter sequence of the PBBD29_05235 gene is mutated.

[0034] In one embodiment, the polynucleotide of this disclosure may comprise the nucleotide sequence of SEQ ID NO: 21, and the nucleotide sequence of SEQ ID NO: 21 may be a sequence in which a portion of the promoter sequence of the PBBD29_14250 gene is mutated.

[0035] In this disclosure, the term "variation" refers to a genetic or non-genetically stable phenotypic change and may be used interchangeably with "mutation".

[0036] Compared to polynucleotides that do not contain mutations (wild-type or pre-mutant polynucleotides), the polynucleotides (mutant promoters) of this disclosure may have altered (increased or decreased) promoter activity. Polynucleotides can regulate (increase or decrease) the expression of a target gene or the protein encoded by that target gene, and can also regulate the expression of other genes besides the target gene.

[0037] "Target gene" refers to a gene whose expression is regulated by the polynucleotides disclosed herein, and in the case of a gene encoding a protein, it can be used interchangeably with "gene encoding a target protein". The protein encoded by the target gene can be described as "target protein", and the gene encoding the "target protein" can be described as "target gene".

[0038] Due to the degeneracy of codons or considering the preferred codons (codon usage frequency) in the organism to which the target gene is to be expressed, the amino acid coding sequence of the target gene can be modified in various ways without changing the protein sequence encoded by the target gene.

[0039] When the polynucleotides of this disclosure are introduced into a suitable host cell together with a target gene operably linked thereto, the polynucleotides may have activity that increases the production capacity (quantity) of the target substance (e.g., the production capacity (quantity) of amino acids) of the host cell.

[0040] In one embodiment, the polynucleotide can be used to increase amino acid production capacity (production volume), and specifically, it can be used to increase L-arginine production capacity (production volume).

[0041] Furthermore, the nucleotide sequence of the polynucleotide can be further modified by conventionally known mutagenesis methods (e.g., directed evolution and site-directed mutagenesis) to maintain the corresponding biological activity (promoter activity) and / or the desired activity (e.g., increasing the activity of target substance production in host cells).

[0042] Therefore, the polynucleotides disclosed herein may comprise a nucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher but less than 100% homology or identity with a nucleotide sequence selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO: 21, or may comprise a sequence having said homology or identity but with some sequence additions, deletions, or modifications.

[0043] In this disclosure, the polynucleotide may consist substantially of any nucleotide sequence selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO: 21. In another embodiment, the polynucleotide of this disclosure may consist of any nucleotide sequence selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO: 21.

[0044] 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.

[0045] 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.

[0046] Whether any two polynucleotide or polypeptide sequences are homologous or identical can be determined, for example, using known computer algorithms, 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 by the Needleman program (version 5.0.0 or later) in 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, BLAST or ClustalW from the National Center for Biotechnology Information (NCBI) database can be used to determine homology or identity.

[0047] 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), JMol Biol. 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.

[0048] In one instance, the polynucleotides containing a specific nucleotide sequence provided in this disclosure can be interpreted as containing not only the specific nucleotide sequence or a substantially equivalent nucleotide sequence, but also polynucleotide fragments containing nucleotide sequences complementary to the specific nucleotide sequence. Specifically, complementary polynucleotides can be identified under conditions described in specific literature. For example, conditions in which genes with high complementarity of 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 98% or higher, 99.5% or higher, or 99.9% or higher hybridize with each other, while genes with lower complementarity do not hybridize, or conditions corresponding to conventional Southern hybridization washing conditions, i.e., 60°C, 1x SSC (saline-sodium citrate buffer) and 0.1% (w / v) SDS (sodium dodecyl sulfate); 60°C, 0.1x SSC and 0.1% SDS (sodium dodecyl sulfate); or 68°C, 0.1x SSC and 0.1% SDS, washing once, specifically two to three times, but not limited to these conditions. Hybridization requires two nucleotides to have complementary sequences, but some base mismatches can be allowed depending on the strictness of the hybridization process. The term "complementarity" can be used to describe the relationship between nucleotides that can hybridize with each other. For example, in the case of DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. The strictness of hybridization between polynucleotides depends on the length and degree of complementarity of the polynucleotides, which is well known in the relevant fields (see Sambrook et al., above, 9.50-9.51, 11.7-11.8).

[0049] Furthermore, the polynucleotides disclosed herein can be operatively linked to genes encoding target proteins, i.e., target genes.

[0050] In this disclosure, the term "operably ligated" refers to the functional ligation of a polynucleotide having the promoter activity of this disclosure to a gene sequence, thereby initiating and mediating transcription of a target gene. Operable ligations can be prepared using gene recombination techniques known in the art, and site-specific DNA splicing and ligation can be produced using splicing and ligases in the art, but are not limited thereto.

[0051] When polynucleotides are operatively linked to a target gene, some nucleotides can be added, deleted, and / or mutated in order to use cleavage and ligases.

[0052] In one embodiment, the target gene may be, but is not limited to, a gene encoding a protein involved in the production of L-arginine according to this disclosure. The protein involved in L-arginine production may be a protein involved in at least one process or step of the intracellular production pathway of L-arginine (e.g., biosynthesis, metabolism, biotransformation, etc.), intracellular transport, and / or extracellular secretion pathway, and may be selected from the group consisting of, for example, enzymes (various synthases, lyases, kinases, carboxylases (e.g., pyruvate carboxylase, etc.), reductases, oxidases, decarboxylases, dehydrogenases, dehydratases, transferases, epimerases, etc.), intermediates, transport proteins, membrane proteins (channels, etc.), etc., but is not limited to.

[0053] In one implementation, the target gene may be, but is not limited to, the PBBD29_12015, PBBD29_07560, PBBD29_12275, PBBD29_01740, PBBD29_04965, PBBD29_05235, or PBBD29_14250 gene.

[0054] Another aspect of this disclosure provides an expression cassette comprising the polynucleotides of this disclosure and the target gene.

[0055] The polynucleotides and target genes are as described above.

[0056] In this disclosure, the term "expression cassette" refers to a unit cassette containing a promoter and a target gene operatively linked thereto, and capable of expressing the target gene downstream of the promoter. Various factors that facilitate the effective expression of the target gene may be included, either inside or outside such a gene expression cassette. In addition to a promoter operatively linked to the target gene, a gene expression cassette may typically contain, but is not limited to, transcription termination signals, ribosome binding sites, and translation termination signals.

[0057] Another aspect of this disclosure provides a vector comprising: the polynucleotide of this disclosure; the polynucleotide and a target gene operatively linked thereto; or the expression cassette.

[0058] The polynucleotide, target gene, and expression cassette are as described above. The vector may contain the target gene operatively linked to the polynucleotide.

[0059] In this disclosure, the term "vector" refers to a DNA formulation containing a polynucleotide base sequence encoding a target protein, operatively linked to a suitable regulatory sequence to enable expression of the target protein in a suitable host. The regulatory sequence may include a promoter capable of initiating transcription, any operon sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and / or a termination sequence regulating transcription and / or translation. Upon transformation into a suitable host cell, the vector may be expressed independently of the host cell's genome or may be integrated into the host cell's genome.

[0060] There are no particular limitations on the vectors that can be used in this disclosure, as long as they are replicable in the host cell and can be selected from all commonly used vectors. Examples of commonly used vectors include plasmids, granules, viruses, bacteriophages, etc., in their natural or recombinant states. For example, as vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or granule vectors, and the pDZ series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used as plasmid vectors. Specifically, examples may include, but are not limited to, pDZ, pDC, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pDC2, and pDC24 vectors.

[0061] The vectors used in this disclosure can be expression vectors or insertion vectors for insertion into host cell chromosomes. The target DNA can be inserted into the host cell chromosome using any method known in the art, such as homologous recombination or the CRISPR system, but not limited thereto. The vector may also include selection markers for confirming whether the vector has been transformed, or further confirming whether the target DNA has been inserted into the chromosome. Selection markers can be selected and used from genes conferring selectable phenotypes, such as drug resistance, nutritional deficiencies, resistance to cytotoxic agents, or expression of surface proteins. In an environment treated with a selection agent, only cells expressing the selection marker can survive or exhibit a different phenotype, thus allowing for the selection of transformed cells.

[0062] In this disclosure, the term "transformation" refers to the introduction of a target polynucleotide into a host cell. The transformed polynucleotide may be inserted into the host cell's chromosome or may be located outside the chromosome. Furthermore, the polynucleotide may be DNA and / or RNA, and the form in which it is introduced is not important, as long as it can be introduced into the host cell and function therein. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette or in the form of a vector containing it, the expression cassette being a gene construct containing all the elements necessary for its own expression.

[0063] The conversion method includes any method of introducing the target polynucleotide into cells, and can be performed by selecting appropriate standard techniques known in the art, depending on the host cell. Examples include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0064] Another aspect of this disclosure provides a microorganism (host cell) comprising: the polynucleotide of this disclosure, the polynucleotide and a target gene operatively linked thereto, or the expression cassette.

[0065] The polynucleotide, target gene, or expression cassette is as described above.

[0066] The polynucleotide, polynucleotide, and target gene or expression cassette operatively linked thereto can be introduced into microorganisms through transformation, but are not limited thereto.

[0067] In this disclosure, the term "microorganism" includes wild-type microorganisms and microorganisms that are naturally or artificially genetically modified, and also includes microorganisms in which a particular mechanism is weakened or enhanced due to reasons such as the insertion of a foreign gene or the enhancement or weakening of the activity of an endogenous gene.

[0068] The microorganism may be a microorganism that naturally expresses the target gene or a microorganism capable of producing the target product, or it may be a microorganism whose ability to express the target gene or produce the target product is conferred by a parent strain that does not naturally express the target gene or has the ability to produce the target product, but is not limited thereto. In one embodiment, the microorganism may be a microorganism that naturally produces amino acids, specifically a microorganism that produces L-arginine.

[0069] In this disclosure, the term "target product" refers to a bioactive substance intended to produce or to regulate (increase or decrease) its production by using a polynucleotide provided in this disclosure, a target gene operatively linked thereto, an expression cassette containing the polynucleotide and the target gene, a vector containing the polynucleotide, and / or a microorganism containing the target gene, and is a concept that includes not only the bioactive substance ultimately produced but also the target protein that can be produced by the microorganism. For example, it can refer to the target protein itself encoded by the target gene, and / or any bioactive substance produced by involving the target protein. The bioactive substance refers to any substance produced or derived from an organism (e.g., a cell) or having a predetermined function in vivo or within a cell, and may be, for example, amino acids (glycine, alanine, valine, arginine, isoleucine, threonine, serine, cysteine, glutamine, methionine, aspartic acid, asparagine, glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, proline, O-acetylhomoserine, etc.), nucleic acids, vitamins (vitamins A, B (B1, B2, B3, B5, B6, B7)). The target proteins (such as B9, B12, C, D, E, K, etc.), proteins (such as hormones, growth factors, cytokines, immunoglobulins (antibodies), antigen proteins, receptors, ligands, their functional fragments (fractions retaining the desired function), fusion proteins of two or more of them), sugars (such as monosaccharides, disaccharides, polysaccharides, sugar alcohols, etc.), fatty acids (myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, transoleic acid, vaccenic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid (EPA), erucic acid, docosahexaenoic acid (DHA), etc.), organic acids (lactic acid, citric acid, oxalic acid, uric acid, butyric acid, stearic acid, propionic acid, etc.), etc., but not limited to these. In addition, if it is a substance produced by involving the target protein, its metabolites (such as polyhydroxyalkanoates (PHA)), precursors, derivatives, etc., that maintain its biological activity can also be included in the target product, in addition to the substances mentioned above.

[0070] In one embodiment, the microorganism comprising the polynucleotide of this disclosure and a target gene operatively linked thereto may be a microorganism with an increased capacity to produce amino acids as the target product, and specifically, a microorganism with an increased capacity to produce L-arginine. The microorganism with increased L-arginine production capacity may be a microorganism with increased L-arginine production capacity compared to a microorganism not comprising the polynucleotide of this disclosure, for example, but not limited to, a microorganism in which the expression of at least one gene selected from the group consisting of BBD29_12015, BBD29_07560, BBD29_12275, BBD29_01740, BBD29_04965, BBD29_05235, and BBD29_14250 is regulated by the polynucleotide prior to the introduction of the mutation. The polynucleotide prior to the introduction of the mutation may be a wild-type polynucleotide, and specifically, may consist of a nucleotide sequence selected from the group consisting of SEQ ID NO: 22 to SEQ ID NO: 28.

[0071] In this disclosure, "unmodified microorganism" does not exclude naturally occurring mutant strains and may refer to wild-type strains or naturally occurring strains themselves, or strains whose characteristics were altered by genetic variation due to natural or artificial factors. For example, an unmodified microorganism may refer to a strain in which the mutant polynucleotide has not been introduced into the promoter region of at least one gene selected from the group consisting of BBD29_12015, BBD29_07560, BBD29_12275, BBD29_01740, BBD29_04965, BBD29_05235, and BBD29_14250 as described in this disclosure, or a strain prior to such introduction. "Unmodified microorganism" may be used interchangeably with "pre-modified strain," "pre-modified microorganism," "unmutated strain," "unmodified strain," "unmutated microorganism," or "reference microorganism."

[0072] The microorganisms disclosed herein may include, but are not limited to, any microorganism in which the polynucleotides of the present disclosure may be introduced and function as promoters.

[0073] In one embodiment, the microorganism may be a microorganism of the genus Corynebacterium, a microorganism of the genus Escherichia, or a microorganism of the genus Bacillus, but is not limited thereto.

[0074] Specifically, the microorganisms may be members of the genus *Corynebacterium*, and more specifically, may include *Corynebacterium glutamicum*, *Corynebacterium stationis*, *Corynebacterium thermoaminogenes*, *Corynebacterium glutamicum*, *Brevibacterium flavum*, *Brevibacterium lactofermentum*, and strains prepared therefrom, but are not limited thereto. Specifically, the *Corynebacterium* genus microorganisms may be *Corynebacterium glutamicum*.

[0075] In one embodiment, the microorganism may be *Corynebacterium glutamicum*, wherein the mutation has been introduced into the promoter of at least one gene selected from the group consisting of BBD29_12015, BBD29_07560, BBD29_12275, BBD29_01740, BBD29_04965, BBD29_05235 and BBD29_14250, but is not limited thereto.

[0076] In one embodiment, the microorganism of this disclosure with increased L-arginine production capacity (production) may have an increased L-arginine production capacity (production) of about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 5.5% or more, or about 5.8% or more (no particular upper limit is specified, and may be, for example, about 200% or less, about 150% or less, about 100% or less, or about 90% or less), but is not limited thereto. In another embodiment, the microorganism of this disclosure with increased L-arginine production capacity (production) may have an increased L-arginine production capacity (production) of about 1.01 times or more, about 1.02 times or more, about 1.03 times or more, 1.04 times or more, about 1.05 times or more, about 1.055 times or more, or about 1.058 times or more (no particular upper limit is imposed, and may 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), but is not limited thereto.

[0077] The term “about” includes the entire range of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes, but is not limited to, all values ​​in the range that are equivalent to or similar to the values ​​following the term “about”.

[0078] Another aspect of this disclosure provides a composition for producing L-arginine, comprising at least one selected from the group consisting of: the polynucleotide of this disclosure, said polynucleotide and a target gene operatively linked thereto, said expression cassette, said vector, and said microorganism. The polynucleotide, target gene, expression cassette, vector, microorganism, etc., are as described above.

[0079] In one example, the composition for producing L-arginine may also contain any suitable excipient conventionally used in compositions for producing L-arginine, and such excipient may be, for example, a preservative, wetting agent, dispersant, suspending agent, buffer, stabilizer or isotonic agent, etc., but is not limited thereto.

[0080] Another aspect of this disclosure provides use for the production of L-arginine by selecting at least one from the group consisting of: the polynucleotide of this disclosure, the polynucleotide and a target gene operatively linked thereto, the expression cassette, the vector, and the microorganism. The polynucleotide, target gene, expression cassette, vector, microorganism, etc., are as described above.

[0081] Another aspect of this disclosure provides use for preparing at least one composition selected from the group consisting of: the polynucleotide of this disclosure, the polynucleotide and a target gene operatively linked thereto, the expression cassette, the vector, and the microorganism. The polynucleotide, target gene, expression cassette, vector, microorganism, etc., are as described above.

[0082] Another aspect of this disclosure provides a method for producing a target product, the method comprising culturing microorganisms in a culture medium containing the polynucleotide of this disclosure, the polynucleotide and a target gene operatively linked thereto, the expression cassette or the vector.

[0083] The polynucleotide, target gene, expression cassette, vector, microorganism, and target product are as described above.

[0084] In one embodiment, the target product may be an L-amino acid. Specifically, the target product may be L-arginine.

[0085] The method may further include a step of recovering the target product from the cultured microorganism, the culture, or both after the culturing step.

[0086] The term "culture" in this disclosure refers to the growth of microorganisms under appropriately controlled and artificially controlled environmental conditions. The culture process of this disclosure can be carried out using suitable culture media and culture 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. These different methods are disclosed, for example, in "Biochemical Engineering" (James M. Lee, Prentice-Hall International Editions, pp. 138-176, 1991).

[0087] In this disclosure, the term "culture medium" refers to a substance prepared by mixing nutrients required for culturing the 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 for the culture medium and other culture conditions without particular limitation; however, the microorganisms of this disclosure can be cultured under aerobic conditions in a conventional culture medium containing suitable carbon, nitrogen, phosphorus, inorganic compounds, amino acids, and / or vitamins, while controlling temperature, pH, etc. Specifically, culture media for microorganisms can be found in literature such as ["Manual of Methods for General Bacteriology", American Bacteriological Society (Washington DC, USA, 1981)].

[0088] The culture medium used for cultivation must meet the requirements of the specific strain in an appropriate manner. For example, cultivation can be carried out under aerobic conditions in a conventional culture medium containing suitable carbon sources, nitrogen sources, amino acids, vitamins, etc., while controlling temperature, pH, etc. In this case, the carbon sources include carbohydrates such as glucose, fructose, and sucrose, and amino acids such as glutamic acid and cysteine. Specifically, natural organic nutrient sources such as starch hydrolysates and molasses can be used, with carbohydrates such as glucose, fructose, and sterilized pretreated molasses (i.e., molasses converted to reducing sugars) being preferred. Other suitable carbon sources can be used in various ways without restriction, but are not limited to these. As nitrogen sources, inorganic nitrogen sources such as ammonia; and amino acids such as glutamic acid and cysteine, and peptone, meat extracts, yeast extracts, etc., can be used as organic nitrogen sources. These nitrogen sources can be used alone or in combination, but are not limited to these. In the culture medium, phosphate, potassium dihydrogen phosphate, or dipotassium hydrogen phosphate, or their corresponding sodium-containing salts, can be used as phosphorus sources, but are not limited to these. As for inorganic compounds, magnesium sulfate, ferric sulfate, manganese sulfate, and calcium chloride can be used. In addition, amino acids, vitamins, and suitable precursors can be included. These mediators or precursors can be added to the culture in batches or continuously, but are not limited to this.

[0089] During cultivation, the pH of the culture can be adjusted by adding compounds such as potassium hydroxide, ammonia, and phosphoric acid in an appropriate manner. Furthermore, antifoaming agents such as polyethylene glycol fatty acids can be used to suppress foam formation during cultivation. Additionally, to maintain an aerobic environment in the culture, oxygen or oxygen-containing gas can be injected into the culture. The cultivation temperature can be between 27°C and 37°C, and specifically, between 30°C and 33°C. The cultivation time can continue until the desired production volume of the useful substance is obtained, specifically between 20 and 160 hours.

[0090] In this disclosure, the term "culture" refers to a substance comprising a culture medium in which microorganisms have grown or have completed their growth under appropriately controlled and artificially controlled environmental conditions. In a narrow sense, the culture does not include grown microorganisms, but it can be included in a broad sense. "Culture" can include various target substances released into the culture medium by microorganisms during their growth, as well as the culture medium components formulated for the cultivation of microorganisms.

[0091] In the cultivation process disclosed herein, the cultivation temperature can be maintained between 20°C and 45°C, specifically between 25°C and 40°C, 25°C and 40°C, 25°C and 37°C, 25°C and 35°C, 27°C and 40°C, 27°C and 37°C, 27°C and 35°C, 30°C and 40°C, 30°C and 37°C, or 30°C and 35°C, and the cultivation can be carried out for approximately 10 to 160 hours, but is not limited thereto.

[0092] The target product produced by the culture method disclosed herein can be secreted into the culture medium or retained inside the cell.

[0093] The steps for recovering the target product can be based on the culture method, such as batch, continuous, or fed-batch culture, using suitable methods known in the relevant art to collect the target product. 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 are not limited thereto, and suitable methods known in the relevant art can be used to recover the desired target product from the culture medium or microorganisms.

[0094] Furthermore, the method for producing the target product of this disclosure may additionally include a purification step. Purification can be performed using suitable methods known in the relevant art. In one instance, when the method for producing the target product of this disclosure includes a recovery step and a purification step, the recovery step and the purification step may be performed at different times (or consecutively) regardless of the order, or they may be performed simultaneously or integrated into a single step, but are not limited thereto.

[0095] Another aspect of this disclosure provides a method for increasing the production capacity of a target product, the method comprising culturing microorganisms containing the polynucleotide of this disclosure, the polynucleotide and the target gene, or an expression cassette containing the polynucleotide and the target gene in a culture medium.

[0096] The polynucleotide, target gene, expression cassette, microorganism, and target product are as described above.

[0097] In one embodiment, the target product may be an L-amino acid, specifically L-arginine.

[0098] Another aspect of this disclosure provides a method for preparing microorganisms with increased production capacity of a target product, the method comprising the step of introducing a polynucleotide of the present disclosure, the polynucleotide and a target gene, or an expression cassette containing the polynucleotide and the target gene into the microorganism.

[0099] The polynucleotide, target gene, expression cassette, microorganism, and target product are as described above.

[0100] In one embodiment, the target product may be an L-amino acid, specifically L-arginine.

[0101] According to another aspect of this disclosure, this disclosure provides compositions, methods, products, processes, or uses characterized by one or more elements disclosed herein.

[0102] [Beneficial Effects]

[0103] This disclosure relates to novel polynucleotides with promoter activity and methods for producing L-arginine using them, and the novel polynucleotides can be effectively used for efficient L-arginine production because the L-arginine production capacity is significantly increased in microorganisms in which the novel polynucleotides of this disclosure are introduced. Detailed Implementation

[0104] The present disclosure will be described in more detail below by way of examples. These examples are intended to illustrate the present disclosure more specifically, and it will be apparent to those skilled in the art that the scope of the present disclosure is obviously not limited to these examples in accordance with the spirit of the disclosure.

[0105] Example

[0106] (Throughout this specification, unless otherwise stated, the term "%" used to express the concentration of a particular substance means (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid.)

[0107] Example 1. Construction of L-arginine production microorganisms

[0108] Example 1-1. Construction of Corynebacterium glutamicum CJR2 strain

[0109] 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).

[0110] 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:43) treated with SmaI restriction enzyme, according to the manual, to obtain plasmids. The prepared vectors were named pDC24-ΔargR and pDC24-argB (M54V), respectively.

[0111] 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.

[0112] Solid agar plate culture medium (pH 7.0)

[0113] 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)

[0114] 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.

[0115] Table 1 below describes the sequence information of the primers used in Examples 1-1.

[0116] Table 1

[0117]

[0118] Examples 1-2. Construction of Corynebacterium glutamicum strain CJR100

[0119] Based on the CJR2 strain prepared in Example 1-1, a CJR100 strain with enhanced N-acetyl-γ-glutamyl-phosphoreductase (hereinafter referred to as argC) gene was prepared.

[0120] 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.

[0121] 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.

[0122] Solid agar plate culture medium (pH 7.0)

[0123] 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)

[0124] Table 2 below describes the sequence information of the primers used in Examples 1-2.

[0125] Table 2

[0126]

[0127] Example 2: Selecting mutant strains with increased arginine production capacity through artificial mutagenesis

[0128] Example 2-1: Random Mutation by UV Radiation

[0129] To select mutant strains with increased arginine production capacity, the arginine-producing strain CJR100 prepared in Example 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.

[0130] <Nutritional medium (pH 7.2)>

[0131] 10g glucose, 5g meat extract, 10g peptone, 2.5g sodium chloride, 5g yeast extract, 20g agar, 2g urea (per liter of distilled water)

[0132] Example 2-2: Selection of strains with increased L-arginine production capacity

[0133] In order to select mutant strains with increased arginine production capacity compared with parent strain CJR100, CJR100 strain and mutant strains in Example 2-1, in which random mutations were induced, were cultured by the following methods.

[0134] 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.

[0135] Seed culture medium (pH 7.0)

[0136] 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)

[0137] To ultimately select strains with reproducibly increased L-arginine production capacity from four selected mutant strains, they were cultured and evaluated using the following methods.

[0138] 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.

[0139] Seed culture medium (pH 7.0)

[0140] 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)

[0141] <Production medium (pH 7.2)>

[0142] 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

[0143] 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 3 below.

[0144] Table 3

[0145]

[0146] As shown in Table 3 above, among the four selected mutant strains, CJR100_mt3 was ultimately selected as the mutant strain with the greatest increase in L-arginine production.

[0147] Example 2: Identification of mutations by whole-genome sequencing (WGS)

[0148] Whole-genome sequencing (WGS) was performed on CJR1000_mt3 selected in Examples 2-2 to analyze the sequence, and variations occurring in seven types of promoter regions were identified by comparing it with the parental strain CJR100 (SEQ ID NO: 22 to SEQ ID NO: 28). The sequences of mutant promoters containing the mutations are shown in Table 4 below.

[0149] Table 4

[0150]

[0151] (Expressed protein: a protein encoded by a gene whose expression is regulated by operative linking to a promoter)

[0152] In the following examples, the effects of each mutant promoter listed in Table 4 on the L-arginine production capacity of Corynebacterium spp. were evaluated to identify the effective factors affecting L-arginine production capacity.

[0153] Example 4: Construction of L-arginine-producing strains with introduced mutant promoters

[0154] Example 4-1: Construction of a recombinant vector introducing a mutant promoter

[0155] To insert each mutant promoter of PBBD29_12015, PBBD29_07560, PBBD29_12275, PBBD29_01740, PBBD29_04965, PBBD29_05235 and PBBD29_14250 from Table 4 into the CJR100 strain, a vector containing the target mutation was constructed.

[0156] 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 5 below.

[0157] Table 5

[0158]

[0159] The mutant insertion fragment obtained above and the pDC24 vector (SEQ ID NO: 43) 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) to obtain recombinant plasmids and vectors pDC24-Pn*_BBD29_12015; pDC24-Pn*_BBD29_07560; pDC24-Pn*_BBD29_12275; pDC24-Pn*_BBD29_01740; pDC24-Pn*_BBD29_04965; pDC24-Pn*_BBD29_05235; and pDC24-Pn*_BBD29_14250.

[0160] Example 4-2: Construction of L-arginine-producing strains with introduced mutant promoters

[0161] The seven vectors prepared in Example 4-1 were transformed into strain CJR100 using electroporation, and strains that inserted the vector into the chromosome through homologous sequence recombination were selected using kanamycin medium. Subsequently, PCR was performed using primers SEQ ID NO:44 and SEQ ID NO:45; or primers SEQ ID NO:46 and SEQ ID NO:47; or primers SEQ ID NO:48 and SEQ ID NO:49; or primers SEQ ID NO:50 and SEQ ID NO:51; or primers SEQ ID NO:52 and SEQ ID NO:53; or primers SEQ ID NO:54 and SEQ ID NO:55; or primers SEQ ID NO:56 and SEQ ID NO:57 to confirm the mutant promoter inoculated in the transformed strains that completed secondary recombination. PCR was performed in the same manner as in Example 4-1. The recombinant strains were named CJR100△Pn_BBD29_12015::Pn*_BBD29_12015, CJR100△Pn_BBD29_07560::Pn*_BBD29_07560, CJR100△Pn_BBD29_12275::Pn*_BBD29_12275, and CJR100△Pn_BBD2, respectively. The primer pairs used for confirmation are: 9_01740::Pn*_BBD29_01740, CJR100△Pn_BBD29_04965::Pn*_BBD29_04965, CJR100△Pn_BBD29_05235::Pn*_BBD29_05235, and CJR100△Pn_BBD29_14250::Pn*_BBD29_14250. The sequences of the primer pairs used for confirmation are shown in Table 6 below.

[0162] Table 6

[0163]

[0164] Example 5. Evaluation of L-arginine production capacity of L-arginine-producing strains with introduced mutant promoters

[0165] 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.

[0166] 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 Example 2-2, and the experiment was repeated three times.

[0167] 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 7 below.

[0168] Table 7

[0169]

[0170] As shown in Table 7, the arginine production of strains with introduced mutant promoters increased or reached levels comparable to those of the parent strains. In particular, “CJR100△Pn_BBD29_14250::Pn*_BBD29_14250” showed a significantly increased L-arginine production compared to the parent strain CJR100.

[0171] This confirms that introducing a mutant promoter that controls the expression of the BBD29_14250 gene can lead to more efficient L-arginine production.

[0172] Based on the above description, those skilled in the art will understand that this disclosure can be implemented in other specific forms without altering its technical concept 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 polynucleotide comprising a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO:

21.

2. The polynucleotide of claim 1, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:

21.

3. An expression cassette comprising the polynucleotide and target gene as described in claim 1.

4. A Corynebacterium microorganism comprising: the polynucleotide according to claim 1 or 2; or the polynucleotide and a target gene operatively linked thereto.

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

6. A method for producing L-arginine, comprising culturing the microorganism according to claim 4 in a culture medium.

7. The method for producing L-arginine according to claim 6, further comprising recovering L-arginine from the culture medium or microorganism.

8. Use of the microorganism according to claim 4 or 5 for the production of L-arginine.

9. Compositions, methods, products, processes, or uses characterized by one or more elements disclosed herein.

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