Novel promoter and method for producing L-isoleucine using same

Abstract

The present disclosure relates to a novel polynucleotide exhibiting promoter activity, and a method for producing L-isoleucine by using the same. The microorganism into which the novel polynucleotide of the present disclosure has been introduced has significantly increased L-isoleucine productivity, and thus the novel polynucleotide can be effectively used for the efficient production of L-isoleucine, in which a specific position of the promoter region of the NCgl2412 gene, NCgl2046 gene, and / or NCgl1896 gene is mutated.

Description

Technical Field

[0001] Cross-references to related applications

[0002] This disclosure claims priority to Korean Patent Application No. 10-2024-0066104, filed on May 21, 2024, and Korean Patent Application No. 10-2024-0140712, filed on October 15, 2024, and the entire contents disclosed in the documents of the respective Korean patent applications are incorporated herein by reference.

[0003] This disclosure references numerous academic papers and patent documents, and their sources are clearly indicated. The contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which this disclosure pertains and the content of the invention.

[0004] This disclosure relates to novel promoters and methods for producing L-isoleucine using the promoters, and more specifically, to novel polynucleotides having promoter activity, expression cassettes containing the polynucleotide and a target gene operatively linked thereto, vectors containing the polynucleotide or the expression cassette, microorganisms containing the polynucleotide or the expression cassette, and methods for producing L-isoleucine using the microorganisms. Background Technology

[0005] L-Isoleucine (Ile, I) is classified as an essential amino acid, one of the twenty essential amino acids, and is a branched-chain amino acid used in the manufacture of various products, such as animal feed, food additives, and pharmaceuticals. L-Isoleucine is increasingly used in intravenous solutions, nutritional supplements, sports nutrition supplements, and animal feed because of its role in post-metabolism energy production, hemoglobin production, blood sugar regulation, muscle building, and repair.

[0006] Based on this trend, various attempts have been made to improve the production capacity of methods for producing L-isoleucine using various microorganisms, including Corynebacterium (US6072083A).

[0007] Despite these efforts, there is still a need to develop technologies to improve L-isoleucine production capacity. Summary of the Invention

[0008] [Technical Issues]

[0009] One object of this disclosure is to provide a novel polynucleotide.

[0010] Another object of this disclosure is to provide an expression cassette containing the polynucleotide and the target gene.

[0011] Another object of this disclosure is to provide microorganisms containing the said polynucleotide or polynucleotide and a target gene operatively linked thereto.

[0012] Another object of this disclosure is to provide a method for producing L-isoleucine, which includes culturing the microorganism in a culture medium.

[0013] Another object of this disclosure is to provide the use of the microorganism for the production of L-isoleucine.

[0014] [Technical Solution]

[0015] 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 specific aspects of this disclosure described herein using only conventional experiments. Furthermore, such equivalents are intended to be included in this disclosure.

[0016] Furthermore, numerous academic papers and patent documents are cited throughout this disclosure, with their sources clearly indicated. The content of the cited papers and patent documents is incorporated into this disclosure in its entirety through citation, in order to more clearly describe the level of the technical field to which this disclosure pertains and the content of this disclosure.

[0017] One aspect of this disclosure provides a polynucleotide comprising:

[0018] A nucleotide sequence wherein, in the nucleotide sequence of SEQ ID NO: 54, the nucleotide at position 99 is replaced by a nucleotide different from the original nucleotide;

[0019] A nucleotide sequence wherein, in the nucleotide sequence of SEQ ID NO: 58, the nucleotide at position 217 is replaced by a nucleotide different from the original nucleotide, the nucleotide at position 218 is replaced by a nucleotide different from the original nucleotide, the nucleotide at position 219 is replaced by a nucleotide different from the original nucleotide, the nucleotide at position 220 is replaced by a nucleotide different from the original nucleotide, the nucleotide at position 221 is replaced by a nucleotide different from the original nucleotide, the nucleotide at position 224 is replaced by a nucleotide different from the original nucleotide, the nucleotide at position 226 is replaced by a nucleotide different from the original nucleotide, and the nucleotide at position 228 is replaced by a nucleotide different from the original nucleotide; or

[0020] A nucleotide sequence wherein, in the nucleotide sequence of SEQ ID NO: 60, the nucleotide at position 155 is replaced by a nucleotide different from the original nucleotide, the nucleotide at position 163 is replaced by a nucleotide different from the original nucleotide, the nucleotide at position 164 is replaced by a nucleotide different from the original nucleotide, and the nucleotide at position 165 is replaced by a nucleotide different from the original nucleotide.

[0021] In this disclosure, the nucleotide sequences of SEQ ID NO: 54, SEQ ID NO: 58, or SEQ ID NO: 60 can be retrieved from the known database NCBI Genbank, and the nucleotide sequences of SEQ ID NO: 54, SEQ ID NO: 58, or SEQ ID NO: 60 can each be derived from the genus *Corynebacterium*, and specifically, can be sequences derived from *Corynebacterium glutamicum*. The nucleotide sequences of SEQ ID NO: 54, SEQ ID NO: 58, or SEQ ID NO: 60 can be used as exemplary sequences to illustrate the mutation sites to be introduced for the preparation of the polynucleotides described in this disclosure, and it is apparent that mutations introduced into the polynucleotides of this disclosure can also be introduced into any sequence functionally corresponding to the nucleotide sequences of SEQ ID NO: 54, SEQ ID NO: 58, or SEQ ID NO: 60. In one instance, the functionally corresponding sequence may be a nucleotide sequence having at least 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, and less than 100% homology or identity with the nucleotide sequences of SEQ ID NO: 54, SEQ ID NO: 58, or SEQ ID NO: 60, or a nucleotide sequence having said homology or identity with partial additions, deletions, or modifications thereof, but is not limited thereto.

[0022] Therefore, in one instance, the polynucleotide of this disclosure may comprise, substantially consist of or be composed of such a nucleotide sequence in which the nucleotide at position 99 of the nucleotide sequence of SEQ ID NO: 54 is replaced by a nucleotide different from the original nucleotide, and has at least 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.35% or more, or 99.39% or more, and less than 100% homology or identity with the nucleotide sequence of SEQ ID NO: 54, or a nucleotide sequence in which a portion of the sequence is added, deleted, or modified and has said homology or identity. In another example, the polynucleotide of this disclosure may comprise, substantially consist of or be composed of such a nucleotide sequence in which the nucleotides at positions 217, 218, 219, 220, 221, 224, 226, and 228 of the nucleotide sequence of SEQ ID NO: 58 are respectively replaced by nucleotides different from the original nucleotides, and have at least 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 96.1% or more, 96.2% or more, 96.3% or more, 96.4% or more, 96.5% or more, 96.55% or more, or 96.59% or more, and less than 100% homology or identity with the nucleotide sequence of SEQ ID NO: 58, or a nucleotide sequence in which a portion of the sequence is added, deleted, or modified with said homology or identity. In another example, the polynucleotide of this disclosure may comprise, substantially consist of or be composed of such a nucleotide sequence in which the nucleotides at positions 155, 163, 164 and 165 of the nucleotide sequence of SEQ ID NO: 60 are respectively replaced by nucleotides different from the original nucleotides, and have at least 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 97.25% or more, 97.5% or more, 97.6% or more, 97.7% or more, 97.8% or more, 97.9% or more, or 98% or more, and less than 100% homology or identity with the nucleotide sequence of SEQ ID NO: 60, or a nucleotide sequence in which a portion of the sequence is added, deleted or modified and has said homology or identity.

[0023] In this disclosure, the nucleotide sequence of SEQ ID NO: 54 may be the promoter sequence of the NCgl2412 gene or a portion thereof.

[0024] In one embodiment, the nucleotide at position 99 of the nucleotide sequence of SEQ ID NO: 54 may be guanine, but is not limited thereto.

[0025] In one embodiment, the polynucleotide may be, but is not limited to, a polynucleotide in which the nucleotide at position 99 of the nucleotide sequence of SEQ ID NO: 54 is replaced by adenine, cytosine or thymine (more specifically by cytosine).

[0026] In this disclosure, the nucleotide sequence of SEQ ID NO: 58 may be the promoter sequence of the gene encoding threonine aminolyase IlvA (NCgl2046 gene, ilvA), or a portion thereof.

[0027] Threonine ammonia lyase IlvA, also known as threonine dehydratase (EC 4.3.1.19), is an enzyme that produces 2-ketobutyrate from threonine and is encoded by the ilvA gene. Microorganisms of the genus Corynebacterium utilize pyruvate and 2-ketobutyrate as precursors to synthesize L-isoleucine through three metabolic intermediates.

[0028] In one embodiment, in the nucleotide sequence of SEQ ID NO: 58, the nucleotide at position 217 may be adenine, the nucleotide at position 218 may be adenine, the nucleotide at position 219 may be guanine, the nucleotide at position 220 may be adenine, the nucleotide at position 221 may be thymine, the nucleotide at position 224 may be cytosine, the nucleotide at position 226 may be cytosine, and / or the nucleotide at position 228 may be adenine, but is not limited thereto.

[0029] In one specific embodiment, the polynucleotide disclosed herein may be a polynucleotide in which the nucleotide at position 217 of the nucleotide sequence of SEQ ID NO: 58 is replaced by cytosine, guanine or thymine (more specifically thymine), but is not limited thereto.

[0030] In one specific embodiment, the polynucleotide disclosed herein may be a polynucleotide in which the nucleotide at position 218 of the nucleotide sequence of SEQ ID NO: 58 is replaced by cytosine, guanine or thymine (more specifically by guanine), but is not limited thereto.

[0031] In one specific embodiment, the polynucleotide disclosed herein may be a polynucleotide in which the nucleotide at position 219 of the nucleotide sequence of SEQ ID NO: 58 is replaced by adenine, cytosine or thymine (more specifically thymine), but is not limited thereto.

[0032] In one specific embodiment, the polynucleotide disclosed herein may be a polynucleotide in which the nucleotide at position 220 of the nucleotide sequence of SEQ ID NO: 58 is replaced by cytosine, guanine or thymine (more specifically by guanine), but is not limited thereto.

[0033] In one specific embodiment, the polynucleotide of this disclosure may be a polynucleotide in which the nucleotide at position 221 of the nucleotide sequence of SEQ ID NO: 58 is replaced by adenine, cytosine or guanine, more specifically, it may be a polynucleotide in which it is replaced by guanine, but is not limited thereto.

[0034] In one specific embodiment, the polynucleotide disclosed herein may be a polynucleotide in which the nucleotide at position 224 of the nucleotide sequence of SEQ ID NO: 58 is replaced by adenine, guanine or thymine (more specifically thymine), but is not limited thereto.

[0035] In one specific embodiment, the polynucleotide disclosed herein may be a polynucleotide in which the nucleotide at position 226 of the nucleotide sequence of SEQ ID NO: 58 is replaced by adenine, guanine or thymine (more specifically adenine), but is not limited thereto.

[0036] In one specific embodiment, the polynucleotide disclosed herein may be a polynucleotide in which the nucleotide at position 228 of the nucleotide sequence of SEQ ID NO: 58 is replaced by cytosine, guanine or thymine (more specifically by guanine), but is not limited thereto.

[0037] In this disclosure, the nucleotide sequence of SEQ ID NO: 60 may be the promoter sequence of or a portion thereof of the gene encoding 4-hydroxy-tetrahydrodipyridinic acid synthase (EC 4.3.3.7) (NCgl1896 gene, dapA).

[0038] 4-Hydroxy-tetrahydrodipyridinecarboxylate synthase is an enzyme that catalyzes the condensation reaction of pyruvate and aspartic acid semialdehyde to produce 4-hydroxy-2,3,4,5-tetrahydrodipyridinecarboxylate (HTPA) and plays an important role in lysine biosynthesis.

[0039] In one embodiment, in the nucleotide sequence of SEQ ID NO: 60, the nucleotide at position 155 may be guanine, the nucleotide at position 163 may be thymine, the nucleotide at position 164 may be adenine, and / or the nucleotide at position 165 may be adenine, but is not limited thereto.

[0040] In one specific embodiment, the polynucleotide disclosed herein may be a polynucleotide in which the nucleotide at position 155 of the nucleotide sequence of SEQ ID NO: 60 is replaced by adenine, cytosine or thymine (more specifically thymine), but is not limited thereto.

[0041] In one specific embodiment, the polynucleotide disclosed herein may be a polynucleotide in which the nucleotide at position 163 of the nucleotide sequence of SEQ ID NO: 60 is replaced by adenine, cytosine or guanine (more specifically by guanine), but is not limited thereto.

[0042] In one specific embodiment, the polynucleotide disclosed herein may be a polynucleotide in which the nucleotide at position 164 of the nucleotide sequence of SEQ ID NO: 60 is replaced by cytosine, guanine or thymine (more specifically by guanine), but is not limited thereto.

[0043] In one specific embodiment, the polynucleotide disclosed herein may be a polynucleotide in which the nucleotide at position 165 of the nucleotide sequence of SEQ ID NO: 60 is replaced by cytosine, guanine or thymine (more specifically by guanine), but is not limited thereto.

[0044] In one embodiment, the polynucleotide may comprise, substantially consist of, or be composed of such a nucleotide sequence having at least 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.35% or more, or 99.39% or more, and less than 100% homology or identity with the nucleotide sequence of SEQ ID NO: 54. More specifically, the polynucleotide may be a polynucleotide in which the nucleotide at position 99 of the nucleotide sequence of SEQ ID NO: 54 is replaced by cytosine (C), and it comprises, is substantially composed of or consists of such a nucleotide sequence having at least 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.35% or more, or 99.39% or more, and less than 100% homology or identity with the nucleotide sequence of SEQ ID NO: 54.

[0045] In one embodiment, the polynucleotide may comprise, consist substantially of, or be composed of, a nucleotide sequence having at least 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 96.1% or more, 96.2% or more, 96.3% or more, 96.4% or more, 96.5% or more, 96.55% or more, or 96.59% or more, and less than 100% homology or identity with the nucleotide sequence of SEQ ID NO: 58. More specifically, the polynucleotide may comprise, substantially consist of, or be composed of, a nucleotide sequence in which, in the nucleotide sequence of SEQ ID NO: 58, the nucleotide at position 217 is replaced by thymine (T), the nucleotide at position 218 is replaced by guanine (G), the nucleotide at position 219 is replaced by thymine (T), the nucleotide at position 220 is replaced by guanine (G), the nucleotide at position 221 is replaced by guanine (G), the nucleotide at position 224 is replaced by thymine (T), the nucleotide at position 226 is replaced by adenine (A), and the nucleotide at position 228 is replaced by guanine (G), and it is consistent with SEQ ID NO: The nucleotide sequence of 58 has at least 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 96.1% or more, 96.2% or more, 96.3% or more, 96.4% or more, 96.5% or more, 96.55% or more, or 96.59% or more, and less than 100% homology or identity.

[0046] In one embodiment, the polynucleotide may comprise, consist substantially of, or be composed of such a nucleotide sequence having at least 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 97.25% or more, 97.5% or more, 97.6% or more, 97.7% or more, 97.8% or more, 97.9% or more, or 98% or more, and less than 100% homology or identity with the nucleotide sequence of SEQ ID NO: 60. More specifically, the polynucleotide may comprise, substantially consist of, or be composed of, a nucleotide sequence in which, in the nucleotide sequence of SEQ ID NO: 60, the nucleotide at position 155 is replaced by thymine, the nucleotide at position 163 is replaced by guanine, the nucleotide at position 164 is replaced by guanine, and the nucleotide at position 165 is replaced by guanine, and it has at least 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 97.25% or more, 97.5% or more, 97.6% or more, 97.7% or more, 97.8% or more, 97.9% or more, or 98% or more, and less than 100% homology or identity with the nucleotide sequence of SEQ ID NO: 60.

[0047] In this disclosure, the polynucleotide may comprise the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 7, or SEQ ID NO: 9. In another embodiment, the polynucleotide of this disclosure may consist substantially of the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 7, or SEQ ID NO: 9. In yet another embodiment, the polynucleotide of this disclosure may consist of the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 7, or SEQ ID NO: 9.

[0048] 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, wherein the nucleotide monomers are covalently linked to form a chain.

[0049] The polynucleotides disclosed herein can have promoter activity and can be used as universal promoters.

[0050] In one embodiment, the polynucleotide may have promoter activity for expressing the target gene in Corynebacterium microorganisms.

[0051] In this disclosure, the polynucleotide with promoter activity can be used interchangeably with the "variant promoter".

[0052] According to one embodiment of this disclosure, a polynucleotide can be used as a synthetic promoter with strong inducible expression activity.

[0053] In this disclosure, the term "promoter" can refer to a DNA region containing a polymerase binding site and initiating transcription of a target gene located downstream therefrom. The promoter can be located upstream of the transcription start site. The promoter can be operatively and / or tunably (enhancing or weakening expression) linked upstream 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 downstream of the target gene in the opposite direction, for example, downstream of a stop codon, particularly between the stop codon and the top of a transcription terminator, it can weaken gene expression by inducing the corresponding gene to transcribe in a direction opposite to the normal transcription direction, thereby causing the RNA polymerase complex to collide with the normally oriented RNA polymerase complex during transcription.

[0054] 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.). The polynucleotide according to one embodiment can be natural or non-natural; for example, it can be a non-natural polynucleotide synthesized chemically or through recombinant synthesis.

[0055] In this disclosure, the term "variation" refers to a genetically or non-genetically stable phenotypic change and may be used interchangeably with "mutation".

[0056] The polynucleotides (variant promoters) disclosed herein may have altered (increased or decreased) promoter activity compared to polynucleotides excluding the variants (wild-type polynucleotides or pre-mutant polynucleotides). These polynucleotides can regulate (increase or decrease) the expression of a target gene operatively linked to them, or the expression or activity of a protein encoded by the target gene, and further, can regulate the expression of other genes besides the target gene.

[0057] "Target gene" refers to the gene whose expression is regulated by the polynucleotides disclosed herein. In the case of a gene encoding a protein, it can be used interchangeably with "gene encoding the 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".

[0058] Due to the degeneracy of codons or taking into account 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.

[0059] When the polynucleotide of this disclosure is introduced into a suitable host cell together with a target gene operatively linked thereto, the polynucleotide may have activity that increases the host cell’s production capacity (production quantity) of the target substance (e.g., the production capacity (production quantity) of amino acids).

[0060] In one embodiment, the polynucleotide can be used to increase the production capacity (production volume) of amino acids, specifically, it can be used to increase the production capacity (production volume) of L-isoleucine.

[0061] Furthermore, the nucleotide sequence of polynucleotides can be further modified using conventional mutagenesis methods, such as directed evolution and site-directed mutagenesis, to the extent that the corresponding biological activity (promoter activity) and / or desired activity (e.g., the activity of increasing the production of target substances in host cells) is maintained.

[0062] Therefore, the polynucleotide of this disclosure can be a polynucleotide comprising a nucleotide sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or higher homology or identity with the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 7, or SEQ ID NO: 9, or a nucleotide sequence having said homology or identity with partial additions, deletions, or modifications thereof; substantially composed of or consisting of said nucleotide sequences.

[0063] In this disclosure, the terms “homology” or “identity” refer to the degree of similarity between two given amino acid sequences or nucleotide sequences, expressed as a percentage. The terms homology and identity are often used interchangeably.

[0064] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms and can be used together with default gap penalties established by the program used. Essentially, homologous or identical sequences can generally hybridize with the entire sequence or a portion thereof under moderately or highly stringent conditions. Clearly, hybridization also includes hybridization with polynucleotides containing universal codons or codons that take into account the degeneracy of codons within the polynucleotide.

[0065] Whether any two polynucleotide or polypeptide sequences are homologous or identical can be determined, for example, by using known computer algorithms such as the “FASTA” program, using default parameters as in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine this, as 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) (including the GCG package (Devereux, J., et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, SF, [et al., J Molec Biol 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] AcademicPress, San Diego, 1994 and [CARILLO] This is performed as described in 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 database.

[0066] Homology or identity of polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, for example Needleman et al. (1970), J Mol Biol. 48:443, and as known in, for example, Smith and Waterman, Adv. Appl. Math (1981) 2:482. In summary, the GAP program can be defined as a value obtained by dividing the number of similarly arranged symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter 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 (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) replacement matrix) as disclosed in Atlas Of Protein Sequence And Structure, edited by Schwartz and Dayhoff, National Biomedical Research Foundation, pp. 353-358 (1979); (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.

[0067] 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 can be listed where 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 the 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; or 68°C, 0.1x SSC and 0.1% SDS) with one wash, especially two or three washes, but not limited to these. 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, adenosine 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., ibid., 9.50–9.51, 11.7–11.8).

[0068] Furthermore, the polynucleotides disclosed herein can be operatively linked to genes encoding target proteins, i.e., target genes.

[0069] In this disclosure, the term "operably linked" or "operatively linked" refers to the functional linking of a polynucleotide having promoter activity of this disclosure to a gene sequence, thereby initiating and mediating transcription of a target gene. Operable links can be prepared using gene recombination techniques known in the art, and site-specific DNA cutting and ligation can be generated using cutting and ligases in the art, but are not limited thereto.

[0070] When polynucleotides are operatively linked to a target gene, some nucleotides can be added, deleted, and / or modified in order to use cleavage and ligase enzymes.

[0071] In one embodiment, the target gene may be a gene encoding a protein involved in the production of L-isoleucine disclosed herein, but is not limited thereto. The protein involved in L-isoleucine production may be a protein involved in at least one process or step of the intracellular production pathway of L-isoleucine (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 thereto.

[0072] In one implementation, the target gene may be the NCgl2412 gene, but is not limited thereto.

[0073] In one embodiment, the target gene may be the NCgl2046 gene, but is not limited thereto. The NCgl2046 gene may be a gene encoding threonine ammonia lyase IlvA (ilvA).

[0074] In one embodiment, the target gene may be the NCgl1896 gene, but is not limited thereto. The NCgl1896 gene may be a gene encoding 4-hydroxy-tetrahydrodipyridinecarboxylic acid synthase (dapA).

[0075] Another aspect of this disclosure provides an expression cassette comprising the polynucleotides of this disclosure and the target gene.

[0076] The polynucleotides and target genes are as described above.

[0077] In this disclosure, the term "expression cassette" refers to a unit box containing a promoter and a target gene operatively linked thereto, and capable of expressing the target gene located downstream of the promoter. Various factors that facilitate the effective expression of the target gene may be included inside or outside such a gene expression cassette. In addition to a promoter operatively linked to the target gene, a gene expression cassette typically includes, but is not limited to, transcription termination signals, ribosome binding sites, and translation termination signals.

[0078] Another aspect of this disclosure provides a vector comprising the polynucleotide; the polynucleotide and a target gene operatively linked thereto; or an expression cassette of this disclosure.

[0079] The polynucleotide, target gene, and expression cassette are as described above. The vector may include the target gene operatively linked to the polynucleotide.

[0080] In this disclosure, the term "vector" refers to a DNA construct containing a nucleotide 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 regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and / or a sequence regulating transcription and / or translation termination. 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.

[0081] There are no particular limitations on the vectors that can be used in this disclosure, as long as they are reproducible 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, pDCM2, and pDC24 vectors.

[0082] Vectors that can be used in this disclosure can be expression vectors or insertion vectors for integration into the host cell chromosome. Insertion of target DNA into the host cell chromosome using an insertion vector can be performed by any method known in the art, such as, but not limited to, homologous recombination or the CRISPR system. The vector may further 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, auxotrophic phenotypes, 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.

[0083] 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 regardless of its form of introduction, it is acceptable 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, which is a gene construct containing all the elements necessary for its own expression, or in the form of a vector containing it.

[0084] The conversion method includes any method for 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.

[0085] Another aspect of this disclosure provides a microorganism (host cell) comprising the polynucleotides of this disclosure, and a target gene or expression cassette operatively linked thereto.

[0086] The polynucleotide, target gene, or expression cassette is as described above.

[0087] The polynucleotide, the polynucleotide, and the target gene or expression cassette operably linked thereto can be introduced into microorganisms through transformation, but are not limited thereto.

[0088] In this disclosure, the term "microorganism" includes wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, 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.

[0089] 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 parent strain that does not naturally express the target gene or have the ability to produce the target product but is endowed with the ability to express the target gene or produce the target product, but is not limited thereto. In one embodiment, the microorganism may be a microorganism that naturally produces amino acids, particularly a microorganism that produces L-isoleucine.

[0090] In this disclosure, the term "target product" refers to a bioactive substance produced or regulated by using a polynucleotide, a target gene operatively linked thereto, an expression cassette containing the polynucleotide and the target gene, a vector containing the polynucleotide and the target gene, and / or a microorganism containing the polynucleotide and the target gene, as provided in this disclosure. It includes not only the ultimately produced bioactive substance 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 involving the target protein. Bioactive substances refer to any substance produced by or derived from an organism (e.g., cells) or that has a predetermined function in or within the body of an organism or cell, and can be, for example, amino acids (glycine, alanine, valine, leucine, 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)). The list includes, but is not limited to, B7, B9, B12, C, D, E, K, proteins (target proteins or other proteins, such as hormones, growth factors, cytokines, immunoglobulins (antibodies), antigen proteins, receptors, ligands, their functional fragments (fragments that retain the desired function), fusion proteins of two or more of them), sugars (e.g., monosaccharides, disaccharides, polysaccharides, sugar alcohols), fatty acids (myristone 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. In addition, if a substance is produced through the participation of a target protein, then in addition to the substances mentioned above, its metabolites (such as polyhydroxyalkanoates (PHAs), its precursors, and derivatives that maintain its biological activity can also be included in the target product.

[0091] In one embodiment, the microorganism comprising the polynucleotide of this disclosure and the 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-isoleucine. The microorganism with increased L-isoleucine production capacity may be a microorganism with increased L-isoleucine 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 the NCgl2412 gene, NCgl2046 gene, or NCgl1896 gene 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, specifically consisting of the nucleotide sequences of SEQ ID NO: 54, SEQ ID NO: 58, or SEQ ID NO: 60.

[0092] 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 have not been altered due to genetic variation caused by natural or artificial factors. For example, unmodified microorganisms may refer to strains in which variant polynucleotides have not yet been introduced into the promoter regions of the NCgl2412, NCgl2046, or NCgl1896 genes described in this disclosure, or strains prior to such introduction. "Unmodified microorganism" may be used interchangeably with "pre-modified strain," "pre-modified microorganism," "non-mutant strain," "unmodified strain," "non-mutant microorganism," or "reference microorganism."

[0093] In one embodiment, the microorganism serving as the host cell may be Corynebacterium glutamicum KCCM12739P (CA10-3101, US 2023-0098971 A1) or Corynebacterium glutamicum KCCM11248P (Korean Patent No. 10-1335789), but is not limited thereto.

[0094] In one embodiment, the microorganism (a variant microorganism or a microorganism serving as a host cell) may be a microorganism in which the biosynthetic pathway of L-isoleucine is additionally enhanced to increase L-isoleucine production, but is not limited thereto.

[0095] In one instance, the microorganism may be one in which feedback inhibition of L-threonine dehydratase is relieved and / or feedback inhibition of homoserine dehydrogenase is relieved.

[0096] In one embodiment, the microorganism may be a microorganism in which feedback inhibition of L-threonine dehydratase is relieved.

[0097] In this disclosure, the term "threonine dehydratase (EC 4.3.1.19)" refers to an enzyme that produces 2-ketobutyrate from threonine, encoded by the ilvA gene, and known to be subject to feedback inhibition by L-isoleucine. Microorganisms of the genus *Corynebacterium* utilize pyruvate and 2-ketobutyrate as precursors to synthesize L-isoleucine via three intermediate metabolites. The amino acid sequence of threonine dehydratase is available from the known database NCBI's GenBank, or from US 2023-0098971 A1 and US 10982244 B2.

[0098] In another embodiment, the microorganism may be a microorganism in which feedback inhibition of homoserine dehydrogenase is relieved.

[0099] In this disclosure, the term "homoserine dehydrogenase (EC:1.1.1.3)" refers to an enzyme encoded by the hom gene that catalyzes the synthesis of homoserine. Homoserine dehydrogenase is known to be subject to feedback inhibition by isoleucine. The amino acid sequence of homoserine dehydrogenase is available from the NCBI GenBank database or from US 10982244 B2.

[0100] In one embodiment, the microorganism may be one in which feedback inhibition of aspartate kinase is relieved. In this disclosure, the term "aspartate kinase (EC: 2.7.2.4)" is encoded by the lysC gene, and the amino acid sequence of aspartate kinase is available from the known database NCBI's GenBank or from US 10662450 B2.

[0101] In this disclosure, the term "relief of feedback inhibition" can refer to an increase or enhancement in the activity of a polypeptide, protein, or enzyme compared to its intrinsic activity, or the removal of inhibition relative to its intrinsic activity. In one embodiment, the microorganism may be a microorganism further comprising one or more of the following mutations: additionally introducing a genetic mutation (R407H) into the hom gene (US 10982244 B2); additionally introducing a genetic mutation (T381A, F383A, and / or V323A) into the ilvA gene (US 2023-0098971 A1, US 10982244 B2); and additionally introducing a genetic mutation (L377K) into the lysC gene encoding aspartate kinase (US 10662450 B2), but is not limited thereto.

[0102] The microorganisms disclosed herein may include, but are not limited to, any microorganism in which the polynucleotides of this disclosure may be introduced and function as promoters.

[0103] In one embodiment, the microorganism may be a Corynebacterium sp., an Escherichia sp., or a Bacillus sp., but is not limited thereto.

[0104] 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*.

[0105] In one embodiment, the microorganism may be Corynebacterium glutamicum, wherein the mutation has been introduced into the promoter of the NCgl2412 gene, NCgl2046 gene, or NCgl1896 gene, but is not limited thereto.

[0106] In one embodiment, the L-isoleucine production capacity (production rate) of the microorganism of this disclosure having increased L-isoleucine production capacity (production rate) can be increased by about 5% or more, about 6% or more, about 8% or more, about 9% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 45% or more, about 50% or more, about 60% or more, about 70% or more, about 75% or more, or about 80% or more (no particular upper limit is specified, for example, it can be 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 L-isoleucine production capacity (production rate) of the microorganism of this disclosure having increased L-isoleucine production capacity (production rate) can be increased by about 1.05 times or more, about 1.06 times or more, about 1.08 times or more, about 1.09 times or more, about 1.1 times or more, about 1.2 times or more, about 1.3 times or more, about 1.4 times or more, about 1.45 times or more, about 1.5 times or more, about 1.6 times or more, about 1.7 times or more, about 1.75 times or more, or about 1.8 times or more (the upper limit is not particularly limited and can be, for example, about 10 times or less, about 5 times or less, about 3 times or less, about 2 times or less, or about 1.5 times or less), but is not limited thereto.

[0107] The term “about” includes all ranges 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 value following the term “about”.

[0108] Another aspect of this disclosure provides a composition for producing L-isoleucine, comprising at least one selected from the group consisting of: the polynucleotide of this disclosure, a polynucleotide and a target gene, expression cassette, vector, and microorganism operably linked thereto. The polynucleotide, target gene, expression cassette, vector, microorganism, etc., are as described above.

[0109] In one example, the composition for producing L-isoleucine may further comprise any suitable excipient conventionally used in compositions for producing L-isoleucine, such excipients may be, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers or isotonic agents, etc., but are not limited thereto.

[0110] Another aspect of this disclosure provides use for the production of L-isoleucine by selecting at least one from the group consisting of: the polynucleotide of this disclosure, the polynucleotide and the target gene, expression cassette, vector, and microorganism operably linked thereto. The polynucleotide, target gene, expression cassette, vector, microorganism, etc., are as described above.

[0111] Another aspect of this disclosure provides use for preparing compositions for the production of L-isoleucine from at least one selected from the group consisting of: the polynucleotide of this disclosure, the polynucleotide and a target gene, expression cassette, vector, and microorganism operably linked thereto. The polynucleotide, target gene, expression cassette, vector, microorganism, etc., are as described above.

[0112] Another aspect of this disclosure provides a method for producing a target product, the method comprising the step of culturing microorganisms in a culture medium, said microorganisms containing the polynucleotides of this disclosure and a target gene, expression cassette or vector operatively linked thereto.

[0113] The polynucleotide, target gene, expression cassette, vector, microorganism, and target product are as described above.

[0114] In one embodiment, the target product may be an L-amino acid. Specifically, the target product may be L-isoleucine.

[0115] The method may also include a step of recovering the target product from the cultured microorganism, culture, or both after the culturing step.

[0116] 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 such a culture process depending on 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).

[0117] In this disclosure, the term "culture medium" refers to a substance prepared by mixing nutrients required as the main component for culturing the microorganisms of this disclosure, 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 microorganisms of this disclosure without particular limitation. However, the microorganisms of this disclosure can be cultured in conventional culture media containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins under aerobic conditions, while controlling temperature, pH, etc. Specifically, culture media for microorganisms can be found in the literature, for example, ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D.C., USA, 1981)].

[0118] 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, while controlling temperature, pH, etc., in a conventional culture medium containing suitable carbon sources, nitrogen sources, amino acids, vitamins, etc. In this case, 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 limitation, but are not limited to these. As nitrogen sources, inorganic nitrogen sources such as ammonia; and amino acids such as glutamic acid and cysteine, as well as 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 corresponding sodium-containing salts, can be used as phosphorus sources, but are not limited to these. As inorganic compounds, magnesium sulfate, ferric sulfate, manganese sulfate, and calcium chloride can be used. In addition, amino acids, vitamins, and appropriate precursors can be included. These culture media or precursors can be added to the culture in batches or continuously, but are not limited to this.

[0119] 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 esters of fatty acids can be used to suppress foam formation during cultivation. Additionally, to maintain the aerobic state of the culture, oxygen or oxygen-containing gas can be injected into the culture. The cultivation temperature is 27°C to 37°C, particularly 30°C to 33°C. The cultivation time can continue until the desired yield of the useful substance is obtained, specifically 10 to 160 hours.

[0120] In this disclosure, the term "culture" refers to a substance comprising a culture medium in which microorganisms grow or have completed their growth under appropriately controlled and artificially controlled environmental conditions. In a narrow sense, a culture does not include grown microorganisms, but in a broad sense includes microorganisms that may have completed their growth. A "culture" can include various target substances released into the culture medium by microorganisms during their growth, as well as the components of the culture medium formulated for the cultivation of microorganisms.

[0121] In the cultivation process disclosed herein, the cultivation temperature can be maintained between 20°C and 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, and the cultivation can be carried out for approximately 10 to 160 hours, 20 to 100 hours, 30 to 90 hours, or 50 to 70 hours, but is not limited thereto.

[0122] The target product produced by the culture of this disclosure can be secreted into the culture medium or retained inside the cells.

[0123] 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 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, 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 art can be used to recover the target product from the culture medium or microorganisms.

[0124] 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 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 separately (or consecutively) without regard to the order, or they may be performed simultaneously or integrated into a single step, but are not limited thereto.

[0125] Another aspect of this disclosure provides a method for increasing the production capacity of a target product, the method comprising culturing microorganisms containing the polynucleotides, polynucleotides and target genes of this disclosure or expression cassettes containing polynucleotides and target genes in a culture medium.

[0126] The polynucleotide, target gene, expression cassette, microorganism, and target product are as described above.

[0127] 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 the polynucleotide, polynucleotide and target gene of this disclosure or an expression cassette containing the polynucleotide and target gene into the microorganism.

[0128] The polynucleotide, target gene, expression cassette, microorganism, and target product are as described above.

[0129] According to another aspect of this disclosure, this disclosure provides compositions, methods, products, processes, or uses characterized by one or more elements disclosed herein.

[0130] [Beneficial Effects]

[0131] This disclosure relates to novel polynucleotides with promoter activity and methods for producing L-isoleucine using the polynucleotides. Furthermore, since the L-isoleucine production capacity is significantly increased in microorganisms in which the novel polynucleotides of this disclosure are introduced, these novel polynucleotides can be effectively used for the efficient production of L-isoleucine. Detailed Implementation

[0132] The present application will be described in more detail below by way of embodiments. These embodiments are merely for illustrating the present application more specifically, and it will be apparent to those skilled in the art that the scope of the present application is obviously not limited to these embodiments in accordance with the spirit of the present application.

[0133] Example

[0134] (Throughout this instruction manual, unless otherwise stated, the term "%" used to indicate the concentration of a particular substance means (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid.)

[0135] The present disclosure is further described below with reference to the following embodiments. However, these embodiments are for illustrative purposes only, and the scope of the present disclosure is not limited to these embodiments.

[0136] Example 1: Selecting mutant strains with increased isoleucine production capacity through artificial mutagenesis

[0137] Example 1-1: Random mutagenesis induced by UV irradiation

[0138] To select mutant strains with increased isoleucine production capacity, the isoleucine-producing strain of *Corynebacterium glutamicum* KCCM12739P was plated on nutrient medium containing agar and incubated at 30°C for 16 hours. Hundreds of colonies were obtained by UV (ultraviolet mutation) irradiation at room temperature to induce random mutations in the strain's genome.

[0139] <Nutritional medium (pH 7.2)>

[0140] 10g glucose, 5g meat extract, 10g polypeptone, 2.5g sodium chloride, 5g yeast extract, 20g agar, 2g urea (based on 1L distilled water)

[0141] Examples 1-2: Selection of strains with increased L-isoleucine production capacity

[0142] To select mutant strains with increased isoleucine production capacity compared to the parent strain KCCM12739P, KCCM12739P strain and the aforementioned mutant strains in which random mutations were induced were cultured by the following method.

[0143] Each of the above-mentioned strains was inoculated into 96-deep well plate-dome (Bioneer) containing 400 µl of seed medium and cultured in a plate shaking incubator (TAITEC) at 32 °C and 1200 rpm for approximately 48 hours. The isoleucine concentration of approximately 3000 cultured strains was measured using near-infrared (NIR) spectroscopy, and the top five mutant strains exhibiting increased isoleucine production compared to the parent strain KCCM12739P were selected.

[0144] Seed culture medium (pH 7.0)

[0145] 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 μg, thiamine hydrochloride 1000 μg, calcium pantothenate 2000 μg, nicotinamide 2000 μg (based on 1L of distilled water)

[0146] To ultimately select the strain with reproducibly increased L-isoleucine production capacity from the five chosen mutant strains, they were cultured and evaluated as follows: The parental strain and the aforementioned mutant strains were inoculated into 250 mL corner-baffled flasks containing 25 mL of isoleucine production medium and then cultured at 32 °C with shaking at 200 rpm for 60 hours. After culture, the L-isoleucine concentration in the culture medium was analyzed using HPLC. The L-isoleucine production concentration of each mutant strain is shown in Table 1 below.

[0147] <Production medium (pH 7.2)>

[0148] Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, ferric sulfate heptahydrate 10 mg / L, manganese sulfate monohydrate 10 mg / L, biotin 200 μg / L (based on 1 L of distilled water)

[0149] [Table 1]

[0150]

[0151] Among the five mutant strains screened, KCCM12739P_mt4 was ultimately selected as the strain with significantly improved L-isoleucine production capacity.

[0152] Example 2: Mutation identification using whole-genome sequencing (WGS)

[0153] Whole-genome sequencing (WGS) was performed on the KCCM12739P_mt4 strain selected in Examples 1-2 to analyze the sequence, and mutations occurring in ten types of promoter regions were identified by comparing it with the parental strain KCCM12739P (SEQ ID NO: 52 to SEQ ID NO: 61). The sequences of the variant promoters containing the mutations are shown in Table 2 below.

[0154] [Table 2]

[0155]

[0156]

[0157] In the following examples, the effects of each variant promoter listed in Table 2 on L-isoleucine production capacity of Corynebacterium spp. were evaluated to identify the effective factors influencing L-isoleucine production capacity.

[0158] Example 3: Construction of L-isoleucine-producing strain with a variant promoter

[0159] Example 3-1: Construction of a recombinant vector for introducing a variant promoter

[0160] To insert the promoter of each variant of the NCgl0867, NCgl1062, NCgl2412, NCgl1224, NCgl2581, NCgl2658, NCgl2046, NCgl0780, NCgl1896 and NCgl2356 genes in Table 2 into KCCM12739P, a vector containing the target mutation was constructed.

[0161] Specifically, using the G-spin Total DNA Extraction Mini Kit (Intron, catalog number 17045), genomic DNA of strain KCCM12739P_mt4 was extracted according to the protocol provided in the kit, and PCR was performed using the genomic DNA as a template. The polymerase used was Solg™ Pfu-X DNA polymerase. The PCR reaction conditions were as follows: denaturation at 95°C for 4 minutes; followed by 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; finally, polymerization was carried out at 72°C for 5 minutes. The following polymerases were used: SEQ ID NO: 12 and SEQ ID NO: 13; or SEQ ID NO: 14 and SEQ ID NO: 15; or SEQ ID NO: 16 and SEQ ID NO: 17; or SEQ ID NO: 18 and SEQ ID NO: 19; or SEQ ID NO: 20 and SEQ ID NO: 21; or SEQ ID NO: 22 and SEQ ID NO: 23; or SEQ ID NO: 24 and SEQ ID NO: 25; or SEQ ID NO: 26 and SEQ ID NO: 27; or SEQ ID NO: 28 and SEQ ID NO: 29; or SEQ ID NO: 30 and SEQ ID NO: 29. 31. Obtain the respective PCR products. The primer sequences used in the above experiments are shown in Table 3 below.

[0162] Recombinant plasmids were obtained by cloning mutant-introducing fragments using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly MasterMix) and by using the pDC24 vector (SEQ ID NO: 11) treated with the restriction enzyme SmaI. The vectors containing each mutant-introducing fragment were named pDC24-Pn*_NCgl0867, pDC24-Pn*_NCgl1062, pDC24-Pn*_NCgl2412, pDC24-Pn*_NCgl1224, pDC24-Pn*_NCgl2581, pDC24-Pn*_NCgl2658, pDC24-Pn*_NCgl2046, pDC24-Pn*_NCgl0780, pDC24- Pn*_NCgl1896 and pDC24-Pn*_NCgl2356.

[0163] [Table 3]

[0164]

[0165]

[0166]

[0167] Example 3-2: Construction of L-isoleucine-producing strains with variant promoters

[0168] The 10 vectors prepared in Example 3-1 were transformed into isoleucine-producing strain KCCM12739P by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), and strains that inserted the vectors into the chromosome through homologous sequence recombination were selected using kanamycin medium. Subsequently, the strain that introduced the variant promoter was confirmed from the transformants that had completed secondary recombination by PCR using the following primer pairs: SEQ ID NO: 32 and SEQ ID NO: 33; SEQ ID NO: 34 and SEQ ID NO: 35; or SEQ ID NO: 36 and SEQ ID NO: 37; or SEQ ID NO: 38 and SEQ ID NO: 39; or SEQ ID NO: 40 and SEQ ID NO: 41; or SEQ ID NO: 42 and SEQ ID NO: 43; or SEQ ID NO: 44 and SEQ ID NO: 45; or SEQ ID NO: 46 and SEQ ID NO: 47; or SEQ ID NO: 48 and SEQ ID NO: 49; or SEQ ID NO: 50 and SEQ ID NO: 51. PCR was performed in the same manner as in Example 3-1. The recombinant strains were named KCCM12739PΔPn::Pn*_NCgl0867, KCCM12739PΔPn::Pn*_NCgl1062, KCCM12739PΔPn::Pn*_NCgl2412, KCCM12739PΔPn::Pn*_NCgl1224, and KCCM12739PΔPn::Pn*_NCgl, respectively. Primer pairs 2581, KCCM12739PΔPn::Pn*_NCgl2658, KCCM12739PΔPn::Pn*_NCgl2046, KCCM12739PΔPn::Pn*_NCgl0780, KCCM12739PΔPn::Pn*_NCgl1896, and KCCM12739PΔPn::Pn*_NCgl2356 were used for confirmation. The primer pairs used for confirmation are shown in Table 4 below.

[0169] [Table 4]

[0170]

[0171] Example 4. Evaluation of L-isoleucine production capacity of L-isoleucine-producing strains with introduced variant promoters

[0172] To evaluate the L-isoleucine production capacity of the strains prepared in Examples 3-2 above at the shake-flask level, the strains were cultured and evaluated using the following method: The parental strain and the mutant strain were inoculated into 250 mL baffled Erlenmeyer flasks containing 25 mL of isoleucine production medium and cultured at 32°C with shaking at 200 rpm for 60 hours.

[0173] After cultivation, the yield of L-isoleucine was measured using high performance liquid chromatography (HPLC), and the results are shown in Table 5 below.

[0174] <Production medium (pH 7.2)>

[0175] Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.1%, ferric sulfate heptahydrate 10 mg / L, manganese sulfate monohydrate 10 mg / L, biotin 200 μg / L (based on 1 L of distilled water)

[0176] [Table 5]

[0177] Comparison of isoleucine production capacity of L-isoleucine-producing strains with introduced variant promoters (flask titer evaluation, 60 hours)

[0178]

[0179] As shown in Table 5 above, the isoleucine production capacity of strains with the introduced variant promoters was increased or showed a comparable level compared to the parental strain. In particular, it was confirmed that the isoleucine production capacity of 'KCCM12739PΔPn::Pn*_NCgl2412', 'KCCM12739PΔPn::Pn*_NCgl2046', and 'KCCM12739PΔPn::Pn*_NCgl1896' was significantly increased compared to the parental strain KCCM12739P.

[0180] This confirms that L-isoleucine can be produced more efficiently by introducing variant promoters that regulate NCgl2412 gene expression, variant promoters that regulate NCgl2046 gene expression, and / or variant promoters that regulate NCgl1896 gene expression.

[0181] Based on the above description, those skilled in the art will understand that this application 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 application should be interpreted to include all variations or modifications derived from the meaning and scope of the above claims and their equivalents, not just the specific embodiments described above.

Claims

1. A polynucleotide comprising: In the nucleotide sequence of SEQ ID NO: 54, the nucleotide at position 99 is replaced by cytosine (C); In the nucleotide sequence of SEQ ID NO: 58, the nucleotide at position 217 is replaced by thymine (T), the nucleotide at position 218 is replaced by guanine (G), the nucleotide at position 219 is replaced by thymine (T), the nucleotide at position 220 is replaced by guanine (G), the nucleotide at position 221 is replaced by guanine (G), the nucleotide at position 224 is replaced by thymine (T), the nucleotide at position 226 is replaced by adenine (A), and the nucleotide at position 228 is replaced by guanine (G); or In the nucleotide sequence of SEQ ID NO: 60, the nucleotide at position 155 is replaced by thymine (T), the nucleotide at position 163 is replaced by guanine (G), the nucleotide at position 164 is replaced by guanine (G), and the nucleotide at position 165 is replaced by guanine (G).

2. The polynucleotide according to claim 1, wherein the polynucleotide has promoter activity.

3. The polynucleotide according to claim 1, wherein the polynucleotide has 80% or more, but less than 100% sequence identity with the nucleotide sequences of SEQ ID NO: 54, SEQ ID NO: 58 or SEQ ID NO: 60 respectively.

4. The polynucleotide of claim 1, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 3, the nucleotide sequence of SEQ ID NO: 7, or the nucleotide sequence of SEQ ID NO:

9.

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

6. A microorganism of the genus Corynebacterium, comprising: The polynucleotide according to any one of claims 1 to 4; or The polynucleotide and the target gene operatively linked thereto.

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

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

9. The method for producing L-isoleucine according to claim 8, further comprising recovering L-isoleucine from the culture medium or the microorganism.

10. Use of the microorganism according to claim 6 or 7 for the production of L-isoleucine.

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