Novel promoters and their applications

Abstract

This application relates to a novel promoter and a method for producing a target product using the same, wherein a polynucleotide, in one specific example, has promoter activity and, when introduced into a microorganism, can increase the expression and activity of a gene operably linked thereto, and can be usefully utilized to efficiently produce a target product affected by the polynucleotide and the gene.

Description

Technical Field

[0001] Cross-Reference to Related Applications This application claims the benefit of priority based on Korean Patent Application No. 10-2024-0035063 filed on March 13, 2024, and all contents disclosed in the document of said Korean patent application are incorporated as a part of the present specification.

[0002] The present application relates to a novel promoter and a method for producing a target product using the same. Background Art

[0003] In order to produce target substances such as amino acids or useful substances that can be used in various applications including feed, pharmaceuticals, and foods with high titer using microorganisms, efforts such as genetic engineering on biosynthetic pathways and / or introduction of exogenous genes have been continuously made. As one of such methods, there is a method of inducing overexpression of a target gene in microorganisms, which requires a highly efficient gene expression system. Since a promoter is one of the elements that are greatly involved in the expression level and expression regulation of a gene, it can be said that development of useful promoters is essential for developing an expression system.

[0004] The E. coli-derived tac promoter is widely known as a strong promoter, and in the case of coryneform microorganisms, strong promoters have been developed by modifying the promoters of their own genes (Gene, 102, 93-98, 1991; Microbiology, 142, 1297-1309, 1996). On the other hand, the promoter sequence for gene expression in coryneform microorganisms is different from that of other industrial microorganisms such as E. coli and Bacillus subtilis, and its general structure is not known. Therefore, promoters have been developed by removing the promoter portion of antibiotic resistance genes such as chloramphenicol, introducing chromosomal DNA isolated from coryneform microorganisms after cutting with appropriate restriction enzymes, and then measuring the antibiotic resistance of the resulting strains obtained by transforming coryneform microorganisms with this modified promoter. In addition, various promoter searches have been conducted to overexpress foreign genes in Bacillus microorganisms, and these have been used to produce enzymes for food, pharmaceutical, and other industrial applications. Numerous vector systems utilizing the expression promoters of α-amylase, protease, and lipase genes are still being used in various Bacillus microorganisms (Schumann 2007. Adv. Appl. Microbiol. 153:813-821).

[0005] However, since a system that exhibits high expression efficiency in various microorganisms, such as those of the genera Escherichia, Corynebacterium, or Bacillus, is still needed, the development of a general-purpose promoter remains a necessity. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] U.S. Patent No. 11041181 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The object of this application is to provide a novel polynucleotide.

[0008] Another object of this application is to provide an expression cassette containing the polynucleotide and the target gene.

[0009] Another object of this application is to provide a recombinant vector (expression vector) comprising the polynucleotide or the expression cassette.

[0010] Another object of this application is to provide a microorganism comprising one or more selected from the group consisting of the polynucleotide, the polynucleotide and the target gene, and the expression cassette and the vector comprising the expression cassette.

[0011] Another object of this application is to provide a method for producing a target product, comprising the step of culturing the microorganism in a culture medium. The method may further include, after the culturing step, a step of recovering the target product from the culture medium or microorganism.

[0012] Another object of this application is to provide a composition for producing a target product comprising the microorganism, a culture medium in which the microorganism is cultured, or a combination thereof.

[0013] Another object of this application is to provide a promoter application for the polynucleotide.

[0014] Another object of this application is to provide applications for producing target products using the microorganism, the culture medium in which the microorganism is cultured, or a combination thereof. [Means for solving the problem]

[0015] Each description and embodiment disclosed herein can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below. The following provides a more detailed explanation.

[0016] One aspect of this application provides a novel polynucleotide.

[0017] Another embodiment provides a promoter application for the polynucleotide.

[0018] In this specification, "polynucleotide" comprises two or more, five or more, ten or more, thirteen or more, twenty or more, or thirty or more nucleotide monomers, and the nucleotide monomers may be covalently linked to form a chain.

[0019] In one example of this application, the polynucleotide may be a polynucleotide comprising a nucleic acid sequence in which the 19th nucleotide, the 22nd nucleotide, or all of these nucleotides from the 5' end of the nucleic acid sequence of Sequence ID No. 1 are substituted with other nucleotides.

[0020] In one example, the polynucleotide may include a nucleic acid sequence in which the 19th nucleotide from the 5' end of the nucleic acid sequence of SEQ ID NO: 1 is substituted with another nucleotide (e.g., adenine (A), thymine (T), or cytosine (C)).

[0021] In one example, the polynucleotide may include a nucleic acid sequence in which the 21st nucleotide from the 5' end of the nucleic acid sequence of SEQ ID NO: 1 is substituted with another nucleotide (e.g., thymine (T), guanine (G), or cytosine (C)). In one example, the polynucleotide is obtained by substituting the 19th nucleotide from the 5' end of the nucleic acid sequence of SEQ ID NO: 1 with another nucleotide (e.g., adenine (A), thymine (T), or cytosine (C)). It may comprise a nucleic acid sequence wherein the 21st nucleotide from the 5' end of the nucleic acid sequence of SEQ ID NO: 1 is substituted with another nucleotide (for example, thymine (T), guanine (G) or cytosine (C)).

[0022] In one example, the polynucleotide may be a polynucleotide comprising any one nucleic acid sequence selected from the group consisting of SEQ ID NO: 39 to 40.

[0023] In one example, the polynucleotide may comprise a nucleic acid sequence wherein the 21st nucleotide from the 5' end of the nucleic acid sequence of SEQ ID NO: 1 is substituted with cytosine.

[0024] The polynucleotide may comprise the nucleic acid sequence of SEQ ID NO: 39.

[0025] In one example, the polynucleotide may comprise a nucleic acid sequence wherein the 19th nucleotide from the 5' end of the nucleic acid sequence of SEQ ID NO: 1 is substituted with adenine.

[0026] The polynucleotide may comprise the nucleic acid sequence of SEQ ID NO: 40.

[0027] In one example, the polynucleotide may comprise a nucleic acid sequence wherein the 19th nucleotide from the 5' end of the nucleic acid sequence of SEQ ID NO: 1 is substituted with adenine, and the 21st nucleotide from the 5' end of the nucleic acid sequence of SEQ ID NO: 1 is substituted with cytosine.

[0028] The polynucleotide may be a polynucleotide in which 3 to 6 nucleotides are added to the 3' end of the nucleic acid sequence of SEQ ID NO: 1.

[0029] In one example, the polynucleotide may be a polynucleotide in which three nucleotides "CAT" (5'-CAT-3') are added to the 3' end of the nucleic acid sequence of SEQ ID NO: 1.

[0030] In one example, the polynucleotide may be a polynucleotide in which six nucleotides of "ACTAGT" (5'-ACTAGT-3') are added to the 3' end of the nucleic acid sequence of SEQ ID NO: 1.

[0031] In one example, the polynucleotide may include the nucleic acid sequence of SEQ ID NO: 41 or SEQ ID NO: 42.

[0032] The polynucleotide may have promoter activity and / or can be used as a general-purpose promoter.

[0033] In one example, the polynucleotide may have promoter activity for the expression of microorganisms of the genus Corynebacterium, microorganisms of the genus Escherichia, and / or microorganisms of the genus Bacillus.

[0034] In this specification, the nucleic acid sequence of the Po2 promoter (U.S. Patent No. 10273491) is shown as Sequence ID No. 1.

[0035] One example of the polynucleotide can be used as a synthetic promoter with strong expression-inducing activity, and can express target genes with significantly higher efficiency than existing Po2 promoters in microorganisms of the genera Corynebacterium, Escherichia, and / or Bacillus.

[0036] The polynucleotides described in one specific example may be natural or non-natural, and may, for example, be non-natural, chemically or recombinantly synthesized.

[0037] In this specification, “promoter” may mean a DNA region containing a polymerase binding site that initiates the transcription of a downstream target nucleotide sequence. The promoter may be located at the 5' end of the transcription initiation site. The polynucleotide may be ligated to the upper (5' end) and / or lower (3' end) of the target gene, and the polynucleotide may be operably and / or regulated (enhance or diminish) to the target gene. For example, the polynucleotide may be ligated to the 5' end of the target gene to enhance (increase) the expression of the target gene, or to the 3' end of the target gene to diminish (decrease) the expression of the target gene.

[0038] In one example, a promoter can be introduced in the reverse direction between the termination codon of the target gene, preferably between the termination codon and the upper end of the transcription terminater, in order to weaken the expression of the target gene. This weakens the expression of the target gene by causing the RNA polymerase complex to collide during the transcription process so that the target gene is transcribed in the reverse direction.

[0039] The polymerase referred to above is also called RNA polymerase or DNA-dependent RNA polymerase, and may refer to an enzyme that synthesizes primary transcript RNA from DNA. The polymerase referred to above is either 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).

[0040] In this specification, "target gene" may mean a gene that is to be expressed. For example, the target gene may mean a gene that codes for a target protein.

[0041] The target protein may be a protein (e.g., an enzyme) involved in the production of the target product.

[0042] In this specification, the "target product" may be one or more bioactive substances selected from the group consisting of, for example, amino acids, amino acid derivatives, nucleic acids, nucleic acid derivatives, vitamins, vitamin derivatives, sugars, sugar derivatives, fatty acids, fatty acid derivatives, proteins, and other metabolites.

[0043] The term “target product” means a biologically active substance that is ultimately produced or whose production is regulated (increased or decreased) using the polynucleotides, expression cassettes, expression vectors, and / or recombinant cells provided herein, for example, the target protein encoded by the target gene, and / or all biologically active substances produced in connection with the target protein. The biologically active substance means all substances that are produced or derived from an organism (e.g., a cell) or that have a predetermined function in vivo or within a cell, for example, amino acids, amino acid derivatives, nucleic acidsAcids (adenine, thymine, guanine, cytosine, uracil, etc.), nucleic acid derivatives, vitamins (vitamin A (retinol), B (B1 (thiamine), B2 (riboflavin), B3 (niacin), B5 (pantothenic acid), B6 ​​(pyridoxine), B7, B9 (folic acid), B12 (cobalamin), etc.), C (ascorbic acid), D (calciferul), E (tocopherol), K (phylloquinone), etc.), vitamin derivatives, proteins (proteins other than the aforementioned target proteins, e.g., hormones, growth factors, cytokines) Immunoglobulins (antibodies), antigen proteins, receptors, ligands, functional fragments of these (fragments possessing the desired function), fusion proteins formed by the fusion of two or more types, sugars (for example, monosaccharides (glyceraldehyde, dihydroxyacetone, erythrose, threose, erythrolose, arabinose, lyxose, ribose, xylose, ribulose, xylulose, deoxyribose, allulose, altrose, galactose, glucose, glucose, idose, mannose, talose, fructose, psyl (Cosose, sorbose, tagatose, fucose, fuculose, rhamnose, mannoheptulose, sedoheptulose, etc.), disaccharides (cellobiose, isomaltose, isomaltulose, lactose, lactulose, maltose, sucrose, trehalose, turanose, etc.), polysaccharides, etc.), sugar derivatives (sugar alcohols, galactosamine, glucosamine, sialic acid, N-acetylglucosamine, sulfoquinovose, ascorbic acid, mannitol, glucuronic acid, etc.), fatty acids (myristoleic acid, palmitoleic acid, saturates, etc.), It may be, but is not limited to, one or more selected from the group consisting of (e.g., pieonic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linole-elaidic acid, arachidonic acid, eicosapentaenoic acid (EPA), erucic acid, docosahexaenoic acid (DHA), etc.), fatty acid derivatives, organic acids (e.g., lactic acid, citric acid, oxalic acid, uric acid, butyric acid, stearic acid, propionic acid, etc.), their metabolites (e.g., polyhydroxyalkanoates (PHAs)), their precursors, and derivatives that maintain their biological activity. In one example, if the target protein is involved in the production of the target product, (1) the target protein may be one or more proteins selected from the group consisting of proteins involved in at least one process or step of the intracellular production pathway (e.g., biosynthesis, metabolism, bioconversion), intracellular transport, and / or extracellular efflux pathway of the target product, such as synthases, degrading enzymes, phosphorylating enzymes, carboxylases (e.g., pyruvate carboxylase), reductases, oxidases, decarboxylases, dehydrogenases, dehydrating enzymes, transferases (e.g., transferase), isomerases (epimerase), etc., intermediates, transport proteins, membrane proteins (channels, etc.), etc., but is not limited thereto. (2) The target gene may be a gene that codes for the target protein described in (1) above.

[0044] The amino acids among the target products may be either proteinogenic amino acids or non-proteinogenic amino acids.

[0045] The protein-producing amino acid may be one or more selected from the group consisting of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, selenocysteine, pyrrolicine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.

[0046] The L-amino acid among the target products is not limited in type. In other words, all L-amino acids that can be produced from microorganisms may be included without restriction, and intermediates of L-amino acids may also be included. The L-amino acid may be one or more selected from the group consisting of L-arginine, L-histidine, L-lysine, L-aspartic acid, L-glutamic acid, L-serine, L-threonine, L-asparagine, L-glutamine, L-tyrosine, L-alanine, L-isoleucine, L-leucine, L-valine, L-phenylalanine, L-methionine, L-tryptophan, L-glycine, L-proline, and L-cysteine, and may, but is not limited to, L-lysine, L-threonine, L-isoleucine, L-leucine, L-valine, L-arginine, or L-glutamic acid. The intermediate of the L-amino acid may, but is not limited to, O-acetylhomoserine.

[0047] The aforementioned non-proteinogenic amino acids are β-alanine, γ-aminobutyric acid (GABA), δ-aminoolevulinic acid, 4-aminobenzoic acid, α-aminoisobutyric acid, dehydroalanine, cystathionine, lanthionine, djenkolic acid, diaminopimelic acid, norvaline, norleucine, alloisoleucine, tert-leucine, α-amino-n-heptanoic acid, and pipecolic acid. It may be one or more selected from the group consisting of (1) acid, α,β-diaminopropionic acid, α,γ-diaminobutyric acid, ornithine, allothreonine, homocysteine, homoserine (or isothreonine), O-acetylhomoserine, etc.

[0048] The aforementioned amino acid may be a D-amino acid or an L-amino acid.

[0049] In one specific example, the target product may be one or more selected from the group consisting of threonine, O-acetylhomoserine, and valine.

[0050] One example of the polynucleotide may, within a cell, regulate (e.g., increase or decrease) the expression of a target gene operably linked to it, the production and / or activity of a target protein encoded by the target gene, and / or the production and / or activity of biologically active substances in which the target protein is involved, compared to a conventional promoter or an in-cellular promoter.

[0051] The nucleic acid sequence of the aforementioned polynucleotide can be modified by conventional mutagenesis methods, such as direct evolution and site-directed mutagenesis. In other words, the polynucleotide may contain or consist of a nucleic acid sequence having 60% or more, 65% or more, 70% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 90.5% or more, 91% or more, 91.5% or more, 92% or more, 92.5% or more, 93% or more, 93.5% or more, 94% or more, 94.5% or more, 95% or more, 95.5% or more, 96% or more, 96.5% or more, 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, 99.5% or more or 99.9% or more homology or identity with respect to the nucleic acid sequence of SEQ ID NO: 1. If a polynucleotide has a sequence homologous to the aforementioned nucleic acid sequence and is substantially identical to or corresponding to the nucleic acid sequence of Sequence ID No. 1 in terms of biological activity, then a polynucleotide having a base sequence in which a portion of the nucleic acid sequence of Sequence ID No. 1 is deleted, modified, substituted, or added may also be included within the scope of this application.

[0052] In this specification, the term "homology" means the degree to which a given nucleic acid sequence or amino acid sequence matches, and can be expressed as a percentage (%). For example, homology can be determined by aligning sequence information and directly aligning the sequence information between two polynucleotide molecules or two polypeptide molecules, such as parameters like score, identity, and similarity, using readily available computer programs. Such computer programs may include BLAST (NCBI), CLC Main Workbench (CLC bio), and MegAlign™ (DNASTAR Inc).

[0053] In one specific example, a polynucleotide comprising a particular nucleic acid sequence provided herein can be interpreted as comprising not only the particular nucleic acid sequence or a substantially equivalent nucleic acid sequence, but also a polynucleotide fragment comprising a nucleic acid sequence complementary to the particular nucleic acid sequence. Specifically, the complementary polynucleotide can be hybridized at a Tm value that can be appropriately adjusted by those skilled in the art depending on the purpose, for example, a Tm value of 55°C, 60°C, 63°C, or 65°C, and analyzed under the conditions described below: such conditions are specifically described in known literature. For example, examples include, but are not limited to, conditions in which genes with high complementarity of 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98% or more, 99.5% or more, or 99.9% or more hybridize with each other, and genes with lower complementarity do not hybridize with each other. Alternatively, conditions in which washing is performed once, specifically two to three times, at salt concentrations and temperatures equivalent to the usual Southern hybridization washing conditions of 60°C, 1xSSC (saline-sodium citrate buffer), and 0.1% (w / v)SDS (Sodium Dodecyl Sulfate); 60°C, 0.1xSSC, and 0.1%SDS; or 68°C, 0.1xSSC, and 0.1%SDS. Hybridization requires that the two nucleotides have complementary sequences, or, depending on the strictness of hybridization, mismatches between bases may be tolerated. The term “complementary” can be used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. The appropriate strictness for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, which is well known in the relevant art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0054] In one specific example, the polynucleotide may contain the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40, and / or consist of or contain the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40 in a proportion of 60% or more, 65% or more, 70% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more. The above may mean a polynucleotide comprising nucleic acid sequences having homology or identity of 90% or more, 90.5% or more, 91% or more, 91.5% or more, 92% or more, 92.5% or more, 93% or more, 93.5% or more, 94% or more, 94.5% or more, 95% or more, 95.5% or more, 96% or more, 96.5% or more, 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, 99.5% or more, or 99.9% or more, and maintaining the original function and / or intended function of the polynucleotide.

[0055] In this specification, the expression "polynucleotide consisting of the nucleic acid sequence of sequence number ~" does not exclude cases such as the addition and / or deletion and / or mutation of nucleotides that may occur during the process of linking to the target gene, such as the use of restriction enzymes, when a polynucleotide, as a specific example, is used as a promoter linked to the target gene.

[0056] In this specification, when a polynucleotide or polypeptide "contains a specific nucleic acid sequence or amino acid sequence," it means that the polynucleotide or polypeptide consists of, or essentially contains, the specific nucleic acid sequence or amino acid sequence, and can be interpreted as including (or not excluding) a "substantially equivalent sequence" to the specific nucleic acid sequence or amino acid sequence, to the extent that mutations (deletions, substitutions, alterations, and / or additions) are made to the extent that the original function and / or intended function of the polynucleotide or polypeptide is maintained. For example, nucleic acid sequences or amino acid sequences provided herein may include those that have been altered by conventional mutagenesis methods, such as direct evolution and / or site-directed mutagenesis, to the extent that their original function or intended function is maintained. In one specific example, a polynucleotide or polypeptide "contains a specific nucleic acid sequence or amino acid sequence" means that the polynucleotide or polypeptide (i) consists of or contains the specific nucleic acid sequence or amino acid sequence as an essential component, or (ii) contains the specific nucleic acid sequence or amino acid sequence in amounts of 60% or more, 65% or more, 70% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more. This may mean that the amino acid sequence consists of, or essentially contains, an amino acid sequence having homology or identity of % or more, 89% or more, 90% or more, 90.5% or more, 91% or more, 91.5% or more, 92% or more, 92.5% or more, 93% or more, 93.5% or more, 94% or more, 94.5% or more, 95% or more, 95.5% or more, 96% or more, 96.5% or more, 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, 99.5% or more, or 99.9% or more, and maintains the original function and / or intended function.

[0057] To give one specific example, the intrinsic and / or intended function of the polynucleotide may be that of a promoter. The statement that the polynucleotide functions as a promoter and contains the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40 may mean that when the polynucleotide is used operably ligated to a target gene as a promoter, it does not preclude mutations such as the addition, and / or deletion, and / or substitution of nucleotides that may occur during the ligation process to the target gene, such as the use of restriction enzymes, from being induced (introduced) into the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40. Furthermore, a polynucleotide containing the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40 and having promoter function may, without limitation, include all polynucleotides that have promoter activity when hybridized under severe conditions with all or part of the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40 or a complementary sequence.

[0058] The aforementioned polynucleotide may be operably linked to a gene encoding a target protein.

[0059] The aforementioned target protein may be one that is involved in the production of the target product, as explained earlier, and examples of such proteins are as follows.

[0060] In one example, the protein involved in the production of arginine among the aforementioned amino acids may be one or more proteins selected from among the proteins involved in arginine biosynthesis (arginine biosynthesis system proteins). For example, the proteins involved in arginine biosynthesis may be, but are not limited to, N-acetylglutamate synthase (argA), N-acetylglutamate kinase (argB), N-acetylglutamyl phosphate reductase (argC), acetylornithine transaminase (argD), acetylornithine deacetylase (argE), ornithine carbamoyltransferase (argF, argI), argininosuccinate synthase (argG), argininosuccinate degrading enzyme (argH), ornithine acetyltransferase (argJ), or carbamoyl phosphate synthase (carAB).

[0061] In one example, the protein involved in the production of histidine among the aforementioned amino acids may be one or more proteins selected from among the proteins involved in histidine biosynthesis (histidine biosynthesis system proteins). For example, the proteins involved in histidine biosynthesis may be, but are not limited to, ATP phosphoribosyltransferase (hisG), phosphoribosyl-AMP cyclohydrolase (hisI), phosphoribosyl-ATP pyrophosphohydrolase (hisI), phosphoribosylpormimino 5-aminoimidazole carboxamidolybotide isomerase (hisA), amide transferase (hisH), histidinol phosphate aminotransferase (hisC), histidinol phosphatase (hisB), and histidinol dehydrogenase (hisD).

[0062] In one example, the protein involved in the production of lysine among the aforementioned amino acids may be one or more proteins selected from among the proteins involved in lysine biosynthesis (lysine biosynthesis system proteins). For example, the proteins involved in lysine biosynthesis include dihydrodipicolinate synthase (dapA), aspartokinase III (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh), phosphoenolpyruvate carboxylase (ppc), aspartate semialdehyde dehydrogenase (asd), aspartate transaminase (aspC), diaminopimelate epimerase (dapF), and tetrahydrodipicolinate succinylase (tetrahydrodipicolinate It may be one or more selected from the group consisting of succinylase (dapD), succinyl-diaminopimelate deacylase (dapE), and aspartase (aspA), but is not limited to these.

[0063] In one example, the protein involved in the production of aspartic acid among the aforementioned amino acids may be one or more proteins selected from among the proteins involved in aspartic acid biosynthesis (aspartic acid biosynthesis system proteins). For example, the protein involved in aspartic acid biosynthesis may be, but is not limited to, aspartate aminotransferase.

[0064] In one example, the protein involved in the production of glutamic acid among the aforementioned amino acids may be one or more proteins selected from among the proteins involved in glutamic acid biosynthesis (glutamic acid biosynthesis proteins). For example, the proteins involved in glutamic acid production include glutamate dehydrogenase (gdhA), glutamine synthase (glnA), glutamate synthase (gltBD), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), methylcitrate synthase (prpC), phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase (pyc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), and It may be one or more enzymes selected from the group consisting of nolase (eno), phosphoglyceromutase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde 3-phosphate dehydrogenase (gapA), triose phosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp), phosphofructokinase (pfkA, pfkB), glucose phosphate isomerase (pgi), 6-phosphogluconate dehydratase (edd), 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), transhydrogenase, etc., but is not limited to these.

[0065] In one example, the protein involved in the production of serine among the aforementioned amino acids may be one or more proteins selected from among the proteins involved in serine biosynthesis (serine biosynthesis proteins). For example, the protein involved in serine biosynthesis may be one or more proteins selected from the group consisting of 3-phosphoglycerate dehydrogenase (serA), phosphoserine transaminase (serC), phosphoserine phosphatase (serB), etc., but is not limited to these.

[0066] In one example, the protein involved in the production of threonine among the aforementioned amino acids may be one or more proteins selected from among the proteins involved in threonine biosynthesis (threonine biosynthesis system proteins). For example, the proteins involved in threonine biosynthesis may be, but are not limited to, aspart kinase III (lysC), aspartate semialdehyde dehydrogenase (asd), aspart kinase I (thrA), homoserine kinase (thrB), threonine synthase (thrC), and aspartate aminotransferase.

[0067] In one example, the protein involved in the production of asparagine among the aforementioned amino acids may be one or more proteins selected from among the proteins involved in asparagine biosynthesis (asparagine biosynthesis system proteins). For example, the protein involved in asparagine biosynthesis may be, but is not limited to, an aminotransferase or an asparagine synthetase.

[0068] In one example, the protein involved in the production of glutamine among the aforementioned amino acids may be one or more proteins selected from among the proteins involved in glutamine biosynthesis (glutamine biosynthesis proteins). For example, the protein involved in glutamine biosynthesis may be one or more proteins selected from the group consisting of glutamate dehydrogenase (gdhA), glutamate setase (glnA), etc., but is not limited to these.

[0069] In one example, the protein involved in the production of cysteine ​​among the aforementioned amino acids may be one or more proteins selected from among the proteins involved in cysteine ​​biosynthesis (cysteine ​​biosynthesis proteins). For example, the protein involved in cysteine ​​biosynthesis may be one or more proteins selected from the group consisting of serine acetyltransferase (cysE), 3-phosphoglycerate dehydrogenase (serA), etc., but is not limited to these.

[0070] In one example, the protein involved in the production of glycine among the aforementioned amino acids may be one or more proteins selected from among the proteins involved in glycine biosynthesis (glycine biosynthesis system proteins). For example, the protein involved in the biosynthesis of glycine may be, but is not limited to, alanine-glyoxylate transaminase.

[0071] In one example, the protein involved in the production of proline among the aforementioned amino acids may be one or more proteins selected from among the proteins involved in proline biosynthesis (proline biosynthesis proteins). For example, the protein involved in the biosynthesis of proline may be one or more proteins selected from the group consisting of glutamate-5-kinase (proB), γ-glutamyl-phosphate reductase, pyrroline-5-carboxylate reductase (putA), etc., but is not limited to these. In another example, the protein involved in the production of alanine among the aforementioned amino acids may be one or more proteins selected from among the proteins involved in alanine biosynthesis (alanine biosynthesis proteins). For example, the protein involved in the biosynthesis of alanine may be alanine transaminase, etc., but is not limited to these.

[0072] In one example, the protein involved in the production of valine among the aforementioned amino acids may be one or more proteins selected from among the proteins involved in valine biosynthesis (valine biosynthesis system proteins). For example, the protein involved in valine biosynthesis may be, but is not limited to, acetohydroxy acid isomeroreductase (IlvC), dihydroxy-acid dehydratase (IlvD), or branched-chain amino acid aminotransferase (IlvE).

[0073] In one example, the protein involved in the production of isoleucine among the aforementioned amino acids may be one or more proteins selected from among the proteins involved in isoleucine biosynthesis (isoleucine biosynthesis system proteins). For example, the proteins involved in the biosynthesis of isoleucine may be, but are not limited to, acetohydroxy acid synthetase (AHAS), acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, valine aminotransferase, etc.

[0074] In one example, the protein involved in the production of leucine among the aforementioned amino acids may be one or more proteins selected from among the proteins involved in leucine biosynthesis (leucine biosynthesis system proteins). For example, the proteins involved in the biosynthesis of leucine may be, but are not limited to, acetolactate synthase, acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, α-isopropylmalic acid synthase, α-isopropylmalate isomerase, or leucine aminotransferase.

[0075] In one example, the protein involved in methionine production among the aforementioned amino acids may be one or more proteins selected from among the proteins involved in methionine biosynthesis (methionine biosynthesis system proteins). For example, the proteins involved in methionine biosynthesis may be, but are not limited to, aspartic acid kinase, aspartic acid-semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine O-succinyltransferase, cystathionine γ-generating enzyme, cystathionine β-lyase, or methionine synthase.

[0076] In one example, the protein involved in the production of phenylalanine among the aforementioned amino acids may be one or more proteins selected from among the proteins involved in phenylalanine biosynthesis (phenylalanine biosynthesis system proteins). For example, the protein involved in phenylalanine biosynthesis may be, but is not limited to, chorismate mutase, prephenate aminotransferase, or aromatate dehydratase.

[0077] In one example, the protein involved in the production of tyrosine among the aforementioned amino acids may be one or more proteins selected from among the proteins involved in tyrosine biosynthesis (tyrosine biosynthesis system proteins). For example, the protein involved in tyrosine biosynthesis may be chorismate mutase, prephenate aminotransferase, or aromatate dehydrogenase, but is not limited to these.

[0078] For example, the protein involved in the production of tryptophan among the aforementioned amino acids may be one or more proteins selected from among the proteins involved in tryptophan biosynthesis (tryptophan biosynthesis proteins). For example, the proteins involved in tryptophan biosynthesis may be, but are not limited to, anthranilate synthase, anthranilate phosphoribosyl transferase, anthranilate isomerase, imidazole glycerol phosphate synthase, or tryptophan synthase.

[0079] In one example, the protein involved in the production of O-acetyl homoserine among the aforementioned amino acids may be one or more proteins selected from among those involved in O-acetyl homoserine biosynthesis. For example, the protein involved in the biosynthesis of O-acetyl homoserine may be, but is not limited to, homoserine O-acetyltransferase.

[0080] For example, the protein involved in the production of β-alanine among the aforementioned amino acids may be one or more proteins selected from among those involved in β-alanine biosynthesis. For example, the protein involved in β-alanine biosynthesis may be, but is not limited to, propionate CoA ligase, medium-chain acyl-CoA dehydrogenase, 3-hydroxypropionyl-CoA dehydratase, 3-hydroxypropionyl-CoA hydrolase, 3-hydroxypropionate dehydrogenase, or beta-alanine-pyruvate transaminase.

[0081] The genes involved in the production of the nucleic acids may be one or more genes selected from the nucleic acid biosynthesis system genes. For example, amidophosphoribosyltransferase (purF), PRA-glycine ligase (purD), phosphoribosylaminoimidazolesuccinocarboxamide synthase (purC), bifunctional AICAR formyltransferase / IMP cyclohydrolase (purH), adenylosuccinate synthase (purA), adenylosuccinate lyase (purB), phosphoribosylaminoimidazole mutase (purE), phosphoribosylaminoimidazole carboxylase (purH) It may be one or more selected from the group consisting of carboxylase, purK, etc., but is not limited to this.

[0082] In one specific example, the target gene may be one or more selected from the group consisting of the pyc gene, the metX gene, and the ilvE gene.

[0083] In one specific example, the pyc gene encodes pyruvate carboxylase, and in one specific example, the polynucleotide and the pyc gene are operably linked and can be used for the production of L-threonine. The pyc gene may be a gene derived from Corynebacterium glutamicum. In one example, the pyc gene can be represented by Ncgl0659 and may include the nucleic acid sequence of SEQ ID NO: 21.

[0084] In one specific example, the metX gene encodes homoserine O-acetyltransferase, and in one specific example, the polynucleotide and the metX gene can be operably linked and used for the production of O-acetylhomoserine. The metX gene may be a gene derived from Corynebacterium glutamicum. In one example, the metX gene can be represented by NCgl0624 and may include the nucleic acid sequence of Sequence ID No. 28.

[0085] In one specific example, the ilvE gene encodes a branched-chain amino acid aminotransferase, and in one specific example, the polynucleotide and the ilvE gene can be functionally linked and used for valine production. The ilvE gene may be derived from Corynebacterium glutamicum. In one example, the ilvE gene can be represented by Ncgl2123 and may include the nucleic acid sequence of Sequence ID No. 32.

[0086] Another embodiment provides an expression cassette containing the polynucleotide and a gene encoding the target protein (hereinafter referred to as the target gene).

[0087] The target protein may be a protein (e.g., an enzyme) involved in the production of the target product.

[0088] The target product may be one or more selected from the group consisting of amino acids, amino acid derivatives, nucleic acids, nucleic acid derivatives, vitamins, vitamin derivatives, sugars, sugar derivatives, fatty acids, fatty acid derivatives, proteins, and other metabolites.

[0089] The expression cassette may contain the polynucleotide as a promoter.

[0090] In this specification, the term "expression cassette" may mean a polynucleotide fragment, the smallest unit expressible in a host cell, that comprises at least the coding gene and expression regulatory sequence (e.g., promoter) of the target protein.

[0091] The polynucleotide may be contained in the 5' or 3' terminal region of the gene encoding the target protein.

[0092] The polynucleotide may be operably linked to the gene encoding the target protein.

[0093] The target gene, target protein, and target product are as described above.

[0094] Another embodiment provides a vector comprising the polynucleotide or the expression cassette.

[0095] The aforementioned vector may be a recombinant vector (expression vector).

[0096] The vector may contain the polynucleotide as a promoter.

[0097] In one specific example, the vector may include a polynucleotide having promoter activity and a gene encoding a target protein operably linked to the polynucleotide (hereinafter referred to as the target gene).

[0098] In one specific example, the target protein may be a single protein (a monoprotein) or a fusion protein formed by the fusion of two or more proteins. If the target protein is a fusion protein containing two or more proteins, the target protein coding gene may be a fusion gene containing genes that code for each of the two or more proteins.

[0099] In one specific example, if the recombinant vector is a fusion gene containing two or more genes that each encode a protein, it can be designed so that all the genes included in the fusion gene are under the control of a single polynucleotide (promoter), or one or more of them are under the control of a separate polynucleotide. For example, the vector may contain a polynucleotide having promoter activity and one or more genes (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) operably linked to the polynucleotide. If it contains two or more genes, it may contain one polynucleotide having promoter activity (i.e., the two or more genes are under the control of a single promoter), or it may contain two or more (in this case, the polynucleotides may be included in a number less than or equal to the number of genes, such that one or more of the two or more genes are under the control of a separate promoter).

[0100] In this specification, the term “vector” refers collectively to artificial DNA molecules that contain genetic material so that a target gene can be expressed in a suitable host cell, and may mean a DNA structure that includes a suitable gene expression regulatory sequence and, optionally, a nucleic acid sequence of the target gene operably linked thereto. The “gene expression regulatory sequence” means elements that perform various regulatory functions in gene expression, and may include, for example, a polynucleotide having promoter activity provided herein, and may mean a nucleic acid sequence capable of expressing a target gene operably linked thereto. Specifically, the gene expression regulatory sequence may include, but is not limited to, a promoter for carrying out gene transcription, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA-ribosome binding site, and / or a nucleic acid sequence that regulates the termination of transcription and decoding. Furthermore, it may include, but is not limited to, a promoter and a ribosome binding site as regulatory sequences suitable for prokaryotes. The polynucleotide having promoter activity of this application can be used by a person skilled in the art to constitute the gene expression regulatory sequences described above as needed.

[0101] In this specification, “operatively linked” means that the polynucleotide having promoter activity is functionally linked to the nucleotide sequence of the target gene so as to initiate and / or mediate the transcription of the target gene. The operative linking may be performed using known genetic engineering techniques in the art, such as site-directed DNA cleavage and / or linking techniques, which may be performed using conventional cleavage enzymes and / or linking enzymes, but are not limited thereto.

[0102] The vector is not particularly limited as long as it is transformable into a host cell and / or expressible in a host cell. Examples of the vector include plasmids, cosmids, viruses, and / or bacteriophages in their native or recombinant state. For example, one or more selected from the group consisting of pWE15, M13, λLB3, λBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, Charon21A, etc., can be used as a phage vector or cosmid vector, and one or more selected from the group consisting of pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc., can be used as a plasmid vector.

[0103] In one example, an endogenous promoter within the chromosome of a host cell can be replaced with a polynucleotide having promoter activity provided herein. In this case, the vector may be a chromosome insertion vector for host cells containing the polynucleotide having promoter activity, and may be, but is not limited to, one or more selected from the group consisting of, for example, pECCG117, pDZ, pACYC177, pACYC184, pCL, pUC19, pBR322, pMW118, pCC1BAC, pCES208, pXMJ19 vectors. Furthermore, the insertion of the polynucleotide into the chromosome can be carried out by any method known in the art, such as homologous recombination or genome editing using a target-specific endonuclease (e.g., RNA-guided endonucleases such as Cas9 and Cpf1).

[0104] In one example, if the vector or the polynucleotides and / or target gene contained therein are to be inserted into a chromosome, the vector may further include a selection marker for confirming the presence or absence of a chromosomal insertion. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of a polynucleotide insertion, and may be a marker that confers a selectable phenotype, such as drug (e.g., antibiotic) resistance, nutritional requirements, resistance to cytotoxic agents, or expression of a surface protein. In an environment treated with a selective agent (drug, cytotoxic agent, etc.), only cells expressing the selection marker will survive or exhibit other phenotypes, thus allowing for the selection of transformed cells.

[0105] Another example provides recombinant cells containing the polynucleotide or the recombinant vector in a host cell. In this specification, the term “recombinant cell” may mean a transformant into which the polynucleotide and / or the recombinant vector has been introduced. The term “transformation” means introducing the polynucleotide or the recombinant vector having promoter activity into a suitable host cell so that a target gene (which may be contained in the recombinant vector or be an endogenous gene in the host cell) that is regulated by the polynucleotide can be expressed in the host cell.

[0106] The host cell can be any cell in which the introduced polynucleotide can act as a promoter, thereby enabling (or increasing) the expression of a target gene (a gene contained in the recombinant vector or a gene in the host cell) that is regulated (operably linked) by the polynucleotide. For example, the host cell or recombinant cell may be one or more selected from the group consisting of various microorganisms, plant cells, animal cells, etc. Specifically, it may be a microorganism such as the genera Escherichia or Corynebacterium, and more specifically, it may be Corynebacterium glutamicum, but is not limited thereto.

[0107] The transformation method includes all methods for introducing the polynucleotide or the recombinant vector into host cells, and can be performed by selecting a suitable standard technique as known in the relevant field, depending on the host cell. For example, the transformation can be performed by electroporation, lipofection, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method, but is not limited thereto.

[0108] In one specific example, if the vector or the polynucleotides and / or target gene contained therein are to be inserted into a chromosome, the vector may further include a selection marker for confirming the presence or absence of chromosomal insertion. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of polynucleotide insertion, and can be a marker that confers a selectable phenotype such as drug (e.g., antibiotic) resistance, nutritional requirements, resistance to cytotoxic agents, or expression of surface proteins. Transformed cells can be selected because only cells expressing the selection marker survive or exhibit other phenotypes in an environment treated with a selective agent (drug, cytotoxic agent, etc.).

[0109] Another embodiment provides a microorganism comprising one or more selected from the group consisting of the polynucleotide, the expression cassette, and the vector.

[0110] In one example, the microorganism may include the polynucleotide and the expression cassette.

[0111] The microorganisms (or strains, recombinant cells) of this application may be microorganisms that have the ability to produce the target product, or microorganisms whose ability to produce the target product has been improved (or increased).

[0112] The microorganisms of this application may be, but are not limited to, microorganisms that do not naturally produce the target product, or microorganisms that have the ability to produce the target product but have been given or improved the ability to produce the target product by introducing one or more selected from the group consisting of the polynucleotide, the expression cassette, and the vector. The microorganisms that have been given or improved the ability to produce the target product may be microorganisms into which one or more selected from the group consisting of the polynucleotide, the expression cassette, and the vector have been introduced.

[0113] To say that the microorganism has improved ability to produce the target product or possesses the ability to produce the target product means that the microorganism has improved ability to produce the target product compared to the non-myxoid microorganism, pre-recombination cells, parent strain, and / or wild-type strain, or that it has been conferred the ability to produce the target product, unlike the non-myxoid microorganism, pre-recombination cells, parent strain, and / or wild-type strain that does not possess the ability to produce the target product.

[0114] Microorganisms into which one or more selected from the group consisting of the polynucleotide, the expression cassette, and the vector have been introduced may have improved ability to produce the target product compared to the microorganism before introduction or before enhancement, i.e., the same species of non-myxoid microorganism. In this application, "non-myxoid microorganism" may mean a wild-type strain or a naturally occurring strain itself, or a strain before its characteristics are altered by genetic mutation due to natural or artificial factors, rather than excluding strains containing mutations that can occur naturally in microorganisms. For example, the non-myxoid microorganism may mean a strain into which one or more selected from the group consisting of the polynucleotide, the expression cassette, and the vector have not been introduced, or a strain before they have been introduced. The "non-myxoid microorganism" can be used interchangeably with "pre-myxoid strain," "pre-myxoid microorganism," "non-mutant strain," "non-myxoid strain," "non-mutant microorganism," or "reference microorganism." The one or more selected from the group consisting of the polynucleotide, the expression cassette, and the vector are as described above.

[0115] In this specification, “recombinant cell” may mean a transformed organism into which the polynucleotide and / or the recombinant vector has been introduced.

[0116] In this specification, "transformation" means introducing the polynucleotide having promoter activity or the recombinant vector into a suitable host cell so that a target gene (which may be contained in the recombinant vector or be an endogenous gene of the host cell) that is regulated by the polynucleotide can be expressed in the host cell.

[0117] The host cells mentioned above may include, without limitation, any cells in which the introduced polynucleotide can act as a promoter, thereby enabling (or increasing) the expression of a target gene (a gene contained in the recombinant vector or a host cell-derived gene) that is regulated (operably linked) by the polynucleotide.

[0118] In one specific example, the host cell or recombinant cell may be one or more species selected from the group consisting of various microorganisms, plant cells, animal cells, etc., and may be, for example, a microorganism of the genera Escherichia, Corynebacterium, or Bacillus, and more specifically, Corynebacterium glutamicum, Escherichia coli, or Bacillus subtilis, but is not limited thereto.

[0119] In one example, the microorganism may be a microorganism of the genus Corynebacterium, Escherichia, or Bacillus.

[0120] The aforementioned Corynebacterium species include Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, and Corynebacterium ammoniagenes. Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium alkanolyticum, Corynebacterium lilium, Corynebacterium melassecola, Corynebacterium samoaminogenes thermoaminogenes), Corynebacterium herculis and Corynebacterium flavecensIt may be one or more species selected from the group consisting of (flavescens), but is not limited to this.

[0121] The aforementioned Bacillus microorganisms may be one or more species selected from the group consisting of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus methylotrophicus, Bacillus licheniformis, Bacillus velezensis, Bacillus sonorensis, and Bacillus valismortis, but are not limited to these.

[0122] The aforementioned Escherichia microorganism may be, but is not limited to, Escherichia coli. As an example, a microorganism into which one or more selected from the group consisting of the polynucleotide, the expression cassette, and the vector has been introduced is newly conferred the ability to produce the target product compared to the pre-mutation parent strain, the non-mutation microorganism, the parent strain, or the wild-type microorganism, or approximately 2% or more, approximately 3% or more, approximately 4% or more, approximately 5% or more, approximately 6% or more, approximately 7% or more, approximately 8% or more, approximately 9% or more, approximately 10% or more, approximately 11% or more, approximately 12% or more, approximately 13% or more, approximately 14% or more, approximately 15% or more, approximately 16% or more, approximately 20% or more, approximately 25% or more. The above could be, but is not limited to, an increase of approximately 30% or more, approximately 40% or more, approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 80% or more, approximately 90% or more, approximately 100% or more, approximately 150% or more, approximately 200% or more, approximately 250% or more, approximately 300% or more, approximately 400% or more, approximately 500% or more, approximately 600% or more, approximately 700% or more, approximately 800% or more, approximately 900% or more, approximately 1,000% or more, approximately 1,500% or more, approximately 2,000% or more, approximately 2,500% or more, or approximately 3,000% or more.

[0123] As another example, the microorganisms with improved target product production ability showed a target product production ability of approximately 1.02 times or more, approximately 1.03 times or more, approximately 1.04 times or more, approximately 1.05 times or more, approximately 1.06 times or more, approximately 1.07 times or more, approximately 1.08 times or more, approximately 1.09 times or more, approximately 1.1 times or more, approximately 1.11 times or more, approximately 1.12 times or more, approximately 1.13 times or more, approximately 1.14 times or more, approximately 1.15 times or more, approximately 1.16 times or more, approximately 1.2 times or more, and approximately 1.3 times or more compared to the pre-mutation parent strain and non-myxogenic microorganisms. It may be more than double, approximately 1.4 times or more, approximately 1.5 times or more, approximately 1.6 times or more, approximately 1.7 times or more, approximately 1.8 times or more, approximately 1.9 times or more, approximately 2 times or more, approximately 2.5 times or more, approximately 3 times or more, approximately 4 times or more, approximately 5 times or more, approximately 6 times or more, approximately 7 times or more, approximately 8 times or more, approximately 9 times or more, approximately 10 times or more, approximately 15 times or more, approximately 20 times or more, approximately 25 times or more, or approximately 30 times or more (the upper limit is not specifically restricted and may be, for example, approximately 1,000 times or less), but is not limited to these.

[0124] As another example, microorganisms with improved target product production capacity, compared to the pre-mutation parent strain and non-myxoid microorganisms, showed target product production capacity of approximately 0.1 g / L or more, approximately 0.2 g / L or more, approximately 0.3 g / L or more, approximately 0.4 g / L or more, approximately 0.5 g / L or more, approximately 0.6 g / L or more, approximately 0.7 g / L or more, approximately 0.8 g / L or more, approximately 0.9 g / L or more, approximately 1 g / L or more, approximately 1.1 g / L or more, approximately 1.2 g / L or more, approximately 1.3 g / L or more, approximately 1.4 g / L or more, approximately 1.5 g / L or more, approximately 1.6 g / L or more, approximately 1.7 g / L or more, and approximately It may be 1.8g / L or more, approximately 1.9g / L or more, approximately 2.0g / L or more, approximately 2.5g / L or more, approximately 3g / L or more, approximately 3.5g / L or more, approximately 4g / L or more, approximately 4.5g / L or more, approximately 5g / L or more, approximately 5.5g / L or more, approximately 6g / L or more, approximately 7g / L or more, approximately 8g / L or more, approximately 9g / L or more, approximately 10g / L or more, approximately 15g / L or more, approximately 20g / L or more, approximately 25g / L or more, approximately 30g / L or more (the upper limit is not specifically restricted and may be, for example, approximately 100g / L or less), but it is not limited to these values.

[0125] The term "about" includes, but is not limited to, all ranges such as ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, and all numerical values ​​within a range equivalent to or similar to the numerical value following the phrase "about". The transformation method includes all methods for introducing the polynucleotide or the recombinant vector into host cells and can be performed by selecting a suitable standard technique as is publicly known in the relevant field, depending on the host cell. For example, the transformation may include, but is not limited to, electroporation, lipofection, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0126] Other embodiments provide compositions for producing a target product, comprising the microorganism, a culture medium in which the microorganism is cultured, or a combination thereof.

[0127] The composition may further contain any suitable excipients commonly used in compositions for producing the target product, such excipients may be, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents.

[0128] Another embodiment provides applications for using the microorganism, the culture medium in which the microorganism is cultured, or a combination thereof, in the production of a target product.

[0129] Another aspect provides an application for using the microorganism in the production of a composition for producing a target product.

[0130] Another embodiment provides a method for producing a target product, which includes the step of culturing the microorganism in a culture medium.

[0131] The method may further include a step of recovering the target product from the culture medium or microorganisms obtained by the culture.

[0132] The culture medium in which the microorganisms are cultured may or may not contain the microorganisms.

[0133] As described above, the microorganism may include one or more selected from the group consisting of a polynucleotide provided in this application, an expression cassette containing the polynucleotide and the target gene, and a vector containing the expression cassette.

[0134] In this specification, “culture” means growing cells under artificially controlled environmental conditions. The methods for producing the target products provided herein can be carried out by means appropriately selected from all methods known in the art. For example, such culture can be carried out in a continuous manner in a batch process, a fed batch, or a repeated fed batch process, but is not limited thereto. The culture medium used may be one that appropriately satisfies the growth requirements of the target cells.

[0135] In this application, "culture medium" means a substance mainly composed of nutrients necessary for culturing the microorganism, such as the Corynebacterium glutamicum strain, and supplies water, which is essential for survival and growth, as well as nutrients and growth factors. Specifically, the culture medium and other culture conditions used for culturing the microorganism in this application can be any culture medium commonly used for culturing microorganisms without any particular restrictions, but the microorganism in this application can be cultured under aerobic conditions in a conventional culture medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, while adjusting the temperature, pH, etc.

[0136] Specifically, for the culture medium for the microorganisms of this application, such as strains of the genus Corynebacterium, refer to the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D. Corynebacterium, USA, 1981)].

[0137] The sugar sources used in the culture or included in the culture medium may include, but are not limited to, one or more selected from the group consisting of sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. The nitrogen sources used in the culture or included in the culture medium may include, but are not limited to, one or more selected from the group consisting of organic nitrogen sources such as peptone, yeast extract, meat juice, malt extract, corn maceration, soybean meal, and urea, and inorganic nitrogen sources such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. The phosphorus sources used in the culture or included in the culture medium may include, but are not limited to, one or more selected from the group consisting of potassium salts of phosphoric acid such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and their corresponding sodium salts. In addition, the culture medium may contain metal salts such as magnesium sulfate or iron sulfate necessary for growth. Furthermore, the culture or medium may contain one or more selected essential growth substances such as amino acids and vitamins. The culture medium may also contain a suitable precursor of the target product as a raw material. The raw material can be added to the culture in a batch and / or continuous manner in a manner appropriate to the culture process.

[0138] During the cell culture, the pH of the culture can be adjusted using appropriate methods with basic compounds such as sodium hydroxide, potassium hydroxide, and ammonia, and / or acidic compounds such as phosphoric acid or sulfuric acid. Furthermore, during the culture, antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble formation. Oxygen or oxygen-containing gases (e.g., air) can be injected into the culture to maintain aerobic conditions. The temperature of the culture medium and / or culture can typically be 20°C to 45°C, or 25°C to 40°C. The culture time can be continued until the production of the target product reaches the desired amount, and can be, for example, about 10 to about 160 hours, but is not limited thereto.

[0139] The step of separating or recovering the target substance from the cultured microorganism or culture medium can be carried out using a suitable method known in the art, depending on the culture method. For example, methods such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), and / or chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion) can be used, but are not limited thereto. The culture medium means a medium in which recombinant cells are cultured.

[0140] In one specific example, the step of separating or recovering the target product can be performed by separating the supernatant obtained by removing biomass from the culture by slow centrifugation using ion exchange chromatography.

[0141] The method for producing the aforementioned target product may further include a step of purifying the target product.

[0142] The target product can be recovered from the culture (medium) by separation methods known to the art. Common methods for separating the target product include centrifugation, filtration, chromatography, and / or crystallization. For example, the culture can be separated by slow centrifugation to remove biomass, and the resulting supernatant can be subjected to ion-exchange chromatography to separate the target product. The recovery step may further include a purification step. [Effects of the Invention]

[0143] A polynucleotide, as a specific example, possesses promoter activity and, when introduced into a microorganism, can increase the expression and activity of genes operably linked to it. This can be usefully utilized to efficiently produce target products affected by the polynucleotide and the gene. [Modes for carrying out the invention]

[0144] The present invention will be described in more detail with reference to the following embodiments, but the scope of the rights is not intended to be limited to the following embodiments.

[0145] Example 1. Preparation of recombinant vectors and transformed strains containing improved promoters. Example 1-1. Preparation of a recombinant vector containing an improved O2 promoter sequence. A mutant promoter was created based on an existing O2 promoter known to exhibit potent activity (U.S. Patent No. 10273491).

[0146] Using the o2 promoter (hereinafter referred to as "Po2") consisting of the nucleotide sequence of Sequence ID No. 1 as a template, we induced random mutations using the Diversify PCR Random Mutagenesis Kit, as per the manufacturer's manual, to obtain two promoters with different sequences.

[0147] Specifically, of the two promoters we secured, the promoter consisting of a nucleic acid sequence in which the 21st nucleotide A is replaced with C in the nucleic acid sequence shown in SEQ ID NO: 1 (SEQ ID NO: 39) was named "Po2.1," and the promoter consisting of a nucleic acid sequence in which the 19th nucleotide G is replaced with A in the nucleic acid sequence shown in SEQ ID NO: 1 (SEQ ID NO: 40) was named "Po2.2."

[0148] Furthermore, we secured two types of promoters by adding a portion of the sequence to the 3' end of promoter SEQ ID NO: 1.

[0149] Specifically, the promoter consisting of a nucleic acid sequence (sequence number 41) in which the nucleotide "CAT" sequence is added to the 3' end of sequence number 1 was named "Po2.3," and the promoter consisting of a nucleic acid sequence (sequence number 42) in which the nucleotide "ACTAGT" sequence is added to the 3' end of the nucleic acid sequence of sequence number 1 was named "Po2.4."

[0150] The nucleic acid sequences of Po2, Po2.1, Po2.2, Po2.3, and Po2.4 are listed in Table 1 below, with substituted or added sequences indicated by an underline.

[0151] [Table 1]

[0152] Example 1-1-1. Preparation of pCES_Po2.1_gfp, pCES_Po2.2_gfp, pCES_Po2.3_gfp, and pCES_Po2.4_gfp vectors To prepare recombinant GFP expression vectors regulated by Po2.1, Po2.2, Po2.3, or Po2.4, PCR was performed using Po2.1 and Po2.2 as templates with primers of SEQ ID NOs. 2 and 3, respectively. PCR was also performed using Po2.3 as a template with primers of SEQ ID NOs. 2 and 4, and PCR was performed using Po2.4 as a template with primers of SEQ ID NOs. 2 and 5. After denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 1 minute was repeated 30 times, followed by polymerization at 72°C for 5 minutes to obtain PCR products for Po2.1, Po2.2, Po2.3, and Po2.4.

[0153] Furthermore, open reading frames (ORFs) of the GFP gene were obtained by PCR using the pGFPuv vector (clontech, USA) as a template, with primers for SEQ ID NOs. 6 and 7, SEQ ID NOs. 8 and 7, and SEQ ID NOs. 9 and 7. Each PCR was performed using the same method as described above.

[0154] Furthermore, using the E. coli-Corynebacterium shuttle vector pCES208 (J. Microbiol. Biotechnol., 18:639-647, 2008) as a template, PCR was performed using primers SEQ ID NO: 10 and SEQ ID NO: 11 to prepare vectors for introducing Po2.1, Po2.2, Po2.3, Po2.4 mutant promoter sequences and GFP expression sequences. PCR was performed by denaturing at 94°C for 5 minutes, followed by denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 5 minutes, and repeating this 30 times, followed by polymerization at 72°C for 5 minutes.

[0155] Subsequently, the Po2.1, Po2.2, Po2.3, and Po2.4 fragments, the ORF fragment of the GFP gene, and the pCES208 vector obtained by the PCR method described above were used to create recombinant vectors in which Po2.1, Po2.2, Po2.3, and Po2.4 were each linked to the ORF of the GFP gene, using an in-fusion enzyme (BD In-Fusion kit). These were named "pCES_Po2.1_gfp", "pCES_Po2.2_gfp", "pCES_Po2.3_gfp", and "pCES_Po2.4_gfp", respectively.

[0156] Example 1-1-2. Preparation of pHT_Po2.1_gfp and pHT_Po2.2_gfp vectors To prepare recombinant vectors for Bacillus expression containing Po2.1 or Po2.2, PCR was performed using Po2.1 and Po2.2 as templates, respectively, with primers SEQ ID NOs. 12 and 13. The PCR involved denaturation at 94°C for 5 minutes, followed by denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 1 minute, repeated 30 times, and then polymerization at 72°C for 1 minute to obtain PCR products for Po2.1 and Po2.2.

[0157] Furthermore, using the pGFPuv vector as a template, PCR was performed using primers SEQ ID NOs. 14 and 15 to obtain gene sections containing GFP ORFs. The PCR was performed using the same method as described above.

[0158] Furthermore, using the pHT43 vector (Mobitec GmbH) as a template, we prepared vectors for introducing the improved Po2.1 and Po2.2 promoter sequences by performing PCR using primers SEQ ID NO: 16 and SEQ ID NO: 17. The PCR procedure involved denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 5 minutes, repeated 30 times, and then polymerization at 72°C for 5 minutes.

[0159] Subsequently, the Po2.1 fragment, the Po2.2 fragment, and the ORF fragment of the GFP gene were ligated into the pHT43 vector obtained by the PCR method using an in-fusion enzyme to create recombinant vectors in which Po2.1 or Po2.2 was ligated to the ORF of the GFP gene, respectively. These were named "pHT_Po2.1_gfp" and "pHT_Po2.2_gfp," respectively.

[0160] Example 1-1-3. Preparation of pCES_Po2_gfp, pHT_Po2_gfp, and pHT_P43_gfp vectors containing a control group promoter. As a control group to confirm the activity of the improved Po2 promoter mutant, a recombinant vector in which the unimproved Po2 (US registered patent US10,273,491B2) and GFP were ligated was used. Using Po2 as a template, PCR was performed using primers of SEQ ID NO: 2 and SEQ ID NO: 3. In the same manner as in Example 1-1-1, the Po2 fragment and the ORF fragment of the GFP gene were operably ligated to the pCES208 vector using the BD In-Fusion kit to create a recombinant vector in which Po2 is ligated to GFP, which was named "pCES_Po2_gfp".

[0161] Furthermore, as a control group to confirm the activity of the improved Po2 promoter mutant in Bacillus subtilis, a recombinant vector in which the unimproved Po2 (US registered patent US10,273,491B2) and GFP were linked was used. Using Po2 as a template, PCR was performed using primers of SEQ ID NO: 12 and SEQ ID NO: 13. In the same manner as in Example 1-1-2, the Po2 fragment and the ORF fragment of the GFP gene were operably linked to the pHT43 vector using the BD In-Fusion kit to create a recombinant vector in which Po2 is linked to GFP, which was named "pHT_Po2_gfp".

[0162] To confirm the activity of the improved Po2 promoter mutant in Bacillus subtilis, a conventionally known strong promoter, P43 (Wang and Doi, Journal of Biological Chemistry, 1984), was used as a template for PCR using primers SEQ ID NO: 18 and SEQ ID NO: 19 to obtain P43. Additionally, pGFPuv vector was used as a template for PCR using primers SEQ ID NO: 20 and SEQ ID NO: 15 to obtain gene sections containing the ORF of the GFP gene. Subsequently, using the same method as described above, the P43 fragment and the GFP gene ORF fragment were ligated operably into the pHT43 vector using the BD In-Fusion Kit to create a recombinant vector in which P43 is ligated to GFP, which was named "pHT_P43_gfp".

[0163] The primers used in the above-mentioned sequence numbers 2-20 are listed in Table 2 below.

[0164] [Table 2]

[0165] Examples 1-2. Preparation of transformed bacterial strains The recombinant vectors pCES_Po2.1_gfp, pCES_Po2.2_gfp, pCES_Po2.3_gfp, and pCES_Po2.4_gfp prepared in Example 1-1, along with pCES_Po2_gfp containing the unimproved promoter Po2, were each transformed into Corynebacterium glutamicum ATCC13032 using the electroconvulsive method (Appl. Microbiol. Biothcenol (1999) 52:541-545). After transformation, Braine heart infusion (BHIS plate medium containing kanamycin 25 mg / L) was performed. Strains transformed with 37 g / l sorbitol, 91 g / l sorbitol, and 2% agar were selected and named "ATCC13032 / pCES_Po2.1_gfp", "ATCC13032 / pCES_Po2.2_gfp", "ATCC13032 / pCES_Po2.3_gfp", "ATCC13032 / pCES_Po2.4_gfp", and "ATCC13032 / pCES_Po2_gfp", respectively.

[0166] Furthermore, the recombinant vectors pCES_Po2.1_gfp, pCES_Po2.2_gfp, pCES_Po2.3_gfp, and pCES_Po2.4_gfp prepared in Example 1-1, along with pCES_Po2_gfp containing the unimproved promoter Po2, were used to transform Escherichia coli DH5α using the thermal shock method. The transformed strains were then selected on LB (Luria-Bertani) agar medium containing 25 mg / L kanamycin, and named "DH5α / pCES_Po2.1_gfp", "DH5α / pCES_Po2.2_gfp", "DH5α / pCES_Po2.3_gfp", "DH5α / pCES_Po2.4_gfp", and "DH5α / pCES_Po2_gfp", respectively.

[0167] Furthermore, the recombinant vectors pHT_Po2.1_gfp and pHT_Po2.2_gfp prepared in Example 1-1, along with pHT_Po2_gfp and pHT_P43_gfp, which contain the unimproved promoter Po2, were used to transform Bacillus subtilis ATCC23857 using the electropulse method (Eppendorf Protocol No. 4308 915 504). After transformation, strains transformed in a selective medium containing 50 mg / L of kanamycin were selected and named "ATCC23857 / pHT_Po2.1_gfp", "ATCC23857 / pHT_Po2.2_gfp", "ATCC23857 / pHT", "ATCC23857 / pHT_Po2_gfp", and "ATCC23857 / pHT_P43_gfp", respectively.

[0168] Examples 1-3. Confirmation of the activity of the improved promoter. Example 1-3-1. Confirmation of expression induction activity of Corynebacterium glutamicum for Po2.1, Po2.2, Po2.3, and Po2.4. To confirm the activity of the Po2 promoter mutant, the transformed strains of Corynebacterium glutamicum obtained in Examples 1-2, "ATCC13032 / pCES_Po2.1_gfp", "ATCC13032 / pCES_Po2.2_gfp", "ATCC13032 / pCES_Po2.3_gfp", "ATCC13032 / pCES_Po2.4_gfp", and "ATCC13032 / pCES_Po2_gfp", were cultured using the method described below, and their GFP activity was measured.

[0169] Specifically, 25 ml of seed medium (pH 7.2) was placed in a 250 ml corner baffle flask, and the transformed Corynebacterium glutamicum strains described above were inoculated in a 1 / 20 ratio (volume ratio). The flasks were then cultured at 30°C with shaking at 200 rp until the mid-stage of culture (OD600 = 10.0). The components of the seed medium are listed in Table 3 below. The bacterial cells were collected from the culture medium by centrifugation (5,000 rpm, 15 minutes), washed twice with 0.1% Tris.HCl (pH 8.0) buffer solution, and then suspended in the same buffer solution until the turbidity at 610 nm was approximately 160. After adding 1.25 g of glass bead per 1.5 ml of suspension, the bacterial cells were lysed using a bead beater for 6 minutes. The supernatant was collected by centrifugation (15,000 rpm, 20 minutes), and the protein concentration was quantified using the Bradford method (Bradford, MM 1976. Anal. Biochem. 72:248-254). The expression level of the GFP gene was measured by irradiating the same amount of bacterial extract with excitation light at 488 nm using a method such as Laure Gory's, and measuring the emitted light at 511 nm using an LS-50B spectrophotometer (Perkin-Elmer), as shown in Table 4 below.

[0170] [Table 3]

[0171] [Table 4]

[0172] As shown in Table 4 above, the four Po2 variants exhibited promoter activity in Corynebacterium glutamicum, and among them, the Po2.1 and Po2.2 promoters showed higher fluorescence intensity than ATCC13032 / pCES_Po2_gfp, which utilizes the existing potent promoter Po2.

[0173] Example 1-3-2. Confirmation of expression induction activity of Po2.1, Po2.2, Po2.3, and Po2.4 in Escherichia coli. To confirm the activity of the Po2 promoter variant in E. coli, the transformed strains obtained in Examples 1-2, "DH5α / pCES_Po2.1_gfp", "DH5α / pCES_Po2.2_gfp", "DH5α / pCES_Po2.3_gfp", "DH5α / pCES_Po2.4_gfp", and "DH5α / pCES_Po2_gfp", were cultured using the method described below, and their GFP activity was measured.

[0174] Specifically, 25 ml of LB medium containing kanamycin was inoculated into 250 ml corner baffle flasks with the transformed E. coli strains in a 1 / 20 ratio, and the cultures were incubated with shaking (200 rpm) at 37°C until the mid-stage (OD600 = 3.0). The cells were collected from the culture medium by centrifugation (5,000 rpm, 15 min), washed twice with 0.1% Tris.HCl (pH 8.0) buffer solution, suspended in the same buffer solution, and then the cells were disrupted by sonication. The supernatant was collected by centrifugation (15,000 rpm, 20 min), and the protein concentration was quantified by the Bradford method. The same amount of cell extract was irradiated with excitation light at 488 nm using a method such as Laure Gory's, and the GFP gene expression level was measured by measuring the emitted light at 511 nm using an LS-50B spectrophotometer (Perkin-Elmer instrument), as shown in Table 5 below.

[0175] [Table 5]

[0176] As shown in Table 5 above, the four Po2 variants exhibited promoter activity in E. coli, and among them, the Po2.1 and Po2.2 promoters showed higher fluorescence sensitivity than the conventionally known strong promoter, Po2.

[0177] Example 1-3-1. Confirmation of Po2.1 and Po2.2 expression induction activity in Bacillus subtilis. To confirm the activity of the o2.1 and o2.2 promoters in Bacillus subtilis, the transformed strains obtained in Examples 1-2, Bacillus subtilis ATCC23857 / pHT_P43_gfp, ATCC23857 / pHT_Po2.1_gfp, ATCC23857 / pHT_Po2.2_gfp, and ATCC23857 / pHT_Po2_gfp, were cultured using the methods described below, and their GFP activity was measured.

[0178] Specifically, 25 ml of seed medium was placed in a 250 ml corner baffle flask, and the transformed Bacillus subtilis strains were inoculated in a 1 / 20 ratio. The flasks were then cultured with shaking (200 rpm) at 37°C until the mid-stage (OD600 = 10.0). The components of the seed medium are listed in Table 6 below. The cells were collected from the culture medium by centrifugation (5,000 rpm, 15 minutes), washed twice with 0.1% Tris.HCl (pH 8.0) buffer solution, and then suspended in the same buffer solution until the turbidity at 610 nm was approximately 160. After adding 1.25 g of glass bead per 1.5 ml of suspension, the bacterial cells were lysed using a bead beater for 6 minutes. The supernatant was collected by centrifugation (15,000 rpm, 20 minutes), and the protein concentration was quantified using the Bradford method (Bradford, MM 1976. Anal. Biochem. 72:248-254). The expression level of the GFP gene was measured by irradiating the same amount of bacterial extract with excitation light at 488 nm using a method such as that of Laure Gory, and measuring the emitted light at 511 nm using an LS-50B spectrophotometer (Perkin-Elmer), as shown in Table 7 below.

[0179] [Table 6]

[0180] [Table 7]

[0181] As shown in Table 7 above, the Po2.1 and Po2.2 promoters exhibited promoter activity in Bacillus subtilis and showed higher fluorescence sensitivity than P43 and Po2, which are conventionally known as strong promoters.

[0182] Example 2. Evaluation of the production capacity of the target product. Example 2-1. Evaluation of L-threonine production capacity Example 2-1-1. Preparation of a pyc expression vector containing Po2 or a Po2 mutant sequence. To confirm the effects of Po2.1 and Po2.2, two of the four mutant promoters of Po2 that exhibit high fluorescence sensitivity, on threonine production capacity, vectors expressing pyc(Ncgl0659, SEQ ID NO: 21), an important gene for supplying precursors necessary for L-threonine production, by Po2.1, Po2.2, and the Po2 promoter were constructed using the method described below.

[0183] Specifically, first, vectors were prepared to replace the original promoter of the pyc gene on the chromosome with a Po2 promoter mutant. Using the DNA of the pCES_Po2.1_gfp or pCES_Po2.2_gfp vector prepared in Example 1-1 as a template, PCR was performed using primers SEQ ID NOs. 22 and 23, respectively. After denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 30 seconds was repeated 30 times, followed by polymerization at 72°C for 7 minutes. As a result, DNA fragments containing the Po2.1 or Po2.2 promoter sequence that can be inserted into the upper end of the pyc gene were obtained. For use as a control group, pCES_Po2_gfp was used as a template, and PCR was performed in the same manner as above to obtain DNA fragments containing the Po2 promoter sequence.

[0184] Furthermore, PCR was performed using Corynebacterium glutamicum ATCC13032 genomic DNA as a template, with primers SEQ ID NOs. 24 and 25, and SEQ ID NOs. 26 and 27. The PCR conditions involved denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 60 seconds, repeated 30 times, and then polymerization at 72°C for 7 minutes. As a result, a 524 bp DNA fragment at the 5' upper end and a 525 bp DNA fragment at the 3' lower end were obtained, centering on the inserted o2.1, o2.2, and o2 promoter sites.

[0185] Using the three PCR products obtained above as templates, PCR was performed using primers SEQ ID NOs. 24 and 27. After denaturation at 95°C for 5 minutes, the following cycles were repeated 30 times: denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 90 seconds, and then polymerization at 72°C for 7 minutes. As a result, an 1114 bp DNA fragment was amplified to replace the original promoter site of the pyc gene with Po2.1, Po2.2, and Po2 promoters.

[0186] Subsequently, the pDC24 (SEQ ID NO: 43) vector and an 1110 bp DNA fragment were treated with the restriction enzyme SmaI, then ligated using the In-Fusion® HD cloning kit (clontech), and finally cloned to obtain the plasmid. The vector with Po2.1 introduced instead of the self-promoter of the pyc gene was named "pDC24_Po2.1_pyc", the vector with Po2.2 introduced was named "pDC24_Po2.2_pyc", and the vector with Po2 introduced in the control group was named "pDC24_Po2_pyc".

[0187] The primers sequence numbers 22-27 used above are listed in Table 8 below.

[0188] [Table 8]

[0189] Example 2-1-2. Preparation of transformed bacterial strains The recombinant vectors pDC24_Po2.1_pyc, pDC24_Po2.2_pyc, and pDC24_Po2_pyc prepared in Example 2-1-1 were transformed into the threonine-producing strain Corynebacterium glutamicum KCCM12120P (US Patent Publication US11,236,374B2) using the electropulse method, respectively. The transformed strains were then obtained in a selective medium containing 25 mg / L of kanamycin, and these were named "KCCM12120P::Po2.1_pyc", "KCCM12120P::Po2.2_pyc", and "KCCM12120P::Po2_pyc", respectively. Example 2-1-3. Evaluation of threonine production capacity of transformed bacterial strains. The transformed bacterial strains prepared in Example 2-1-2 were cultured using the method described below, and the amount of threonine produced was measured. The KCCM12120P::Po2_pyc strain was used as a control group.

[0190] Specifically, each transformed strain was inoculated into a 250 ml corner baffle flask containing 25 ml of seed medium and cultured at 30°C for 20 hours with shaking at 200 rpm. Subsequently, 1 ml of seed culture solution was inoculated into a 250 ml corner baffle flask containing 24 ml of production medium and cultured at 30°C for 48 hours with shaking at 200 rpm. The compositions of the seed medium and production medium used are shown in Table 9.

[0191] [Table 9]

[0192] After the culturing was complete, the L-threonine production capacity was measured by HPLC.

[0193] The percentage increase in L-threonine concentration in the culture medium for each of the experimentally tested bacterial strains is shown in Table 10 below.

[0194] [Table 10]

[0195] As shown in Table 10 above, we confirmed that introducing a mutant promoter of Po2 increased pyc activity and thus increased threonine production. This indicates that Po2.1 and Po2.2 are effective in increasing threonine production.

[0196] Example 2-2. Evaluation of O-acetylhomoserine production capacity Example 2-2-1. Preparation of a metX expression vector containing Po2 or a Po2 mutant sequence. To confirm the effect of the Po2 mutant promoter on O-acetylhomoserine production capacity, vectors expressing metX (NCgl0624, SEQ ID NO: 28), a gene important for O-acetylhomoserine production, regulated by Po2.1, Po2.2, and the Po2 promoter were constructed using the following method.

[0197] Specifically, PCR was performed using the DNA of the pCES_Po2.1_gfp or pCES_Po2.2_gfp vector prepared in Example 1-1 as a template, and using primers SEQ ID NOs. 2 and 29, respectively. The PCR involved denaturation at 94°C for 5 minutes, followed by denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 1 minute, repeated 30 times, and then polymerization at 72°C for 5 minutes to obtain DNA fragments containing the o2.1 or o2.2 promoter sequence. For use as a control group, PCR was performed using pCES_Po2_gfp as a template in the same manner as described above to obtain DNA fragments containing the Po2 promoter sequence.

[0198] Furthermore, using Corynebacterium glutamicum ATCC13032 genomic DNA as a template, PCR was performed using primers SEQ ID NOs. 30 and 31 to secure DNA fragments containing metX. The PCR process involved denaturation at 94°C for 5 minutes, followed by denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 2 minutes, repeated 30 times, and then polymerization at 72°C for 5 minutes.

[0199] Furthermore, to introduce Po2.1, Po2.2, or Po2 and metX into the E. coli-Corynebacterium shuttle vector pCES208, the pCES208 vector was used as a template, and PCR was performed using SEQ ID NOs. 10 and 11 to prepare the vectors. The PCR involved denaturation at 94°C for 5 minutes, followed by denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 5 minutes, repeated 30 times, and then polymerization at 72°C for 5 minutes.

[0200] Subsequently, the Po2.1, Po2.2, or Po2 DNA fragments and the ORF of the metX gene obtained above were ligated into the pCES208 vector obtained by the method described above using the In-Fusion® HD cloning kit (clontech) to create recombinant vectors in which Po2.1, Po2.2, or Po2 were ligated to metX, respectively. These were named "pCES_Po2.1_MetX", "pCES_Po2.2_MetX", and "pCES_Po2_MetX".

[0201] The primers with sequence numbers 29-31 used above are listed in Table 11 below.

[0202] [Table 11]

[0203] Example 2-2-2. Preparation of transformed bacterial strains The recombinant vectors "pCES_Po2.1_MetX", "pCES_Po2.2_MetX", and "pCES_Po2_MetX" prepared in Example 2-2-1 were transformed into the acetylhomoserine-producing strain Corynebacterium glutamicum KCCM12634P (European published patent EP4050022A1) using the electrophoretic pulse method. The transformed strains were then obtained in a selective medium containing 25 mg / L of kanamycin, and these were named "KCCM12634P::Po2.1_metX", "KCCM12634P::Po2.2_metX", and "KCCM12634P::Po2_metX", respectively.

[0204] Example 2-2-3. Evaluation of O-acetylhomoserine production capacity of transformed bacterial strains. The transformed bacterial strains prepared in Example 2-2-2 were cultured using the method described below, and the production of O-acetylhomoserine was measured. The KCCM12634P::Po2_metX strain was used as a control group.

[0205] Specifically, the bacterial strain was inoculated into a 250 ml corner baffle flask containing 25 ml of production medium using one platinum loop (inoculation loop), and cultured at 37°C for 20 hours with shaking at 200 rpm. The composition of the production medium is shown in Table 12 below.

[0206] [Table 12]

[0207] After the culturing was complete, the O-acetylhomoserine concentration was analyzed using HPLC, and the percentage increase in O-acetylhomoserine concentration for each strain is shown in Table 13 below.

[0208] [Table 13]

[0209] As shown in Table 13 above, we confirmed that introducing a mutant promoter of Po2 increased metX activity and thus increased acetylhomoserine production. This indicates that Po2.1 and Po2.2 are effective in increasing O-acetylhomoserine production.

[0210] Example 2-3. Evaluation of L-valine production capacity Example 2-3-1. Preparation of an ilvE expression vector containing Po2 or a Po2 mutant sequence. To confirm the effect of the Po2 mutant promoter on valine production capacity, vectors expressing ilvE (Ncgl2123, SEQ ID NO: 32), which encodes branched-chain amino acid aminotransferase, a major gene in valine biosynthesis, under the Po2.1, Po2.2, and Po2 promoters were constructed using the following method.

[0211] Specifically, first, vectors were prepared to replace the original promoter of the ilvE gene on the chromosome with the Po2.1 or Po2.2 promoter. Using the DNA of the pCES_Po2.1_gfp or pCES_Po2.2_gfp vector prepared in Example 1-1 as a template, PCR was performed using primers SEQ ID NOs. 33 and 34, respectively. After denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 30 seconds was repeated 30 times, followed by polymerization at 72°C for 7 minutes. As a result, DNA fragments containing the Po2.1 or Po2.2 promoter sequence that can be inserted into the upper end of the ilvE gene were obtained. For use as a control group, pCES_Po2_gfp was used as a template, and PCR was performed in the same manner as above to obtain DNA fragments containing the Po2 promoter sequence.

[0212] Furthermore, PCR was performed using the Corynebacterium glutamicum ATCC14067 chromosome as a template, with primers numbered 35 and 36, and 37 and 38. The PCR conditions were as follows: denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 60 seconds, repeated 30 times, followed by polymerization at 72°C for 7 minutes. As a result, a 529 bp DNA fragment at the 5' upper end and a 525 bp DNA fragment at the 3' lower end were obtained, centering on the inserted o2.1, o2.2, and o2 promoter sites.

[0213] Using the three PCR products obtained above as templates, PCR was performed using primers SEQ ID NOs. 35 and 38. After denaturation at 95°C for 5 minutes, the following cycles were repeated 30 times: denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 90 seconds, and then polymerization at 72°C for 7 minutes. As a result, an 1114 bp DNA fragment was amplified to replace the original promoter site of the ilvE gene with the o2.1, o2.2, and o2 promoters.

[0214] Subsequently, the pDC24 vector (SEQ ID NO: 43) and an 1114 bp DNA fragment were treated with the restriction enzyme SmaI, then ligated using the In-Fusion® HD cloning kit (clontech), and finally cloned to obtain the plasmid. The vector with Po2.1 introduced instead of the ilvE gene's self-promoter was named "pDC24_Po2.1_ilvE", the vector with Po2.2 introduced was named "pDC24_Po2.2_ilvE", and the control vector with Po2 introduced was named "pDC24_Po2_ilvE".

[0215] The primers numbered 33-38 used above are listed in Table 14 below.

[0216] [Table 14]

[0217] Example 2-3-2. Preparation of transformed bacterial strains The recombinant vectors pDC24_Po2.1_ilvE, pDC24_Po2.2_ilvE, and pDC24_Po2_ilvE prepared in Example 2-3-1 were transformed into the valine-producing strain Corynebacterium glutamicum KCCM11201P (US Registered Patent US8,465,962B2) using the electropulse method, respectively. The transformed strains were then obtained in a selective medium containing 25 mg / L of kanamycin, and these were named "KCCM11201P::Po2.1_ilvE", "KCCM11201P::Po2.2_ilvE", and "KCCM11201P::Po2_ilvE", respectively.

[0218] Example 2-3-3. Evaluation of valine production capacity of transformed bacterial strains. The transformed bacterial strains prepared in Example 2-3-2 were cultured using the method described below, and the amount of valine produced was measured. The KCCM12120P::Po2_ilvE strain was used as a control group.

[0219] Specifically, each bacterial strain was inoculated into a 250 ml corner baffle flask containing 25 ml of production medium, and incubated at 30°C for 72 hours with shaking at 200 rpm. The composition of the production medium used is shown in Table 15.

[0220] [Table 15]

[0221] After the culture was completed, the L-valine production capacity was measured by HPLC.

[0222] The percentage increase in L-valine concentration in the culture medium for each of the experimentally tested bacterial strains is shown in Table 16 below.

[0223] [Table 16]

[0224] As shown in Table 16 above, we confirmed that introducing a mutant promoter of Po2 increased ilvE activity and thus increased valine production. This indicates that Po2.1 and Po2.2 are effective in increasing valine production.

[0225] From the above description, those skilled in the art in which the present invention pertains will understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, which are described below, and their equivalent concepts, rather than from the above detailed description.

Claims

1. A polynucleotide containing any one nucleic acid sequence selected from the group consisting of sequence numbers 39-40.

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

3. An expression cassette comprising the polynucleotide and target gene according to claim 1.

4. A microorganism comprising the polynucleotide described in claim 1 or the expression cassette described in claim 3.

5. The microorganism according to claim 4, wherein the microorganism is a microorganism of the genus Corynebacterium, Escherichia, or Bacillus.

6. A method for producing a target product, comprising the step of culturing the microorganism described in claim 4 in a culture medium.

7. The method for producing a target product according to claim 6, further comprising the step of recovering the target product from the culture medium or microorganisms obtained by the culture.

8. The method for producing the target product according to claim 6, wherein the target product is one or more selected from the group consisting of amino acids, amino acid derivatives, nucleic acids, nucleic acid derivatives, vitamins, vitamin derivatives, proteins, fatty acids, fatty acid derivatives, organic acids, and other metabolites.

9. The method for producing the target product according to claim 6, wherein the target product is one or more selected from the group consisting of threonine, O-acetylhomoserine, and valine.

10. The method for producing the target product according to claim 6, wherein the microorganism is a microorganism of the genus Corynebacterium, Escherichia, or Bacillus.

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