Novel promoter and use thereof

Abstract

The present application relates to a novel promoter, and a method for producing a target product using the same, and a polynucleotide according to one specific embodiment has a promoter activity and is introduced into a microorganism so as to improve the expression and activity of a gene operably linked thereto, thereby improving the expression and activity of a gene operably linked thereto. And the method can be effectively used for producing target products influenced by polynucleotides and genes.

Description

Technical Field

[0001] Cross-reference to related applications

[0002] This application claims the benefit of priority based on Korean Patent Application Nos. 10-2024-0072573, filed on June 3, 2024, and 10-2024-0147026, filed on October 24, 2024, and the entire contents disclosed in the corresponding Korean patent application documents are incorporated as part of this specification.

[0003] Throughout this application, a large number of papers and patent documents are referenced and their citation sources are indicated. For a clearer description of the level of the technical field related to the present invention and the content of the present invention, the disclosed contents of the referenced papers and patent documents are incorporated into this application by reference in their entirety.

[0004] This application relates to a novel promoter and a method for producing a target product using the promoter. Background Art

[0005] In order to produce target substances such as amino acids or useful substances that can be used for various purposes such as feed, medicine, and food at high titers using microorganisms, attempts have been continuously made to perform gene manipulation and / or introduction of foreign genes in the biosynthetic pathway. One of these methods is a method of inducing overexpression of a target gene in a microorganism, and for this, an efficient gene expression system is required. A promoter is one of the factors that greatly participate in the expression level and expression regulation of a gene, and thus it can be said that the development of useful promoters is crucial for the development of an expression system.

[0006] The tac promoter derived from Escherichia coli is a well-known strong promoter, while in 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, different from other industrial microorganisms such as Escherichia coli or Bacillus subtilis, the general structure of the promoter sequence for gene expression in coryneform microorganisms is not known. Therefore, promoters have been developed by measuring the antibiotic resistance of strains obtained by removing the promoter portion of an antibiotic resistance gene (such as chloramphenicol), introducing the chromosomal DNA by cutting the chromosomal DNA isolated from coryneform microorganisms with an appropriate restriction enzyme, and transforming coryneform microorganisms with it. In addition, to date, various promoters have been explored for overexpressing foreign genes in Bacillus microorganisms, and enzymes for food, pharmaceuticals, and other industrial uses have been produced using this method. To date, many vector systems using expression promoters of α-amylase, protease, and lipase genes have continuously emerged in various microorganisms of the genus Bacillus (Schumann 2007. Adv. Appl. Microbiol. 153: 813-821)

[0007] However, since there is still a need for a system that can exhibit high expression efficiency in various microorganisms (i.e., even in microorganisms of the genus Escherichia, Corynebacterium, or Bacillus), there is still a need to develop a universal promoter.

[0008] [Prior Art]

[0009] [Patent Document]

[0010] (Patent Document 1) US Granted Patent (US11,041,181B2) Summary of the Invention

[0011] [Technical Problem]

[0012] An object of the present application is to provide a novel polynucleotide. The polynucleotide may have promoter activity.

[0013] Another object of the present application is to provide an expression cassette containing the polynucleotide and a target gene.

[0014] Other objects of the present application are to provide a recombinant vector (expression vector) containing the polynucleotide; or an expression cassette containing the polynucleotide and a target gene.

[0015] Another object of the present application is to provide a microorganism comprising at least one selected from the group consisting of: a polynucleotide, an expression cassette comprising the polynucleotide and a target gene, and a vector comprising the expression cassette. The microorganism may have the ability to produce a target product.

[0016] Another object of the present application is to provide a method for producing a target product, which comprises culturing the microorganism in a culture medium. The method may further comprise recovering the target product from the culture medium or the microorganism produced by the culture after culturing.

[0017] Another object of the present application is to provide a composition for producing a target product, which composition comprises the microorganism, the culture medium in which the microorganism is cultured, or a combination thereof.

[0018] Another object of the present application is to provide the use of the polynucleotide as a promoter.

[0019] Another object of the present application is to provide the use of the microorganism, the culture medium in which the microorganism is cultured, or a combination thereof for producing a target product.

[0020] [Technical Solution]

[0021] The various descriptions and embodiments disclosed in this specification are also applicable to other descriptions and embodiments. In other words, all combinations of the various elements disclosed in this specification fall within the scope of the present application. In addition, the scope of the present application should not be regarded as being limited by the specific embodiments described below.

[0022] A more detailed description will be given below.

[0023] One aspect of the present application provides a novel polynucleotide.

[0024] Another aspect provides the use of the polynucleotide as a promoter.

[0025] In this specification, "polynucleotide" comprises 2 or more, 5 or more, 10 or more, 13 or more, 20 or more, or 30 or more nucleotide monomers, and the nucleotide monomers can be covalently bonded to form a chain.

[0026] In one embodiment of the present application, the polynucleotide may be a polynucleotide comprising any one nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.

[0027] In one embodiment, the polynucleotide may comprise the nucleic acid sequence of SEQ ID NO:1.

[0028] In one embodiment, the polynucleotide may comprise the nucleic acid sequence of SEQ ID NO:2.

[0029] In one embodiment, the polynucleotide may comprise the nucleic acid sequence of SEQ ID NO:3.

[0030] In one embodiment, the polynucleotide may comprise the nucleic acid sequence of SEQ ID NO:4.

[0031] In one embodiment, the polynucleotide may comprise the nucleic acid sequence of SEQ ID NO:5.

[0032] In one embodiment, the polynucleotide may comprise the nucleic acid sequence of SEQ ID NO:6.

[0033] In one embodiment, the polynucleotide may comprise the nucleic acid sequence of SEQ ID NO:7.

[0034] The polynucleotide may have promoter activity. For example, the polynucleotide can be used as a general promoter.

[0035] In one embodiment, the polynucleotide may have promoter activity for expression in a Corynebacterium microorganism, an Escherichia microorganism, and / or a Bacillus microorganism.

[0036] The polynucleotide according to a specific embodiment can be used as a synthetic promoter with strong expression induction activity. For example, compared with a known promoter (such as the Pbetp promoter (SEQ ID NO:8), etc.), it can express a target gene with extremely high efficiency in a Corynebacterium, Escherichia microorganism, and / or a Bacillus microorganism.

[0037] The polynucleotide according to a specific embodiment can be natural or non-natural. For example, it can be non-natural, chemically synthesized, or recombinantly synthesized.

[0038] In this specification, a "promoter" may refer to a DNA region that contains a binding site for a polymerase and allows initiation of transcription of a downstream target sequence. The promoter may be located in the 5' region of the transcription start site. The promoter may be operably linked upstream (towards the 5' end) of the target sequence, and / or capable of regulating (enhancing or reducing expression) of the linkage upstream (towards the 5' end) of the target sequence. For example, a promoter is linked forward to the 5' end of any gene to enhance (increase) the expression of that gene, or linked backward to the 3' end of any gene to weaken (reduce) the expression of the corresponding gene. When a promoter is introduced backward at the 3' end of any gene, for example downstream of a stop codon, preferably between the stop codon and upstream of a transcription terminator, the expression of the gene may be weakened because the RNA polymerase complex causes a collision during transcription, by inducing transcription in a direction opposite to the normal transcription direction of the corresponding gene.

[0039] The polymerase, also known as RNA polymerase or DNA-dependent RNA polymerase, may refer to an enzyme that synthesizes primary transcript RNA. The polymerase may be an RNA polymerase of a prokaryotic cell, or an RNA polymerase of a eukaryotic cell (e.g., RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, or RNA polymerase V, etc.).

[0040] In this specification, a "target sequence" may be a "target gene" that undergoes expression. In one embodiment, the target gene may be a gene encoding a target protein. The target protein may be a protein (such as an enzyme) involved in the production of a target product.

[0041] In this specification, the term "target product" may refer to a bioactive substance that is ultimately produced or regulated (increased or decreased) using the polynucleotides, expression cassettes, expression vectors, and / or recombinant cells provided in this specification. For example, it may refer to the target protein encoded by the target gene itself and / or all bioactive substances involved in the production of the target protein. A bioactive substance refers to all substances produced or derived from an organism (such as a cell) or having a certain function in a living body or cell. For example, it may be at least one selected from the group consisting of: amino acids, amino acid derivatives, nucleic acids (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 (calciferol), E (tocopherol), K (phylloquinone), etc.), vitamin derivatives, proteins (proteins other than the target protein, such as hormones, growth factors, cytokines, immunoglobulins (antibodies), antigenic proteins, receptors, ligands, their functional fragments (fragments having the target function), fusion proteins that fuse at least two proteins, etc.), saccharides (e.g., monosaccharides (glyceraldehyde, dihydroxyacetone, erythrose, threose, erythrulose, arabinose, lyxose, ribose, xylose, ribulose, xylulose, deoxyribose, allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, tagatose, trehalose, fucose, rhamnose, mannoheptulose, sedoheptulose, etc.), disaccharides (cellobiose, isomaltose, palatinose, lactose, lactulose, maltose, sucrose, trehalose, turanose, etc.), polysaccharides, etc.), saccharide derivatives (sugar alcohols, galactosamine, glucosamine, sialic acid, N-acetylglucosamine, sulfoquinovose, ascorbic acid, mannitol, glucuronic acid, etc.), fatty acids (myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, elaidic linoleic acid, arachidonic acid, eicosapentaenoic acid (EPA), erucic acid, docosahexaenoic acid (DHA), etc.), fatty acid derivatives, organic acids (lactic acid, citric acid, oxalic acid, uric acid, butyric acid, stearic acid, propionic acid, etc.), their metabolites (polyhydroxyalkanoates (PHA), etc.), their precursors, derivatives that maintain their biological activity, etc., but not limited thereto.

[0042] In one embodiment, when the target protein is involved in the production of the target product, (1) the target protein can be a protein that participates in at least one process or step of the intracellular pathway (such as biosynthesis, metabolism, biotransformation, etc.), intracellular transport, and / or efflux pathway of the target product. For example, it can be at least one selected from the group consisting of: synthase, lyase, kinase, carboxylase (such as pyruvate carboxylase, etc.), reductase, oxidase, decarboxylase, dehydrogenase, dehydratase, transferase (such as transferase, epimerase, etc.), intermediate product, carrier protein, membrane protein (channel, etc.), etc., but not limited thereto, and

[0043] (2) the target gene can be a gene encoding the target protein described in (1) above.

[0044] The amino acids in the target product can be proteinogenic amino acids or non-proteinogenic amino acids.

[0045] Proteinogenic amino acids can be at least one selected from the group consisting of: arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, selenocysteine, pyrrolysine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In addition, it can also be an intermediate thereof.

[0046] Proteinogenic amino acids can be L-amino acids. For example, it can be at least one 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, or an intermediate thereof. Specifically, it can be L-lysine, L-threonine, L-isoleucine, L-leucine, L-valine, L-arginine, or L-glutamic acid, but not limited thereto. The intermediate can be, for example, O-acetylhomoserine, which is an intermediate of L-methionine, but not limited thereto.

[0047] The protein source amino acids can be at least one selected from the group consisting of: β-alanine, γ-aminobutyric acid (GABA), δ-aminolevulinic acid, 4-aminobenzoic acid, α-aminobutyric acid (α-aminoisobutyric acid), dehydroalanine, cystathionine, lanthionine, djenkolic acid, diaminopimelic acid, norvaline, norleucine, alloisoleucine, t-leucine (tert-leucine), α-amino-n-heptanoic acid, pipecolic acid, α,β-diaminopropionic acid, α,γ-diaminobutyric acid, ornithine, allothreonine, homocysteine, homoserine (or isothreonine), O-acetylhomoserine, etc., but not limited thereto.

[0048] The amino acids can be D-amino acids or L-amino acids.

[0049] In a specific embodiment, the target product can be at least one selected from the group consisting of threonine, O-acetylhomoserine, and valine.

[0050] According to a specific embodiment, compared with a conventional promoter or an endogenous promoter of a cell, the polynucleotide having promoter activity in the present application can regulate (such as increase or decrease) the expression of a target gene operably linked thereto in a cell, the production and / or activity of a target protein encoded by the target gene, and / or the production and / or activity of a bioactive substance involved in the production of the target protein.

[0051] The nucleic acid sequence of the polynucleotide with promoter activity of the present application can be modified by conventionally known mutagenesis methods (such as direct evolution and site-directed mutagenesis, etc.). In other words, the polynucleotide may contain 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 (the upper limit can be 100% or less than 100%) homology or identity with the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, and at the same time having the same or corresponding biological activity as the nucleic acid sequence of the polynucleotide or a nucleic acid sequence composed of the corresponding nucleic acid sequence. In addition, as long as it is a polynucleotide having substantially the same or corresponding biological activity as the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, it is a sequence having homology or identity with the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, and polynucleotides having a base sequence in which some sequences are deleted, modified, substituted or added in the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7 can also be included within the scope of the polynucleotides with promoter activity of the present application.

[0052] In this specification, the terms "homology" or "identity" refer to the degree of match with a given nucleic acid sequence or amino acid sequence, which can be expressed as a percentage (%). For example, homology can be determined by directly aligning the sequence information (such as parameters like score, identity, and similarity) between two polynucleotide molecules or two polypeptide molecules using a computer program that readily available alignment sequence information. The computer program can be BLAST (NCBI), CLC Main Workbench (CLC bio), MegAlignTM (DNASTAR Inc), etc.

[0053] In a specific embodiment, the polynucleotides containing a specific nucleic acid sequence provided in this specification can be interpreted as including not only the specific nucleic acid sequence or a nucleic acid sequence substantially equivalent thereto, but also a nucleic acid sequence complementary to the specific nucleic acid sequence. Specifically, the complementary polynucleotides can hybridize at a Tm value appropriately adjusted by those skilled in the art according to the purpose, for example, at a Tm value of 55°C, 60°C, 63°C, or 65°C, and analyzed under the conditions described later: these conditions are specifically described in known literature. For example, it can be listed that the conditions where polynucleotides 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 while polynucleotides with lower complementarity do not hybridize, or the conditions of washing once, specifically twice or three times, at a salt concentration and temperature corresponding to 60°C, 1x SSC (saline-sodium citrate buffer) and 0.1% (w / v) SDS (sodium dodecyl sulfate); 60°C, 0.1x SSC and 0.1% SDS; or 68°C, 0.1x SSC and 0.1% SDS, which are the common washing conditions for southern hybridization, etc., but not limited thereto. Hybridization requires that the two nucleotides have complementary sequences, but depending on the stringency of hybridization, mismatches between bases may be allowed. The term "complementary" can be used to describe the relationship between nucleobases that can hybridize with each other. For example, in the case of DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. The appropriate stringency of polynucleotide hybridization depends on the length and degree of complementarity of the polynucleotide, which is well known in the relevant technical field (see Sambrook et al., ibid., 9.50 - 9.51, 11.7 - 11.8).

[0054] According to one specific embodiment, the polynucleotide may comprise the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, and may be a polynucleotide consisting of or substantially comprising the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

[0055] In this specification, a polynucleotide or polypeptide "comprising a specific nucleic acid sequence or amino acid sequence" may mean that the polynucleotide or polypeptide consists of or substantially comprises the specific nucleic acid sequence or amino acid sequence, and may be interpreted to include "substantially equivalent sequences", in which mutations (deletions, substitutions, modifications, and / or additions) are made to the specific nucleic acid sequence or amino acid sequence within the range that maintains the original function and / or desired function of the polynucleotide or polypeptide (or mutations are not excluded). In one embodiment, the nucleic acid sequences or amino acid sequences provided in this specification may include nucleic acid sequences or amino acid sequences modified by common mutagenesis such as directed evolution and / or site-directed mutagenesis within the range that maintains their original function or desired function. In a specific embodiment, a polynucleotide or polypeptide "comprising a specific nucleic acid sequence or amino acid sequence" may mean that the polynucleotide or polypeptide (i) consists of or substantially comprises the specific nucleic acid sequence or amino acid sequence, or (ii) consists of or substantially comprises an amino acid sequence having a homology or identity 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, 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 (the upper limit may be 100% or less than 100%) with the specific nucleic acid sequence or amino acid sequence, and maintains the original function and / or desired function.

[0056] According to a specific embodiment, the original function and / or desired function of the polynucleotides provided herein may be the function as a promoter. The polynucleotide can be operably linked to a target gene as a promoter, and when linked, additions, deletions, and / or substitutions of nucleotides for restriction enzymes can be introduced at the 5'-end and / or 3'-end of the corresponding polynucleotide. The target gene can be a gene encoding a target protein, and the target protein can be involved in the production of the target product as described above, and examples thereof are as follows.

[0057] In one embodiment, the protein involved in the production of arginine in an amino acid can be at least one selected from the proteins involved in arginine biosynthesis (arginine biosynthesis proteins). For example, the proteins involved in arginine biosynthesis can be N-acetylglutamate synthase (argA), N-acetylglutamate kinase (argB), N-acetylglutamyl phosphate reductase (argC), acetylornithine aminotransferase (argD), acetylornithine deacetylase (argE), ornithine carbamoyltransferase (argF, argI), argininosuccinate synthase (argG), argininosuccinate lyase (argH), ornithine acetyltransferase (argJ), carbamoyl phosphate synthetase (carAB), but are not limited thereto.

[0058] In one embodiment, the protein involved in the production of histidine in an amino acid can be at least one selected from the proteins involved in histidine biosynthesis (histidine biosynthesis proteins). For example, the proteins involved in histidine biosynthesis can be ATP phosphoribosyltransferase (hisG), phosphoribosyl-AMP cyclohydrolase (hisl), phosphoribosyl-ATP pyrophosphohydrolase (hisI), phosphoribosylformimino-5-aminoimidazole carboxamide ribonucleotide isomerase (hisA), amidotransferase (hisH), histidinol phosphate aminotransferase (hisC), histidinol phosphatase (hisB), histidinol dehydrogenase (hisD), but are not limited thereto.

[0059] In one embodiment, the protein involved in the production of lysine in an amino acid can be at least one selected from the proteins involved in lysine biosynthesis (lysine biosynthesis proteins). For example, the proteins involved in lysine biosynthesis can be at least one selected from the group consisting of dihydropicolinate synthase (dapA), aspartokinase III (lysC), dihydropicolinate reductase (dapB), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh), phosphoenolpyruvate carboxylase (ppc), aspartate semialdehyde dehydrogenase (asd), aspartate aminotransferase (aspC), diaminopimelate epimerase (dapF), succinyl-diaminopimelate desuccinylase (dapD), succinyl-diaminopimelate desacylase (dapE), and aspartase (aspA), but are not limited thereto.

[0060] In one embodiment, the protein involved in the production of aspartic acid in an amino acid can be at least one selected from the proteins involved in aspartic acid biosynthesis (aspartic acid biosynthesis proteins). For example, the proteins involved in aspartic acid biosynthesis can be aspartate aminotransferase and the like, but are not limited thereto.

[0061] In one embodiment, the protein involved in the production of glutamate in amino acids can be at least one selected from the proteins involved in glutamate biosynthesis (glutamate biosynthesis proteins). For example, the protein involved in glutamate biosynthesis can be at least one selected from the group consisting of: glutamate dehydrogenase (ghdhA), glutamine synthetase (glnA), glutamate synthase (gltBD), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), citramalate synthase (prpC), phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase (pyc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglycerate mutase (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 not limited thereto.

[0062] In one embodiment, the protein involved in the production of serine in amino acids can be at least one selected from the proteins involved in serine biosynthesis (serine biosynthesis proteins). For example, the protein involved in serine biosynthesis can be at least one selected from the group consisting of: 3-phosphoglycerate dehydrogenase (serA), phosphoserine transaminase (serC), phosphoserine phosphatase (serB), etc., but not limited thereto.

[0063] In one embodiment, the protein involved in the production of threonine in amino acids can be at least one selected from the proteins involved in threonine biosynthesis (threonine biosynthesis proteins). For example, the protein involved in threonine biosynthesis can be aspartokinase III (lysC), aspartate semialdehyde dehydrogenase (asd), aspartokinase I (thrA), homoserine kinase (thrB), threonine synthase (thrC), aspartate aminotransferase, but not limited thereto.

[0064] In one embodiment, the protein involved in the production of asparagine in amino acids can be at least one selected from the proteins involved in asparagine biosynthesis (asparagine biosynthesis proteins). For example, the protein involved in asparagine biosynthesis can be transaminase, asparagine synthetase, etc., but not limited thereto.

[0065] In one embodiment, the protein involved in the production of glutamine in amino acids can be at least one selected from the proteins involved in glutamine biosynthesis (glutamine biosynthesis proteins). For example, the protein involved in glutamine biosynthesis can be at least one selected from the group consisting of glutamate dehydrogenase (ghdhA), glutamine synthetase (glnA), etc., but is not limited thereto.

[0066] In one embodiment, the protein involved in the production of cysteine in amino acids can be at least one selected from the proteins involved in cysteine biosynthesis (cysteine biosynthesis proteins). For example, the protein involved in cysteine biosynthesis can be at least one selected from the group consisting of serine acetyltransferase (cysE), 3-phosphoglycerate dehydrogenase (serA), etc., but is not limited thereto.

[0067] In one embodiment, the protein involved in the production of glycine in amino acids can be at least one selected from the proteins involved in glycine biosynthesis (glycine biosynthesis proteins). For example, the protein involved in glycine biosynthesis can be alanine-glyoxylate aminotransferase, etc., but is not limited thereto.

[0068] In one embodiment, the protein involved in the production of proline in amino acids can be at least one selected from the proteins involved in proline biosynthesis (proline biosynthesis proteins). For example, the protein involved in proline biosynthesis can be at least one selected from the group consisting of glutamate-5-kinase (proB), γ-glutamyl-phosphate reductase, pyrroline-5-carboxylate reductase (putA), etc., but is not limited thereto. In one embodiment, the protein involved in the production of alanine in amino acids can be at least one selected from the proteins involved in alanine biosynthesis (alanine biosynthesis proteins). For example, the protein involved in alanine biosynthesis can be alanine aminotransferase, etc., but is not limited thereto.

[0069] In one embodiment, the protein involved in the production of valine in amino acids can be at least one selected from the proteins involved in valine biosynthesis (valine biosynthesis proteins). For example, the protein involved in valine biosynthesis can be acetohydroxy acid isomeroreductase (IlvC), dihydroxy acid dehydratase (IlvD), branched-chain amino acid aminotransferase (IlvE), but is not limited thereto.

[0070] In one embodiment, the protein involved in the production of isoleucine in an amino acid can be at least one selected from the proteins involved in isoleucine biosynthesis (isoleucine biosynthesis proteins). For example, the proteins involved in isoleucine biosynthesis can be acetohydroxyacid synthase (AHAS), acetohydroxyacid isomeroreductase, dihydroxyacid dehydratase, valine aminotransferase, etc., but are not limited thereto.

[0071] In one embodiment, the protein involved in the production of leucine in an amino acid can be at least one selected from the proteins involved in leucine biosynthesis (leucine biosynthesis proteins). For example, the proteins involved in leucine biosynthesis can be acetolactate synthase, acetohydroxyacid isomeroreductase, dihydroxyacid dehydratase, α-isopropylmalate synthase, α-isopropylmalate isomerase, leucine aminotransferase, etc., but are not limited thereto.

[0072] In one embodiment, the protein involved in the production of methionine in an amino acid can be at least one selected from the proteins involved in methionine biosynthesis (methionine biosynthesis proteins). For example, the proteins involved in methionine biosynthesis can be aspartokinase, aspartate-semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine O-succinyltransferase, cystathionine γ-synthase, cystathionine β-lyase, methionine synthase, etc., but are not limited thereto.

[0073] In one embodiment, the protein involved in the production of phenylalanine in an amino acid can be at least one selected from the proteins involved in phenylalanine biosynthesis (phenylalanine biosynthesis proteins). For example, the proteins involved in phenylalanine biosynthesis can be chorismate mutase, prephenate aminotransferase, arogenate dehydratase, etc., but are not limited thereto.

[0074] In one embodiment, the protein involved in the production of tyrosine in an amino acid can be at least one selected from the proteins involved in tyrosine biosynthesis (tyrosine biosynthesis proteins). For example, the proteins involved in tyrosine biosynthesis can be chorismate mutase, prephenate aminotransferase, arogenate dehydrogenase, etc., but are not limited thereto.

[0075] In one embodiment, the protein involved in the production of tryptophan in an amino acid can be at least one selected from the proteins involved in tryptophan biosynthesis (tryptophan biosynthesis proteins). For example, the proteins involved in tryptophan biosynthesis can be anthranilate synthase, anthranilate phosphoribosyltransferase, anthranilate isomerase, imidazoleglycerol phosphate synthase, tryptophan synthase, etc., but are not limited thereto.

[0076] In one embodiment, the protein involved in the production of O-acetylhomoserine in an amino acid can be at least one selected from the proteins involved in the biosynthesis of O-acetylhomoserine. For example, the protein involved in the biosynthesis of O-acetylhomoserine can be homoserine O-acetyltransferase or the like, but is not limited thereto.

[0077] In one embodiment, the protein involved in the production of β-alanine in an amino acid can be at least one selected from the proteins involved in the biosynthesis of β-alanine. For example, the proteins involved in the biosynthesis of β-alanine can be propionate CoA ligase, medium-chain acyl-CoA dehydrogenase, 3-hydroxypropionyl-CoA dehydratase, 3-hydroxypropionyl-CoA hydrolase, 3-hydroxypropionate dehydrogenase, β-alanine-pyruvate transaminase or the like, but is not limited thereto.

[0078] The gene involved in nucleic acid production can be at least one or more genes selected from nucleic acid biosynthesis genes. For example, it can be at least one selected from the group consisting of: amidophosphoribosyltransferase (purF), PRA-glycine ligase (purD), phosphoribosylaminoimidazole succinocarboxamide synthetase (purC), bifunctional AICAR formyltransferase / IMP cyclohydrolase (purH), adenylosuccinate synthetase (purA), adenylosuccinate lyase (purB), phosphoribosylaminoimidazole mutase (purE), phosphoribosylaminoimidazole carboxylase (purK) or the like, but is not limited thereto.

[0079] In a specific embodiment, the target gene can be at least one selected from the group consisting of the pyc gene, the metX gene, and the ilvE gene.

[0080] According to a specific embodiment, the pyc gene encodes pyruvate carboxylase, and according to a specific embodiment, the polynucleotide and the pyc gene are operably linked and can be used for the production of L-threonine. The pyc gene can be a gene derived from Corynebacterium glutamicum.

[0081] According to a specific embodiment, the metX gene encodes homoserine O-acetyltransferase, and according to a specific embodiment, the polynucleotide and the metX gene are operably linked and can be used for the production of O-acetylhomoserine. The metX gene can be a gene derived from Corynebacterium glutamicum.

[0082] According to a specific embodiment, the ilvE gene encodes a branched-chain amino acid aminotransferase, and according to a specific embodiment, the polynucleotide and the ilvE gene are operably linked and can be used for the production of valine. The ilvE gene can be a gene derived from Corynebacterium glutamicum.

[0083] On the other hand, an expression cassette is provided, which comprises the polynucleotide provided herein and a target gene.

[0084] The target gene can be a gene encoding a target protein, and the target protein can be a protein (such as an enzyme) involved in the production of a target product.

[0085] The target product can be at least one selected from the group consisting of: amino acids, amino acid derivatives, nucleic acids, nucleic acid derivatives, vitamins, vitamin derivatives, saccharides, saccharide derivatives, fatty acids, fatty acid derivatives, proteins, and other metabolites, etc.

[0086] The expression cassette may comprise a polynucleotide as a promoter.

[0087] In this specification, the term "expression cassette" may refer to a construct comprising at least a target gene and an expression regulatory sequence (such as a promoter), which is the smallest unit of nucleic acid fragment capable of being expressed in a host cell.

[0088] The polynucleotide may be contained in the 5'-end or 3'-end region of the target gene.

[0089] The polynucleotide may be operably linked to the target gene.

[0090] The polynucleotide, target gene, target protein, and target protein are as described above.

[0091] Other aspects provide a vector, which comprises the polynucleotide or expression cassette provided herein.

[0092] The vector can be a recombinant vector.

[0093] The recombinant vector can be an expression vector or an insertion vector.

[0094] The vector may comprise a polynucleotide as a promoter.

[0095] According to a specific embodiment, the vector may comprise a polynucleotide having promoter activity and a target gene operably linked to the polynucleotide. The target gene can be a gene encoding a target protein, and the target protein can be a protein (such as an enzyme) involved in the production of a target product.

[0096] According to a specific embodiment, the target protein can be a fusion protein, which is fused with one protein (a single protein), or two or more proteins. When the target protein is a fusion protein containing two or more proteins, the gene encoding the target protein can be a fusion gene containing genes encoding the two or more proteins respectively.

[0097] According to a specific embodiment, when the gene encoding the target protein in the recombinant vector is a fusion gene containing genes encoding two or more proteins respectively, it can be designed such that all genes contained in the fusion gene are controlled by one polynucleotide (promoter), or at least one of the genes is controlled by a separate polynucleotide. For example, the vector can contain a polynucleotide having promoter activity and one gene or two or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) genes operably linked to the polynucleotide, and when containing two or more genes, the vector can contain one polynucleotide having promoter activity (i.e., the two or more genes are controlled by one promoter), or contain two or more polynucleotides (in this case, the number of polynucleotides can be less than the number of genes so that at least one of the two or more genes is controlled by a separate promoter).

[0098] In this specification, the term "vector" refers to a general term for DNA molecules used to deliver nucleic acids of a target sequence to a suitable host or cell, which can contain appropriate gene expression regulatory sequences; and optionally, the nucleic acid sequence of the target gene operably linked thereto. "Gene expression regulatory sequences" are elements that perform various regulatory functions in gene expression. In one embodiment, it can include the polynucleotides having promoter activity provided in this specification, and can refer to nucleic acid sequences capable of expressing the target gene operably linked thereto. Specifically, in addition to the promoter for gene transcription, gene expression regulatory sequences can also include any operon sequences regulating transcription, sequences encoding appropriate mRNA ribosome binding sites, and / or nucleic acid sequences regulating transcription and translation termination, etc., but are not limited thereto. In addition, as regulatory sequences applicable to prokaryotes, it can include promoters and ribosome binding sites, but are not limited thereto. Those skilled in the art can construct gene expression regulatory sequences containing the polynucleotides having promoter activity in this application as needed.

[0099] In this specification, "operably linked" means that a polynucleotide having promoter activity is functionally linked to the nucleotide sequence of the target gene to initiate and / or mediate transcription of the target gene. Operable linkage can be carried out using gene recombination techniques known in the art, for example, site-specific DNA cleavage and / or ligation techniques, and site-specific DNA cleavage and ligation can be carried out using common cleavage enzymes and / or ligases, etc., but are not limited thereto.

[0100] There are no particular limitations as long as the vector can be transformed into the host cell and / or expressed in the host cell. Examples of vectors can include plasmids, cosmids, viruses, and / or phages in their natural or recombinant states. For example, as a phage vector or cosmid vector, at least one selected from the group consisting of pWE15, M13, λLB3, λBL4, λⅨII, λASHII, λAPII, λt10, λt11, Charon4A, Charon21A, etc. can be used. As a plasmid vector, at least one selected from the group consisting of pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, gene pCL-based, pET-based plasmid vectors, etc. can be used.

[0101] In one embodiment, the endogenous promoter in the host cell chromosome can be replaced with a polynucleotide having promoter activity provided in this specification. In this case, the vector can be a vector for inserting into the chromosome in the host cell, which contains a polynucleotide having promoter activity. For example, at least one selected from the group consisting of pECCG117, pDZ, pDC, pACYC177, pACYC184, pCL, pUC19, pBR322, pMW118, pCC1BAC, pCES208, pXMJ19 vectors, etc. can be used, but not limited thereto. In addition, the insertion of the polynucleotide into the chromosome can be carried out by any method known in the art, such as homologous recombination, genome editing by target-specific endonucleases (such as RNA-guided endonucleases like Cas9, Cpf1, etc.).

[0102] In one embodiment, when the vector or the polynucleotide having promoter activity and / or the target gene contained therein is inserted into the chromosome, the vector can further contain a selection marker for confirming the chromosomal insertion. As a selection marker, a marker that can produce a selectable phenotype can be used, such as drug (e.g., antibiotic) resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins. In an environment treated with a selection agent (drug, cytotoxic agent, etc.), only the cells expressing the selection marker can survive or exhibit other phenotypes, so the cells in which the sequence related to the selection marker is inserted into the chromosome can be selected.

[0103] The polynucleotide, target gene, target protein, target product, and expression cassette are as described above.

[0104] Other aspects provide a host cell or recombinant cell that contains at least one selected from the group consisting of the polynucleotide, expression cassette, and vector provided herein.

[0105] In this specification, the term "recombinant cell" may refer to a transformant into which a polynucleotide and / or a recombinant vector has been introduced. The term "transformation" refers to the alteration of the genetic characteristics of a host cell (microorganism) by introducing a target nucleic acid (DNA or RNA) into the host cell (microorganism). The transformation method can be carried out by selecting appropriate standard techniques known in the art according to the host cell. For example, transformation includes, but is not limited to, electroporation, liposome infection, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method, etc.

[0106] The host cell can include any one without limitation, as long as it is a cell in which the polynucleotide provided herein functions as a promoter to allow (or increase) the expression of a target gene (a gene contained in a recombinant vector or an endogenous gene of the host cell) (operatively linked). In one embodiment, the host cell can be a microorganism, a plant cell, or an animal cell.

[0107] The microorganism (or strain) of the present application can be a microorganism having the ability to produce a target product, or a microorganism having an improved (or enhanced) ability to produce a target product.

[0108] The microorganism of the present application can be a microorganism that does not naturally have the ability to produce a target product, or a microorganism that has been endowed with or has an improved ability to produce a target product, because at least one selected from the group consisting of a polynucleotide, an expression cassette, and a vector has been introduced into a microorganism having the ability to produce a target product, but is not limited thereto.

[0109] That the microorganism has an improved ability to produce a target product or has the ability to produce a target product can mean that the microorganism has an improved ability to produce a target product compared to an unmodified microorganism, a pre-recombinant cell, a parental strain, and / or a wild-type strain, or has been endowed with an ability to produce a target product different from that of an unmodified microorganism, a pre-recombinant cell, a parental strain, and / or a wild-type strain, which does not have the ability to produce a target product.

[0110] A microorganism into which at least one selected from the group consisting of a polynucleotide, an expression cassette, and a vector is introduced may have an improved target product-producing ability as compared with the microorganism before introduction or the microorganism before enhancement (i.e., a homologous non-modified microorganism). In the present text, the "non-modified microorganism" does not exclude a strain containing mutations that may occur naturally in the microorganism, and may refer to a wild-type strain or a natural strain itself, or a strain before a change in traits due to gene mutations caused by natural or artificial factors. For example, the non-modified microorganism may refer to a strain in which at least one selected from the group consisting of a polynucleotide, an expression cassette, and a vector is not introduced or a strain before introduction. The "non-modified microorganism" may be used interchangeably with "pre-modification strain", "pre-modification microorganism", "non-mutant strain", "non-modified strain", "non-mutant strain", "non-mutant microorganism", or "reference microorganism". The polynucleotide, expression cassette, and vector are as described above.

[0111] In one specific embodiment, the microorganism may be, for example, an Escherichia sp. microorganism, a Corynebacterium sp. microorganism, or a Bacillus sp. microorganism, etc. More specifically, it may be Corynebacterium glutamicum, Escherichia coli, or Bacillus subtilis, but is not limited thereto.

[0112] In one embodiment, the microorganism may be a Corynebacterium sp. microorganism, an Escherichia sp. microorganism, and / or a Bacillus sp. microorganism.

[0113] The coryneform bacterium may be at least one selected from the group consisting of: Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium alkanolyticum, Corynebacterium lilium, Corynebacterium melassecola, Corynebacterium thermoaminogenes, Corynebacterium herculis, and Corynebacterium flavescens, but is not limited thereto.

[0114] The Bacillus microorganism can be at least one selected from the group consisting of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus methylotrophicus, Bacillus licheniformis, Bacillus velezensis, Bacillus sonorensis, and Bacillus valismortis, but is not limited thereto.

[0115] The Escherichia microorganism can be Escherichia coli, but is not limited thereto.

[0116] In one embodiment, a microorganism into which at least one selected from the group consisting of a polynucleotide, an expression cassette, and a vector is introduced can be newly endowed with the ability to produce a target product, or its production ability is increased by about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 16% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, about 150% or more, about 200% or more, about 250% or more, about 300% or more, about 400% or more, about 500% or more, about 600% or more, about 700% or more, about 800% or more, about 900% or more, about 1,000% or more, about 1,500% or more, about 2,000% or more, about 2,500% or more, or about 3,000% or more as compared with the parental strain before mutation, the unmodified microorganism, the parental strain, or the wild-type microorganism, but is not limited thereto.

[0117] As another example, compared to the parental strain before mutation and non-modified microorganisms, in a microorganism with improved target product production ability, the target product production ability can be about 1.01-fold or more, about 1.02-fold or more, about 1.03-fold or more, about 1.04-fold or more, about 1.05-fold or more, about 1.06-fold or more, about 1.07-fold or more, about 1.08-fold or more, about 1.09-fold or more, about 1.1-fold or more, about 1.11-fold or more, about 1.12-fold or more, about 1.13-fold or more, about 1.14-fold or more, about 1.15-fold or more, about 1.16-fold or more, about 1.2-fold or more, about 1.3-fold or more, about 1.4-fold or more, about 1.5-fold or more, about 1.6-fold or more, about 1.7-fold or more, about 1.8-fold or more, about 1.9-fold or more, about 2-fold or more, about 2.5-fold or more, about 3-fold or more, about 4-fold or more, about 5-fold or more, about 6-fold or more, about 7-fold or more, about 8-fold or more, about 9-fold or more, about 10-fold or more, about 15-fold or more, about 20-fold or more, about 25-fold or more, or about 30-fold or more (the upper limit is not particularly limited, for example, it can be about 1,000-fold or less), but is not limited thereto.

[0118] The term "about" encompasses all ranges among ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values within a range equivalent or similar to the value after the term "about".

[0119] The polynucleotide, expression cassette, and vector are as described above.

[0120] In other aspects, there is provided a composition for producing a target product, which composition comprises a microorganism, a culture medium in which the microorganism is cultured, or a combination thereof.

[0121] The composition may further comprise any suitable excipient commonly used in a composition for producing a target product, and the excipient can be, for example, a preservative, a wetting agent, a dispersing agent, a buffering agent, a stabilizing agent, or a tonicifying agent, but is not limited thereto.

[0122] In other aspects, there is provided the use of a microorganism, a culture medium in which the microorganism is cultured, or a combination thereof in the production of a target product.

[0123] In other aspects, there is provided the use of a microorganism, a culture medium in which the microorganism is cultured, or a combination thereof in the preparation of a composition for producing a target product.

[0124] In other aspects, there is provided a method for producing a target product, which comprises culturing a microorganism in a culture medium.

[0125] The method may further include recovering the target product from the culture medium or the microorganisms produced by the culture.

[0126] The culture medium for culturing the microorganisms may or may not contain microorganisms.

[0127] As described above, the microorganisms may include at least one selected from the group consisting of the polynucleotides provided herein, expression cassettes comprising the polynucleotides and target genes, and vectors comprising the expression cassettes.

[0128] In the present specification, "culturing" refers to culturing cells under artificially controlled environmental conditions. The method for producing the target product provided in the present specification can be carried out by a method appropriately selected from all methods widely known in the art. For example, the culturing can adopt continuous culturing methods such as batch processes, fed-batch or repeated fed-batch processes, but is not limited thereto. The culture medium for culturing can meet the growth requirements of the cells cultured by an appropriate method.

[0129] In the present context, "culture medium" refers to a substance in which nutrients required for culturing microorganisms (such as Corynebacterium glutamicum strains) are mixed as the main components, and which provides nutrients and growth factors necessary for survival and development, including water. Specifically, for the culture medium and other culture conditions for culturing the microorganisms of the present application, any one can be used as long as it is a culture medium for culturing ordinary microorganisms, without particular limitation, but the microorganisms of the present application can be cultured in a common culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins, while adjusting the temperature, pH, etc. under aerobic conditions.

[0130] Specifically, for the culture medium of the microorganisms of the present application, such as Corynebacterium microorganisms, reference can be made to the document of the American Society for Bacteriology ["Manual of Methods for General Bacteriology" (Washington D.C., Corynebacterium, USA, 1981)].

[0131] The sugar sources that can be used for culturing or included in the culture medium may include at least one selected from the group consisting of sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, cellulose, etc.; oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, etc.; fatty acids such as palmitic acid, stearic acid and linoleic acid; alcohols such as glycerol, ethanol, etc.; organic acids such as acetic acid, etc., but not limited thereto. The nitrogen sources that can be used for culturing or included in the culture medium may include at least one selected from the group consisting of organic nitrogen sources such as peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal, urea, etc., and inorganic nitrogen sources such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc., but not limited thereto. The phosphorus sources that can be used for culturing or included in the culture medium may include at least one selected from the group consisting of potassium salts of phosphoric acid such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate, etc., and the corresponding sodium salts, etc., but not limited thereto. In addition to this, the culture medium may contain metal salts required for growth such as magnesium sulfate or iron sulfate. Furthermore, in addition to this, the culture or culture medium may further use or include at least one selected from essential growth substances such as amino acids and vitamins. In addition, the culture medium may contain a suitable precursor of the target product as a raw material. The above raw materials may be added to the culture medium batchwise and / or continuously during the culturing process.

[0132] During the cell culturing process, alkali compounds such as sodium hydroxide, potassium hydroxide and ammonia and / or acid compounds such as phosphates or sulfates may be used to adjust the pH of the culture in a suitable manner. In addition, during the culturing process, an antifoaming agent such as fatty acid polyethylene glycol ester may be used to inhibit the generation of foam. In addition, in order to maintain an aerobic state, oxygen or an oxygen-containing gas (such as air) may be injected into the culture. The temperature of the culture medium and / or the culture is usually 20°C to 45°C or 25°C to 40°C. The culturing time may continue until the yield of the target product reaches the required amount, for example, it may continue for about 10 to about 160 hours, but not limited thereto.

[0133] Separation or recovery of the target substance from the cultured microorganism or the culture medium can be carried out by appropriate methods known in the art according to the culturing method. For example, methods such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (such as ammonium sulfate precipitation) and / or chromatography (such as ion exchange, affinity, hydrophobic and size exclusion), but not limited thereto. The culture medium refers to the culture medium in which recombinant cells are cultured.

[0134] According to a specific embodiment, separation or recovery of the target product may be separation of the supernatant obtained by centrifuging the culture at a low speed and removal of the biomass by ion exchange chromatography.

[0135] The method for producing the target product may further include a process of purifying the target product.

[0136] Recovery of the target product from the culture (culture medium) can be carried out by separation methods through conventional means known in the art. As conventional means that can be used for separating the target product, centrifugation, filtration, chromatography, and / or crystallization methods, etc. can be exemplified. In one embodiment, the target product can be separated by ion exchange chromatography of the supernatant obtained by removing biomass from the low-speed centrifuged culture, but it is not limited thereto. The recovery can further include a purification process.

[0137] [Advantageous Effects]

[0138] The polynucleotide according to a specific embodiment has promoter activity and is introduced into a microorganism, thereby increasing the expression and activity of the gene operably linked thereto, and can be effectively used for highly efficient production of the target product affected by the polynucleotide and the gene.

[0139] [Modes of the Invention]

[0140] The present application will be described in more detail below by way of examples. It is obvious to those skilled in the art that these examples are only intended to describe the present invention more specifically, but according to the gist of the present application, the scope of the present application is not limited by these examples. Detailed Description of the Embodiments

[0141] Examples

[0142] (Throughout the specification, unless otherwise specified, "%" used to represent the concentration of a specific substance is (weight / weight)% when used for solid / solid, (weight / volume)% when used for solid / liquid, and (volume / volume)% when used for liquid / liquid.)

[0143] Example 1. Construction of a Recombinant Vector Using a Novel Promoter

[0144] To synthesize a novel promoter that induces the expression of a target gene in a microorganism, various promoter sequences of microorganisms of the genus Corynebacterium and Escherichia were analyzed. As a result of this analysis, polynucleotides containing the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 were synthesized and named Psbd, Psbe, Psbf, Psbg, Psbh, Psbi, and Psbj, respectively.

[0145] To confirm the activity of the promoter, recombinant vectors for eGFP (enhanced green fluorescent protein) expression regulated by Psbd, Psbe, Psbf, Psbg, Psbh, Psbi, Psbj or the Pbetp promoter (SEQ ID NO:8) which is usually used as a control group were generated, and their fluorescence sensitivities were compared.

[0146] First, using the pCES208 (J. Microbiol. Biotechnol., 18:639 - 647, 2008) vector, an Escherichia coli - Corynebacterium shuttle vector, an expression vector containing the eGFP gene was prepared. The nucleic acid sequence of the eGFP gene was obtained from the NIH nucleic acid sequence database of Genbank (GenBank: MN832871.1), and the ORF of the eGFP gene was amplified using the gene synthesis service of Bionics. Using the eGFP gene as a template, PCR was performed with the primer sequences of SEQ ID NO:9 and SEQ ID NO:10. Denaturation was carried out at 94°C for 5 minutes, and then repeated for 30 seconds at 94°C, annealing at 60°C for 30 seconds, and polymerization at 72°C for 30 seconds, for a total of 30 cycles, and then polymerization at 72°C for 5 minutes to perform PCR.

[0147] The amplified eGFP fragment was cloned into the pCES208 vector digested with the restriction enzyme EcoRV by Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix). The recombinant vector was named pCES208 - eGFP.

[0148] To introduce each promoter into the eGFP expression vector, the primer sequences of SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26 were used. Using the PbetP promoter (SEQ ID NO:8), synthetic Psbd (SEQ ID NO:1), Psbe (SEQ ID NO:2), Psbf (SEQ ID NO:3), Psbg (SEQ ID NO:4), Psbh (SEQ ID NO:5), Psbi (SEQ ID NO:6), and Psbj (SEQ ID NO:7) fragments as templates, PCR was performed. Denaturation was carried out at 94°C for 5 minutes, and then repeated 30 times with denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 30 seconds, followed by polymerization at 72°C for 5 minutes for PCR.

[0149] The synthetic polynucleotide sequences, the PbetP promoter used, the eGFP sequence, and the primer sequences are shown in Table 1 below.

[0150] [Table 1]

[0151]

[0152]

[0153]

[0154] By Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix), the amplified fragments were cloned into the pCES208-eGFP vector digested with the restriction enzyme EcoRV. The recombinant vectors were named pCES208-PbetP-eGFP, pCES208-Psbd-eGFP, pCES208-Psbe-eGFP, pCES208-Psbf-eGFP, pCES208-Psbg-eGFP, pCES208-Psbh-eGFP, pCES208-Psbi-eGFP, pCES208-Psbj-eGFP, respectively.

[0155] Example 2. Generation of recombinant strains containing novel promoters

[0156] The vectors generated in Example 1 were separately introduced into Corynebacterium glutamicum ATCC13032 and ATCC13869 using electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541 - 545). Then, the transformed strains were screened in Luria - Bertani (LB) medium containing 25 mg / l kanamycin. The recombinant strains screened from Corynebacterium glutamicum ATCC13032 were named ATCC13032 / pCES - eGFP, ATCC13032 / pCES - PbetP - eGFP, ATCC13032 / pCES - Psbd - eGFP, ATCC13032 / pCES - Psbe - eGFP, ATCC13032 / pCES - Psbf - eGFP, ATCC13032 / pCES - Psbg - eGFP, ATCC13032 / pCES - Psbh - eGFP, ATCC13032 / pCES - Psbi - eGFP and ATCC13032 / pCES - Psbj - eGFP, and the recombinant strains screened from Corynebacterium glutamicum ATCC13869 were named ATCC13869 / pCES - eGFP, ATCC13869 / pCES - PbetP - eGFP, ATCC13869 / pCES - Psbd - eGFP, ATCC13869 / pCES - Psbe - eGFP, ATCC10869 / pCES - Psbf - eGFP, ATCC13869 / pCES - Psbg - eGFP, ATCC13869 / pCES - Psbh - eGFP, ATCC13869 / pCES - Psbi - eGFP and ATCC13869 / pCES - Psbj - eGFP.

[0157] Example 3. Evaluation of GFP expression induction activity of novel promoters

[0158] The recombinant strains generated in Example 2 were cultured as described below, and the GFP fluorescence of the recombinant strains was measured. The experiment was repeated three times, and the average values are shown in Table 2.

[0159] The recombinant strains were separately inoculated into flasks containing 25 ml of culture solution and cultured in a shaker at 30 °C for 24 hours.

[0160] Glucose medium (pH 7.2)

[0161] 20 g of glucose, 5 g of ammonium sulfate, 5 g of yeast extract, 1.5 g of urea, 4 g of KH2PO4, 8 g of K2HPO4, 0.5 g of MgSO4·7H2O, 150 μg of biotin, 1.5 mg of thiamine hydrochloride, 3 mg of calcium pantothenate, 3 mg of nicotinamide (based on 1 L of distilled water).

[0162] Microbial cells were collected from the culture solution by centrifugation (5,000 rpm, 15 minutes), washed with 50 mM Tris-HCl (pH 8.0) buffer solution, and then resuspended in the same buffer solution. After adding 1.25 g of glass beads to each 1.5 ml of suspension, the microbial cells were lysed with a bead beater for 6 minutes, and the supernatant was collected by centrifugation (15,000 rpm, 20 minutes), and the protein concentration was quantified by the Bradford method (Bradford, M.M 1976. Anal. Biochem. 72:248 - 254). For the same amount of microbial cell extract, the method of Laure Gory et al. (FEMS Microbiology Letters 194, 127 - 133, 2001) was used to measure the expression level of the GFP gene by irradiating excitation light at 488 nm and measuring the emitted light using an LS-50B spectrophotometer (Perkin-Elmer) device.

[0163] Table 2

[0164]

[0165]

[0166] As can be confirmed in Table 2 above, all of the Psbd, Psbe, Psbf, Psbg, Psbh, Psbi, and Psbj promoters showed promoter activity and exhibited higher eGFP fluorescence sensitivity than PbetP, which is generally known as a Corynebacterium glutamicum promoter.

[0167] To confirm whether the carbon source of the medium would affect the promoter activity, the recombinant strain in Example 2 was cultured in a medium containing sucrose instead of glucose, and the experiment was carried out in the same manner, and the results are shown in Table 3 below.

[0168] Sucrose medium (pH 7.2)

[0169] 20 g of sucrose, 5 g of ammonium sulfate, 5 g of yeast extract, 1.5 g of urea, 4 g of KH2PO4, 8 g of K2HPO4, 0.5 g of MgSO4·7H2O, 150 μg of biotin, 1.5 mg of thiamine hydrochloride, 3 mg of calcium pantothenate, 3 mg of nicotinamide (based on 1 L of distilled water).

[0170] Table 3

[0171]

[0172]

[0173] As can be verified in Table 3 above, the promoters Psbd, Psbe, Psbf, Psbg, Psbh, Psbi, and Psbj show higher eGFP fluorescence sensitivity than the PbetP promoter.

[0174] This result indicates that the promoters Psbd, Psbe, Psbf, Psbg, Psbh, Psbi, and Psbj are all strong promoters that can induce the expression of target genes regardless of the type of microorganism introduced and the carbon source of the culture medium.

[0175] From the above description, those skilled in the art to which the present invention pertains can understand that the present invention can be implemented in other specific forms without changing its technical spirit or basic characteristics. In this regard, it should be understood that the above embodiments are exemplary and not restrictive in all aspects. The scope of the present invention should be interpreted to include all changes or modifications derived from the meaning and scope of the appended claims and their equivalent concepts rather than the above specific embodiments.

Claims

1. A polynucleotide comprising any one of the nucleic acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:

7.

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

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

4. A microorganism comprising the polynucleotide according to claim 1; or an expression cassette comprising the polynucleotide and a target gene.

5. The microorganism according to claim 4, wherein the microorganism produces a target product.

6. The microorganism according to claim 4, wherein the target product is at least one 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.

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

8. A method for producing a target product, comprising culturing the microorganism according to claim 4 in a medium.

9. The method for producing a target product according to claim 8, further comprising recovering the target product from the medium or the microorganism produced by the culture.

10. The method for producing a target product according to claim 8, wherein the target product is at least one 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.

11. The method for producing a target product according to claim 8, wherein the microorganism is a microorganism of the genus Corynebacterium, Escherichia or Bacillus.

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