Novel promoter and use thereof

Abstract

The present application relates to a novel promoter and a method for producing a target product using same, wherein a polynucleotide according to one embodiment has promoter activity, when introduced into a microorganism can increase the expression and activity of an actively linked gene, and can be useful for efficiently producing a target product affected by the gene and the polynucleotide.

Description

Novel promoters and their uses

[0001] Cross-citation with related application(s)

[0002] This application claims the benefit of priority to Republic of Korea Patent Application No. 10-2024-0072573, dated June 3, 2024, and Republic of Korea Patent Application No. 10-2024-0147026, dated October 24, 2024, the entire contents of which are incorporated herein by reference.

[0003] Numerous papers and patents are referenced and cited throughout this application. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the present invention.

[0004] The present application relates to a novel promoter and a method for producing a target product using the same.

[0005]

[0006] Efforts to utilize microorganisms to produce high-yield target substances, such as amino acids or other useful compounds for various applications, such as feed, pharmaceuticals, and food, through genetic manipulation and / or the introduction of exogenous genes into biosynthetic pathways have been ongoing. One such method involves inducing overexpression of target genes in microorganisms, which requires a highly efficient gene expression system. Since promoters are a key factor in determining the level and regulation of gene expression, developing a useful promoter is essential for developing expression systems.

[0007] The tac promoter derived from Escherichia coli 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 general structure of the promoter sequence for gene expression in coryneform microorganisms is not known, unlike that of other industrial microorganisms such as E. coli or Bacillus subtilis. Therefore, promoters have been developed by removing the promoter portion of an antibiotic resistance gene such as chloramphenicol, introducing chromosomal DNA isolated from coryneform microorganisms after digestion with an appropriate restriction enzyme, and then transforming coryneform microorganisms with this promoter and measuring the antibiotic resistance of the resulting strain. In addition, various promoters have been explored to overexpress foreign genes in Bacillus microorganisms, and these promoters are being used to produce enzymes for food, pharmaceutical, and other industrial purposes. Many vector systems using promoters for the expression of alpha-amylase, protease, and lipase genes from various Bacillus genus microorganisms are still being developed to this day (Schumann 2007. Adv. Appl. Microbiol. 153:813-821).

[0008] However, there is still a need for a system that exhibits high expression efficiency in various microorganisms, such as Escherichia genus microorganisms, Corynebacterium genus microorganisms, or Bacillus genus microorganisms, and thus the need for the development of a universal promoter is still emerging.

[0009]

[0010] [Prior Art Literature]

[0011] [Patent Document]

[0012] (Patent Document 1) U.S. Patent Publication (US 11,041,181 B2)

[0013] An example of the present application is to provide a novel polynucleotide. The polynucleotide may have promoter activity.

[0014] Another example is to provide an expression cassette comprising the polynucleotide and a gene of interest.

[0015] Another example is to provide a recombinant vector (expression vector) comprising the polynucleotide; or the expression cassette comprising the polynucleotide and the gene of interest.

[0016] Another object of the present application is to provide a microorganism comprising at least one member selected from the group consisting of the 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.

[0017] Another object of the present application is to provide a method for producing a target product, comprising a step of culturing the microorganism in a medium. The method may further comprise a step of recovering the target product from the medium or microorganism resulting from the cultivation, after the culturing step.

[0018] Another object of the present application is to provide a composition for producing a target product comprising the microorganism, a medium in which the microorganism is cultured, or a combination thereof.

[0019] Another object of the present application is to provide a promoter use of the polynucleotide.

[0020] Another object of the present application is to provide a use for producing a desired product using the microorganism, a medium in which the microorganism is cultured, or a combination thereof.

[0021]

[0022] Each description and embodiment disclosed in this specification may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this specification fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0023] Below, it is explained in more detail.

[0024]

[0025] One aspect of the present application provides a novel polynucleotide.

[0026] Another aspect provides for the use of the polynucleotide as a promoter.

[0027] As used herein, a “polynucleotide” comprises 2 or more, 5 or more, 10 or more, 13 or more, 20 or more, or 30 or more nucleotide monomers, wherein the nucleotide monomers may be covalently linked to form a chain.

[0028] In one example 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.

[0029] In one example, the polynucleotide may comprise a nucleic acid sequence of SEQ ID NO: 1.

[0030] In one example, the polynucleotide may comprise a nucleic acid sequence of SEQ ID NO: 2.

[0031] In one example, the polynucleotide may comprise a nucleic acid sequence of SEQ ID NO: 3.

[0032] In one example, the polynucleotide may comprise a nucleic acid sequence of SEQ ID NO: 4.

[0033] In one example, the polynucleotide may comprise a nucleic acid sequence of SEQ ID NO: 5.

[0034] In one example, the polynucleotide may comprise a nucleic acid sequence of SEQ ID NO: 6.

[0035] In one example, the polynucleotide may comprise a nucleic acid sequence of SEQ ID NO: 7.

[0036]

[0037] The polynucleotide may have promoter activity. For example, the polynucleotide may be used as a universal promoter.

[0038] In one example, the polynucleotide may have promoter activity for expression in microorganisms of the Corynebacterium genus, microorganisms of the Escherichia genus, and / or microorganisms of the Bacillus genus.

[0039] The polynucleotide according to one specific example can be utilized as a synthetic promoter having strong expression inducing activity, and can express a target gene with a significantly higher efficiency compared to known promoters (e.g., Pbetp promoter (SEQ ID NO: 8) etc.) in, for example, microorganisms of the genus Corynebacterium, microorganisms of the genus Escherichia, and / or microorganisms of the genus Bacillus.

[0040] The polynucleotide according to one embodiment may be natural or non-natural, for example, it may be non-natural, synthesized chemically or recombinantly.

[0041] As used herein, a "promoter" may refer to a DNA region that includes a binding site for a polymerase and initiates transcription of a downstream target sequence. The promoter may be located 5' of the transcription start site. The promoter may be operably and / or regulatorily linked (enhanced or weakened in expression) upstream (toward the 5' end) of the target sequence. For example, the promoter may be forwardly linked to the 5' end of any gene to enhance (increase) the expression of the gene, or reversely linked to the 3' end of any gene to weaken (decrease) the expression of the gene. When a promoter is introduced in the reverse direction at the 3' end of any gene, for example, downstream of the stop codon, or preferably between the stop codon and the transcription terminator, it can induce transcription in the opposite direction to the normal transcription direction of the gene, thereby causing collisions with the RNA polymerase complex during the transcription process, thereby attenuating the expression of the gene.

[0042] The polymerase may refer to an enzyme that synthesizes primary transcript RNA from DNA, also called RNA polymerase or DNA-dependent RNA polymerase. The polymerase may be a prokaryotic RNA polymerase or a eukaryotic RNA polymerase (e.g., RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV, or RNA polymerase V, etc.).

[0043] In this specification, the "target sequence" may be a "target gene" that is the target of expression. In one example, the target gene may be a gene encoding a target protein. The target protein may be a protein (e.g., an enzyme) involved in the production of the target product.

[0044] The term "target product" in this specification refers to a biologically active substance whose production is ultimately to be produced or whose production is to be controlled (increased or decreased) using the polynucleotide provided herein, the expression cassette containing the same, the expression vector, and / or the recombinant cell, and may refer to, for example, the target protein itself encoded by the target gene, and / or all biologically active substances produced with the participation of the target protein. The above biologically active substance means all substances produced or derived from an organism (e.g., a cell) or having a predetermined function in a living body or in a cell, for example, amino acids, amino acid derivatives, nucleic acids (nucleic acid, adenine, thymine, guanine, cytosine, uracil, etc.), nucleic acid derivatives, vitamins (vitamins 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 proteins, for example, hormones, growth factors, cytokines, immunoglobulins (antibodies), antigen proteins, receptors, ligands, functional fragments thereof (fragments having the target function), fusion proteins in which two or more are fused, etc.), sugars (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, 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, sulfoquinobose, ascorbic acid, mannitol, glucuronic acid, etc.), fatty acids (myristoleic acid, palitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linolelaidic 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.), metabolites thereof (polyhydroxyalkanoates (PHAs), etc.), precursors thereof, derivatives that maintain their biological activity, etc., but are not limited thereto.

[0045] In one example, when the target protein is involved in the production of the target product, (1) the target protein may be at least one 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, etc.), intracellular transport, and / or extracellular excretion pathway of the target product, such as synthetase, decomposition enzyme, phosphorylation enzyme, carboxylase (e.g., pyruvate carboxylase, etc.), reductase, oxidase, decarboxylase, dehydrogenase, dehydratase, transferase (e.g., transferase, epimerase, etc.), intermediate, transport protein, membrane protein (channel, etc.), but is not limited thereto.

[0046] (2) The above target gene may be a gene encoding the target protein described in (1) above.

[0047] Among the above target products, the amino acids may be proteinogenic amino acids or non-proteinogenic amino acids.

[0048] The above protein-forming amino acids may 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, and may also be intermediates thereof.

[0049] The above protein-forming amino acid may be at least one selected from the group consisting of L-amino acids, for example, 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, and specifically may be L-lysine, L-threonine, L-isoleucine, L-leucine, L-valine, L-arginine, or L-glutamic acid, but is not limited thereto. The intermediate may be, for example, O-acetyl homoserine, which is an intermediate of L-methionine, but is not limited thereto.

[0050] The above non-proteinogenic amino acids are beta-alanine, gamma-aminobutyric acid (GABA), 5-aminolevulinic acid (δ-Aminolevulinic acid), p-aminobenzoic acid (4-Aminobenzoic acid), α-aminoisobutyric acid, dehydroalanine, cystathionine, lanthionine, djenkolic acid, diaminopimelic acid, norvaline, norleucine, alloisoleucine, tert-Leucine, α-amino-n-heptanoic acid, pipecolic acid, alpha, It may be at least one selected from the group consisting of, but is not limited to, α,β-diaminopropionic acid, α,γ-diaminobutyric acid, ornithine, allothreonine, homocysteine, homoserine (or isothreonine), O-acetyl homoserine, etc.

[0051] The above amino acid may be a D-amino acid or an L-amino acid.

[0052] In one specific example, the target product may be at least one selected from the group consisting of threonine, O-acetyl homoserine, and valine.

[0053] According to one specific example, a polynucleotide having promoter activity of the present application may, in a cell, regulate (e.g., increase or decrease) the expression of a target gene operably linked thereto, the production and / or activity of a target protein encoded by the target gene, and / or the production and / or activity of a biologically active substance involved in the production of the target protein, compared to an existing promoter or a cell-endogenous promoter.

[0054] The nucleic acid sequence of the polynucleotide having the promoter activity of the present application can be modified by conventionally known mutagenesis methods, such as directed evolution and site-directed mutagenesis. That is, the polynucleotide has a biological activity identical to or corresponding to 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 has an activity of at least 60%, at least 65%, at least 70%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93% It may comprise or consist of a nucleic acid sequence having a homology or identity of at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or at least 99.9% (the upper limit may be 100% or less than 100%) to the nucleic acid sequence.In addition, if it is a polynucleotide having a biological activity substantially identical to or corresponding to 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 a sequence having the homology or identity with 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, a polynucleotide having a base sequence in which a part of the sequence is deleted, modified, substituted, or added in 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 may also be included within the scope of polynucleotides having promoter activity of the present application.

[0055] As used herein, the term 'homology' or 'identity' refers to the degree of identity with a given nucleic acid sequence or amino acid sequence, and may be expressed as a percentage (%). For example, homology may be determined by aligning sequence information and directly aligning parameters such as score, identity, and similarity between two polynucleotide molecules or two polypeptide molecules using a readily available computer program. The computer program may be BLAST (NCBI), CLC Main Workbench (CLC bio), MegAlignTM (DNASTAR Inc), etc.

[0056] In one specific embodiment, a polynucleotide comprising a specific nucleic acid sequence provided herein may be interpreted to include not only the specific nucleic acid sequence or a nucleic acid sequence substantially equivalent thereto, but also a polynucleotide fragment comprising a nucleic acid sequence complementary to the specific nucleic acid sequence. Specifically, the polynucleotide having the complementarity can be hybridized at a Tm value that can be appropriately adjusted by one 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, conditions in which polynucleotides having a 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 polynucleotides having a lower complementarity do not hybridize with each other, or washing conditions of conventional Southern hybridization: 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, conditions such as washing once, specifically two to three times, at a salt concentration and temperature equivalent to 68°C, 0.1x SSC, and 0.1% SDS, etc., can be listed, but are not limited thereto. Hybridization requires that two nucleotides have complementary sequences, but mismatches between bases may be allowed depending on the stringency of hybridization. The term "complementary" can be used to describe the relationship between nucleotide bases 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 for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, and is well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0057] According to one specific example, the polynucleotide may comprise a 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, or may be a polynucleotide consisting of or essentially comprising a 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.

[0058] In the present specification, the phrase "a polynucleotide or polypeptide comprises a specific nucleic acid sequence or amino acid sequence" may mean that the polynucleotide or polypeptide consists of or essentially includes the specific nucleic acid sequence or amino acid sequence, and may be interpreted as including (or not excluding) a "substantially equivalent sequence" in which a mutation (deletion, substitution, modification, and / or addition) is added to the specific nucleic acid sequence or amino acid sequence within the scope of maintaining the original function and / or desired function of the polynucleotide or polypeptide. In one example, the nucleic acid sequence or amino acid sequence provided herein may include one that has been modified by a conventional mutagenesis method, such as directed evolution and / or site-directed mutagenesis, within the scope of maintaining the original function or desired function thereof. In one embodiment, a polynucleotide or polypeptide "comprises a particular nucleic acid sequence or amino acid sequence" means that the polynucleotide or polypeptide (i) consists of or essentially comprises the particular nucleic acid sequence or amino acid sequence, or (ii) is at least 60%, at least 65%, at least 70%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, 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.It may mean that it consists of or essentially contains an amino acid sequence having a homology or identity of 9% or more (the upper limit may be 100% or less than 100%) and that it maintains the original function and / or the intended function.

[0059] According to one specific example, the original function and / or the intended function of the polynucleotide provided in the present application may be a function as a promoter. The polynucleotide may be operably linked to a target gene as a promoter, and during the linkage, additions, deletions, and / or substitutions of nucleotides for use with restriction enzymes may be introduced at the 5' end and / or 3' end of the polynucleotide. The target gene may be a gene encoding a target protein, and the target protein may be involved in the production of the target product as described above, examples of which are as follows.

[0060] In one example, the protein involved in the production of arginine among the amino acids may be at least one selected from proteins involved in arginine biosynthesis (arginine biosynthesis proteins). For example, the protein involved in arginine biosynthesis may be, but is not limited to, N-acetylglutamic acid synthetase (argA), N-acetylglutamic acid kinase (argB), N-acetylglutamyl phosphate reductase (argC), acetylornithine transaminase (argD), acetylornithine deacetylase (argE), ornithine carbamoyltransferase (argF, argI), argininosuccinate synthetase (argG), argininosuccinate lyase (argH), ornithine acetyltransferase (argJ), and carbamoyl phosphate synthetase (carAB).

[0061] In one example, the protein involved in the production of histidine among the amino acids may be at least one selected from proteins involved in histidine biosynthesis (histidine biosynthesis proteins). For example, the protein involved in histidine biosynthesis may be, but is not limited to, ATP phosphoribosyl transferase (hisG), phosphoribosyl-AMP cyclohydrolase (hisI), phosphoribosyl-ATP pyrohydrolase (hisI), phosphoribosylformimino-5-aminoimidazocarboxamidribotide isomerase (hisA), amide transferase (hisH), histidine phosphate aminotransferase (hisC), histidine phosphatase (hisB), and histidine dehydrogenase (hisD).

[0062] In one example, the protein involved in the production of lysine among the amino acids may be one or more selected from proteins involved in lysine biosynthesis (lysine biosynthesis proteins). For example, the proteins involved in the above lysine biosynthesis are 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 ( The enzyme may be at least one selected from the group consisting of, but is not limited to, tetrahydrodipicolinate succinylase (dapF), tetrahydrodipicolinate succinylase (dapD), succinyl-diaminopimelate deacylase (dapE), and aspartase (aspA).

[0063] In one example, the protein involved in the production of aspartic acid among the amino acids may be at least one selected from proteins involved in aspartic acid biosynthesis (aspartic acid biosynthesis 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 amino acids may be at least one selected from proteins involved in glutamic acid biosynthesis (glutamic acid biosynthesis proteins). For example, the proteins involved in the production of glutamic acid include glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthetase (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), enolase (eno), phosphoglycero mutase (pgmA, pgmI), phosphoglycerate kinase (pgk), It may be at least one selected from the group consisting of glyceraldehyde 3-phosphate dehydrogenase (gapA), triose phosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp), phosphofructokinase (pfkA, pfkB), glucose phosphate isomerase (pgi), 6-phosphogluconic acid dehydratase (edd), 2-keto-3-deoxy-6-phosphogluconic acid aldolase (eda), transhydrogenase, etc., but is not limited thereto.

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

[0066] In one example, the protein involved in the production of threonine among the amino acids may be at least one selected from proteins involved in threonine biosynthesis (threonine biosynthesis proteins). For example, the protein involved in the biosynthesis of threonine may be, but is not limited to, aspartokinase III (lysC), aspartic semialdehyde dehydrogenase (asd), aspartokinase 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 amino acids may be at least one selected from proteins involved in asparagine biosynthesis (asparagine biosynthesis proteins). For example, the protein involved in the biosynthesis of asparagine may be, but is not limited to, transaminase, asparagine synthetase, etc.

[0068] In one example, the protein involved in the production of glutamine among the amino acids may be at least one selected from proteins involved in glutamine biosynthesis (glutamine biosynthesis system proteins). For example, the protein involved in glutamine biosynthesis may be at least one selected from the group consisting of glutamate dehydrogenase (gdhA), glutamate thetase (glnA), and the like, but is not limited thereto.

[0069] In one example, the protein involved in the production of cysteine ​​among the amino acids may be at least one selected from among proteins involved in cysteine ​​biosynthesis (cysteine ​​biosynthetic proteins). For example, the protein involved in cysteine ​​biosynthesis may be at least one selected from the group consisting of serine acetyltransferase (cysE), 3-phosphoglycerate dehydrogenase (serA), and the like, but is not limited thereto.

[0070] In one example, the protein involved in the production of glycine among the amino acids may be at least one selected from 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 amino acids may be at least one selected from the proteins involved in proline biosynthesis (proline biosynthesis proteins). For example, the protein involved in the biosynthesis of proline may 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 example, the protein involved in the production of alanine among the amino acids may be at least one selected from the proteins involved in alanine biosynthesis (alanine biosynthesis proteins). For example, the protein involved in the biosynthesis of alanine may be at least one selected from the group consisting of alanine transaminase, etc., but is not limited thereto.

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

[0073] In one example, the protein involved in the production of isoleucine among the amino acids may be at least one selected from proteins involved in isoleucine biosynthesis (isoleucine biosynthesis proteins). For example, the protein involved in the biosynthesis of isoleucine may be, but is 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 amino acids may be at least one selected from proteins involved in leucine biosynthesis (leucine biosynthesis proteins). For example, the protein involved in the biosynthesis of leucine may be, but is not limited to, acetolactate synthase, acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, α-isopropylmalic acid synthase, α-isopropylmalate isomerase, leucine aminotransferase, etc.

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

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

[0077] In one example, the protein involved in the production of tyrosine among the amino acids may be at least one selected from proteins involved in tyrosine biosynthesis (tyrosine biosynthesis proteins). For example, the protein involved in the biosynthesis of tyrosine may be, but is not limited to, chorismate mutase, prephenate aminotransferase, arogenate dehydrogenase, etc.

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

[0079] In one example, the protein involved in the production of O-acetyl homoserine among the amino acids may be at least one selected from proteins 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-acetyl transferase.

[0080] In one example, the protein involved in the production of beta-alanine among the amino acids may be at least one selected from proteins involved in beta-alanine biosynthesis. For example, the protein involved in the biosynthesis of beta-alanine 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, beta alanine-pyruvate transaminase, etc.

[0081] The gene involved in the production of the above nucleic acid may be one or more genes selected from nucleic acid biosynthesis 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), and phosphoribosylaminoimidazole carboxylase. (phosphoribosylaminoimidazole carboxylase, purK), but is not limited thereto.

[0082] In one specific example, the target gene may be at least one selected from the group consisting of a pyc gene, a metX gene, and an ilvE gene.

[0083] According to one specific example, the pyc gene encodes pyruvate carboxylase, and according to one specific example, the polynucleotide and the pyc gene are operably linked and can be used to produce L-threonine. The pyc gene may be a gene derived from Corynebacterium glutamicum.

[0084] According to one specific example, the metX gene encodes homoserine O-acetyltransferase, and according to one specific example, the polynucleotide and the metX gene are operably linked to each other and can be used to produce O-acetyl homoserine. The metX gene may be a gene derived from Corynebacterium glutamicum.

[0085] According to one specific example, the ilvE gene encodes a branched-chain amino acid aminotransferase, and according to one specific example, the polynucleotide and the ilvE gene are operably linked to each other and can be used for producing valine. The ilvE gene may be a gene derived from Corynebacterium glutamicum.

[0086]

[0087] Another aspect provides an expression cassette comprising the polynucleotide and the target gene provided in the present application.

[0088] The above target gene may be a gene encoding a target protein, and the target protein may be a protein (e.g., an enzyme) involved in the production of the target product.

[0089] The above target product may be at least one 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.

[0090] The above expression cassette may comprise the above polynucleotide as a promoter.

[0091] As used herein, the term “expression cassette” may mean a structure that is the smallest unit of nucleic acid fragment capable of being expressed in a host cell and includes at least a target gene and an expression control sequence (e.g., a promoter).

[0092] The above polynucleotide may be included in the 5' end or 3' end region of the target gene.

[0093] The above polynucleotide may be operably linked to the target gene.

[0094] The polynucleotide, target gene, target protein and target product are as described above.

[0095]

[0096] Another aspect provides a vector comprising the polynucleotide or the expression cassette provided in the present application.

[0097] The above vector may be a recombinant vector.

[0098] The above recombinant vector may be an expression vector or an insertion vector.

[0099] The above vector may comprise the above polynucleotide as a promoter.

[0100] According to one specific example, the vector may comprise a polynucleotide having the promoter activity and a target gene operably linked to the polynucleotide. The target gene may be a gene encoding a target protein, and the target protein may be a protein (e.g., an enzyme) involved in the production of the target product.

[0101] According to one specific example, the target protein may be a single protein (a single protein) or a fusion protein comprising two or more proteins. If the target protein is a fusion protein comprising two or more proteins, the target protein encoding gene may be a fusion gene comprising genes encoding each of the two or more proteins.

[0102] According to one specific example, in the recombinant vector, when the target protein encoding gene is a fusion gene comprising genes each encoding two or more proteins, all genes included in the fusion gene may be designed to be under the control of one polynucleotide (promoter), or one or more of them may be under the control of a separate polynucleotide. For example, the vector may comprise a polynucleotide having the promoter activity and one gene or two or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) genes operably linked to the polynucleotide, and when comprising two or more genes, the vector may comprise one polynucleotide having the promoter activity (i.e., the two or more genes are under the control of one promoter), or may comprise two or more (in which case, the polynucleotide may comprise less than or equal to the number of genes such that at least one of the two or more genes is under the control of a separate promoter).

[0103] As used herein, the term "vector" refers to a DNA molecule for delivering a nucleic acid of a target sequence into a suitable host or cell, and may include a suitable gene expression control sequence; and, optionally, a nucleic acid sequence of a target gene operably linked thereto. The "gene expression control sequence" refers to elements that perform various regulatory functions in gene expression, and may include, for example, a polynucleotide having promoter activity provided herein, and may refer to a nucleic acid sequence capable of expressing a target gene operably linked thereto. Specifically, the gene expression control sequence may include, but is not limited to, a promoter for transcription of a gene, an arbitrary operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and / or a nucleic acid sequence for regulating the termination of transcription and translation. In addition, the gene expression control sequence may include, but is not limited to, a promoter and a ribosome binding site as regulatory sequences suitable for prokaryotes. A person skilled in the art can construct the above gene expression control sequence comprising a polynucleotide having the promoter activity of the present application as needed.

[0104] As used herein, "operably linked" means that a polynucleotide having the promoter activity is functionally linked to a nucleotide sequence of a target gene so as to initiate and / or mediate transcription of the target gene. Operable linking can be performed using genetic recombination techniques known in the art, such as site-specific DNA cleavage and / or ligation techniques, and the site-specific DNA cleavage and ligation can be performed using conventional cleavage enzymes and / or ligation enzymes, but is not limited thereto.

[0105] The above vector is not particularly limited as long as it is capable of transforming into a host cell and / or expressing in a host cell. Examples of the above vector include plasmids, cosmids, viruses and / or bacteriophages in a natural or recombinant state. For example, as a phage vector or cosmid vector, one or more selected from the group consisting of pWE15, M13, λLB3, λBL4, λⅨII, λASHII, λAPII, λt10, λt11, Charon4A, Charon21A, etc. may be used, and as a plasmid vector, one or more selected from the group consisting of pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, pET series, etc. may be used.

[0106] In one example, an endogenous promoter in 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 vector for chromosomal insertion into a host cell including the polynucleotide having the promoter activity, and examples thereof include, but are not limited to, one or more selected from the group consisting of pECCG117, pDZ, pDC, pACYC177, pACYC184, pCL, pUC19, pBR322, pMW118, pCC1BAC, pCES208, and pXMJ19 vectors. In addition, the insertion of the polynucleotide into the chromosome can be performed by any method known in the art, for example, homologous recombination, genome editing by a target-specific endonuclease (e.g., RNA-guided endonuclease such as Cas9 or Cpf1), etc.

[0107] In one example, if the vector or the polynucleotide having the promoter activity contained therein and / or the target gene is inserted into a chromosome, the vector may further include a selection marker to confirm whether the chromosome has been inserted. The selection marker may be a marker that confers a selectable phenotype, such as drug (e.g., antibiotic) resistance, nutrient requirement, cytotoxic agent resistance, or expression of a surface protein. In an environment treated with a selective agent (e.g., drug, cytotoxic agent, etc.), only cells expressing the selection marker survive or exhibit other phenotypic characteristics, so that cells in which the selection marker-related sequence has been inserted into the chromosome can be selected.

[0108] The polynucleotide, target gene, target protein, target product, and expression cassette are as described above.

[0109]

[0110] Another aspect provides a host cell or recombinant cell comprising at least one selected from the group consisting of the polynucleotide provided in the present application, the expression cassette and the vector.

[0111] In this specification, the term "recombinant cell" may refer to a transformant into which the polynucleotide and / or the recombinant vector has been introduced. The term "transformation" refers to changing 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 may be performed by selecting a suitable standard technique known in the relevant field depending on the host cell. For example, the transformation may be performed by, 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.

[0112] The above host cell may include, without limitation, any cell capable of (or increasing) expression of a target gene (a gene included in a recombinant vector or a host cell-endogenous gene) (operably linked) by the polynucleotide provided in the present application acting as a promoter. In one example, the host cell may be a microorganism, a plant cell, or an animal cell.

[0113] The microorganism (or strain) of the present application may be a microorganism having the ability to produce a target product or having an improved (or increased) ability to produce a target product.

[0114] The microorganism of the present application may be a microorganism that naturally does not have the ability to produce the target product, or a microorganism that has the ability to produce the target product, but is not limited thereto, by introducing at least one selected from the group consisting of the polynucleotide, the expression cassette, and the vector into a microorganism that has the ability to produce the target product, thereby imparting or improving the ability to produce the target product.

[0115] The fact that the above microorganism has an improved ability to produce a target product or has an ability to produce a target product may mean that the microorganism has an improved ability to produce a target product compared to a non-modified microorganism, a cell before recombination, a parent strain, and / or a wild-type strain, or that the microorganism has been granted an ability to produce a target product unlike a non-modified microorganism, a cell before recombination, a parent strain, and / or a wild-type strain that does not have an ability to produce a target product.

[0116] A microorganism into which at least one selected from the group consisting of the polynucleotide, the expression cassette, and the vector has been introduced may have an improved ability to produce a target product compared to a microorganism before introduction or a microorganism before enhancement, i.e., an unmodified microorganism of the same species. In the present application, the term "unmodified microorganism" does not exclude a strain containing a mutation that may occur naturally in a microorganism, and may refer to a wild-type strain or a natural strain itself, or a strain before its characteristics have been changed due to a genetic mutation caused by natural or artificial factors. For example, the unmodified microorganism may refer to a strain into which at least one selected from the group consisting of the polynucleotide, the expression cassette, and the vector has not been introduced, or before it has been introduced. The term "unmodified microorganism" may be used interchangeably with "pre-modified strain," "pre-modified microorganism," "unmutated strain," "unmodified microorganism," or "reference microorganism." The polynucleotide, the expression cassette, and the vector are as described above.

[0117] In one specific example, the microorganism may be a microorganism of the genus Escherichia sp., Corynebacterium sp., or Bacillus sp., and more specifically, may be, but is not limited to, Corynebacterium glutamicum, Escherichia coli, or Bacillus subtilis.

[0118] In one example, the microorganism may be a microorganism of the genus Corynebacterium, a microorganism of the genus Escherichia, or a microorganism of the genus Bacillus.

[0119] The above-mentioned Corynebacterium genus microorganisms are Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, and Corynebacterium pollutisoli. Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium alkanolyticum, Corynebacterium lilium, Corynebacterium melassecola, Corynebacterium thermoaminogenes, Corynebacterium herculis, and Corynebacterium flavescens. There may be one or more types selected from the group formed, butBut it is not limited to this.

[0120] The above Bacillus genus microorganism may 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.

[0121] The above Escherichia genus microorganism may be, but is not limited to, Escherichia coli.

[0122] For example,

[0123] A microorganism into which at least one selected from the group consisting of the polynucleotide, the expression cassette and the vector is introduced has a newly granted ability to produce a target product, or is 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, or about It may be increased by, but is not limited to, 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.

[0124] As another example, the microorganism with improved target product production ability has a target product production ability of about 1.01 times or more, about 1.02 times or more, about 1.03 times or more, about 1.04 times or more, about 1.05 times or more, about 1.06 times or more, about 1.07 times or more, about 1.08 times or more, about 1.09 times or more, about 1.1 times or more, about 1.11 times or more, about 1.12 times or more, about 1.13 times or more, about 1.14 times or more, about 1.15 times or more, about 1.16 times or more, about 1.2 times or more, about 1.3 times or more, about 1.4 times or more, about 1.5 times or more, about 1.6 times or more, about 1.7 times or more, about 1.8 times or more, about 1.9 times or more, about It may be, but is not limited to, 2 times or more, about 2.5 times or more, about 3 times or more, about 4 times or more, about 5 times or more, about 6 times or more, about 7 times or more, about 8 times or more, about 9 times or more, about 10 times or more, about 15 times or more, about 20 times or more, about 25 times or more, or about 30 times or more (the upper limit is not particularly limited, and may be, for example, about 1,000 times or less).

[0125] The term “about” above includes all ranges including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all ranges of values ​​equal to or similar to the value following the term “about,” but is not limited thereto.

[0126] The polynucleotide, the expression cassette, and the vector are as described above.

[0127]

[0128] Another aspect provides a composition for producing a target product comprising the microorganism, a medium in which the microorganism is cultured, or a combination thereof.

[0129] The composition may further comprise any suitable excipients commonly used in compositions for producing the desired product, and such excipients may be, for example, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizers, or isotonic agents.

[0130] Another aspect provides a use of the microorganism, the medium in which the microorganism is cultured, or a combination thereof, for producing a desired product.

[0131] Another aspect provides a use of the microorganism, the medium in which the microorganism is cultured, or a combination thereof, for preparing a composition for producing a desired product.

[0132] Another aspect provides a method for producing a target product, comprising the step of culturing the microorganism in a medium.

[0133] The above method may further include a step of recovering the target product from the medium or microorganism according to the above culture.

[0134] The medium in which the above microorganism is cultured may or may not contain the above microorganism.

[0135] The above microorganism may include at least one selected from the group consisting of a polynucleotide provided in the present application, an expression cassette including the polynucleotide and a target gene, and a vector including the expression cassette, as described above.

[0136] As used herein, "cultivation" refers to growing cells under artificially controlled environmental conditions. The method for producing the target product provided herein can be performed by any method appropriately selected from among all methods widely known in the art. For example, the cultivation can be continuously cultured in a batch process, a fed batch process, or a repeated fed batch process, but is not limited thereto. The medium used for the cultivation can be one that satisfies the growth requirements of the target cells in an appropriate manner.

[0137] In the present application, "medium" means a material containing nutrients as a main component necessary for culturing the microorganism, for example, a Corynebacterium glutamicum strain, and supplies nutrients and growth factors, including water essential for survival and growth. Specifically, the medium and other culture conditions used for culturing the microorganism of the present application may be any medium used for culturing general microorganisms without particular limitation, but the microorganism of the present application may be cultured under aerobic conditions while controlling temperature, pH, etc. in a general medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin.

[0138] Specifically, culture media for the microorganisms of the present application, such as strains of the genus Corynebacterium, can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D.Corynebacterium, USA, 1981)].

[0139] The sugar source that can be used for the above culture or included in the medium may include at least one selected from the group consisting of sugars and carbohydrates such as glucose, saccharose, 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, linoleic acid; alcohols such as glycerol, ethanol, etc.; organic acids such as acetic acid, etc., but is not limited thereto. The nitrogen source that can be used for the above culture or included in the medium may include at least one selected from the group consisting of organic nitrogen sources such as peptone, yeast extract, meat juice, 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 is not limited thereto. The phosphorus that may be used for the above culture or included in the medium may include, but is not limited to, one or more selected from the group consisting of potassium salts of phosphoric acid such as potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and their corresponding sodium salts. In addition, the medium may contain metal salts such as magnesium sulfate or iron sulfate necessary for growth. In addition to the above, the culture or medium may additionally use or include one or more selected from essential growth substances such as amino acids and vitamins. In addition, the medium may contain an appropriate precursor of the desired product as a raw material. The above-mentioned raw materials may be added to the culture in a batch and / or continuous manner in an appropriate manner during the culture process.

[0140] During the culture of the above cells, the pH of the culture can be adjusted by appropriately using basic compounds such as sodium hydroxide, potassium hydroxide, and ammonia, and / or acid compounds such as phosphoric acid or sulfuric acid. In addition, during the culture, an antifoaming agent such as fatty acid polyglycol ester can be used to suppress bubble formation. In addition, oxygen or an oxygen-containing gas (e.g., air) can be injected into the culture to maintain an aerobic state. The temperature of the medium and / or culture can be typically set to 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.

[0141] The step of isolating or recovering the target substance from the cultured microorganism or culture medium can be performed using a suitable method known in the art depending on the culture method. For example, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g., ammonium sulfate precipitation), and / or chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion) can be used, but is not limited thereto. The culture medium refers to a medium in which recombinant cells are cultured.

[0142] According to one specific example, the step of isolating or recovering the target product may include removing biomass by low-speed centrifugation of the culture and separating the obtained supernatant through ion exchange chromatography.

[0143] The method for producing the above target product may additionally include a process for purifying the target product.

[0144] The recovery of the target product from the culture medium (medium) can be performed by a separation method using conventional means known in the art. Examples of conventional means available for the separation of the target product include centrifugation, filtration, chromatography, and / or crystallization. In one example, the culture medium may be centrifuged at low speed to remove biomass, and the resulting supernatant may be subjected to ion exchange chromatography to separate the target product, but is not limited thereto. The recovery step may additionally include a purification process.

[0145]

[0146] A polynucleotide according to one specific example has promoter activity and can increase the expression and activity of a gene introduced into a microorganism and operably linked thereto, and can be usefully utilized to efficiently produce a target product influenced by the polynucleotide and the gene.

[0147]

[0148] Hereinafter, the present application will be described in more detail through examples. These examples are intended solely to illustrate the present application more specifically, and it will be apparent to those skilled in the art that the scope of the present application is not limited by these examples, in accordance with the gist of the present application.

[0149]

[0150] Example

[0151]

[0152] (Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight) % for solid / solid, (weight / volume) % for solid / liquid, and (volume / volume) % for liquid / liquid, unless otherwise stated.)

[0153]

[0154] Example 1. Construction of a recombinant vector using a novel promoter

[0155] In order to synthesize a novel promoter that induces target gene expression in a microorganism, various promoter sequences of microorganisms of the genus Corynebacterium and the genus Escherichia were analyzed. As a result of this analysis, polynucleotides comprising 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 were named Psbd, Psbe, Psbf, Psbg, Psbh, Psbi, and Psbj, respectively.

[0156] To confirm the activity of the above promoter, eGFP (enhanced green fluorescent protein) expression recombinant vectors regulated by Psbd, Psbe, Psbf, Psbg, Psbh, Psbi, Psbj or the previously known Pbetp promoter (SEQ ID NO: 8) as a control were prepared, and their fluorescence sensitivities were compared.

[0157]

[0158] First, an expression vector containing the eGFP gene was constructed using the pCES208 vector, which is an E. coli-Corynebacterium shuttle vector (J. Microbiol. Biotechnol., 18:639-647, 2008). 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. PCR was performed using the eGFP gene as a template and the primer sequences of SEQ ID NO: 9 and SEQ ID NO: 10. The PCR was performed at 94°C for 5 minutes, followed by 30 cycles of 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.

[0159]

[0160] The amplified eGFP fragment was cloned into the pCES208 vector cut with the restriction enzyme EcoRV using the 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.

[0161] To introduce each promoter into the eGFP expression vector, PCR was performed using the PbetP promoter (SEQ ID NO: 8), the synthesized 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, and 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, respectively. PCR was performed by denaturing at 94°C for 5 minutes, then repeating 30 cycles of 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.

[0162] The synthesized polynucleotide sequence, the PbetP promoter used, the eGFP sequence, and the primer sequences are shown in Table 1 below.

[0163]

[0164] The amplified fragments were cloned into the pCES208-eGFP vector cut with the restriction enzyme EcoRV using the Gibson Assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix). 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, and pCES208-Psbj-eGFP, respectively.

[0165] Example 2. Production of a recombinant strain containing a novel promoter

[0166] The vector prepared in Example 1 was introduced into Corynebacterium glutamicum ATCC13032 and ATCC13869 using electroporation (Appl. Microbiol. Biotechnol. (1999) 52: 541-545), respectively. The transformed strains were then selected on Luria-Bertani (LB) medium containing 25 mg / l of kanamycin. The recombinant strains selected 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, respectively, and the recombinant strains selected 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, They were named ATCC13869 / pCES-Psbi-eGFP and ATCC13869 / pCES-Psbj-eGFP.

[0167]

[0168] Example 3. Evaluation of the GFP expression induction activity of the novel promoter.

[0169] The recombinant strain produced in Example 2 was cultured as described below, and the GFP fluorescence of the recombinant strain was measured. The experiment was repeated three times, and the average values ​​are shown in Table 2.

[0170] The recombinant strains were each inoculated into flasks containing 25 ml of culture medium and cultured in a shaking incubator at 30°C for 24 hours.

[0171]

[0172] Glucose medium (pH 7.2)

[0173] Glucose 20 g, ammonium sulfate 5 g, yeast extract 5 g, urea 1.5 g, KH2PO44 g, K2HPO48 g, MgSO47 & H2O 0.5 g, biotin 150 μg, thiamine hydrochloride 1.5 mg, calcium pantothenate 3 mg, nicotinamide 3 mg (based on 1 L distilled water)

[0174]

[0175] The cells were collected from the culture medium by centrifugation (5,000 rpm, 15 min), washed with 50 mM Tris-HCl (pH 8.0) buffer, and resuspended in the same buffer. After adding 1.25 g of glass beads per 1.5 ml of the suspension, the cells were disrupted using a bead beater for 6 minutes, and the supernatant was collected by centrifugation (15,000 rpm, 20 min), and the protein concentration was quantified by 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 mycelial extract with excitation light at 488 nm and measuring the emission light at 511 nm using a LS-50B spectrophotometer (Perkin-Elmer) using the method of Laure Gory et al. (FEMS Microbiology Letters 194, 127-133, 2001).

[0176] Strain promoter fluorescence sensitivity Relative fluorescence sensitivity (%)ATCC13032 / pCES208-eGFPNegative control9846.854.7ATCC13032 / pCES208-PbetP-eGFPbetP17985.4100.0ATCC13032 / pCES208-Psbd-eGFPsbd35335 .9196.5ATCC13032 / pCES208-Psbe-eGFPsbe27121.6150.8ATCC13032 / pCES208-Psbf-eGFPsbf25677.2142.8ATCC1 3032 / pCES208-Psbg-eGFPsbg23919.8133.0ATCC13032 / pCES208-Psbh-eGFPsbh21727.7120.8ATCC13032 / pCES208 -Psbi-eGFPsbi19352.2107.6ATCC13032 / pCES208-Psbj-eGFPsbj19084.7106.1ATCC13869 / pCES208-eGFPNegative control8233.646.1ATCC13869 / pCES208-PbetP-eGFPbetP17850.8100.0ATCC13869 / pCES208-Psbd-eGFP sbd25047.1140.3ATCC13869 / pCES208-Psbe-eGFPsbe21439.9120.1ATCC13869 / pCES208-Psbf-eGFPsbf19 173.1107.4ATCC13869 / pCES208-Psbg-eGFPsbg21283.7119.2ATCC13869 / pCES208-Psbh-eGFPsbh18075.5 101.3ATCC13869 / pCES208-Psbi-eGFPsbi18829.5105.5ATCC13869 / pCES208-Psbj-eGFPsbj18190.0101.9

[0177] As can be confirmed in Table 2 above, the Psbd, Psbe, Psbf, Psbg, Psbh, Psbi, and Psbj promoters all exhibited promoter activity in Corynebacterium glutamicum strains, and exhibited higher eGFP fluorescence sensitivity than PbetP, which is known as a promoter of Corynebacterium glutamicum.

[0178] To determine whether the carbon source of the medium affects promoter activity, the recombinant strain of Example 2 was cultured in a medium containing sucrose instead of glucose, and the experiment was performed in the same manner as above, and the results are shown in Table 3 below.

[0179]

[0180] Sucrose medium (pH 7.2)

[0181] Sucrose 20 g, ammonium sulfate 5 g, yeast extract 5 g, urea 1.5 g, KH2PO44 g, K2HPO48 g, MgSO47 & H2O 0.5 g, biotin 150 μg, thiamine hydrochloride 1.5 mg, calcium pantothenate 3 mg, nicotinamide 3 mg (based on 1 L distilled water)

[0182]

[0183] Strain promoter fluorescence sensitivity Relative fluorescence sensitivity (%)ATCC13032 / pCES208-eGFPNegative control7520.437.07ATCC13032 / pCES208-PbetP-eGFPbetP20285.0100.00ATCC13032 / pCES208-Psbd-eGFPsbd31496 .2155.27ATCC13032 / pCES208-Psbe-eGFPsbe28014.5138.10ATCC13032 / pCES208-Psbf-eGFPsbf26800.1132.12ATCC1 3032 / pCES208-Psbg-eGFPsbg26644.7131.35ATCC13032 / pCES208-Psbh-eGFPsbh24894.0122.72ATCC13032 / pCES208 -Psbi-eGFPsbi22010.2108.50ATCC13032 / pCES208-Psbj-eGFPsbj21477.7105.88ATCC13869 / pCES208-eGFPNegative control6934.743.33ATCC13869 / pCES208-PbetP-eGFPbetP16004.5100.00ATCC13869 / pCES208-Psbd-eGFPs bd25693.6160.54ATCC13869 / pCES208-Psbe-eGFPsbe20083.1125.48ATCC13869 / pCES208-Psbf-eGFPsbf204 26.1127.63ATCC13869 / pCES208-Psbg-eGFPsbg19871.9124.16ATCC13869 / pCES208-Psbh-eGFPsbh18796.81 17.45ATCC13869 / pCES208-Psbi-eGFPsbi18604.3116.24ATCC13869 / pCES208-Psbj-eGFPsbj17821.1111.35

[0184] As can be seen in Table 3 above, the Psbd, Psbe, Psbf, Psbg, Psbh, Psbi, and Psbj promoters showed higher eGFP fluorescence sensitivity than the PbetP promoter.

[0185] These results indicate that the Psbd, Psbe, Psbf, Psbg, Psbh, Psbi, and Psbj promoters are strong promoters that induce the expression of target genes regardless of the type of microorganism introduced and the carbon source of the medium.

[0186]

[0187] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A polynucleotide comprising any one nucleic acid sequence selected from the group consisting of sequence number 1, sequence number 2, sequence number 3, sequence number 4, sequence number 5, sequence number 6, and sequence number 7.

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

3. An expression cassette comprising the polynucleotide of paragraph 1 and the target gene.

4. The polynucleotide of paragraph 1; or A microorganism comprising an expression cassette comprising the above polynucleotide and a target gene.

5. A microorganism according to paragraph 4, wherein the microorganism produces a target product.

6. In paragraph 5, the target product is a microorganism 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. In the fourth paragraph, the microorganism is a microorganism of the genus Corynebacterium, a microorganism of the genus Escherichia, or a microorganism of the genus Bacillus.

8. A method for producing a target product, comprising a step of culturing the microorganism of clause 4 in a medium.

9. A method for producing a target product, further comprising a step of recovering the target product from a medium or microorganism according to the culture in paragraph 8.

10. A method for producing a target product in 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. A method for producing a target product in claim 8, wherein the microorganism is a microorganism of the genus Corynebacterium, a microorganism of the genus Escherichia, or a microorganism of the genus Bacillus.