Novel promoter variants of bacillus and methods of producing proteins using the same

Abstract

The present application relates to a novel Bacillus promoter variant and a protein production method using the same. When the novel Bacillus promoter variant of the present application is used, expression of a target protein in a host cell can be significantly increased, and thus the novel Bacillus promoter variant can be effectively used to produce proteins useful in biotechnology, medicine, and industry by mass-producing proteins and target proteins such as polypeptide drugs.

Description

Technical Field

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority based on Korean Patent Application No. 10-2024-0044220, filed on April 1, 2024, and the entire contents disclosed in the corresponding Korean patent application are incorporated in this specification.

[0002] This application references numerous papers and patent documents, and their citations are acknowledged. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the level of the technical field to which this application pertains and the content of this application.

[0003] This application relates to novel Bacillus promoter variants and methods for protein production using them. Background Technology

[0004] Bacillus is an industrial microorganism suitable as a host for the production of proteins used in traditional fermented foods and pharmaceuticals. The advantages of Bacillus as a protein production host include its status as a GRAS (Generally Recognized As Safe) microorganism, meaning it is harmless and safe for humans; its superior ability to secrete intracellularly biosynthesized proteins extracellularly compared to other microorganisms; and its lack of endotoxin production. Furthermore, Bacillus possesses well-developed genomic information, genetic manipulation methods and materials, and a wealth of accumulated biological research data, making it easy to develop hosts for large-scale protein culture and production. Industrial enzymes produced using Bacillus as a host account for more than half of the global market, and various Bacillus strains and expression systems are being actively developed to increase the productivity of these enzymes.

[0005] The most crucial step in increasing protein production in microorganisms is the transcription of the target gene, and protein yield varies depending on promoter activity. The promoter is located upstream of the structural gene's nucleotide sequence and initiates transcription for mRNA production when RNA polymerase binds to the promoter region. Promoter strength is a key factor determining the amount of mRNA produced by RNA polymerase; the efficiency with which RNA polymerase binds to the promoter to rapidly and stably initiate transcription determines the level of protein production within the cell. In other words, a strong promoter increases the expression ratio of the target protein in the total protein produced by the host cell and facilitates the isolation and purification of the target protein. Therefore, ensuring a highly active promoter is essential for developing efficient protein production systems. Summary of the Invention

[0006] [Technical Issues] One aspect of this application provides a promoter variant in which Bacillus subtilis (Bacillus subtilis )of aprE The promoter variant has its -35 region replaced by TTGACA and its -10 region replaced by TATAAT. The promoter variant includes a spacer region between the -35 and -10 regions, the spacer region being 19 bp or 17 bp in length. The TAT at the 3' end of the spacer region is replaced by TGN, and N is selected from A, T, G, or C.

[0007] Another aspect of this application provides an expression cassette comprising the aforementioned promoter variant and the target gene.

[0008] Another aspect of this application provides a vector comprising the aforementioned promoter variant, or an expression cassette comprising the promoter variant and a target gene.

[0009] Another aspect of this application provides a microorganism comprising one or more selected from the group consisting of said promoter variant, an expression cassette comprising said promoter variant and a target gene, and a vector comprising said expression cassette. The microorganism may be capable of producing the target product.

[0010] Another aspect of this application provides a method for producing a target product, comprising culturing the aforementioned microorganisms in a culture medium.

[0011] Another aspect of this application provides a composition for producing a target product, the composition comprising the microorganism, a culture medium comprising the microorganism, or a combination thereof.

[0012] Another aspect of this application provides the use of the aforementioned microorganisms in the production of the target product.

[0013] [Technical Solution] This will be described in detail below. Furthermore, the various descriptions and embodiments disclosed herein can also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed herein fall within the scope of this application. Moreover, the scope of this application should not be construed as limited to the specific embodiments described below.

[0014] According to one aspect of this application, this application provides a promoter variant in which Bacillus subtilis... aprE The promoter variant has its -35 region replaced by TTGACA and its -10 region replaced by TATAAT. The promoter variant includes a spacer region between the -35 and -10 regions, the spacer region being 19 bp or 17 bp in length. The TAT at the 3′ end of the spacer region is replaced by TGN, and N is A, T, G, or C.

[0015] The "Bacillus subtilis" in this application is a Gram-positive rod-shaped bacterium, also known as haybacillus. Bacillus subtilis is generally non-pathogenic and widely used in various industrial fields, such as enzyme production, antibiotic production, and probiotics for animal feed.

[0016] In this specification, examples of Bacillus subtilis include subspecies of Bacillus subtilis, such as Bacillus subtilis var. subtilis (… B. subtilis subsp. subtilis ), Bacillus subtilis subsp. stearothermia ( B. subtilis subsp. spizizenii ), Bacillus subtilis desert subspecies ( B. subtilis subsp. inaquosorum However, the sources of promoter variants in this application are not limited to these instances.

[0017] In this specification, the term "promoter" refers to a nucleic acid sequence upstream (5' direction) of a coding region that contains an RNA polymerase binding site and has transcription initiation activity to transcribe a downstream (3' direction) target sequence into mRNA. In prokaryotes, promoters are generally defined as binding sites located near the transcription start site (TSS) for RNA polymerase binding. In this specification, promoters may be entirely derived from natural genes, may consist of different elements derived from different promoters found in nature, or may contain synthetic nucleic acid segments.

[0018] In prokaryotes, promoters consist of two short nucleotide sequences crucial for promoter activity, typically located approximately 10 bp (base pairs) and 35 bp upstream of the transcription start site, respectively. The nucleotide sequence located approximately 10 bp upstream, as described above, is called the Pribnow box or -10 region (-10 element) and is essential for initiating transcription in prokaryotes. Simultaneously, the nucleotide sequence located approximately 35 bp upstream, as described above, is called the -35 element (-35 region) and plays a role in promoting active transcription. The exact locations of the -10 and -35 elements can vary depending on the biological species and can be regulated due to complex interactions with various elements involved in transcriptional regulation.

[0019] In this specification, the term "spacer region" refers to the region between the aforementioned -35 and -10 regions. The length of the spacer region in a prokaryotic promoter is typically 17 to 19 bp. The spacer region affects RNA polymerase binding, and transcription efficiency can vary depending on the length and sequence of the spacer region. The length and sequence of the spacer region vary depending on the specific promoter or biological species.

[0020] In this specification, the term " aprE"" refers to the gene encoding subtilisin, a serine alkaline protease of Bacillus subtilis. aprE promoter (P) aprE Subject to factor A (σ) A RNA polymerase regulation by sig A). Subtilisin is also known as subtilisin E or AprE.

[0021] In this specification, the term "shared sequence" refers to a sequence in which a specific nucleotide occurs at a high frequency at a specific position within all or part of the -35 region, -10 region, or spacer region of a prokaryotic promoter. In other words, a shared sequence is a theoretically representative nucleotide sequence, referring to the sequence that occurs most frequently at the corresponding position among various sequences existing in nature. Specifically, given that... aprE Promoter-dependent σ of RNA polymerase A This fact aprE promoter (P) aprE The shared sequence of ) can be used interchangeably with "sigA shared sequence".

[0022] In one embodiment of this application, compared with the cell’s original promoter or endogenous promoter, the promoter variant of this application can regulate (e.g., increase or decrease) the expression of the target gene that is operatively linked to it in the cell, the production and / or activity of the target protein encoded by the target gene, and / or the production and / or activity of the bioactive substance in which the target protein participates in production.

[0023] As shown in the following examples, when P aprE When the -35 region is replaced by the conservative sequence TTGACA, and / or when the -10 region is replaced by the conservative sequence TATAAT, P aprE Increased transcriptional activity leads to increased expression levels of the target protein.

[0024] In one embodiment of this application, when the TAT at the end of the aforementioned interval 3' is replaced by TGN, N can be selected from A, T, G or C, for example, it can be T, A or G, but is not limited thereto.

[0025] As shown in the following examples, when the TAT at the 3' end of the aforementioned spacer region is replaced by TGN, the nucleotide represented by N can affect P. aprE transcriptional activity.

[0026] In one embodiment of this application, the aforementioned -35 zone may be located in aprE 37 or 35 bp upstream of the transcription start site (TSS), but not limited to this.

[0027] aprE Transcription start site (TSS) refers to the wild-typeaprE The transcription start site. Specifically, the -35 region can be from... aprE It begins 37 or 35 bp upstream of the transcription start site and consists of a 6 bp nucleotide sequence in the 5' to 3' direction, but is not limited to this.

[0028] In one instance, the -35 region could be wild-type P from SEQ ID NO: 21. aprE A nucleotide sequence of 6 bp in length, starting at position 114 from the 5' end and extending from the 5' to 3' direction, or from the wild-type P of SEQ ID NO: 21. aprE It is a nucleotide sequence of 6 bp in length, starting from position 116 at the 5' end and running from the 5' to the 3' direction, but is not limited to this.

[0029] In one embodiment of this application, the aforementioned -10 zone may be located in aprE The transcription start site is 12 bp upstream, but not limited to this. Specifically, the -10 region can be from... aprE It begins 12 bp upstream of the transcription start site and consists of a 6 bp nucleotide sequence in the 5' to 3' direction, but is not limited to this.

[0030] In one instance, the -10 region could be wild-type P from SEQ ID NO: 21. aprE It is a nucleotide sequence of 6 bp in length, starting from position 139 at the 5' end and running from the 5' to the 3' direction, but is not limited to this.

[0031] As shown in the following embodiments, P aprE The location of the -35 zone can affect P aprE transcriptional activity.

[0032] In one embodiment of this application, the -35 region of the aforementioned promoter variant may be located in aprE The transcription start site is 37 bp upstream, and the aforementioned spacer region can be a 19 bp sequence located between the -35 and -10 regions, but is not limited to this.

[0033] In one embodiment of this application, the -35 region of the aforementioned promoter variant may be located in aprE The transcription start site is 35 bp upstream, and the aforementioned spacer region can be a 17 bp sequence located between the -35 and -10 regions, but is not limited to this.

[0034] In one embodiment of this application, the -35 region of the promoter variant may be located in aprE 37 bp upstream of the transcription start site, and the spacer region can be the AA sequence at its 5' end (more specifically, located in the wild type).aprE The spacer region can be 17 bp in length by deleting the AA sequence from the 19 bp sequence between the -35 and -10 regions (30 bp and 31 bp upstream of the transcription start site).

[0035] As shown in the following embodiments, P aprE The length of the interval region can affect P aprE transcriptional activity.

[0036] In this specification, "promoter transcriptional activity" refers to a qualitative or quantitative measurement of the function of a promoter in promoting the transcription of a downstream target sequence. Methods for measuring promoter transcriptional activity are typically based on the expression of a reporter gene (e.g., green fluorescent protein (GFP)), wherein the target promoter sequence is located upstream of and operatively linked to the reporter gene (e.g., GFP). For example, promoter transcriptional activity can be measured by operatively linking the target promoter sequence (or a variant thereof, e.g., containing one or more mutations, deletions, or substitutions) upstream of the reporter gene (e.g., GFP) and detecting or measuring changes in reporter gene expression. Furthermore, promoter transcriptional activity can be assessed by introducing a target protein expression cassette into a suitable host cell (e.g., Bacillus bacteria) and expressing the target protein.

[0037] In this specification, the term "target sequence" can refer to the "target gene" to be expressed. In one instance, the target gene can be a gene encoding a target protein. The target protein can be a protein (e.g., an enzyme) involved in the production of the target product.

[0038] In one implementation, the target protein may be a serine protease, but is not limited thereto.

[0039] In this specification, the term "target product" means a bioactive substance that is ultimately produced or whose production is regulated (increased or reduced) using the promoter variants provided in this specification, expression cassettes containing such a product, expression vectors and / or recombinant cells (microorganisms), and may, for example, refer to the target protein itself encoded by the target gene, and / or any bioactive substance produced by means of the target protein. Bioactive substances are any substances produced by or derived from an organism (e.g., cells) or that have a predetermined function in the body or cells, and may be, for example, one or more selected from the group consisting of: amino acids, amino acid derivatives, nucleic acids (e.g., adenine, thymine, guanine, cytosine, uracil), nucleic acid derivatives, vitamins (e.g., vitamin A (retinol), B (B1 (thiamine), B2 (riboflavin), B3 (niacin), B5 (pantothenic acid), B6 ​​(pyridoxine), B7, B9 (folic acid), B12 (cobalamin)), C (ascorbic acid), D (calciferol), E (tocopherol), K (phylloquinone)), vitamin derivatives, proteins (e.g., proteins other than the target protein, such as hormones, growth factors, cytokines, immunoglobulins (antibodies), antigen proteins, receptors, ligands, and their functional fragments (having the target function)). (fragments), and fusion proteins fused from two or more of these types), sugars (e.g., monosaccharides (glyceraldehyde, dihydroxyacetone, erythrose, threose, erythritol, arabinose, lythose, ribose, xylose, ribulose, xylulose, deoxyribose, allose, azoose, galactose, glucose, gulose, idose, mannose, tarose, fructose, allulose, sorbose, tagatose, fucose, fucose, fucoidan, rhamnose, mannoheptulose, sedum Heptanulose), disaccharides (cellobiose, isomaltose, isomaltulose, lactose, lactulose, maltose, sucrose, trehalose, menobiose), polysaccharides), sugar derivatives (e.g., sugar alcohols, galactosamine, glucosamine, sialic acid, N-acetylglucosamine, sulfoquinovose, ascorbic acid, mannitol, glucuronic acid), fatty acids (e.g., myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, transoleic acid, isoleic acid, linoleic acid, translinoleic acid, arachidonic acid, eicosapentaenoic acid (EPA), erucic acid, docosahexaenoic acid (DHA)), fatty acid derivatives, organic acids (e.g., lactic acid, citric acid, oxalic acid, uric acid, butyric acid, stearic acid, propionic acid), their metabolites (e.g., polyhydroxyalkanoates (PHA)), their precursors and their bioactive derivatives, but not limited thereto.

[0040] In one instance, where the target protein is involved in the production of the target product, (1) the target protein may be at least one of the following groups of proteins selected from at least one process or step of the intracellular production pathway (e.g., biosynthesis, metabolism, biotransformation, etc.), intracellular transport and / or extracellular export pathway of the target product, such as synthases, lyases, kinases, carboxylases (e.g., pyruvate carboxylase, etc.), reductases, oxidases, decarboxylases, dehydrogenases, dehydratases, transferases, isomerases (e.g., epimerases), intermediates, transport proteins, membrane proteins (channels, etc.), etc., but not limited thereto, and (2) the target gene may be a gene encoding the target protein described in (1) above.

[0041] In the target product, the amino acid can be a protein amino acid or a non-protein amino acid.

[0042] Protein amino acids can be selected from one or more of the following groups: arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, selenocysteine, pyrrolidone, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan, or they can be intermediates.

[0043] The protein amino acid can be one or more selected from the group consisting of L-amino acids, such as 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 intermediates thereof, specifically L-lysine, L-threonine, L-isoleucine, L-leucine, L-valine, L-arginine, or L-glutamic acid, but not limited thereto. Intermediates can be, for example, O-acetylhomoserine, which is an intermediate of L-methionine, but not limited thereto.

[0044] Non-protein amino acids may be selected from one or more of the following groups: β-alanine, γ-aminobutyric acid (GABA), 5-aminolevulinic acid (δ-aminolevulinic acid), para-aminobenzoic acid (4-aminobenzoic acid), α-aminobutyric acid, dehydroalanine, cystathionine, lanethionine, djenkolic acid, diaminopimelic acid, valine, leucine, isoleucine, t-leucine (tertiary leucine), α-amino-n-heptanoic acid, pipecolic acid, α,β-diaminopropionic acid, α,γ-diaminobutyric acid, ornithine, allothreonine, homocysteine, homoserine (or isothreonine), and O-acetylhomoserine, but are not limited thereto.

[0045] Amino acids can be D-amino acids or L-amino acids.

[0046] In one embodiment of this application, the promoter variant may comprise a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NO: 22 to SEQ ID NO: 28 shown in Table 1 below, for example, a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NO: 23, SEQ ID NO: 26 and SEQ ID NO: 28, but is not limited thereto.

[0047] [Table 1] The nucleotide sequence of the promoter variant of this application can be modified by conventionally known mutagenesis methods, such as directed evolution and site-directed mutagenesis. That is, the promoter variant may comprise or consist of the following nucleotide sequences: the nucleotide sequence has the same or equivalent biological activity as any nucleotide sequence selected from the group consisting of SEQ ID NO: 22 to SEQ ID NO: 28, and has 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, or 91.5% of any nucleotide sequence selected from the group consisting of SEQ ID NO: 22 to SEQ ID NO: 28. Homology or identity of 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). Furthermore, any polynucleotide that has the aforementioned homology or identity with any nucleotide sequence in the group consisting of nucleotide sequences selected from SEQ ID NO: 22 to SEQ ID NO: 28, and has substantially the same or equivalent biological activity with any nucleotide sequence in the group consisting of nucleotide sequences selected from SEQ ID NO: 22 to SEQ ID NO: 28, including polynucleotides having partially deleted, modified, or substituted sequences, or polynucleotide sequences with one or more nucleotides added to any nucleotide sequence in the group consisting of nucleotide sequences selected from SEQ ID NO: 22 to SEQ ID NO: 28, may also be included within the scope of promoter variants of this application.

[0048] In one implementation, the wild-type P of SEQ ID NO: 21 aprE It can be adjacent to the wild type aprE The upstream sequence of the transcription start site, but not limited to this.

[0049] In this specification, the terms "homology" or "identity" refer to the degree of correspondence with a given nucleic acid or amino acid sequence and can be expressed as a percentage (%). For example, homology can be determined by aligning sequence information and directly comparing parameters (such as score, identity, and similarity) between two polynucleotide molecules or two polypeptide molecules using readily available computer programs. Computer programs may include BLAST (NCBI), CLC Main Workbench (CLC bio), and MegAlign. TM (DNASTAR Inc.), etc.

[0050] In one specific embodiment, a promoter variant comprising a specific nucleotide sequence provided in this specification can be interpreted as comprising not only the specific nucleotide sequence or a substantially equivalent nucleotide sequence, but also a multinucleotide fragment comprising a nucleotide sequence complementary to the specific nucleotide sequence. Specifically, a multinucleotide having such complementarity can hybridize with the specific nucleotide sequence at a Tm value that can be appropriately adjusted by those skilled in the art for the purpose, for example, a Tm value of 55°C, 60°C, 63°C, or 65°C, and analysis can be performed under conditions specifically described in known literature. For example, the conditions may include conditions where multinucleotides with high complementarity, such as 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, while multinucleotides with lower complementarity do not hybridize with each other. The conditions may also include common Southern hybridization washing conditions, including washing once, or particularly two to three times, at salt concentrations and temperatures corresponding to 60°C, 1× SSC (saline-sodium citrate buffer), and 0.1% (w / v) SDS (sodium dodecyl sulfate); 60°C, 0.1× SSC, and 0.1% SDS; or 68°C, 0.1× SSC, and 0.1% SDS. Hybridization requires that the two nucleotides have complementary sequences, but mismatches between bases may be permitted depending on the stringency of the hybridization. The term “complementary” can be used to describe the relationship between the bases of nucleotides that are capable of hybridizing 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 hybridization of polynucleotides depends on the length and degree of complementarity of the polynucleotide, as is well known in the relevant field (see Sambrook et al., above, 9.50-9.51, 11.7-11.8).

[0051] According to one specific embodiment, the promoter variant may comprise any nucleotide sequence selected from the group consisting of nucleotide sequences selected from SEQ ID NO: 22 to SEQ ID NO: 28, and may consist of or substantially consist of any nucleotide sequence selected from the group consisting of nucleotide sequences selected from SEQ ID NO: 22 to SEQ ID NO: 28.

[0052] In one embodiment of this application, the promoter variant may be isolated. As used herein, "isolation" or "purification" refers to the alteration of the native state of a biomolecule (e.g., a polypeptide or polynucleotide) by separating it from some or all of its naturally occurring components associated with it in nature. Such isolation or purification can be achieved using isolation techniques recognized in the art, such as ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, ammonium sulfate precipitation or other salt precipitation methods, centrifugation, size exclusion chromatography, filtration, microfiltration, gel electrophoresis, or gradient separation, to remove unwanted whole cells, cell debris, impurities, foreign proteins, or enzymes from the final composition. Components that provide additional benefits, such as activators, agents to counteract inhibitors, desired ions, compounds for pH control, or other enzymes or chemicals, may also be added to the purified or isolated biomolecule composition.

[0053] According to another aspect of this application, this application provides an expression cassette comprising the promoter variant of this application and the target gene.

[0054] The target gene can be a gene that encodes a target protein, and the target protein can be a protein (e.g., an enzyme) that participates in the production of the target product.

[0055] The target product can be one or more selected from the group consisting of amino acids, amino acid derivatives, nucleic acids, nucleic acid derivatives, vitamins, vitamin derivatives, sugars, sugar derivatives, fatty acids, fatty acid derivatives, proteins, and other metabolites.

[0056] In this specification, the term "expression cassette" may refer to a construct, which is the smallest unit of a nucleic acid fragment that can be expressed in a host cell and may contain at least a target gene and an expression regulatory sequence (e.g., a promoter).

[0057] The promoter variant may be contained in the 5' or 3' end region of the target gene.

[0058] The promoter variant can be operatively linked to the target gene.

[0059] The promoter variant, target gene, target protein, and target product are as described above.

[0060] According to another aspect of this application, this application provides a carrier comprising the aforementioned promoter variants or expression boxes.

[0061] In this specification, the term "vector" refers to an expression vector capable of expressing a target protein in a suitable host cell (microorganism), and specifically to a gene construct containing essential regulatory elements operatively linked to a gene insert, enabling the gene insert (expression cassette) to be expressed. There are no particular limitations on the expression vectors used in this application, as long as they can replicate in a host cell (microorganism) and can be selected from any commonly used vector. Examples of commonly used vectors include, but are not limited to, plasmids, granules, viruses, and bacteriophages in their native or recombinant states. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, or Charon21A can be used as phage vectors or granule vectors, and pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, or pET-based vectors can be used as plasmid vectors. Specifically, examples include pDG1730, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, or pCC1BAC vectors, but are not limited to these.

[0062] In this specification, the term "regulatory element" refers to a non-translated nucleic acid sequence that assists or influences the transcription, translation, or expression enhancement of a nucleic acid sequence encoding a protein. The expression vector of this application must contain the promoter variant of this application as a regulatory element and may contain expression regulatory sequences capable of influencing protein expression, such as start codons, stop codons, polyadenylation signals, enhancers, signal sequences for membrane targeting or secretion, etc.

[0063] In one embodiment of this application, the aforementioned vector may additionally include a gene encoding the target protein downstream of the aforementioned promoter variant sequence, and the gene encoding the target protein may be operatively linked to the aforementioned promoter variant.

[0064] In this specification, "operably ligated" means that the aforementioned promoter variant is functionally ligated to the nucleotide sequence of the gene encoding the target protein, thereby initiating and / or mediating the transcription of the gene encoding the target protein. Operable ligation can be performed using genetic recombination techniques known in the art, such as site-specific DNA cutting and / or ligation techniques, which can be performed using conventional cutting enzymes and / or ligases, but are not limited thereto.

[0065] In this specification, the term "target protein" refers to any protein whose expression is increased using the promoter variant of this application, and may be homologous or heterologous.

[0066] In this specification, the term "gene" refers to a polynucleotide that encodes a specific amino acid sequence, including all or part of a protein-coding sequence, and may include, for example, regulatory (non-transcribed) DNA sequences, such as promoter sequences that determine gene expression conditions.

[0067] In one embodiment of this application, the vector containing the aforementioned promoter variant may additionally contain a tag for protein purification. The tag for protein purification is not limited, as long as it facilitates the purification of the target protein recovered in this application; examples include glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and 6xHis (hexahistidine; Qiagen, USA).

[0068] In one embodiment of this application, the vector containing the aforementioned promoter variant may further include a selection marker for confirming integration into the host cell (microorganism) chromosome. The selection marker is used to select cells transformed by the vector, i.e., to confirm whether the nucleotide sequence of the promoter variant of this application has been inserted, and can be selected from a set of genes conferring selectable phenotypes (such as drug resistance, auxotrophic phenotype, resistance to cytotoxic agents, or surface protein expression). In an environment treated with a selection agent, only cells expressing the selection marker survive or exhibit different phenotypic traits, thus allowing for the selection of transformed cells.

[0069] According to another aspect of this application, this application provides a host cell or recombinant cell comprising at least one selected from the group consisting of the aforementioned promoter variants, promoter variants and target genes, expression cassettes and vectors operatively linked thereto.

[0070] The promoter variant, target gene, expression cassette, or vector is as described above.

[0071] In this specification, the term "recombinant cell" can refer to a transformant in which polynucleotides and / or recombinant vectors have been introduced. The term "transformation" refers to the introduction of a target nucleic acid (DNA or RNA) into a host cell (microorganism) to alter the genetic traits of the host cell (microorganism). Transformation methods can be performed by selecting appropriate standard techniques known in the art, depending on the host cell. For example, transformation includes, but is not limited to, electroporation, lipid transfection, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0072] The host cell is not particularly restricted, as long as the polynucleotide provided in this application functions as a promoter, enabling (or increasing) expression of the target gene operatively linked to it, wherein the target gene may be a gene contained in a recombinant vector or an endogenous gene of the host cell.

[0073] In one instance, the host cell can be a microorganism, a plant cell, or an animal cell.

[0074] The microorganisms in this application may be microorganisms capable of producing the target product or capable of improving the production of the target product.

[0075] The microorganisms in this application can be microorganisms that are endowed or enhanced with the ability to produce the target product by introducing one or more of the group consisting of promoter variants, expression cassettes and vectors into a microorganism that naturally lacks the ability to produce the target product, or a microorganism that has the ability to produce the target product, but are not limited thereto.

[0076] The ability of a microorganism to produce the target product or the ability to produce the target product may mean that the microorganism has an enhanced ability to produce the target product compared to unmodified microorganisms, pre-recombination cells, parental strains and / or wild-type strains, or that the microorganism is different from unmodified microorganisms, pre-recombination cells, parental strains and / or wild-type strains (each of which lacks the ability to produce the target product) and is endowed with the ability to produce the target product.

[0077] Microorganisms introduced with one or more of the group consisting of promoter variants, expression cassettes, and vectors may have an enhanced ability to produce the target product compared to the microorganism before introduction or such pre-enhancement microorganisms (i.e., unmodified microorganisms of the same species). In this application, "unmodified microorganism" does not exclude microorganisms with mutations that may occur naturally in microorganisms, and may refer to wild-type microorganisms or naturally occurring microorganisms themselves, or microorganisms whose traits have been altered due to genetic variation caused by natural or artificial factors. For example, unmodified microorganisms may refer to strains that have not yet been introduced with one or more of the group consisting of promoter variants, expression cassettes, and vectors, or such pre-introduction strains. "Unmodified microorganism" may be used interchangeably with "pre-modified strain," "pre-modified microorganism," "unmutated strain," "unmodified strain," "unmutated microorganism," or "reference microorganism." The promoter variants, expression cassettes, and vectors are as described above.

[0078] In one embodiment of this application, a microorganism having been introduced with at least one selected from the group consisting of promoter variants, expression cassettes, and vectors may have a newly conferred or increased ability to produce the target product compared to the unmodified parental strain, the unmodified microorganism, the parental strain, or the wild-type microorganism, but is not limited thereto.

[0079] As another example, a microorganism with an enhanced ability to produce the target product may have a production capacity of about 2 times or more, about 10 times or more, about 50 times or more, about 80 times or more, about 95 times or more, about 100 times or more, about 110 times or more, about 120 times or more, about 130 times or more, about 140 times or more, about 150 times or more, about 160 times or more, about 175 times or more, about 200 times or more, about 230 times or more, about 250 times or more, about 280 times or more (with no particular upper limit, and for example, about 1,000 times or less) of the production capacity of the target product, but is not limited thereto.

[0080] The term “about” covers a range of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes, but is not limited to, all values ​​that are equivalent to or similar to the value following the term “about”.

[0081] In one embodiment of this application, the host cell of this application as described above can be isolated.

[0082] As the host cell or cell line capable of stably and continuously cloning and expressing the vector of this application, any host cell known in the art can be used, examples of which include Bacillus strains, such as Bacillus subtilis and Bacillus thuringiensis. Bacillus thuringiensis ); Enterobacterial strains, such as Salmonella typhimurium ( Salmonella typhimurium ) and Serratia marcescens ( Serratia marcescens ); various species of the genus *Pseudomonas*; and *Escherichia coli* ( E. coli (E. coli) strains, such as E. coli Origami2, E. coli JM109, E. coli BL21(DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776 and E. coli W3110, but not limited to these.

[0083] In one embodiment of this application, the host cell described above can be a Gram-positive bacterium.

[0084] In one embodiment of this application, the Gram-positive bacteria may be selected from the group consisting of: Bacillus subtilis, Bacillus licheniformis (…Bacillus licheniformis ), Bacillus megaterium ( Bacillus megaterium ), Bacillus cereus ( Bacillus cereus ), Bacillus thuringiensis, Bacillus brevis ( Bacillus brevis ) and Lactobacillus genus ( Lactobacillus ) or Bacillus spp. ( Paenibacillus (bacteria, but not limited to)

[0085] In this specification, "transformation" or "transfection" refers to the introduction of a gene into a host cell so that the gene can be expressed therein. Methods of transforming or transfecting host cells with a vector containing the target gene include any method of introducing nucleic acids into cells, and can be performed by selecting appropriate standard techniques known in the art.

[0086] Methods for delivering the vectors of this application into host cells include CaCl2 precipitation, the Hanahan method (which improves efficiency by using the reducing agent DMSO (dimethyl sulfoxide) in the CaCl2 method), electroporation, calcium phosphate precipitation, protoplast fusion, stirring using silicon carbide fibers, Agrobacterium-mediated transformation, polyethylene glycol (PEG)-mediated transformation, and transformation methods mediated by dextran sulfate, Lipofectamine, and drying / inhibition. The methods for transforming or transfecting host cells with the vectors of this application are not limited to the examples described above; methods commonly used in the art can be used without limitation.

[0087] According to another aspect of this application, this application provides a composition for producing a target product, comprising the microorganism, a culture medium for culturing the microorganism, or a combination thereof.

[0088] The composition may also contain any suitable excipient commonly used in compositions for producing the target product, such excipients as, but not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents.

[0089] According to another aspect of this application, this application provides the use of the aforementioned microorganisms in the production of the target product.

[0090] According to another aspect of this application, this application provides the use of the aforementioned microorganisms in preparing compositions for the production of the target product.

[0091] According to another aspect of this application, this application provides a method for producing a target product, which includes culturing the aforementioned microorganisms in a culture medium.

[0092] The method may also include recovering the target product from the culture medium or microorganism after culturing.

[0093] Culture media for culturing microorganisms may or may not contain microorganisms.

[0094] As described above, the microorganism may contain one or more selected from the group consisting of: the polynucleotide provided in this application; an expression cassette containing the polynucleotide and the target gene; and a vector containing the expression cassette.

[0095] In this specification, "culture" means the growth of cells under artificially controlled environmental conditions. The methods for producing the target product provided in this specification can be carried out by means of any appropriate selection from all methods widely known in the art. For example, culturing can be carried out in a batch process, a fed-batch process, or a repeatedly fed-batch process, but is not limited thereto. The culture medium used for culturing can be a culture medium that appropriately meets the growth requirements of the cells to be cultured.

[0096] In this application, "culture medium" refers to a substance in which nutrients required for the cultivation of microorganisms are mixed as the main components, and which provides nutrients and growth factors, including water necessary for survival and growth. Specifically, any culture medium and other culture conditions used for culturing the microorganisms of this application can be used without particular limitation, as long as it is a culture medium conventionally used for culturing microorganisms; however, the microorganisms of this application can be cultured under aerobic conditions in a common culture medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and / or vitamins, while controlling temperature, pH, etc.

[0097] Carbon sources used for cultivation or inclusion in the culture medium may include one or more selected from the group consisting of: sugars and carbohydrates, such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats, such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids, such as palmitic acid, stearic acid, and linoleic acid; alcohols, such as glycerol and ethanol; and organic acids, such as acetic acid, but not limited thereto. Nitrogen sources used for cultivation or inclusion in the culture medium may include one or more selected from the group consisting of: organic nitrogen sources, such as peptone, yeast extract, meat extract, malt extract, corn steep liquor, soybean flour, and urea; and inorganic nitrogen sources, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate, but not limited thereto. Phosphorus sources used for cultivation or inclusion in the culture medium may include one or more selected from the group consisting of: potassium salts of phosphate, such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and their corresponding sodium salts, but not limited thereto. In addition, the culture medium may contain metal salts required for growth, such as magnesium sulfate or ferric sulfate. In addition to the above, one or more of essential growth substances such as amino acids and vitamins may be additionally used or included in the culture or culture medium. Furthermore, the culture medium may contain appropriate precursors of the target product as raw materials. These raw materials may be added to the culture in batches and / or continuously during the culture process using suitable methods.

[0098] During cell culture, the pH of the culture can be adjusted by using alkaline compounds such as sodium hydroxide, potassium hydroxide, and ammonia, and / or acidic compounds such as phosphoric acid or sulfuric acid in an appropriate manner. Furthermore, antifoaming agents such as polyethylene glycol esters of fatty acids can be used to suppress foaming during culture. Additionally, to maintain an aerobic environment, oxygen or an oxygen-containing gas (e.g., air) can be injected into the culture. The temperature of the culture medium and / or the culture itself is typically between 20°C and 45°C, or between 25°C and 40°C. Culture can continue until the desired yield of the target product is achieved, for example, from about 10 to about 160 hours, but is not limited to this.

[0099] The isolation or recovery of a target substance from cultured microorganisms or culture media can be performed using suitable methods known in the art, depending on the culturing method. For example, methods such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), and / or chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic chromatography, and size exclusion chromatography) can be used, but are not limited thereto. Culture media refers to a medium in which recombinant cells have already been cultured.

[0100] According to one implementation, the separation or recovery of the target product may include removing biomass by low-speed centrifugation of the culture and separating the obtained supernatant by ion exchange chromatography.

[0101] The method for producing the target product may also include the process of purifying the target product.

[0102] Recovery of the target product from the culture (culture medium) can be performed using separation methods known in the art as conventional means. Examples of conventional means that can be used to separate the target product include centrifugation, filtration, chromatography, and / or crystallization. In one example, biomass is removed by low-speed centrifugation of the culture, and the resulting supernatant is subjected to ion-exchange chromatography to separate the target product, but this is not limited to this. Recovery may also include purification processes.

[0103] According to another aspect of this application, this application provides uses for producing a target product, and / or manufacturing microorganisms that produce a target product, and / or conferring and / or increasing the ability of microorganisms to produce a target product, selected from the promoter variants of this application described above; a vector containing a promoter variant; and one or more microorganisms that contain a promoter variant and / or a vector containing a promoter variant.

[0104] In the context of the production of a target product, and / or the manufacture of microorganisms for producing a target product, and / or the conferring and / or increasing of the ability of microorganisms to produce a target product, the promoter variants, vectors, and microorganisms, etc., are as described in the other aspects above; therefore, their common description is incorporated herein by reference, and their detailed description is omitted to avoid excessive complexity of this specification.

[0105] According to another aspect of this application, this application provides compositions, methods, products, processes, or uses characterized by one or more elements disclosed in this application. Attached Figure Description

[0106] Figure 1 Is it to display included aprE promoter (P) aprE A diagram of the pDG1730_PaprE_protease vector containing the protease gene.

[0107] Figure 2 It displays P aprE A diagram of a portion of the sequence and the corresponding portion of the promoter variant sequence.

[0108] Figure 3 It displays P aprE A diagram of a portion of the sequence and a specific mutation in the corresponding portion of the promoter variant. Detailed Implementation

[0109] The present application will be described in more detail below through embodiments. These embodiments are only used to explain 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 to these embodiments.

[0110] Example (Throughout this instruction manual, unless otherwise stated, the "%" used to express the concentration of a particular substance is (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid.) Example 1. Construction of pDG1730_PaprE_protease vector Vectors for evaluating promoter transcriptional activity were constructed using the following method.

[0111] To assess the promoter's transcriptional activity, an expression cassette containing the promoter and serine protease gene was inserted into the Bacillus subtilis chromosome using the pDG1730 plasmid (BGSC, Bacillus Genetic Depository). amyE The pDG1730 plasmid was used to analyze protease expression levels at specific sites. This plasmid is associated with Bacillus subtilis. amyEThe site sequence is homologous to the sequence; therefore, when the plasmid is linearized using restriction enzymes and introduced into the strain, the gene can be inserted into Bacillus subtilis via homologous recombination. amyE Site.

[0112] Specifically, to construct the pDG1730_PaprE_protease vector, pDG1730 was prepared by digestion with BamH1; using a polynucleotide with the sequence of SEQ ID NO: 29 as a template and primer pairs of SEQ ID NO: 1 and 2 (Table 2), P was prepared by PCR. aprE Fragments; using a polynucleotide sequence having SEQ ID NO: 30 as a template and primer pairs SEQ ID NO: 3 and 4, a protease-encoding fragment was prepared by PCR. The three fragments prepared in this manner were ligated using the In-Fusion HD Cloning Kit (Clontech) and then transformed into DH5α (E. coli) to complete vector cloning for assessing promoter transcriptional activity. Figure 1 ).

[0113] [Table 2] Example 2. Construction aprE promoter variants Bacillus subtilis is known aprE promoter (P) aprE The transcriptional activity of RNA polymerase depends on the σ factor A (σ) of RNA polymerase. A In order to analyze P aprE The configuration, considering that the length of the promoter spacer region is typically 17 bp to 19 bp, was carefully analyzed for P. aprE The DNA sequence. As a result, i) confirmed the hypothesis that it was... aprE The TCTACT region at -35, 37 bp upstream of the transcription start site (TSS) (hereinafter referred to as the "37 bp upstream position"), and the TACAAT region at -10, 19 bp downstream of the -35 region (i.e., the 19 bp spacer region), and ii) confirmed the hypothesis that it was... aprE TACTAA, located 35 bp upstream of the transcription start site (hereinafter referred to as "35 bp upstream position"), and TACAAT, located 17 bp downstream of the -35 bp upstream position (i.e., the 17 bp spacer region).

[0114] Based on the above analysis of the promoter sequence, the following were analyzed: whether the transcriptional activity of the promoter depends on i) the position of the -35 region at the start of the promoter sequence, ii) optimizing the length of the spacer region to 17 bp by deleting the AA sequence at the 5' end of the spacer region, and iii) mutating the TAT sequence at the 3' end of the spacer region to TGT.

[0115] Specifically, a total of 7 variants were constructed as follows, and as... Figure 2 and Figure 3 As shown. TTGACA is used as the common sequence for the -35 region, and TATAAT is used as the common sequence for the -10 region. The constructed P... aprE The variations are as follows: 1) P aprE sigA1 (SEQ ID NO: 22, hereinafter referred to as "PsigA1"), wherein the -35 region (TCTACT) at the 37 bp upstream position is replaced by the common sequence (TTGACA), the TAT sequence at the 3' end of the 19 bp spacer region is replaced by TGT, and the -10 region (TACAAT) 19 bp away from the -35 region at the 37 bp upstream position is replaced by the common sequence (TATAAT); 2) P aprE sigA2 (SEQ ID NO: 23, hereinafter referred to as "PsigA2"), wherein the -35 region (TCTACT) at the 37 bp upstream position is replaced by the common sequence (TTGACA), the AA sequence at the 5' end of the original 19 bp spacer is deleted, the TAT sequence at the 3' end of the original 19 bp spacer is replaced by TGT, and due to the deletion of the AA sequence, the -10 region (TACAAT) 17 bp away from the -35 region at the 37 bp upstream position is replaced by the common sequence (TATAAT); 3) P aprE sigA3 (SEQ ID NO: 24, hereinafter referred to as "PsigA3"), wherein the -35 region (TACTAA) at the upstream position of 35 bp is replaced by the common sequence (TTGACA), and the -10 region (TACAAT) 17 bp away from the -35 region at the upstream position of 37 bp is replaced by the common sequence (TATAAT); 4) P aprE sigA4 (SEQ ID NO: 25, hereinafter referred to as "PsigA4"), wherein the wild-type -35 region (TACTAA) at the upstream position of 35 bp is replaced by the shared sequence (TTGACA), the -10 region (TACAAT) 17 bp away from the -35 region at the upstream position of 37 bp is replaced by the shared sequence (TATAAT), and the TAT sequence at the 3' end of the 17 bp spacer region is replaced by TGT; 5) PaprE sigA5 (SEQ ID NO: 26, hereinafter referred to as "PsigA5"), wherein the -35 region (TCTACT) at the 37 bp upstream position is replaced by the common sequence (TTGACA), the AA sequence at the 5' end of the original 19 bp spacer region is deleted, the TAT sequence at the 3' end of the original 19 bp spacer region is replaced by TGA, and due to the deletion of the AA sequence, the -10 region (TACAAT) 17 bp away from the -35 region at the 37 bp upstream position is replaced by the common sequence (TATAAT); 6) P aprE sigA6 (SEQ ID NO: 27, hereinafter referred to as "PsigA6"), wherein the -35 region (TCTACT) at the 37 bp upstream position is replaced by the common sequence (TTGACA), the AA sequence at the 5' end of the original 19 bp spacer is deleted, the TAT sequence at the 3' end of the original 19 bp spacer is replaced by TGC, and due to the deletion of the AA sequence, the -10 region (TACAAT) 17 bp away from the -35 region at the 37 bp upstream position is replaced by the common sequence (TATAAT); 7) P aprE sigA7 (SEQ ID NO: 28, hereinafter referred to as "PsigA7"), wherein the -35 region (TCTACT) at the 37 bp upstream position is replaced by the common sequence (TTGACA), the AA sequence at the 5' end of the original 19 bp spacer is deleted, the TAT sequence at the 3' end of the original 19 bp spacer is replaced by TGG, and due to the deletion of the AA sequence, the -10 region (TACAAT) 17 bp away from the -35 region at the 37 bp upstream position is replaced by the common sequence (TATAAT).

[0116] Using pDG1730_PaprE_ protease as template DNA, partial mutations were introduced through site-directed mutagenesis to construct a P-based... aprE Promoter variants. Primers used to induce partial mutations are shown in Table 3 below.

[0117] [Table 3] Example 3. Constructing a P-based system aprE and P aprE Variants (P) aprE *) protease expression strains Linear DNA was prepared by digesting the pDG1730_PaprE(PaprE*)_protease vector constructed in Examples 1 and 2 with SpeI. The prepared linear DNA was transformed into Bacillus subtilis strain LB700, and the amyE gene locus was inserted via a homologous recombination induction cassette. Strains containing the inserted target gene were obtained by selecting colonies resistant to spectinomycin. Finally, colony PCR was performed using primers listed in Table 4 to detect a band of approximately 1,700 bp, followed by sequencing, to confirm the insertion of the target gene.

[0118] [Table 4] Example 4. Comparison of P aprE and P aprE Variant (PaprE*) transcriptional activity P was evaluated using the following method. aprE Does the variant act as a promoter? If so, P aprE Does the substitution of each component or optimization of the spacer length affect transcriptional activity?

[0119] In the promoter expression strain constructed in Example 3, wild-type P aprE The expression strains, including PsigA1, PsigA2, PsigA3, and PsigA4, were cultured overnight in LB medium at 37°C. Then, 1% of each culture was inoculated into 25 ml of potency medium (10 g / L glucose, 15 g / L CSL, 20 g / L yeast extract, 2 g / L MgCl2, 2 g / L NaSO4, 2 g / L (NH4)2SO4, 1.5 g / L K2HPO4) and cultured at 37°C and 200 rpm for 24 hours. Protease expression levels were measured using Suc-AAPF-pNA kinetic assays. After 24 hours, the cultures were centrifuged to obtain a supernatant containing the enzyme. 10 μl of the supernatant diluted in buffer was mixed with 190 μl of Suc-AAPF-pNA substrate dissolved in buffer to a final concentration of 0.5 mM and reacted in 96-well plates at 25°C. The degree of substrate degradation was determined by comparing the absorbance at 410 nm using a microplate reader (Biotek, Synergy HTX).

[0120] [Table 5] Comparison of promoter transcriptional activity As shown in Table 5, the variant PsigA1, in which the -35 region (TCTACT) at the 37 bp upstream position is replaced by the shared sequence (TTGACA) of the -35 region, the TAT sequence at the 3' end of the original 19 bp spacer is replaced by TGT, and the -10 region (TACAAT) is replaced by the shared sequence (TATAAT) of the -10 region, exhibits a relative activity 108 times that of the wild-type promoter. The variant PsigA2, in which the AA sequence at the 5' end of the original 19 bp spacer is additionally deleted from PsigA1, exhibits a relative activity 206 times that of the wild-type promoter. Therefore, the substitutions of the -35 region, the 3' end of the spacer, and the -10 region confirm that these substitutions increase promoter transcriptional activity. Furthermore, when the spacer length is optimized from 19 bp (PsigA1) to 17 bp (PsigA2) by deleting the AA sequence at the 5' end of the original 19 bp spacer, the relative activity increases by approximately 91%, indicating that spacer length optimization is an additional factor enhancing promoter transcriptional activity. Meanwhile, PsigA1, defined based on a 37 bp upstream position in the -35 and -10 regions, showed approximately 11% higher transcriptional activity than PsigA3, also defined based on a 35 bp upstream position in the -35 and -10 regions. This confirms that promoter position affects transcriptional activity, and that defining promoter elements based on a 37 bp upstream position is beneficial for improving transcriptional efficiency.

[0121] The variant PsigA3, in which the -35 region (TACTAA) at the 35 bp upstream position is replaced by the shared sequence (TTGACA) of the -35 region, and the -10 region (TACAAT) at the 37 bp upstream position is replaced by the shared sequence (TATAAT) of the -10 region, exhibits a relative activity 98 times that of the wild-type promoter. The variant PsigA4, in which the TAT sequence at the 3' end of the spacer is additionally replaced by TGT compared to PsigA3, exhibits a relative activity 134 times that of the wild-type promoter, indicating that the replacement of the TAT sequence at the 3' end of the spacer is an additional factor enhancing promoter transcriptional activity.

[0122] Therefore, it was confirmed that i) the promoter position (starting from relative to) aprE The optimization of the promoter transcriptional activity was significantly affected by factors including: ii) replacement of the 37 bp or 35 bp upstream position of the transcription start site; iii) replacement of the 35 bp region with a shared sequence; iv) replacement of the 10 bp region with a shared sequence; and iv) optimization of the spacer length. Finally, PsigA2, in which Bacillus subtilis P... aprEThe -35 region (TCTACT) 37 bp upstream of the promoter is replaced by the shared sequence (TTGACA) of the -35 region. The AA sequence at the 5' end of the original 19 bp spacer is deleted, and the TAT sequence at the 3' end of the original 19 bp spacer is replaced by TGT. Furthermore, due to the deletion of the AA sequence, the -10 region (TACAAT) 17 bp upstream of the -35 region is replaced by the shared sequence (TATAAT) of the -10 region, exhibiting a higher performance than the wild-type P. aprE The promoter exhibits more than 200-fold higher transcriptional activity, thus demonstrating the greatest increase in transcriptional activity.

[0123] Example 5. Comparison of transcriptional activity based on substitutions of the 3' TAT sequence in the spacer region. In Example 4 above, the promoter PsigA2 was confirmed, in which Bacillus subtilis P aprE The -35 region (TCTACT) located 37 bp upstream of the original spacer was replaced by the shared sequence (TTGACA), the AA sequence at the 5' end of the original 19 bp spacer was deleted, the TAT sequence at the 3' end of the original 19 bp spacer was replaced by TGT, and due to the deletion of the AA sequence, the -10 region (TACAAT) 17 bp upstream of the -35 region was replaced by the shared sequence (TATAAT), showing the most significant increase in transcriptional activity.

[0124] Furthermore, the transcriptional activities of PsigA5, PsigA6, and PsigA7 were compared. Like PsigA2, these variants were prepared by: i) replacing the -35 region (TCTACT) 37 bp upstream with a shared sequence (TTGACA), ii) deleting the AA sequence at the 5' end of the original 19 bp spacer region, and iii) replacing the -10 region (TACAAT) 17 bp upstream of the -35 region with a shared sequence (TATAAT) due to the deletion of the AA sequence. However, unlike PsigA2, the TAT sequence at the 3' end of the original 19 bp spacer region was replaced by TGA, TGC, or TGG, resulting in PsigA5, PsigA6, or PsigA7, respectively.

[0125] The transcriptional activity of the PsigA2, PsigA5, PsigA6 and PsigA7 expression strains constructed in Example 3 was measured in the same manner as in Example 4.

[0126] [Table 6] As shown in Table 6, substitution of the TAT sequence at the 3' end of the original 19 bp spacer region with TGA instead of TGT (PsigA5) resulted in increased transcriptional activity, while substitution with TGG (PsigA7) also led to a slight increase in transcriptional activity. In contrast, substitution of the TAT sequence at the 3' end of the original 19 bp spacer region with TGC (PsigA6) resulted in lower transcriptional activity than PsigA2. However, considering that PsigA2 exhibits more than 200-fold higher transcriptional activity than the wild-type promoter, PsigA6 was also confirmed to retain high transcriptional activity.

[0127] Therefore, it was confirmed that high transcriptional activity can be achieved through the following: i) Bacillus subtilis P aprE The following substitutions were performed: ii) The -35 region (TCTACT) 37 bp upstream of the promoter was replaced by the shared sequence (TTGACA) of the -35 region; iii) The AA sequence at the 5' end of the original 19 bp spacer region was deleted; iii) Due to the deletion of the AA sequence, the -10 region (TACAAT) 17 bp upstream of the -35 region was replaced by the shared sequence (TATAAT) of the -10 region; and iv) The TAT sequence at the 3' end of the original 19 bp spacer region was replaced by TGT, TGA, TGG, or TGC. In particular, the replacement of the TAT sequence with TGT, TGA, or TGG resulted in a more significant increase in transcriptional activity.

Claims

1. A promoter variant in which Bacillus subtilis aprE The promoter variant has its -35 region replaced by TTGACA and its -10 region replaced by TATAAT. The promoter variant includes a spacer region between the -35 and -10 regions, the spacer region having a length of 19 bp or 17 bp. The TAT at the 3' end of the spacer region is replaced by TGN, and N is A, T, G, or C.

2. The promoter variant of claim 1, wherein the -35 region is located in aprE 37 or 35 bp upstream of the transcription start site (TSS).

3. The promoter variant of claim 2, wherein the -35 region is located in aprE 37 bp upstream of the transcription start site.

4. The promoter variant of claim 1, wherein the -35 region is located in aprE The transcription start site is 37 bp upstream, and the spacer region is the AA sequence at the 5' end that is missing from the 19 bp sequence between the -35 and -10 regions.

5. The promoter variant of claim 1, wherein the promoter variant comprises any nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NO: 22 to SEQ ID NO:

28.

6. An expression cassette comprising a promoter variant and a target gene as described in claim 1.

7. A microorganism comprising a promoter variant according to claim 1; or an expression cassette comprising the promoter variant and a target gene.

8. The microorganism according to claim 7, wherein the microorganism is a Gram-positive bacterium.

9. The microorganism according to claim 8, wherein the Gram-positive bacterium is any Gram-positive bacterium selected from the group consisting of: Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus cereus, Bacillus thuringiensis, Bacillus brevis, Lactobacillus spp. and Bacillus-like bacteria.

10. A method for producing a target product, comprising culturing the microorganism according to claim 7.

11. The method of claim 10, further comprising recovering the target product from the culture medium or the microorganism after culturing.

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