Novel promoter variants for constitutive expression and their uses

Abstract

The present invention provides a novel promoter variant in which some nucleotides are inserted, deleted or substituted in the tatA (twin arginine translocase A) gene promoter of Escherichia coli. The novel promoter variant according to the present invention can constitutively express a target protein, particularly an enzyme, at a high level in E. coli. Therefore, a recombinant strain transformed with an expression vector containing the novel promoter variant according to the present invention can be used to economically mass-produce a target protein, particularly an enzyme. As an example, a recombinant strain transformed with an expression vector containing the novel promoter variant according to the present invention can be used to economically mass-produce allulose epimerization enzyme or to economically mass-produce allulose from fructose.

Description

Technical Field

[0001] The present invention relates to novel promoter mutants and the like, and more particularly to novel promoter mutants capable of constantly highly expressing a target protein and various uses thereof.

Background Art

[0002] With the development of molecular biology, various mechanisms for regulating gene expression have been revealed. Gene expression refers to a series of processes in which proteins are synthesized according to the codes input into genes through transcription and translation that occur within cells. In particular, the transcription process is the initial stage of gene expression, which is initiated by RNA polymerase binding to the promoter sequence existing upstream of the gene with the help of numerous cofactors. A transcription factor (TF) is one of such cofactors and is known to directly bind to the promoter sequence. In particular, since the regulation of gene expression in prokaryotes mainly occurs at the transcription stage, new transcription factors and promoters have been continuously revealed by the present researchers.

[0003] For industrially producing foreign proteins such as enzymes, a transformant prepared by transforming a prokaryote such as Escherichia coli with a pET-type expression vector mainly containing a foreign protein gene is used as an expression system. In a prokaryotic expression system transformed with a pET-type expression vector, generally, an expensive expression inducer such as IPTG (isopropyl-β-D-thiogalactopyranoside) is required, and there are disadvantages that the inducer concentration, equipment, expression induction time, etc. must be finely adjusted.

[0004] On the one hand, in order to mass-produce psicose epimerase (or allulose epimerase) having an activity of converting fructose into allulose (or psicose), an expression system using a Corynebacterium strain, which is a GRAS (Generally Recognized As Safe) strain, as a host cell has been presented. In relation to the Corynebacterium strain-based psicose epimerase (or allulose epimerase) expression system, Korean Registered Patent No. 10-1656063 discloses a nucleotide sequence encoding psicose epimerase, and a regulatory sequence that is operably linked upstream thereof and regulates the expression of the psicose epimerase in a Corynebacterium strain, and the regulatory sequence is composed of a transcriptional promoter, a first ribosome binding region (RBS) sequence and a first spacer sequence, a linker sequence, a second RBS sequence, and the like. In addition, Korean Registered Patent No. 10-1695830 discloses a gene expression cassette including a nucleic acid sequence encoding psicose epimerase and a regulatory sequence that is operably linked upstream thereof and regulates the expression of the psicose epimerase in a Corynebacterium strain, and the regulatory sequence includes a transcriptional promoter derived from Escherichia coli (E. coli). However, the Corynebacterium-based psicose epimerase (or allulose epimerase) expression system has a low expression level of the promoter that can be used, a narrow selection range, and is not suitable for a large-scale enzyme expression system.

[0005] Therefore, for the mass production of psicose epimerase (or allulose epimerase) or the mass production of allulose from fructose using psicose epimerase (or allulose epimerase), not limited to the case of using Corynebacterium as a host cell, there is a need to develop a constitutive expression promoter capable of stably expressing a foreign protein at a high level without growth inhibition under general culture conditions of Escherichia coli host cells, and a constitutive expression system containing the same.

Summary of the Invention

Problems to be Solved by the Invention

[0006] The present invention has been derived under the background of the prior art. The object of the present invention is to provide a novel promoter variant capable of constitutively overexpressing a target protein. Another object of the present invention is to provide various uses of the novel promoter variant.

Means for Solving the Problems

[0007] The inventors of the present invention operably linked the tatA gene promoter, which is a promoter for expressing the tatA (twin arginine translocase A) gene of Escherichia coli, to a polynucleotide encoding an allulose epimerase to prepare a recombinant expression vector, which was introduced into Escherichia coli for transformation. At this time, random mutations occurred in the tatA promoter or the allulose epimerase gene sequence, and the inventors of the present invention secured recombinant Escherichia coli transformed with various recombinant expression vectors. Among the recombinant expression vectors secured by the random mutations, the allulose epimerase expression activities of the recombinant Escherichia coli transformed with a recombinant expression vector in which no mutation occurred in the allulose epimerase gene sequence and a mutation occurred only in the tatA gene promoter were compared. As a result, in the nucleotide sequence of the tatA gene promoter, a promoter mutant in which cytosine (C) was added after thymine (T), which is the 160th nucleotide, or in the nucleotide sequence of the tatA gene promoter, cytosine (C), which is the 146th nucleotide, was replaced with guanine (G), cytosine (C), which is the 148th nucleotide, was replaced with adenine (A), adenine (A), which is the 149th nucleotide, was replaced with guanine (G), and guanine (G) and thymine (T), which are the 159th and 160th nucleotides, were deleted, were confirmed to be able to constantly and highly express allulose epimerase, and the present invention was completed.

[0008] To achieve the above object, an example of the present invention provides a promoter variant composed of the nucleotide sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 10. Further, an example of the present invention provides a recombinant vector containing a promoter variant composed of the nucleotide sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 10. Further, an example of the present invention provides an expression vector containing a polynucleotide encoding a target protein and operably linked thereto, and a promoter variant composed of the nucleotide sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 10. Further, an example of the present invention provides a recombinant strain transformed with the above expression vector. Further, an example of the present invention provides a method for producing a target protein using the above recombinant strain. Further, a preferred example of the present invention provides a method for producing an enzyme conversion reaction product from a substrate, which includes adding the above recombinant strain to a substrate-containing solution and allowing the enzyme conversion reaction to proceed.

Effect of the Invention

[0009] The novel promoter variant according to the present invention can constantly and highly express a target protein, particularly an enzyme, in Escherichia coli. Therefore, when using a recombinant strain transformed with an expression vector containing the novel promoter variant according to the present invention, a target protein, particularly an enzyme, can be economically mass-produced. As an example, when using a recombinant strain transformed with an expression vector containing the novel promoter variant according to the present invention, allose epimerase can be economically mass-produced, or allose can be economically mass-produced from fructose.

Brief Description of the Drawings

[0010]

Figure 1

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Figure 2

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Figure 3

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Figure 4

Mode for Carrying Out the Invention

[0014] Hereinafter, the present invention will be specifically described.

[0015] The term "promoter" used in the present invention means the minimum nucleic acid sequence that is operably linked to the target nucleotide sequence to be transcribed and regulates the transcription of the target nucleotide sequence. Further, the promoter may include a promoter configuration sufficient to express a regulatable promoter-dependent gene induced by cell type specificity or an external signal or agent, and such a configuration can be located in the 5' or 3' region of the gene. The promoter includes both conserved promoters and inducible promoters. The promoter sequence can be derived from prokaryotes, eukaryotes, or viruses. The promoter in prokaryotes is generally defined as the binding site in the vicinity of the transcription start site where RNA polymerase binds.

[0016] The term "promoter variant" used in the present invention is defined as a promoter in which some nucleotides are deleted, added, or substituted in the nucleic acid sequence of the basic promoter and which has a different or improved target protein expression activity from the basic promoter.

[0017] The term "homology" as used in the present invention refers to identity with the nucleic acid sequence of a wild type or a mutant having the same activity. The homology comparison can be carried out visually or using a readily available comparison program to calculate the homology percentage (%) between two or more sequences.

[0018] The term "target protein" as used in the present invention refers to a foreign protein that cannot normally exist in a transformed strain (or host cell) expressing the protein.

[0019] The term "polynucleotide" as used in the present invention means all non-modified or modified polyribonucleotides (RNA) or polydeoxyribonucleotides (DNA). The polynucleotide includes, but is not limited to, single-stranded or double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA that is a mixture of single-stranded and double-stranded regions, or hybrid molecules thereof.

[0020] The term "operably linked" as used in the present invention is defined as a state in which a promoter sequence and a nucleotide sequence encoding a target protein are functionally linked and the promoter can regulate the expression of the target protein. For example, when a promoter can control the expression of a coding sequence (i.e., when the coding sequence is under the transcriptional regulation of the promoter), the promoter is operably linked to the coding sequence and functions, or if the ribosome binding site is positioned such that it can promote translation, the ribosome binding site is operably linked to the coding sequence and functions. The coding sequence is operably linked to the regulatory sequence in the sense or antisense direction.

[0021] The term "recombinant vector" used in the present invention is defined as recombinant DNA prepared by excising a promoter variant or a target gene using a restriction enzyme and inserting the excised DNA into a vector.

[0022] The term "cloning vector" used in the present invention is defined as a substance that can carry a DNA fragment into a host cell and reproduce it. The cloning vector can contain a polyadenylation signal, a transcription termination sequence, and a multiple cloning site. The multiple cloning site contains at least one endonuclease restriction site. Further, the cloning vector can further contain a promoter. Also, the polynucleotide encoding the target protein within the cloning vector can be located upstream of the polyadenylation signal and the transcription termination sequence.

[0023] As used herein, the term "expression vector" is defined as a DNA sequence necessary for the transcription and translation of DNA cloned in a suitable host. Specifically, when present in the cells of an individual, it means a gene construct containing essential regulatory elements operably linked to an insert so that the insert is expressed. Expression vectors can be manufactured and purified using standard recombinant DNA techniques. The type of the expression vector is not particularly limited as long as it functions to express a desired gene and produce a desired protein in various host cells of prokaryotic and eukaryotic cells. The expression vector includes at least a promoter, a start codon, a gene encoding a desired protein, and a termination codon terminator. In addition, the expression vector can also appropriately contain, among others, DNA encoding a signal peptide, additional expression regulatory sequences, untranslated regions on the 5' side and 3' side of the desired gene, a selection marker region, or a replicable unit. The selection marker region can be a selection marker gene for an antibiotic for screening the target vector.

[0024] As used herein, the term "recombinant strain" means a cell in which a polynucleotide encoding one or more target proteins or an expression vector having the same is introduced into a host cell and transformed. Examples of methods for introducing the expression vector into a host cell to produce a transformant include, but are not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation, electroinjection, chemical treatment methods such as PEG, methods using a gene gun, heat shock methods, and the like.

[0025] As used herein, the term "substrate" refers to any substance or compound that is or is to be converted into another compound by the action of an enzyme. The term includes not only a single compound, but also combinations of compounds such as solvents, mixtures, and other materials containing at least one substrate, as well as derivatives thereof.

[0026] One aspect of the present invention relates to a novel promoter variant capable of constantly highly expressing a target protein. The novel promoter variant according to an example of the present invention is composed of the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 10. The inventor of the present invention named the promoter variant composed of the nucleotide sequence of SEQ ID NO: 9 as "tatAm1" and the promoter variant composed of the nucleotide sequence of SEQ ID NO: 10 as "tatAm2". The novel promoter variant tatAm1 according to an example of the present invention is a variant in which the tatA (twin arginine translocase A) gene promoter derived from Escherichia coli composed of the nucleotide sequence of SEQ ID NO: 1 is mutated. Specifically, in the nucleotide sequence of SEQ ID NO: 1, cytosine (C) is added after thymine (T), which is the 160th nucleotide. Further, in the novel promoter variant tatAm2 according to an example of the present invention, cytosine (C), which is the 146th nucleotide in the nucleotide sequence of SEQ ID NO: 1, is substituted with guanine (G), cytosine (C), which is the 148th nucleotide, is substituted with adenine (A), adenine (A), which is the 149th nucleotide, is substituted with guanine (G), and guanine (G) and thymine (T), which are the 159th and 160th nucleotides, are deleted. The promoter variant composed of the nucleotide sequence of SEQ ID NO: 9 is generated by a Ribosome-Binding Site (RBS) spacer mutation, and the promoter variant composed of the nucleotide sequence of SEQ ID NO: 10 is generated by a sequence mutation around the Ribosome-Binding Site (RBS). An expression system containing the novel promoter variant according to an example of the present invention can constantly highly express a target protein in Escherichia coli. Therefore, the novel promoter variant according to an example of the present invention can be used as a promoter for constitutive expression. The novel promoter variant according to an example of the present invention is composed of the nucleotide sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 10, but the equivalent range of the novel promoter variant according to an example of the present invention is not necessarily limited thereto.For example, the equivalent range of the novel promoter variant according to an example of the present invention includes a promoter in which some nucleotides are substituted, inserted, and / or deleted in the nucleotide sequence of SEQ ID NO: 9 within the range where the function of constantly highly expressing the target protein is maintained. Further, the equivalent range of the novel promoter variant according to an example of the present invention includes a sequence having substantial identity to the nucleotide sequence of SEQ ID NO: 9. The above-mentioned substantial identity means that the nucleotide sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 10 is aligned with any other sequence to maximize correspondence, the sequence is analyzed, and the any other sequence has a sequence homology of 70% or more, 90% or more, or 98% or more with the nucleotide sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 10. A person skilled in the art having ordinary knowledge in the art can easily understand that by using gene recombination techniques and the like known in the art, one or more bases in the nucleotide sequence of the novel promoter variant are substituted, added, or deleted to produce a polynucleotide having the same or similar activity within the range having substantial homology. Such homology comparison can be performed by using a commercially available computer program to calculate the homology between two or more sequences as a percentage (%). Therefore, the equivalent range of the novel promoter variant according to an example of the present invention can include a nucleotide sequence having a homology of 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more with the nucleotide sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 10 within the range where the function of constantly highly expressing the target protein is maintained.

[0027] Another aspect of the present invention relates to various uses of the novel promoter variant according to an example of the present invention. Examples of the uses of the novel promoter variant according to an example of the present invention include recombinant vectors, expression vectors, recombinant strains, methods for producing target proteins, methods for exerting the functions of target proteins using recombinant strains, and the like, and are not necessarily limited thereto.

[0028] The recombinant vector according to an example of the present invention includes a promoter variant composed of the nucleotide sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 10.

[0029] The recombinant vector may be a cloning vector. The cloning vector may include an origin of replication, a multi-cloning site (MCS) for cloning the target protein gene, a transcription termination sequence, and a selection marker. The selection marker is for screening cells transformed with the vector, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins can be used. Since only cells expressing the selection marker survive in an environment treated with a selective agent, it is possible to screen transformed cells. For example, the selection marker may be a drug resistance gene such as a kanamycin antibiotic resistance gene or an ampicillin antibiotic resistance gene.

[0030] Alternatively, the recombinant vector may be an expression vector. The expression vector contains a polynucleotide encoding a target protein and a promoter variant composed of the nucleotide sequence of SEQ ID NO: 9, which is operably linked thereto. The promoter variant is preferably located upstream of the polynucleotide encoding the target protein. The type of the target protein expressed through the expression vector according to an example of the present invention is not greatly limited, and can be selected, for example, from proteins involved in the biosynthesis or metabolism of carbohydrates, proteins involved in the biosynthesis or metabolism of lipids and fatty acids, proteins involved in the biosynthesis or metabolism of proteins and peptides, etc. Also, when considering the activity of regulating the expression of the promoter variant, the target protein is preferably an enzyme. The type of the enzyme is not greatly limited and can be selected from D-allulose 3-epimerase, D-tagatose 3-epimerase, (1→4)-α-D-glucan 1-α-D-glucosylmutase, 4-α-D-{(1→4)-α-D-glucano}trehalose trehalohydrolase, L-rhamnose isomerase, fructose-6-phosphate-3-epimerase, etc., and is preferably selected from D-allulose 3-epimerase, (1→4)-α-D-glucan 1-α-D-glucosylmutase, 4-α-D-{(1→4)-α-D-glucano}trehalose trehalohydrolase.In the examples of the present invention, although not specifically described, when the novel promoter variant of the present invention is compared with the tatA (twin arginine translocase A) gene promoter derived from Escherichia coli composed of the nucleotide sequence of SEQ ID NO: 1, (1→4)-α-D-glucan 1-α-D-glucosylmutase, 4-α-D-{(1→4)-α-D-glucano}trehalose trehalohydrolase, etc. were expressed at higher levels. The allulose epimerase is not greatly limited in its type as long as it is an enzyme having an activity of converting fructose into allulose. For example, it can be composed of the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence of SEQ ID NO: 7. Further, the polynucleotide encoding the allulose epimerase can be composed of the nucleotide sequence of SEQ ID NO: 4, the nucleotide sequence of SEQ ID NO: 6, or the nucleotide sequence of SEQ ID NO: 8. Further, the present invention relates to an allulose epimerase and a polynucleotide encoding the same, and includes the contents disclosed in Korean Patent Registration Publication No. 10-1919713, Korean Patent Registration Publication No. 10-2187354, Korean Patent Registration Publication No. 10-1656063, Korean Patent Registration Publication No. 10-1695830, Korean Patent Registration Publication No. 10-2189458, Korean Patent Registration Publication No. 10-1539097, Korean Patent Registration Publication No. 10-1539096, Korean Patent Registration Publication No. 10-1455759, Korean Patent Registration Publication No. 10-1318422, etc. A preferred example of the expression vector according to the present invention has the vector map of FIG. 1 or the vector map of FIG. 2.The expression vector having the vector map of FIG. 1 has a structure in which a replication origin (ori) derived from pUC, a promoter mutant (PtatAm1) consisting of the nucleotide sequence of SEQ ID NO: 9, a polynucleotide [FpDPE (W29K / A77S / G216S / M234I)] encoding allulose epimerase consisting of the nucleotide sequence of SEQ ID NO: 8, a kanamycin resistance gene marker (KanR), etc. are sequentially linked. Further, the expression vector having the vector map of FIG. 2 has a structure in which a replication origin (ori) derived from pUC, a promoter mutant (PtatAm2) consisting of the nucleotide sequence of SEQ ID NO: 10, a polynucleotide [FpDPE (W29K / A77S / G216S / M234I)] encoding allulose epimerase consisting of the nucleotide sequence of SEQ ID NO: 8, a kanamycin resistance gene marker (KanR), etc. are sequentially linked.

[0031] A recombinant strain according to an example of the present invention is one in which a host cell is transformed by the introduction of an expression cassette consisting of a polynucleotide encoding a target protein and a promoter mutant operably linked thereto and composed of the nucleotide sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 10, or an expression vector containing the expression cassette. In the present invention, the host cell that can be transformed with the expression vector is not greatly limited as long as the novel promoter mutant according to an example of the present invention can operate smoothly. It is preferably a prokaryote. When considering the activity of regulating the expression of the novel promoter mutant, the DNA introduction efficiency, the expression efficiency of the introduced DNA, etc., Escherichia coli is more preferable. Examples of the Escherichia coli include, but are not limited to, BL21, JM109, K-12, LE392, RR1, DH5α, or W3110.

[0032] A method for producing a target protein according to an example of the present invention includes culturing the aforementioned recombinant strain to express the target protein, and separating the target protein from the culture broth of the recombinant strain or the cells of the recombinant strain. Depending on its type, the target protein can be present inside the cells of the recombinant strain or secreted outside the cells of the recombinant strain after expression. For example, when the target protein is an allose epimerase, the allose epimerase produced by the recombinant strain will be present inside the cells of the recombinant strain. A method for producing an allose epimerase according to a preferred example of the present invention includes culturing a recombinant strain transformed by the introduction of an expression cassette consisting of a polynucleotide encoding an allose epimerase and a promoter variant composed of the nucleotide sequence of SEQ ID NO: 9 and operably linked thereto, or an expression vector containing the expression cassette, to express the allose epimerase, and separating the allose epimerase from the disrupted product of the recombinant strain in which the allose epimerase has been expressed. Since the novel promoter variant according to an example of the present invention is a constitutive expression vector, expression can be induced without using a protein expression inducer such as IPTG (isopropyl-1-thio-β-D-galactopyranoside). The allose epimerase in the present invention can be recovered from the disrupted product of the recombinant strain. The cells used for protein expression can be disrupted by various physical or chemical means such as repeated freeze-thaw cycles, sonication, mechanical disruption, or cell lysing agents, and can be separated or purified by ordinary biochemical separation techniques (Sambrook et al., Molecular Cloning: A laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989; Deutscher, M., Guide to Protein Purification Methods Enzymology, Vol. 182. Academic Press. Inc., San Diego, CA, 1990).For example, methods for separating or purifying a protein expressed by a host cell include, but are not limited to, electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, reverse phase HPLC, gel permeation HPLC), isoelectric focusing, and various variations or combined methods thereof. On the other hand, in the present invention, the step of separating allulose epimerase from the disrupted product of the recombinant strain can preferably be carried out by affinity chromatography using a peptide tag. As the peptide tag, various known tags such as HA tag, FLAG tag, His tag, BCCP (biotin carboxyl carrier protein), c-myc tag, V5 tag, glutathione-S-transferase (GST), or MBP (maltose binding protein) can be used, among which, the His tag is preferred. The protein tagged with His-tag can be specifically trapped on a column of Ni-NTA (nickel-nitrilotriacetic acid) resin and eluted with EDTA or imidazole.

[0033] A recombinant strain according to an example of the present invention can be used not only for producing a target protein but also for indirectly exerting the function of the target protein. For example, when the target protein is an enzyme, the recombinant strain according to an example of the present invention can be used to produce a product by an enzyme conversion reaction from a substrate. Specifically, when the target protein is an allose epimerase, the present invention provides a method for producing allose from fructose, which includes the step of adding the recombinant strain to a fructose-containing solution and reacting. The recombinant strain is a host cell transformed by introducing an expression cassette consisting of a polynucleotide encoding an allose epimerase and a promoter variant operably linked thereto and composed of the nucleotide sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 10, or an expression vector containing the expression cassette, and preferably, it is recombinant Escherichia coli. Also, the fructose-containing solution can further contain metal ions such as Ca 2+ , Mn 2+ to promote the activity of the allose epimerase. In the method for producing allose from fructose, the reaction temperature is 50 to 70°C, preferably 55 to 65°C, and more preferably in the range of 55 to 60°C when considering the smooth enzyme expression, enzyme stability and maximum activity of the recombinant strain. The reaction pH is in the range of 6.5 to 8, preferably 6.5 to 7.5, and more preferably 6.5 to 7. The concentration of fructose in the method for producing allose from fructose is not particularly limited, but preferably 1 to 75% (w / w) based on all reactants, and more preferably 35 to 45% (w / w) when considering productivity or economy.

[0034] Hereinafter, the present invention will be described more specifically through examples. However, the following examples are only for clearly exemplifying the technical features of the present invention and do not limit the protection scope of the present invention.

Examples

[0035] Example 1: Securing a promoter for enzyme gene expression 1.1. Securing the tatA (twin arginine translocase A) gene promoter The tatA (twin arginine translocase A) gene promoter is a promoter for expressing the tatA (twin arginine translocase A) gene of Escherichia coli. It is composed of the nucleotide sequence of SEQ ID NO: 1 and is known as a promoter that induces constitutive expression in Escherichia coli.

[0036] To secure a polynucleotide fragment corresponding to the promoter region of the tatA (twin arginine translocase A) gene from Escherichia coli, genomic DNA of Escherichia coli MG1655 was used as a template, and PCR was performed using the primer set shown in Table 1 below. The obtained amplification product was cloned using the pGEM-Teasy vector (Promega Co., USA), and the nucleotide sequence was analyzed. As a result, it was confirmed that the polynucleotide fragment had a length of 160 bp and was composed of the nucleotide sequence of SEQ ID NO: 1.

[0037]

Table 1

[0038] 1.2. Securing the BLMA gene promoter The BLMA gene promoter is a promoter for expressing the maltogenic amylase gene of Bacillus licheniformis, which is composed of the nucleotide sequence of SEQ ID NO: 2 and is known as a promoter that induces stable high expression of foreign genes in Escherichia coli [see TAE-JIP KIM et al (1999) Modes of Action of Acarbose Hydrolysis and Transglycosylation Catalyzed by a Thermostable Maltogenic Amylase, the Gene for Which Was Cloned from a Thermus Strain].

[0039] To obtain a polynucleotide fragment corresponding to the promoter region of the maltogenic amylase gene from Bacillus licheniformis, genomic DNA of Bacillus licheniformis ATCC14580 was used as a template, and PCR was performed using the primer set described in Table 2 below. The obtained amplification product was cloned using pGEM-Teasy vector (Promega Co., USA), and the nucleotide sequence was analyzed. As a result, it was confirmed that the polynucleotide fragment had a length of 115 bp and was composed of the nucleotide sequence of SEQ ID NO: 2.

[0040]

Table 2

[0041] Example 2: Obtaining an allulose epimerase mutant cloning vector The applicant of the present invention disclosed a wild-type D-allose epimerase derived from Flavonifractor plautii and a polynucleotide encoding the same through Korean Patent Registration Publication No. 10-14739180. The wild-type D-allose epimerase is composed of the amino acid sequence of SEQ ID NO: 3, and the polynucleotide encoding the same is composed of the nucleotide sequence of SEQ ID NO: 4.

[0042] In addition, the applicant of the present invention disclosed a D-allose epimerase mutant W29K / G216S / M234I with improved conversion rate of fructose to allose and thermal stability and a polynucleotide encoding the same through Korean Patent Publication No. 10-2021-0132405. The D-allose epimerase mutant W29K / G216S / M234I is such that tryptophan (Trp) at the 29th position in the amino acid sequence of the wild-type D-allose epimerase derived from Flavonifractor plautii is replaced by lysine (Lys), glycine (Gly) at the 216th position is replaced by serine (Ser), and at the same time methionine (Met) at the 234th position is replaced by isoleucine (Ile), and it is composed of the amino acid sequence of SEQ ID NO: 5, and the polynucleotide encoding the same is composed of the nucleotide sequence of SEQ ID NO: 6.

[0043] In addition, the applicant of the present invention conceived of a D-allose epimerase mutant W29K / A77S / G216S / M234I that has extremely excellent thermal stability under high-temperature conditions and filed an application on December 14, 2021 (Korean Patent Application No. 10-2021-0178690, in an unpublished state). The D-allose epimerase mutant W29K / A77S / G216S / M234I is such that tryptophan (Trp) present at the 29th position in the amino acid sequence of the wild-type D-allose epimerase derived from Flavonifractor plautii is replaced with lysine (Lys), alanine (Als) present at the 77th position is replaced with serine (Ser), glycine (Gly) present at the 216th position is replaced with serine (Ser), and simultaneously methionine (Met) present at the 234th position is replaced with isoleucine (Ile), and it is composed of the amino acid sequence of SEQ ID NO: 7, and the polynucleotide encoding this is composed of the base sequence of SEQ ID NO: 8.

[0044] Based on the polynucleotide of D-allose epimerase variant W29K / G216S / M234I (SEQ ID NO: 6), a polynucleotide fragment encoding the amino acid sequence of D-allose epimerase variant W29K / A77S / G216S / M234I was prepared using the overlap extension polymerase chain reaction method. Specifically, 1 pM of the oligonucleotide primers (A77S forward primer, A77S reverse primer) shown in Table 3 below and 100 ng of the polynucleotide of D-allose epimerase variant W29K / G216S / M234I (SEQ ID NO: 6) used as a template were mixed in a reaction solution supplemented with 100 μM of deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP). Using a Thermocycler (TP600, TAKARA BIO Inc., JAPAN), a PCR reaction was carried out with 25 to 30 cycles in the presence of 1 unit of pfu-X DNA polymerase mixture (Bioneer). After amplifying the mutant fragment by the primer combination, using the amplified fragment as a template and the oligonucleotide primers (NdeI forward primer, XhoI reverse primer) into which the sequences of the NdeI and XhoI restriction enzyme recognition sites shown in Table 3 below were introduced, a polynucleotide fragment (SEQ ID NO: 8) encoding the amino acid sequence of D-allose epimerase variant W29K / A77S / G216S / M234I was finally prepared by overlap extension PCR. Then, the prepared polynucleotide fragment was inserted into the same restriction enzyme sites of the pET28a vector (Novagen) using the restriction enzymes NdeI and XhoI to secure a cloning vector.

[0045]

Table 3

[0046] Example 3: Production of a Ligated Fragment of a Promoter and a Gene of an Allose Epimerase Variant 3.1. Production of a DNA fragment in which the tatA promoter and the D-allose epimerase mutant gene are linked To amplify the tatA promoter, PCR was performed using the pGEM-Teasy vector cloned with the tatA promoter secured in Example 1 as a template and the primer sets (XhoI-PtatA, PtatA-FpDPE_R) shown in Table 4 below.

[0047] Also, to amplify the gene of W29K / A77S / G216S / M234I, a D-allose epimerase mutant derived from Flavonifractor plautii, PCR was performed using the pET28a vector (Novagen) cloned with the gene of the D-allose epimerase mutant W29K / A77S / G216S / M234I secured in Example 2 as a template and the primer sets (PtatA-FpDPE_F, PstI-FpDPE) shown in Table 4 below.

[0048]

Table 4

[0049] The tatA promoter fragment and the D-allose epimerase mutant gene fragment amplified in this way can be ligated into one fragment by the complementary sequences of the primers used during amplification. Using the two fragments as templates and the primers (XhoI-PtatA, PstI-FpDPE) into which the sequences of the XhoI and PstI restriction enzyme recognition sites shown in Table 4 above were introduced, overlap extension PCR was performed to obtain one amplified fragment. The DNA fragment in which the secured tatA promoter and the gene of the allose epimerase mutant W29K / A77S / G216S / M234I were linked was named "PtatA-FpDPE".

[0050] 3.2. Production of a DNA fragment in which the BLMA promoter and the D-allose epimerase gene are linked To amplify the BLMA promoter, PCR was performed using the pGEM-Teasy vector cloned with the BLMA promoter secured in Example 1 as a template and the primer sets (XhoI-PblmA, Pblma-FpDPE_R) shown in Table 5 below.

[0051] Also, to amplify the gene of the D-allose epimerase mutant W29K / A77S / G216S / M234I, which is a D-allose epimerase mutant derived from Flavonifractor plautii, PCR was performed using the pET28a vector (Novagen) cloned with the gene of the D-allose epimerase mutant W29K / A77S / G216S / M234I secured in Example 2 as a template and the primer sets (Pblma-FpDPE_F, PstI-FpDPE) shown in Table 5 below.

Table 5

[0052] Example 4: Production of D-allose epimerase mutant expression vector 4.1. Production of D-allose epimerase mutant expression vector having the tatA promoter A recombinant plasmid vector containing an origin of replication derived from pUC that can replicate in Escherichia coli by genetic recombination from the pTrc99A vector (Pharmacia, US), a multi-cloning site (MCS) such as restriction enzyme XhoI and PstI sites, a transcription terminator, and a kanamycin antibiotic resistance gene was constructed. Then, after cleaving the polynucleotide fragment PtatA-FpDPE prepared in Example 3 with restriction enzymes XhoI and PstI, it was ligated with the recombinant plasmid vector having the same restriction enzyme sites to produce the D-allose epimerase mutant expression vector pPtatA:FpDPE (W29K / A77S / G216S / M234I). Then, the D-allose epimerase mutant expression vector was transformed into Escherichia coli DH5α by the heat shock method (see Sambrook and Russell: Molecular Cloning), colonies with kanamycin resistance were secured, five colonies were selected, and five types of the recombinant expression vector pPtatA:FpDPE (W29K / A77S / G216S / M234I) were recovered and then the nucleotide sequences were analyzed. As a result of the sequence analysis of five types of the pPtatA:FpDPE (W29K / A77S / G216S / M234I) recombinant expression vectors, random mutations were confirmed in the introduced tatA promoter or the allose epimerase mutant W29K / A77S / G216S / M234I gene sequence, and the random mutation details are shown in Table 6 below.

[0053]

Table 6

[0054] As shown in Table 6 above, in the case of the recombinant expression vectors recovered from colonies 2, 3, and 5, mutations occurred in the allulose epimerase mutant gene sequence, and it was determined that translation for producing the target allulose epimerase mutant could not occur. On the contrary, in the case of the recombinant expression vector recovered from colony 1, a mutation occurred in the spacer sequence located between the ribosome-binding site (RBS) of the tatA promoter and the translation start codon. In the case of the recombinant expression vector recovered from colony 4, mutations occurred in the sequences around the ribosome-binding site (RBS) of the tatA promoter, and since the enzyme gene sequences were identical, it was determined that there was a possibility of expressing the target enzyme. The mutant promoter in the recombinant expression vector recovered from colony 1 was named "tatAm1", and the mutant promoter in the recombinant expression vector recovered from colony 4 was named "tatAm2". The tatAm1 promoter is composed of the nucleotide sequence of SEQ ID NO: 9, and the tatAm2 promoter is composed of the nucleotide sequence of SEQ ID NO: 10. Also, the recombinant expression vector recovered from colony 1 was renamed "pPtatapAm1:FpDPE(W29K / A77S / G216S / M234I)", and the recombinant expression vector recovered from colony 4 was renamed "pPtatAm2:FpDPE(W29K / A77S / G216S / M234I)". Figure 1 is a vector map of the recombinant expression vector pPtatAm1:FpDPE(W29K / A77S / G216S / M234I) produced in the example of the present invention. Figure 2 is a vector map of the recombinant expression vector pPtatAm2:FpDPE(W29K / A77S / G216S / M234I) produced in the example of the invention.

[0055] 4.2. Production of D-allulose epimerase mutant expression vector having BLMA promoter A recombinant plasmid vector containing an origin of replication (replication origin) derived from pUC that can be replicated in E. coli through genetic recombination from the pTrc99A vector (Pharmacia, US), a multi-cloning site (Multi cloning site, MCS) such as restriction enzyme XhoI and PstI sites, a transcription terminator, and a kanamycin antibiotic resistance gene was prepared. Then, after cleaving the polynucleotide fragment Pblma-FpDPE prepared in Example 3 with restriction enzymes XhoI and PstI, it was ligated to the recombinant plasmid vector having the same restriction enzyme positions to produce the D-allose epimerase mutant expression vector pPblma:FpDPE (W29K / A77S / G216S / M234I). Thereafter, the D-allose epimerase mutant expression vector was transformed into E. coli DH5α by the heat shock method (see Sambrook and Russell: Molecular Cloning), colonies having kanamycin resistance were secured, six colonies were selected, and six types of the recombinant expression vector pPblma:FpDPE (W29K / A77S / G216S / M234I) were recovered and then the nucleotide sequence was analyzed. As a result of the sequence analysis of the six types of the recombinant expression vector pPblma:FpDPE (W29K / A77S / G216S / M234I), it was confirmed that all the intended BLMA promoter and allose epimerase mutant gene sequences were present. Figure 3 is a vector map of the recombinant expression vector pPblma:FpDPE (W29K / A77S / G216S / M234I) produced in the example of the present invention.

[0056] Example 5: Production of transformants using a D-allose epimerase mutant expression vector Each of the recombinant expression vectors pPtatAm1:FpDPE(W29K / A77S / G216S / M234I), pPtatAm2:FpDPE(W29K / A77S / G216S / M234I), and pPblma:FpDPE(W29K / A77S / G216S / M234I) produced in Example 4 was introduced into Escherichia coli (E. coli) W3110 by the heat shock method. Subsequently, the presence or absence of kanamycin antibiotic resistance was confirmed, and recombinant strains transformed with the recombinant expression vector were selected. A glycerol solution was added to the prepared recombinant E. coli so that the final concentration was 20% (v / v), and it was stored frozen at -70°C before culturing for enzyme expression.

[0057] Example 6: Measurement of the conversion rate of fructose to allulose by recombinant strains and comparison of the enzyme expression intensity of promoters Since the D-allulose epimerase can convert fructose to allulose, the strength of enzyme expression induction of each promoter in the recombinant strain was compared through measurement of the conversion rate of fructose to allulose, which is proportional to the enzyme expression level of the recombinant strain.

[0058] To culture the recombinant Escherichia coli transformed with the recombinant expression vector, 100 ml of LB medium containing kanamycin at a final concentration of 50 μg / ml was placed in a 1 L flask, and 1 ml of the recombinant Escherichia coli prepared in Example 5 was inoculated therein. Then, the flask was transferred to a shaking incubator, and the recombinant Escherichia coli was cultured for 14 hours (hr) while maintaining the temperature condition of 30°C and the shaking condition of 140 rpm. The culture solution was centrifuged to recover the cells. Thereafter, the recovered cells were added at a concentration of 1 mg / ml to a 50 mM PIPES buffer solution (pH 7.0) containing 30% (w / w) fructose and 1 mM metal ions of manganese sulfate (MnSO4). After allowing the reaction to proceed for a predetermined time at 62°C, the temperature of the reaction product solution was lowered to 4°C to stop the reaction, and the solution was centrifuged under the conditions of 16,600×g and 4°C to recover the supernatant. Thereafter, using high performance liquid chromatography (HPLC), the concentrations of allulose and fructose in the supernatant were measured, and the conversion rate of fructose to allulose was calculated from the measured results. Then, the conversion rate was used as an index of enzyme activity. FIG. 4 shows the conversion rate according to the reaction time when the conversion reaction of fructose to allulose was carried out using the recombinant Escherichia coli prepared in the examples of the present invention. In FIG. 4, "PtatAm1" indicates the recombinant Escherichia coli transformed with the recombinant expression vector pPtatAm1:FpDPE (W29K / A77S / G216S / M234I), "PtatAm2" indicates the Escherichia coli transformed with the recombinant expression vector pPtatAm2:FpDPE (W29K / A77S / G216S / M234I), and "Pblma" indicates the Escherichia coli transformed with the recombinant expression vector pPblma:FpDPE (W29K / A77S / G216S / M234I). As shown in FIG. 4, the recombinant Escherichia coli into which the tatAm1 promoter was introduced and the recombinant Escherichia coli into which the tatAm2 promoter was introduced showed a very high conversion rate of fructose to allulose compared to the recombinant Escherichia coli into which the BLMA promoter was introduced. From such results, it can be seen that the enzyme expression induction effects of the tatAm1 promoter and the tatAm2 promoter are very strong compared to the BLMA promoter.On the other hand, if the expression intensity of the target gene by the constitutive promoter is too strong, it can impose a burden on the recombinant strain. Therefore, even if the expression intensity by the tatAm1 promoter is somewhat lower than that by the tatAm2 promoter, from a commercial perspective, the tatAm1 promoter is judged to be more advantageous.

[0059] As described above, the present invention has been described through the above embodiments, but the present invention is not necessarily limited thereto, and various modifications can be made without departing from the scope and spirit of the present invention. Therefore, the protection scope of the present invention should be interpreted as including all embodiments belonging to the scope of the claims attached to the present invention.

Claims

1. A promoter variant composed of the nucleotide sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO:

10.

2. A recombinant vector comprising the promoter variant according to Claim 1.

3. An expression vector comprising a polynucleotide encoding a target protein and the promoter variant according to Claim 1 operably linked thereto.

4. The expression vector according to Claim 3, wherein the target protein is an enzyme.

5. The expression vector according to Claim 4, wherein the enzyme is an allulose epimerase.

6. The expression vector according to Claim 5, wherein the allulose epimerase is composed of the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence of SEQ ID NO:

7.

7. The expression vector according to Claim 5, wherein the polynucleotide encoding the allulose epimerase is composed of the nucleotide sequence of SEQ ID NO: 4, the nucleotide sequence of SEQ ID NO: 6, or the nucleotide sequence of SEQ ID NO:

8.

8. A recombinant strain transformed with the expression vector according to Claim 5.

9. A method for producing allulose from fructose, comprising the step of adding the recombinant strain according to Claim 8 to a fructose-containing solution and reacting them.

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