Novel promoter variant for constitutive expression and its use

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 a novel promoter mutant and the like, and more particularly to a novel promoter mutant capable of constitutively and highly expressing a target protein and various uses thereof. [Background technology]

[0002] With the development of molecular biology, various mechanisms that regulate gene expression have been revealed. Gene expression refers to a series of processes in which proteins are synthesized according to the code entered in genes through transcription and translation that occur in cells. In particular, the transcription process is the initial stage of gene expression, and is initiated when RNA polymerase binds to the promoter sequence located upstream of the gene with the help of various cofactors. Transcription factors (TFs) are one of such cofactors, and are known to bind directly to the promoter sequence. In particular, regulation of gene expression in prokaryotes occurs mainly at the transcription stage, and new transcription factors and promoters are continually being identified by our researchers.

[0003] To industrially produce foreign proteins such as enzymes, a transformant prepared by transforming a prokaryote such as E. coli with a pET-type expression vector containing a foreign protein gene is used as an expression system. Prokaryote expression systems transformed with pET-type expression vectors generally require expensive expression inducers such as IPTG (isopropyl-β-D-thiogalactopyranoside), and have the disadvantage of requiring detailed adjustment of the inducer concentration, equipment, and expression induction time.

[0004] Meanwhile, an expression system using a Corynebacterium sp. strain, which is a generally recognized as safe (GRAS) strain, as a host cell has been proposed to mass-produce a cyclose epimerase enzyme (or allulose epimerase enzyme) having an activity of converting fructose to allulose (or cyclose). Korean Patent No. 10-1656063, which relates to a cyclose epimerase enzyme (or allulose epimerase enzyme) expression system based on a Corynebacterium sp. strain, discloses a gene expression cassette including a nucleotide sequence encoding a cyclose epimerase enzyme and a regulatory sequence operably linked to the upstream thereof and regulating the expression of the cyclose epimerase enzyme in a Corynebacterium sp. strain, and the regulatory sequence is composed of a transcription promoter, a first ribosome binding region (RBS) sequence, a first spacer sequence, a linker sequence, a second RBS sequence, etc. In addition, Korean Patent Registration No. 10-1695830 discloses a gene expression cassette including a nucleic acid sequence encoding a cyclase epimerization enzyme and a regulatory sequence operably linked to the upstream thereof to regulate the expression of the cyclase epimerization enzyme in a Corynebacterium strain, and the regulatory sequence includes a transcription promoter derived from E. coli. However, the expression system for the cyclase epimerization enzyme (or allulose epimerization enzyme) based on Corynebacterium has a low expression level of the promoters that can be used, and the range of selection is narrow, so it is not suitable for a large-scale expression system for the enzyme.

[0005] Therefore, for mass production of cyclose epimerization enzyme (or allulose epimerization enzyme) or mass production of allulose from fructose using cyclose epimerization enzyme (or allulose epimerization enzyme), it is necessary to develop a constitutive expression promoter and a constitutive expression system including the same that can stably express foreign proteins at high levels without growth inhibition under general culture conditions of E. coli host cells, not limited to when Corynebacterium is used as a host cell. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention has been devised under the conventional technical background, and an object of the present invention is to provide a novel promoter mutant capable of constitutively and highly expressing a target protein, and to provide various uses of the novel promoter mutant. [Means for solving the problem]

[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 allulose epimerase to prepare a recombinant expression vector, which was then introduced into Escherichia coli to transform it. At this time, random mutations occurred in the tatA promoter or the allulose epimerase gene sequence, and the inventors of the present invention obtained recombinant Escherichia coli transformed with various recombinant expression vectors. Among the recombinant expression vectors obtained by the random mutation, the allulose epimerization enzyme expression activity of recombinant E. coli transformed with recombinant expression vectors in which no mutation occurred in the allulose epimerization enzyme gene sequence, but only in the tatA gene promoter, was compared. As a result, it was confirmed that a promoter variant in which cytosine (C) is added after thymine (T), the 160th nucleotide in the base sequence of the tatA gene promoter, or a promoter variant in which cytosine (C), the 146th nucleotide, in the base sequence of the tatA gene promoter, is replaced with guanine (G), cytosine (C), the 148th nucleotide, in the base sequence of the tatA gene promoter, is replaced with adenine (A), the 149th nucleotide, in the base sequence of the tatA gene promoter, is replaced with guanine (G), and the 159th and 160th nucleotides, guanine (G) and thymine (T), are deleted, are capable of constitutively expressing high levels of allulose epimerization enzyme, thereby completing the present invention.

[0008] In order to achieve the above object, one example of the present invention provides a promoter variant consisting of the nucleotide sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 10. Another example of the present invention provides a recombinant vector comprising the promoter variant consisting of the nucleotide sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 10. Another example of the present invention provides an expression vector comprising a polynucleotide encoding a target protein and a promoter variant consisting of the nucleotide sequence of SEQ ID NO: 9 or the nucleotide sequence of SEQ ID NO: 10, operably linked thereto. Another example of the present invention provides a recombinant strain transformed with the expression vector. Another example of the present invention provides a method for producing a target protein using the recombinant strain. Another preferred example of the present invention provides a method for producing an enzyme conversion reaction product from a substrate, comprising the steps of adding the recombinant strain to a substrate-containing solution and proceeding with an enzyme conversion reaction. Effect of the Invention

[0009] The novel promoter variant according to the present invention can stably and highly express a target protein, particularly an enzyme, 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. For 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. [Brief description of the drawings]

[0010] [Figure 1] 1 shows a vector map of the recombinant expression vector pPtatAm1:FpDPE (W29K / A77S / G216S / M234I) produced in an example of the present invention.

[0011] [Diagram 2]1 is a vector map of the recombinant expression vector pPtatAm2:FpDPE (W29K / A77S / G216S / M234I) prepared in an embodiment of the present invention.

[0012] [Diagram 3] 1 shows a vector map of the recombinant expression vector pPblma:FpDPE (W29K / A77S / G216S / M234I) produced in an example of the present invention.

[0013] [Figure 4] 1 shows the conversion rate as a function of reaction time when the conversion reaction of fructose to allulose is carried out using the recombinant E. coli prepared in the examples of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention will be specifically described below.

[0015] The term "promoter" as used herein refers to a minimal nucleic acid sequence that is operably linked to a nucleotide sequence of interest that is to be transcribed and that regulates the transcription of the nucleotide sequence of interest. The promoter may also include a promoter structure sufficient to express a regulatable promoter-dependent gene that is cell type specific or induced by an external signal or agent, and such structure may be located in the 5' or 3' region of the gene. The promoter includes both conserved and inducible promoters. The promoter sequence may be derived from a prokaryote, a eukaryote, or a virus. In prokaryotes, a promoter is generally defined as a binding site immediately adjacent to the transcription start site where RNA polymerase binds.

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

[0017] The term "homology" as used herein indicates identity with a wild type or a mutant nucleic acid sequence having the same activity, and homology can be compared by naked eye or by using a readily available comparison program, and the homology between two or more sequences can be calculated as a percentage (%).

[0018] The term "target protein" as used herein means a foreign protein that is not normally present in the transformed strain (or host cell) expressing said protein.

[0019] The term "polynucleotide" as used herein refers to any polyribonucleotide (RNA) or polydeoxyribonucleotide (DNA), whether non-modified or modified, including, but 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 herein is defined as a state in which a promoter sequence and a nucleotide sequence encoding a protein of interest are functionally linked, and the promoter can regulate the expression of the protein of interest. For example, a promoter is operatively linked to a coding sequence if it is capable of controlling the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter), and a ribosome binding site is operatively linked to a coding sequence if the ribosome binding site is positioned so as to facilitate translation. The coding sequence is operably linked to a regulatory sequence in either the sense or antisense orientation.

[0021] The term "recombinant vector" as used herein is defined as a recombinant DNA prepared by cutting out a promoter mutant or a gene of interest using a restriction enzyme and inserting it into a vector.

[0022] The term "cloning vector" as used herein is defined as a substance capable of carrying and reproducing a DNA fragment in a host cell. The cloning vector may include a polyadenylation signal, a transcription termination sequence, and a multiple cloning site. The multiple cloning site includes at least one endonuclease restriction site. The cloning vector may further include a promoter. In the cloning vector, a polynucleotide encoding a target protein may be located upstream of the polyadenylation signal and the transcription termination sequence.

[0023] The term "expression vector" as used herein is defined as a DNA sequence required for transcription and translation of cloned DNA in a suitable host, specifically a genetic construct that includes essential regulatory elements operably linked to an insert such that the insert is expressed when present in an individual's cells. Expression vectors can be produced and purified using standard recombinant DNA techniques. The type of expression vector is not particularly limited as long as it functions to express a desired gene and produce a desired protein in various prokaryotic and eukaryotic host cells. The expression vector includes at least a promoter, an initiation codon, a gene encoding the desired protein, and a stop codon terminator. In addition, the expression vector may also include DNA encoding a signal peptide, additional expression regulatory sequences, untranslated regions 5' and 3' of the desired gene, a selection marker region, or a replicable unit, as appropriate. The selection marker region may be an antibiotic selection marker gene for selecting the desired vector.

[0024] The term "recombinant strain" as used herein refers to a cell transformed by introducing a polynucleotide encoding one or more target proteins or an expression vector having the same into a host cell. Methods for producing a transformant by introducing the expression vector into a host cell 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, and heat shock.

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

[0026] One aspect of the present invention relates to a novel promoter mutant capable of constitutively and highly expressing a target protein. The novel promoter mutant according to one embodiment of the present invention is composed of the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 10. The inventors of the present invention have named the promoter mutant composed of the nucleotide sequence of SEQ ID NO: 9 as "tatAm1" and the promoter mutant composed of the nucleotide sequence of SEQ ID NO: 10 as "tatAm2". The novel promoter mutant tatAm1 according to one embodiment of the present invention is a mutant of the tatA (twin arginine translocase A) gene promoter derived from Escherichia coli composed of the nucleotide sequence of SEQ ID NO: 1, and specifically, cytosine (C) is added after thymine (T), which is the 160th nucleotide in the nucleotide sequence of SEQ ID NO: 1. In addition, the novel promoter mutant tatAm2 according to one embodiment of the present invention is a mutant in which the 146th nucleotide, cytosine (C), is replaced with guanine (G), the 148th nucleotide, cytosine (C), is replaced with adenine (A), the 149th nucleotide, adenine (A), is replaced with guanine (G), and the 159th and 160th nucleotides, guanine (G) and thymine (T), are deleted in the base sequence of SEQ ID NO: 1. The promoter mutant composed of the base sequence of SEQ ID NO: 9 is generated by a spacer mutation in the ribosome-binding site (RBS), and the promoter mutant composed of the base sequence of SEQ ID NO: 10 is generated by a sequence mutation around the ribosome-binding site (RBS). An expression system including the novel promoter mutant according to one embodiment of the present invention can constitutively express a target protein at a high level in E. coli. Therefore, the novel promoter mutant according to one embodiment of the present invention can be used as a constitutive expression promoter. A novel promoter variant according to one embodiment of the present invention is composed of the base sequence of SEQ ID NO: 9 or the base sequence of SEQ ID NO: 10, but the equivalent range of the novel promoter variant according to one embodiment of the present invention is not necessarily limited thereto.For example, the equivalent range of the novel promoter variant according to one embodiment of the present invention includes a promoter in which some nucleotides are substituted, inserted and / or deleted in the base sequence of SEQ ID NO: 9 within a range in which the function of constitutively expressing a high level of a target protein is maintained. In addition, the equivalent range of the novel promoter variant according to one embodiment of the present invention includes a sequence having substantial identity to the base sequence of SEQ ID NO: 9. The above-mentioned substantial identity means that the base sequence of SEQ ID NO: 9 or the base sequence of SEQ ID NO: 10 is aligned with any other sequence so as to correspond to the maximum extent possible, and 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 base sequence of SEQ ID NO: 9 or the base sequence of SEQ ID NO: 10. A technician having ordinary skill in the art will easily understand that a polynucleotide having the same or similar activity can be produced within a range of substantial homology by substituting, adding or deleting one or more bases in the base sequence of the novel promoter variant using gene recombination techniques known in the art. Such a comparison of homology can be performed by calculating the homology between two or more sequences in percentage (%) using a commercially available computer program. Therefore, the equivalent range of the novel promoter mutant according to one embodiment of the present invention can include a base sequence having 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more homology with the base sequence of SEQ ID NO: 9 or the base sequence of SEQ ID NO: 10 within a range that maintains the function of constantly expressing a target protein at a high level.

[0027] Another aspect of the present invention relates to various uses of the novel promoter variant according to one embodiment of the present invention, including, but not limited to, recombinant vectors, expression vectors, recombinant strains, methods for producing a target protein, and methods for exerting the function of a target protein using recombinant strains.

[0028] A recombinant vector according to one embodiment of the present invention comprises a promoter mutant consisting 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 a replication origin, a multicloning site (MCS) for cloning a gene of interest, a transcription termination sequence, and a selection marker. The selection marker is used to select cells transformed with the vector, and may be a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to a cytotoxic agent, or expression of a surface protein. Only cells expressing the selection marker survive in an environment treated with a selective agent, making it possible to select 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] The recombinant vector may be an expression vector. The expression vector includes a polynucleotide encoding a target protein and a promoter variant operably linked thereto and consisting of the base sequence of SEQ ID NO: 9. The promoter variant is preferably located upstream of the polynucleotide encoding the target protein. The type of target protein expressed through the expression vector according to one embodiment of the present invention is not limited to a large extent and may be selected from, for example, proteins involved in the biosynthesis or metabolism of carbohydrates, proteins involved in the biosynthesis or metabolism of lipids and fatty acids, and proteins involved in the biosynthesis or metabolism of proteins and peptides. In addition, the target protein is preferably an enzyme, taking into consideration the expression regulation activity of the promoter variant. The type of the enzyme is not particularly limited, and examples thereof include D-allulose 3-epimerase, D-tagatose 3-epimerase, (1→4)-alpha-D-glucan 1-alpha-D-glucosylmutase, 4-alpha-D-{(1→4)-alpha-D-glucano}trehalose trehalohydrolase, L-rhamnose isomerase, and the like. The enzyme may be selected from D-allulose 3-epimerase, (1→4)-α-D-glucan 1-α-D-glucosylmutase, and 4-α-D-{(1→4)-α-D-glucano}trehalose trehalohydrolase.Although not specifically described in the examples of the present invention, the novel promoter variant of the present invention expressed (1→4)-α-D-glucan 1-α-D-glucosylmutase, 4-α-D-{(1→4)-α-D-glucano}trehalose trehalohydrolase, etc. at higher levels when compared with the tatA (twin arginine translocase A) gene promoter derived from Escherichia coli composed of the base sequence of SEQ ID NO: 1. The allulose epimerization enzyme is not limited to a particular type as long as it is an enzyme having the activity of converting fructose to allulose, and may be composed of, for example, 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. Furthermore, the polynucleotide encoding the allulose epimerization enzyme can be composed of the base sequence of SEQ ID NO:4, the base sequence of SEQ ID NO:6, or the base sequence of SEQ ID NO:8. In addition, the present invention relates to allulose epimerization enzymes and polynucleotides encoding the same, and includes the contents disclosed in Korean Patent Publication No. 10-1919713, Korean Patent Publication No. 10-2187354, Korean Patent Publication No. 10-1656063, Korean Patent Publication No. 10-1695830, Korean Patent Publication No. 10-2189458, Korean Patent Publication No. 10-1539097, Korean Patent Publication No. 10-1539096, Korean Patent Publication No. 10-1455759, Korean Patent Publication No. 10-1318422, etc. An expression vector according to a preferred embodiment of 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 Figure 1 has a structure in which a replication origin (ori) derived from pUC, a promoter mutant (PtatAm1) consisting of the base sequence of SEQ ID NO: 9, a polynucleotide encoding an allulose epimerase consisting of the base sequence of SEQ ID NO: 8 [FpDPE (W29K / A77S / G216S / M234I)], a kanamycin resistance gene marker (KanR), etc. are sequentially linked. The expression vector having the vector map of Figure 2 has a structure in which a replication origin (ori) derived from pUC, a promoter mutant (PtatAm2) consisting of the base sequence of SEQ ID NO: 10, a polynucleotide encoding an allulose epimerase consisting of the base sequence of SEQ ID NO: 8 [FpDPE (W29K / A77S / G216S / M234I)], a kanamycin resistance gene marker (KanR), etc. are sequentially linked.

[0031] The recombinant strain according to one embodiment of the present invention is a host cell transformed by introducing an expression cassette comprising a polynucleotide encoding a target protein and a promoter variant operably linked thereto and consisting 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 particularly limited as long as the novel promoter variant according to one embodiment of the present invention can smoothly operate, and is preferably a prokaryote, and more preferably Escherichia coli in consideration of the expression regulation activity of the novel promoter variant, the introduction efficiency of DNA, the expression efficiency of the introduced DNA, etc. Examples of the Escherichia coli include, but are not limited to, BL21, JM109, K-12, LE392, RR1, DH5α, and W3110.

[0032] A method for producing a target protein according to an embodiment of the present invention includes culturing the recombinant strain to express the target protein, and isolating the target protein from the culture solution or cells of the recombinant strain. The target protein may be present in the recombinant strain or secreted outside the recombinant strain after expression depending on its type. For example, when the target protein is an allulose epimerization enzyme, the allulose epimerization enzyme produced by the recombinant strain is present in the recombinant strain. A method for producing an allulose epimerization enzyme according to a preferred embodiment of the present invention includes culturing a recombinant strain transformed by introducing an expression cassette comprising a polynucleotide encoding an allulose epimerization enzyme and a promoter variant comprising the base sequence of SEQ ID NO: 9 operably linked thereto, or an expression vector comprising the expression cassette, to express the allulose epimerization enzyme, and isolating the allulose epimerization enzyme from a disrupted product of the recombinant strain in which the allulose epimerization enzyme is expressed. Since the novel promoter variant according to one embodiment 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 allulose epimerization enzyme of the present invention can be recovered from the lysate of the recombinant strain. The cells used for protein expression can be disrupted by various physical or chemical means such as repeated freezing and thawing, sonication, mechanical disruption, or cell lysing agents, and can be separated or purified by conventional biochemical separation techniques (Sambrook et al., Molecular Cloning: A laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989; Deuscher, M., Guide to Protein Purification Methods Enzymology, Vol. 182. Academic Press. Inc., San Diego, CA, 1990).For example, methods for isolating or purifying proteins expressed by host cells include, but are not limited to, electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immunoadsorption affinity chromatography, reversed-phase HPLC, gel permeation HPLC), isoelectric focusing, and various variations or combinations thereof. Meanwhile, in the present invention, the step of isolating allulose epimerization enzyme from the recombinant strain lysate can be preferably 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 His tag is preferred. His-tagged proteins can be specifically trapped on a column of Ni-NTA (nickel-nitrilotriacetic acid) resin and eluted with EDTA or imidazole.

[0033] The recombinant strain according to an embodiment of the present invention can be used to indirectly exert the function of a target protein in addition to being used to produce a target protein. For example, when the target protein is an enzyme, the recombinant strain according to an embodiment of the present invention can be used to produce a product from a substrate by an enzymatic conversion reaction. Specifically, when the target protein is an allulose epimerization enzyme, the present invention provides a method for producing allulose from fructose, comprising the steps of adding the recombinant strain to a fructose-containing solution and reacting the solution. The recombinant strain is a host cell transformed by introducing an expression cassette comprising a polynucleotide encoding the allulose epimerization enzyme and a promoter variant operably linked thereto and consisting of the base sequence of SEQ ID NO: 9 or the base sequence of SEQ ID NO: 10, or an expression vector comprising the expression cassette, and is preferably a recombinant Escherichia coli. In addition, the fructose-containing solution may contain Ca in order to promote the activity of the allulose epimerization enzyme. 2+ , Mn 2+ The reaction temperature in the method for producing allulose from fructose is 50 to 70°C, preferably 55 to 65°C, more preferably 55 to 60°C in consideration of smooth enzyme expression of the recombinant strain, enzyme stability and maximum activity, and the reaction pH is 6.5 to 8, preferably 6.5 to 7.5, more preferably 6.5 to 7. In the method for producing allulose from fructose, the concentration of fructose is not particularly limited, but is preferably 1 to 75% (w / w) based on the total reaction product in consideration of productivity and economic efficiency, and more preferably 35 to 45% (w / w).

[0034] The present invention will be described in more detail with reference to the following examples. However, the following examples are merely intended to clearly illustrate the technical features of the present invention and are not intended to limit the scope of the present invention. EXAMPLES

[0035] Example 1: Securing a promoter for enzyme gene expression 1.1. Establishment of 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, and is composed of the base sequence of SEQ ID NO: 1. It is known as a promoter that induces constitutive expression in E. coli.

[0036] In order to obtain a polynucleotide fragment corresponding to the promoter site of the tatA (twin arginine translocase A) gene from E. coli, PCR was performed using the genomic DNA of E. coli MG1655 as a template and the primer set shown in Table 1 below. The resulting amplified product was cloned using pGEM-Teasy vector (Promega Co., USA) and analyzed for its nucleotide sequence, confirming that it was a polynucleotide fragment having a length of 160 bp and 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, and is composed of the base sequence of SEQ ID NO: 2. It is known to be a promoter that induces stable high expression of foreign genes in E. 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] In order to obtain a polynucleotide fragment corresponding to the promoter region of the maltogenic amylase gene from Bacillus licheniformis, PCR was performed using the genomic DNA of Bacillus licheniformis ATCC14580 as a template and the primer set shown in Table 2 below. The resulting amplified product was cloned using pGEM-Teasy vector (Promega Co., USA) and analyzed for its base sequence, confirming that it was a polynucleotide fragment having a length of 115 bp and consisting of the base sequence of SEQ ID NO:2.

[0040] [Table 2]

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

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

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

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

[0045] [Table 3]

[0046] Example 3: Preparation of a ligated fragment of a promoter and an allulose epimerization enzyme mutant gene 3.1. Preparation of DNA fragment containing tatA promoter and mutant allulose epimerase gene To amplify the tatA promoter, PCR was performed using the pGEM-Teasy vector into which the tatA promoter secured in Example 1 had been cloned as a template and the primer set (XhoI-PtatA, PtatA-FpDPE_R) in Table 4 below.

[0047] In addition, to amplify the gene for the D-allulose epimerization enzyme mutant W29K / A77S / G216S / M234I derived from Flavonifractor plautii, PCR was performed using the pET28a vector (Novagen) into which the gene for the D-allulose epimerization enzyme mutant W29K / A77S / G216S / M234I obtained in Example 2 was cloned as a template and the primer set (PtatA-FpDPE_F, PstI-FpDPE) in Table 4 below.

[0048] [Table 4]

[0049] The amplified tatA promoter fragment and D-allulose epimerase mutant gene fragment can be linked into a single fragment by the complementary sequences of the primers used during amplification. Using the two fragments as templates, overlap extension PCR was performed using primers (XhoI-PtatA, PstI-FpDPE) containing the XhoI and PstI restriction enzyme recognition site sequences shown in Table 4 to obtain a single amplified fragment. The obtained DNA fragment in which the tatA promoter and allulose epimerase mutant W29K / A77S / G216S / M234I genes were linked was named "PtatA-FpDPE".

[0050] 3.2. Preparation of DNA fragment containing BLMA promoter and allulose epimerization enzyme gene To amplify the BLMA promoter, PCR was performed using the pGEM-Teasy vector into which the BLMA promoter secured in Example 1 was cloned as a template and the primer set (XhoI-PblmA, Pblma-FpDPE_R) in Table 5 below.

[0051] In addition, to amplify the gene for the D-allulose epimerization enzyme mutant W29K / A77S / G216S / M234I derived from Flavonifractor plautii, PCR was performed using the pET28a vector (Novagen) into which the gene for the D-allulose epimerization enzyme mutant W29K / A77S / G216S / M234I obtained in Example 2 was cloned as a template and the primer set (Pblma-FpDPE_F, PstI-FpDPE) in Table 5 below. [Table 5] The amplified BLMA promoter fragment and D-allulose epimerase mutant gene fragment can be linked into a single fragment by the complementary sequences of the primers used during amplification. Using the two fragments as templates, overlap extension PCR was performed using primers (XhoI-PblmA, PstI-FpDPE) containing the XhoI and PstI restriction enzyme recognition site sequences shown in Table 5 to obtain a single amplified fragment. The DNA fragment in which the obtained BLMA promoter and allulose epimerase mutant W29K / A77S / G216S / M234I genes were linked was named "Pblma-FpDPE".

[0052] Example 4: Preparation of D-allulose epimerization enzyme mutant expression vector 4.1. Construction of expression vector for D-allulose epimerization enzyme mutant having tatA promoter A recombinant plasmid vector was prepared by genetic recombination from pTrc99A vector (Pharmacia, US), which contains a pUC-derived replication origin capable of replicating in E. coli, a multicloning site (MCS) such as the restriction enzyme XhoI site and PstI site, a transcription terminator, and a kanamycin antibiotic resistance gene.Then, the polynucleotide fragment PtatA-FpDPE prepared in Example 3 was cleaved with the restriction enzymes XhoI and PstI, and then ligated to the recombinant plasmid vector having the same restriction enzyme site to prepare the D-allulose epimerization enzyme mutant expression vector pPtatA:FpDPE(W29K / A77S / G216S / M234I). Then, the D-allulose epimerization enzyme mutant expression vector was transformed into E. coli DH5α by the heat shock method (see Sambrook and Russell: Molecular Cloning), colonies with kanamycin resistance were obtained, five colonies were selected, and five recombinant expression vectors pPtatA:FpDPE(W29K / A77S / G216S / M234I) were recovered and their base sequences were analyzed. As a result of the sequence analysis of the five pPtatA:FpDPE(W29K / A77S / G216S / M234I) recombinant expression vectors, random mutations were confirmed in the introduced tatA promoter or allulose epimerization enzyme mutant W29K / A77S / G216S / M234I gene sequence, and the random mutation contents are shown in Table 6 below.

[0053] [Table 6]

[0054] As shown in Table 6, in the case of the recombinant expression vectors recovered from colonies 2, 3, and 5, a mutation occurred in the gene sequence of the allulose epimerization enzyme mutant, and it was determined that translation to produce the target allulose epimerization enzyme mutant could not be performed. On the other hand, 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 initiation codon, and in the case of the recombinant expression vector recovered from colony 4, a mutation occurred in the sequence surrounding the ribosome-binding site (RBS) of the tatA promoter, and the enzyme gene sequence was consistent, so it was determined that there was a possibility of expression of 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 base sequence of SEQ ID NO: 9, and the tatAm2 promoter is composed of the base sequence of SEQ ID NO: 10. 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 an embodiment of the present invention. Figure 2 is a vector map of the recombinant expression vector pPtatAm2:FpDPE(W29K / A77S / G216S / M234I) produced in an embodiment of the present invention.

[0055] 4.2. Preparation of expression vector for D-allulose epimerization enzyme mutant having BLMA promoter A recombinant plasmid vector was prepared from pTrc99A vector (Pharmacia, US) through genetic recombination, which contains a pUC-derived replication origin capable of replicating in E. coli, a multicloning site (MCS) such as the restriction enzyme XhoI site and PstI site, a transcription terminator, and a kanamycin antibiotic resistance gene. The polynucleotide fragment Pblma-FpDPE prepared in Example 3 was then cleaved with the restriction enzymes XhoI and PstI, and ligated to the recombinant plasmid vector having the same restriction enzyme sites to prepare a D-allulose epimerization enzyme mutant expression vector, pPblma:FpDPE(W29K / A77S / G216S / M234I). Then, the D-allulose epimerase mutant expression vector was transformed into E. coli DH5α by the heat shock method (see Sambrook and Russell: Molecular Cloning), colonies with kanamycin resistance were obtained, six colonies were selected, and six types of recombinant expression vectors pPblma:FpDPE (W29K / A77S / G216S / M234I) were recovered and their base sequences were analyzed. As a result of sequence analysis of the six types of recombinant expression vectors pPblma:FpDPE (W29K / A77S / G216S / M234I), it was confirmed that all of them contained the intended BLMA promoter and allulose epimerase mutant gene sequences. Figure 3 is a vector map of the recombinant expression vector pPblma:FpDPE (W29K / A77S / G216S / M234I) prepared in the examples of the present invention.

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

[0057] Example 6: Determination of the conversion rate of fructose to allulose by recombinant strains and comparison of the enzyme expression strength of promoters Since D-allulose epimerization enzyme can convert fructose to allulose, the strength of induction of enzyme expression by each promoter in the recombinant strain was compared by measuring the conversion rate of fructose to allulose, which is proportional to the amount of enzyme expression in the recombinant strain.

[0058] In order to cultivate the recombinant E. 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 E. coli prepared in Example 5 was inoculated therein. The flask was then transferred to a shaking incubator, and the recombinant E. coli was cultivated for 14 hours (hr) while maintaining a temperature condition of 30°C and a shaking condition of 140 rpm, and the culture solution was centrifuged to recover the bacterial cells. The recovered bacterial cells were then 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 manganese sulfate (MnSO4) metal ion, and the reaction was allowed to proceed for a predetermined time at 62°C, after which the temperature of the reaction solution was lowered to 4°C to stop the reaction, and the reaction was centrifuged at 16,600×g and 4°C to recover the supernatant. Then, the concentration of allulose and the concentration of fructose in the supernatant were measured by high performance liquid chromatography (HPLC), and the conversion rate of fructose to allulose was calculated from the measurement results, and the conversion rate was used as an index of enzyme activity. Figure 4 shows the conversion rate as a function of reaction time when the conversion reaction of fructose to allulose is carried out using the recombinant E. coli prepared in the examples of the present invention. In FIG. 4, "PtatAm1" represents recombinant E. coli transformed with the recombinant expression vector pPtatAm1:FpDPE(W29K / A77S / G216S / M234I), "PtatAm2" represents E. coli transformed with the recombinant expression vector pPtatAm2:FpDPE(W29K / A77S / G216S / M234I), and "Pblma" represents E. coli transformed with the recombinant expression vector pPblma:FpDPE(W29K / A77S / G216S / M234I). As shown in FIG. 4, the recombinant E. coli containing the tatAm1 promoter and the recombinant E. coli containing the tatAm2 promoter showed a much higher conversion rate of fructose to allulose than the recombinant E. coli containing the BLMA promoter. These results indicate that the enzyme expression induction effect of the tatAm1 promoter and the tatAm2 promoter is much stronger than that of the BLMA promoter.On the other hand, if the expression strength of the target gene by a constitutive expression promoter is too strong, it may place a burden on the recombinant strain. Therefore, even if the expression strength by the tatAm1 promoter is somewhat lower than that by the tatAm2 promoter, the tatAm1 promoter is considered to be more advantageous from a commercial point of view.

[0059] As described above, the present invention has been described through the above examples, 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 scope of protection of the present invention should be interpreted as including all embodiments falling within the scope of the claims attached to the present invention.

Claims

1. A promoter mutant consisting of the base sequence of SEQ ID NO:9 or the base sequence of SEQ ID NO:

10.

2. A recombinant vector comprising the promoter mutant of claim 1.

3. An expression vector comprising a polynucleotide encoding a target protein and the promoter mutant of 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 epimerization enzyme.

6. The expression vector according to claim 5, wherein the allulose epimerization enzyme 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 epimerization enzyme is composed of the base sequence of SEQ ID NO: 4, the base sequence of SEQ ID NO: 6, or the base 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 described in claim 8 to a fructose-containing solution and reacting it.

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