Allulose epimerase expression cassette and manufacturing method of allulose using the same

Abstract

To provide: an allulose epimerase expression cassette capable of highly expressing an allulose enzyme in a stationary phase after a logarithmic phase of a Corynebacterium host microorganism without inducing growth inhibition of the Corynebacterium genus host microorganism because of a low expression level of the allulose enzyme in an early stage of culture; and uses thereof.SOLUTION: An exemplary embodiment of the present disclosure provides an allulose epimerase expression cassette including a polynucleotide encoding allulose epimerase and a promoter which is operably linked thereto and regulates the expression of the allulose epimerase in a Corynebacterium genus strain. The promoter consists of a specific base sequence.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to an allulose epimerization enzyme expression cassette, and more particularly, to an allulose epimerization enzyme expression cassette that can highly express allulose enzyme during the stagnant phase after the logarithmic growth phase of a Corynebacterium host microorganism without inducing growth inhibition of the Corynebacterium host microorganism since the expression level of the allulose enzyme is low in the early stages of culture, a recombinant Corynebacterium strain containing the same, and a method for producing allulose using the same. [Background technology]

[0002] D-allulose is the epimer of the 3-carbon of fructose, and is also called D-psicose. Allulose has 70% of the sweetness of sugar (Oshima, 2006), but only 0.3% of the energy, making it a functional monosaccharide that can be used as a low-calorie sweetener in diet foods (Matsuo et al., 2002). Allulose also has the function of suppressing glucose absorption and lowering blood sugar, so it can be used in foods for diabetics and slimming foods, and it has the function of suppressing the activity of enzymes involved in lipid synthesis in the liver, which can suppress abdominal fat accumulation, so it can be used in various functional foods such as health foods (Matsuo et al., 2001; Iida et al., 2008; Hayashi et al., 2010; Hossain et al., 2011). Due to the above characteristics, allulose is a good source that can replace sugar. However, since it is a rare sugar, a monosaccharide that is found very rarely in nature, an efficient method for producing allulose is required for its application in the food industry.

[0003] Representative biological methods for producing allulose include a method of directly reacting fructose with D-allulose 3-epimerizing enzyme to convert it into allulose, and a method of reacting fructose with the cells of a strain that produces D-allulose 3-epimerizing enzyme as an intracellular enzyme to convert it into allulose. A recombinant strain transformed by introducing a D-allulose 3-epimerizing enzyme expression cassette is used as the strain that produces the D-allulose 3-epimerizing enzyme, and Escherichia coli is generally used as the host cell. However, since the transformed recombinant Escherichia coli is not a GRAS (Generally Recognized As Safe) strain, it is not suitable for use as a strain for producing food materials. To solve this problem, it is necessary to develop a D-allulose 3-epimerizing enzyme expression cassette suitable for Corynebacterium sp. strains, which are GRAS (Generally Recognized As Safe) strains.

[0004] For example, Korean Patent Publication No. 10-1656063 discloses an expression cassette for D-allulose 3-epimerization enzyme suitable for transformation of Corynebacterium sp. strains and a method for producing D-allulose using the same. The transcription promoter used in the prior art contains both the region expressing the entire gene for superoxide dismutase (sod) and peptide methionine sulfoxide reductase (MsrA) present in the full-length base sequence of Corynebacterium glutamicum strains, and therefore, the use of an expression cassette containing the transcription promoter may result in unintended reverse transcription, and may further adversely affect cell growth due to reduced expression of the target protein or inefficient energy consumption. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention has been developed under the conventional technical background, and the object of the present invention is to provide an allulose epimerization enzyme expression cassette that can highly express allulose enzyme during the stagnant phase after the logarithmic growth phase of a Corynebacterium host microorganism without inducing growth inhibition of the Corynebacterium host microorganism due to the low expression level of allulose enzyme in the early stages of culture, and various uses thereof. [Means for solving the problem]

[0006] The inventors of the present invention cloned a 200 bp long promoter region for superoxide dismutase (sod) gene expression from the genomic DNA of Corynebacterium glutamicum ATCC 13032 strain, sequentially linked the promoter region to allulose epimerization enzyme to prepare an expression cassette, which was then inserted into a recombinant shuttle vector to prepare a recombinant allulose epimerization enzyme expression vector. The inventors then introduced the recombinant allulose epimerization enzyme expression vector into Corynebacterium glutamicum, a GRAS (Generally Recognized As Safe) strain, and transformed it to prepare a recombinant Corynebacterium glutamicum strain. As a result of culturing the recombinant Corynebacterium glutamicum strain, it was confirmed that the growth rates during the logarithmic and stationary phases were similar to those of the wild-type Corynebacterium glutamicum strain, and that the D-allulose 3-epimerization enzyme was highly expressed in response to environmental stress from the stationary phase after the logarithmic growth phase, thereby completing the present invention.

[0007] In order to solve the above problems, one example of the present invention provides an allulose epimerization enzyme expression cassette comprising a polynucleotide encoding an allulose epimerization enzyme and a promoter operably linked thereto, the promoter being composed of the nucleotide sequence of SEQ ID NO: 1, 9, 10, or 11.

[0008] In order to solve the above problems, one example of the present invention provides a recombinant expression vector into which an allulose epimerization enzyme expression cassette is inserted.

[0009] In order to solve the above problems, one example of the present invention provides a recombinant Corynebacterium strain, which is obtained by transforming a Corynebacterium host strain by introducing an allulose epimerization enzyme expression cassette or a recombinant expression vector into which the expression cassette is inserted.

[0010] To solve the above problems, one embodiment of the present invention provides a method for producing allulose from fructose, which includes the step of adding a recombinant Corynebacterium sp. strain to a fructose-containing solution and reacting the mixture. Effect of the Invention

[0011] The allulose epimerization enzyme expression cassette according to one embodiment of the present invention hardly functions in the normal environment in which the recombinant Corynebacterium strain is cultured, and does not induce growth inhibition of the recombinant Corynebacterium strain. Therefore, the recombinant Corynebacterium strain transformed with the allulose epimerization enzyme expression cassette according to one embodiment of the present invention can grow and proliferate normally up to the logarithmic growth phase, and stably expresses the allulose epimerization enzyme at a high level after the logarithmic growth phase. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a schematic diagram showing the process of constructing a recombinant plasmid vector, pDS vector, based on a commercial shuttle vector, pJC1 vector.

[0013] [Diagram 2] FIG. 1 is a schematic diagram showing a process for constructing a recombinant expression vector, pDS_FDPE, from a recombinant plasmid vector, pDS vector.

[0014] [Diagram 3] 1 is a vector map of the recombinant expression vector pDS_FDPE.

[0015] [Figure 4] 1 shows the results of comparing the growth rates of a wild-type Corynebacterium glutamicum strain (referred to as WT) and a recombinant Corynebacterium glutamicum strain (referred to as DS00001) in Example 5 of the present invention.

[0016] [Diagram 5] In Example 6 of the present invention, recombinant Corynebacterium glutamicum was inoculated into a substrate solution containing fructose and cultured. The D-allulose 3-epimerization enzyme activity of the recombinant Corynebacterium glutamicum strain is shown as a conversion rate of fructose to allulose according to the culture time. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention will be specifically described below.

[0018] 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.

[0019] The term "homology" as used herein indicates identity with a wild type or a mutant nucleic acid sequence having the same activity, and the homology can be compared by visual inspection or by using a readily available comparison program, and the homology between two or more sequences can be calculated as a percentage (%). Also, "homology" is used to indicate identity with an amino acid sequence of a wild type or a mutant having the same activity.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] The term "expression cassette" as used herein refers to a regulatory sequence operably linked to a nucleotide sequence to be expressed, for example, a polynucleotide sequence encoding an allulose epimerization enzyme. Thus, unlike an expression unit, an expression cassette includes not only nucleotide sequences that regulate transcription and translation, but also nucleotide sequences that are expressed as proteins as a result of transcription and translation.

[0024] 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 appropriately 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. The selection marker region may be an antibiotic selection marker gene for selecting the desired vector.

[0025] The term "recombinant strain" as used herein means 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, gene gun methods, and heat shock methods. In addition, the host cell of the recombinant strain is not particularly limited in type as long as the promoter present in the expression vector can operate smoothly, and is preferably a prokaryote.

[0026] One aspect of the present invention relates to an expression cassette that can induce high expression of a target protein after the logarithmic growth phase of a host cell without inducing growth inhibition of the host cell in the early stage of culture.

[0027] An allulose epimerization enzyme expression cassette according to one embodiment of the present invention comprises a polynucleotide encoding an allulose epimerization enzyme and a promoter operably linked thereto. The promoter is preferably located upstream of the polynucleotide encoding the allulose epimerization enzyme, which is a target protein. The promoter also regulates the expression of the allulose epimerization enzyme in a Corynebacterium strain.

[0028] The promoter, which is a component of the allulose epimerization enzyme expression cassette, is an SOD promoter obtained by cloning a specific portion from the genome of Corynebacterium glutamicum ATCC 13032 strain, and is a polynucleotide corresponding to -200 bp based on the superoxide dismutase (sod) gene. The promoter is composed of the nucleotide sequence of SEQ ID NO: 1. The promoter also includes an analog of the SOD promoter. The analog of the SOD promoter may be composed of the nucleotide sequence of SEQ ID NO: 9, the nucleotide sequence of SEQ ID NO: 10, or the nucleotide sequence of SEQ ID NO: 11. In the present invention, the specific SOD promoter cloned has a length of 200 bp, but the nucleotides from the 191st nucleotide to the 200th nucleotide correspond to a spacer sequence, and the spacer sequence is a sequence present between genes and is presumed not to have a significant effect on the expression of the target protein, so it can be partially modified. The nucleotide sequence of SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11 is a partial modification of the spacer sequence corresponding to the 191st to 200th nucleotides in the nucleotide sequence of SEQ ID NO:1. The promoter, which is a component of the allulose epimerization enzyme expression cassette according to one embodiment of the present invention, is composed of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, but the equivalent range of the promoter is not necessarily limited thereto. For example, the equivalent range of the SOD promoter of the present invention includes a sequence having substantial identity to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11. The above-mentioned substantial identity means that the nucleotide sequence of SEQ ID NO:1, the nucleotide sequence of SEQ ID NO:9, the nucleotide sequence of SEQ ID NO:10 or the nucleotide sequence of SEQ ID NO:11 is aligned with any other sequence to maximize the 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:1, the nucleotide sequence of SEQ ID NO:9, the nucleotide sequence of SEQ ID NO:10 or the nucleotide sequence of SEQ ID NO:11.A person skilled in the art will easily understand that a polynucleotide having the same or similar activity within the range of substantial homology can be produced by substituting, adding or deleting one or more bases in the SOD promoter nucleotide sequence using recombinant gene technology known in the art, etc. 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.

[0029] The polynucleotide encoding the allulose epimerase, which is a component of the allulose epimerase expression cassette, is not particularly limited in type as long as it is a polynucleotide encoding an enzyme having the activity of converting fructose to allulose. For example, the allulose epimerase may be derived from Flavonifractor plautii, Clostridium scindens, Treponema primitia, Ensifer adhaerens, or Ruminococcus torques, and is preferably derived from Flavonifractor plautii in consideration of the activity of converting fructose to allulose. Specifically, the allulose epimerase may be composed of the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 13, or the amino acid sequence of SEQ ID NO: 15. The allulose epimerase having the amino acid sequence of SEQ ID NO: 3 is a wild-type enzyme derived from Flavonifractor plautii. The allulose epimerase having the amino acid sequence of SEQ ID NO: 13 has tryptophan (Trp) at position 29 substituted with lysine (Lys), glycine (Gly) at position 216 substituted with serine (Ser), and methionine (Met) at position 234 substituted with isoleucine (Ile) in the amino acid sequence of SEQ ID NO: 3. The allulose epimerization enzyme consisting of the amino acid sequence of SEQ ID NO:15 is an amino acid sequence of SEQ ID NO:3 in which the tryptophan (Trp) at position 29 is replaced with lysine (Lys), the alanine (Ala) 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).The polynucleotide encoding the allulose epimerization enzyme is not limited to a particular type, and may preferably be composed of the base sequence of SEQ ID NO: 2, or may have 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: 2. The base sequence of SEQ ID NO: 2 is a polynucleotide encoding an allulose epimerization enzyme composed of the amino acid sequence of SEQ ID NO: 3. The polynucleotide encoding the allulose epimerization enzyme may be composed of the base sequence of SEQ ID NO: 12 or the base sequence of SEQ ID NO: 14. The base sequence of SEQ ID NO: 12 is a polynucleotide encoding an allulose epimerization enzyme composed of the amino acid sequence of SEQ ID NO: 13, and the base sequence of SEQ ID NO: 14 is a polynucleotide encoding an allulose epimerization enzyme composed of the amino acid sequence of SEQ ID NO: 15. The present invention relates to an allulose epimerization enzyme and a polynucleotide 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.

[0030] The allulose epimerization enzyme expression cassette according to one embodiment of the present invention may further include one or more sequences selected from the group consisting of a replication origin, a multicloning site (MCS) for cloning a target protein gene, a transcription termination sequence, and a selection marker. The selection marker is for selecting cells transformed with the vector, and may be a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface protein. For example, the selection marker may be an antibiotic resistance gene marker such as a kanamycin antibiotic resistance gene or an ampicillin antibiotic resistance gene.

[0031] One aspect of the present invention relates to various uses of the allulose epimerization enzyme expression cassette. The allulose epimerization enzyme expression cassette can be used in recombinant vectors, recombinant strains, and the production of allulose from fructose.

[0032] A recombinant vector according to an embodiment of the present invention is a recombinant expression vector into which the above-mentioned allulose epimerization enzyme expression cassette is inserted. The recombinant expression vector preferably has a vector map as shown in Figure 3. The recombinant expression vector having the vector map as shown in Figure 3 has a structure in which a replication origin, a promoter (SOD) consisting of the base sequence of SEQ ID NO: 1, a polynucleotide (FDPE) encoding the allulose epimerization enzyme consisting of the base sequence of SEQ ID NO: 2, a transcription terminator, a kanamycin resistance gene marker (NeoR / KanR) consisting of the base sequence of SEQ ID NO: 4, etc. are sequentially linked.

[0033] A recombinant strain according to one embodiment of the present invention is a recombinant Corynebacterium strain, which is a host cell transformed by introducing the allulose epimerization enzyme expression cassette or a recombinant expression vector into which the expression cassette is inserted. The host strain used to prepare the recombinant Corynebacterium strain is not particularly limited as long as it is a Corynebacterium strain, and may be selected from the group consisting of, for example, Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoacidophilum, Corynebacterium thermoaminogenes, Corynebacterium melassecola, and Corynebacterium efficiens.

[0034] A method for producing allulose from fructose according to an embodiment of the present invention includes the step of adding a recombinant Corynebacterium sp. strain to a fructose-containing solution and reacting the solution. The fructose-containing solution is added with Ca to promote the activity of 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 60 to 65°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 fructose concentration of the fructose-containing solution is not particularly limited, but is preferably 5 to 75% (w / w) based on the total weight of the fructose-containing solution in consideration of productivity and economic efficiency, and is preferably 10 to 55% (w / w).

[0035] 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

[0036] Example 1: Amplification and securing of promoter sequence for overexpression of D-allulose 3-epimerization enzyme In order to obtain a polynucleotide fragment of the promoter region for the expression of the superoxide dismutase (sod) gene from Corynebacterium glutamicum ATCC 13032 strain, genomic DNA was extracted from Corynebacterium glutamicum ATCC 13032 strain. Then, PCR was performed using the extracted genomic DNA as a template and the primer set shown in Table 1 below to obtain an amplified promoter fragment. The amplified promoter fragment was cloned into a cloning vector and analyzed for its base sequence, confirming that it was a polynucleotide fragment having a length of 200 bp and composed of the base sequence of SEQ ID NO:1. [Table 1]

[0037] In general, the exact positional relationship of the SOD promoter site relative to the superoxide dismutase (sod) gene is not specified, but the site obtained using the primer set in Table 1 was confirmed to be -200bp from the superoxide dismutase (sod) gene. On the other hand, the expression regulatory sequence described in Korean Patent Publication No. 10-1656063 is -300bp from the superoxide dismutase (sod) gene.

[0038] Example 2: Amplification and isolation of polynucleotide sequences encoding D-allulose 3-epimerization enzymes In order to clone the gene encoding the D-allulose 3-epimerase, genomic DNA was extracted from Flavonifractor plautii KCTC 5970 strain. Then, PCR was performed using the extracted genomic DNA as a template and the primer set shown in Table 2 below to obtain an amplified D-allulose 3-epimerase gene fragment. The amplified gene fragment was cloned into a cloning vector and analyzed for its base sequence, confirming that it was a polynucleotide fragment having a length of 885 bp and composed of the base sequence of SEQ ID NO: 2, and named FDPE gene. The D-allulose 3-epimerase decoded by the polynucleotide composed of the base sequence of SEQ ID NO: 2 is a wild-type enzyme and is composed of the amino acid sequence of SEQ ID NO: 3. [Table 2]

[0039] Example 3: Construction of an expression vector into which an SOD promoter fragment and a D-allulose 3-epimerization enzyme gene are inserted The antibiotic resistance gene neomycin phospho-transferase I (NTPI) of the commercial shuttle vector pJC1 vector was replaced with the NTPII gene derived from E. coli K-12, and a multicloning site (MCS) and other elements were inserted to prepare a recombinant plasmid vector pDS vector. FIG. 1 is a schematic diagram showing the process of preparing a recombinant plasmid vector pDS vector based on the commercial shuttle vector pJC1 vector. The pDS vector contains a replication initiation protein (repA) gene, an antibiotic (kanamycin) resistance gene derived from E. coli K-12 composed of the base sequence of SEQ ID NO: 4, and the like.

[0040] Then, the 200 bp SOD promoter DNA fragment and the FDPE gene fragment obtained above were mixed, and PCR was performed for 10 cycles. Then, the SOD promoter forward primer shown in Table 1 and the FDPE reverse primer shown in Table 2 were added, and PCR was performed for 15 cycles to obtain a fusion DNA fragment combining the SOD promoter and the FDPE gene. Then, the ends of the fusion DNA fragment were cut using the restriction enzymes PstI and BamHI, and the fusion DNA fragment was inserted into the PstI and BamHI restriction enzyme sites, which are the multicloning site (MCS) of the pDS vector, to prepare the recombinant expression vector pDS_FDPE. Figure 2 is a schematic diagram showing the process of preparing the recombinant expression vector pDS_FDPE from the recombinant plasmid vector pDS vector, and Figure 3 is a vector map of the recombinant expression vector pDS_FDPE. As shown in FIG. 3, the recombinant expression vector pDS_FDPE contains a replication initiation protein (repA) gene, a replication origin, multicloning sites (MCS) such as restriction enzyme PstI, BamHI, and NdeI sites, an SOD promoter, a gene of interest (FDPE), a transcription terminator, and an antibiotic resistance gene marker.

[0041] Example 4: Preparation of recombinant strain transformed by introduction of recombinant expression vector pDS_FDPE and confirmation of D-allulose 3-epimerization enzyme activity The recombinant expression vector pDS_FDPE prepared above was introduced into Corynebacterium glutamicum competent cells by electroporation, and the transformed recombinant Corynebacterium glutamicum strain was selected by checking whether it had antibiotic resistance. The selected recombinant Corynebacterium glutamicum strain was inoculated into 2YT medium (containing 16 g / L Trytone, 10 g / L Yeast Extract, and 5 g / L NaCl) and cultured at 30°C for 40 hours. The culture medium was removed by centrifugal separation, and the cells were suspended in 0.85% (w / w) NaCl solution and centrifuged again to obtain the recombinant Corynebacterium glutamicum strain. The obtained recombinant Corynebacterium glutamicum strain cells were added to 50 mM Tris-HCl buffer (pH 7.0) with 1 mM MnSO4 at 0.06% (w / w) to prepare a cell suspension. Then, 50 mM Tris-HCl buffer (pH 7.0) with 20% (w / w) fructose and 1 mM MnSO4 was mixed with the recombinant Corynebacterium glutamicum strain cell suspension at a volume ratio of 1:1, and the enzyme conversion reaction was carried out at 65°C for 1 hour (hr), and then 1M hydrochloric acid solution was added to lower the pH to 2 or less to terminate the reaction. The reaction product solution was then separated by elongation to collect the supernatant from which the cells and foreign matter had been removed, and the supernatant was filtered through a 0.45 μm syringe filter to prepare the sample. Then, the allulose and fructose concentrations in the samples were measured using high performance liquid chromatography (HPLC) to confirm the D-allulose 3-epimerization enzyme activity of the transformed recombinant Corynebacterium glutamicum strain. The HPLC analysis conditions were as follows: * Column: 87C (BIO-RAD) * Column temperature: 80℃ * Mobile phase: Ion-exchanged water *Flow rate: 0.6ml / min * Detector: Refractive Index Detector (Agilent 1260 TID)

[0042] Example 5: Comparison of growth rates between wild-type and recombinant Corynebacterium glutamicum strains The wild-type Corynebacterium glutamicum strain and the recombinant Corynebacterium glutamicum strain were inoculated into 2YTG medium (containing 16 g / L Trytone, 10 g / L Yeast Extract, 5 g / L NaCl, and 20 g / L dextrose; pH 7) and cultured as seed culture at 30°C for 16 hours (hr). The seed culture containing the bacteria was then inoculated into 2YTG medium at 1% (v / v) and cultured at 30°C, with sampling being performed at regular intervals. The optical density at 600 nm of the sampled samples was measured to evaluate the growth rate of the strains.

[0043] 4 shows the results of comparing the growth rates of the wild-type Corynebacterium glutamicum strain (referred to as WT) and the recombinant Corynebacterium glutamicum strain (referred to as DS00001) in Example 5 of the present invention. As shown in FIG. 4, there was no significant difference in the growth rates of the wild-type Corynebacterium glutamicum strain and the recombinant Corynebacterium glutamicum strain. In the recombinant Corynebacterium glutamicum strain prepared in the present invention, overexpression of D-allulose 3-epimerization enzyme driven by SOD promoter did not occur in the early stage of cultivation, and showed logarithmic growth phase and plateau phase at similar levels to those of wild-type Corynebacterium glutamicum strain.

[0044] Example 6: D-Allulose 3-epimerization enzyme activity according to the culture time of recombinant Corynebacterium glutamicum strain The recombinant Corynebacterium glutamicum strain was inoculated into 2YT medium (containing 16 g / L Trytone, 10 g / L Yeast Extract, and 5 g / L NaCl) and cultured at 30°C for 40 hours. The culture medium was removed by centrifugal separation, and the cells were suspended in 0.85% (w / w) NaCl solution and centrifuged again to obtain the recombinant Corynebacterium glutamicum strain cells. The obtained recombinant Corynebacterium glutamicum strain cells were added to 50 mM Tris-HCl buffer (pH 7.0) containing 1 mM MnSO4 at a concentration of 0.06% (w / w) to prepare a cell suspension. Then, 50 mM Tris-HCl buffer (pH 7.0) with a fructose concentration of 20% (w / w) and MnSO4 concentration of 1 mM was mixed with the bacterial cell suspension of the recombinant Corynebacterium glutamicum strain in a volume ratio of 1:1, and the enzyme conversion reaction was carried out at 65°C, while measuring the D-allulose 3-epimerization enzyme activity of the recombinant Corynebacterium glutamicum strain as a function of reaction time. When the preset reaction time was reached, a 1M hydrochloric acid solution was added to the reaction product to lower the pH to below 2 and terminate the reaction. The reaction product was then separated by elongation, and the supernatant from which the bacterial cells and foreign matter had been removed was collected, and the supernatant was filtered through a 0.45 μm syringe filter to prepare the sample. Then, the allulose and fructose concentrations in the samples were measured using high performance liquid chromatography (HPLC), and the conversion rate of fructose to allulose was calculated.

[0045] 5 shows the D-allulose 3-epimerization enzyme activity of the recombinant Corynebacterium glutamicum strain as a conversion rate of fructose to allulose according to the culture time when the recombinant Corynebacterium glutamicum strain was inoculated and cultured in a substrate solution containing fructose in Example 6 of the present invention. As shown in FIG 5, the recombinant Corynebacterium glutamicum strain did not show growth inhibition due to overexpression of D-allulose 3-epimerization enzyme at the early stage of culture, and after sufficient growth up to the logarithmic growth phase, it was shown to highly express D-allulose 3-epimerization enzyme from the stagnant phase.

[0046] Example 7: Production of allulose using recombinant Corynebacterium glutamicum strains in commercial substrate-containing solutions 500g (corresponding to about 380mL) of high fructose syrup (HFCS; sugar composition: fructose 55% by weight, glucose 45% by weight) with a solid concentration of about 75 Brix, 500g of a suspension of recombinant Corynebacterium glutamicum strain, and 0.2g of MnSO4·4H2O were mixed, and the pH was adjusted to about 7 using 4% NaOH aqueous solution, and the enzymatic conversion reaction was carried out at 65℃ with stirring at 200 rpm. During the enzymatic conversion reaction, the pH of the reaction system was maintained at 7 using 4% NaOH aqueous solution. Sampling was performed at each enzymatic conversion reaction time, and 10% HCl aqueous solution was added to the sampled sample to stop the enzymatic conversion reaction. The sampled sample was then diluted about 25 times with distilled water and filtered through a 0.45μm syringe filter, and the filtrate was analyzed by high performance liquid chromatography (HPLC) to measure the conversion rate. The conversion rate was calculated by the following formula: TIFF2025069921000004.tif26137

[0047] The following Table 3 summarizes the results of measuring the conversion rate of fructose to allulose according to reaction time when the recombinant Corynebacterium glutamicum strain of the present invention was inoculated into high fructose syrup, a commercial substrate, and the enzymatic conversion reaction was carried out. [Table 3]

[0048] As shown in Table 3, when allulose was produced from high fructose syrup using the recombinant Corynebacterium glutamicum strain of the present invention, a conversion rate of 30% was achieved within about 12 hours (hr).

[0049] As described above, the present invention has been described through the above examples, but the scope of protection of 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 not be limited to the specific embodiment disclosed as the best mode, but should be interpreted as including all embodiments falling within the scope of the claims attached to the present invention.

Claims

1. An expression cassette comprising a polynucleotide encoding an allulose epimerization enzyme and a promoter operably linked thereto, The promoter is composed of the base sequence of SEQ ID NO: 1, the base sequence of SEQ ID NO: 9, the base sequence of SEQ ID NO: 10, or the base sequence of SEQ ID NO:

11.

2. The allulose epimerization enzyme expression cassette according to claim 1, wherein the allulose epimerization enzyme is derived from Flavonifractor plautii, Clostridium scindens, Treponema primitia, Ensifer adhaerens or Ruminococcus torques.

3. The allulose epimerization enzyme expression cassette of claim 1, wherein the allulose epimerization enzyme is composed of the amino acid sequence of SEQ ID NO:

3.

4. The allulose epimerization enzyme expression cassette of claim 1, wherein the polynucleotide encoding the allulose epimerization enzyme is composed of the base sequence of SEQ ID NO:

2.

5. The allulose epimerization enzyme expression cassette according to claim 1, further comprising one or more sequences selected from the group consisting of a replication origin, a multicloning site (MCS) for cloning a target protein gene, a transcription termination sequence, and a selection marker.

6. A recombinant expression vector into which the expression cassette according to any one of claims 1 to 5 has been inserted.

7. 7. The recombinant expression vector of claim 6, having the vector map of FIG.

8. A recombinant Corynebacterium strain, which is obtained by transforming a Corynebacterium host strain by introducing an expression cassette according to any one of claims 1 to 5 or a recombinant expression vector into which the expression cassette is inserted.

9. 9. The recombinant Corynebacterium strain of claim 8, wherein the Corynebacterium host strain is selected from the group consisting of Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoacidophilum, Corynebacterium thermoaminogenes, Corynebacterium melassecola, and Corynebacterium efficiens.

10. A method for producing allulose from fructose, comprising the step of adding the recombinant Corynebacterium strain according to claim 9 to a fructose-containing solution and reacting the mixture.

Images