Allulose epimerization enzyme expression cassette and method for producing 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 due to low expression of the allulose enzyme 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 fructose at the C3 position and is also known as D-psicose. Compared to sugar, allulose has 70% of the sweetness (Oshima, 2006) but only 0.3% of the energy content, making it a functional monosaccharide suitable for use as a low-calorie sweetener in diet foods (Matsuo et al., 2002). Furthermore, allulose inhibits glucose absorption and has hypoglycemic properties, making it suitable for use in foods for diabetics and weight loss. It also inhibits the activity of enzymes involved in hepatic lipid synthesis, potentially suppressing abdominal fat accumulation, making it suitable for use in a variety of functional foods, including 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 rarely found 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 directly reacting fructose with a D-allulose 3-epimerizing enzyme to convert it to allulose, and reacting fructose with the cells of a bacterial strain that produces the D-allulose 3-epimerizing enzyme as an intracellular enzyme to convert it to allulose. The D-allulose 3-epimerizing enzyme-producing strain is a recombinant strain transformed with a D-allulose 3-epimerizing enzyme expression cassette, and Escherichia coli is typically used as the host cell. However, transformed recombinant E. coli is not a GRAS (Generally Recognized As Safe) strain and is therefore unsuitable for use as a bacterial strain for producing food ingredients. To address this issue, it is necessary to develop a D-allulose 3-epimerizing enzyme expression cassette suitable for Corynebacterium sp., a GRAS (Generally Recognized As Safe) strain.

[0004] For example, Korean Patent Publication No. 10-1656063 discloses a D-allulose 3-epimerization enzyme expression cassette suitable for transforming Corynebacterium strains and a method for producing D-allulose using the same. The transcription promoter used in the prior art contains both the superoxide dismutase (sod) gene and the peptide methionine sulfoxide reductase (MsrA) gene, both of which are present in the full-length nucleotide sequence of Corynebacterium glutamicum. Therefore, using an expression cassette containing this transcription promoter can result in unintended reverse transcription, which can 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 was developed against the background of the prior art, and an 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 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 recombinant allulose epimerization enzyme expression vector was then transformed into Corynebacterium glutamicum, a GRAS (Generally Recognized As Safe) strain, 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, leading to the completion of the present invention.

[0007] To achieve the above object, one example of the present invention provides an allulose epimerization enzyme expression cassette comprising a polynucleotide encoding the allulose epimerization enzyme and a promoter operably linked thereto, wherein the promoter is composed of the nucleotide sequence of SEQ ID NO: 1, 9, 10, or 11.

[0008] 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] 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 with an allulose epimerization enzyme expression cassette or a recombinant expression vector into which the expression cassette has been inserted.

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

[0011] The allulose epimerization enzyme expression cassette according to one embodiment of the present invention is barely active in the normal environment in which a recombinant Corynebacterium strain is cultured and does not induce growth inhibition of the recombinant Corynebacterium strain. Therefore, a 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 high levels after the logarithmic growth phase. [Brief explanation 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 commercially available shuttle vector, pJC1 vector.

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

[0014] [Figure 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] [Figure 5] In Example 6 of the present invention, a recombinant Corynebacterium glutamicum strain was inoculated into a substrate solution containing fructose and cultured. This shows the D-allulose 3-epimerization enzyme activity of the recombinant Corynebacterium glutamicum strain as a conversion rate of fructose to allulose at each culture time. DETAILED DESCRIPTION OF THE INVENTION

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

[0018] As used herein, the term "promoter" refers to a minimal nucleic acid sequence operably linked to a target nucleotide sequence to be transcribed and regulating the transcription of the target nucleotide sequence. The promoter may also include a promoter structure sufficient to express a regulatable promoter-dependent gene that is inducible by cell-type-specific or external signals or agents, and such a structure may be located in the 5' or 3' region of the gene. The promoter includes both conserved promoters and inducible promoters. Promoter sequences can be derived from prokaryotes, eukaryotes, or viruses. 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 refers to the identity of a nucleic acid sequence with a wild type or a mutant having the same activity. Homology can be compared visually or by using a readily available comparison program, and the homology between two or more sequences can be calculated as a percentage (%). Furthermore, "homology" refers to the identity of an amino acid sequence with a wild type or a mutant having the same activity.

[0020] As used herein, the term "polynucleotide" 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] As used herein, the term "operably linked" is defined as a state in which a promoter sequence and a nucleotide sequence encoding a protein of interest are functionally linked, such that the promoter can regulate the expression of the protein of interest. For example, a promoter is operably linked to a coding sequence if it is capable of controlling the expression of the coding sequence (i.e., if the coding sequence is under the transcriptional control of the promoter), and a ribosome binding site is operably linked to a coding sequence if the ribosome binding site is positioned so as to promote translation. The coding sequence can be 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 the transcription and translation of cloned DNA in a suitable host. Specifically, it refers to a genetic construct containing essential regulatory elements operably linked to an insert so that the insert is expressed when present in the cells of an individual. 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. An expression vector contains at least a promoter, an initiation codon, a gene encoding the desired protein, and a stop codon terminator. Expression vectors can also appropriately contain DNA encoding a signal peptide, additional expression regulatory sequences, 5' and 3' untranslated regions of the desired gene, a selectable marker region, or a replicable unit. The selectable marker region can be an antibiotic selectable marker gene for selecting the desired vector.

[0025] The term "recombinant strain" as used herein refers to cells transformed by introducing a polynucleotide encoding one or more target proteins or an expression vector carrying the same into a host cell. 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 treatments such as PEG, gene gun methods, and heat shock. Furthermore, the host cell of a recombinant strain is not particularly limited as long as it can smoothly operate the promoter present in the expression vector, and prokaryotes are preferred.

[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 host cells without inducing growth inhibition of the host cells in the early stages of culture.

[0027] An allulose epimerization enzyme expression cassette according to one embodiment of the present invention comprises a polynucleotide encoding the 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 the target protein. The promoter also regulates the expression of the allulose epimerization enzyme in a Corynebacterium strain.

[0028] The promoter, a component of the allulose epimerization enzyme expression cassette, is an SOD promoter obtained by cloning a specific portion from the genome of the Corynebacterium glutamicum ATCC 13032 strain and is a polynucleotide corresponding to -200 bp relative to 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 SOD promoter analog can be composed of the nucleotide sequence of SEQ ID NO: 9, 10, or 11. In the present invention, the specific SOD promoter cloned has a length of 200 bp, with the nucleotides from 191 to 200 corresponding to a spacer sequence. This spacer sequence is a sequence present between genes and is expected not to significantly affect the expression pattern of the target protein, so some modifications are possible. The nucleotide sequence of SEQ ID NO: 9, 10, or 11 is a modified version of the spacer sequence corresponding to the 191st to 200th bases in the nucleotide sequence of SEQ ID NO: 1. The promoter, 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, 9, 10, or 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, 9, 10, or 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 correspondence, and the sequence is analyzed, and the any other sequence has 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.Those skilled in the art will readily understand that polynucleotides 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 DNA techniques known in the art. Such homology comparisons can be performed by calculating the percentage (%) of homology between two or more sequences using commercially available computer programs.

[0029] The polynucleotide encoding the allulose epimerization enzyme, a component of the allulose epimerization enzyme expression cassette, is not particularly limited as long as it encodes an enzyme capable of converting fructose to allulose. For example, the allulose epimerization enzyme may be derived from Flavonifractor plautii, Clostridium scindens, Treponema primitia, Ensifer adhaerens, or Ruminococcus torques. Considering the activity of converting fructose to allulose, the allulose epimerization enzyme is preferably derived from Flavonifractor plautii. Specifically, the allulose epimerization enzyme may be composed of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 13, or SEQ ID NO: 15. The allulose epimerization enzyme having the amino acid sequence of SEQ ID NO: 3 is a wild-type enzyme derived from Flavonifractor plautii. The allulose epimerization enzyme having the amino acid sequence of SEQ ID NO: 13 has the tryptophan (Trp) at position 29 of the amino acid sequence of SEQ ID NO: 3 substituted with lysine (Lys), the glycine (Gly) at position 216 with serine (Ser), and the methionine (Met) at position 234 with isoleucine (Ile). The allulose epimerization enzyme consisting of the amino acid sequence of SEQ ID NO: 15 is the 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 comprise the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more homology to the nucleotide sequence of SEQ ID NO: 2. The nucleotide sequence of SEQ ID NO: 2 is a polynucleotide encoding an allulose epimerization enzyme consisting of the amino acid sequence of SEQ ID NO: 3. The polynucleotide encoding the allulose epimerization enzyme may also comprise the nucleotide sequence of SEQ ID NO: 12 or the nucleotide sequence of SEQ ID NO: 14. The nucleotide sequence of SEQ ID NO: 12 is a polynucleotide encoding an allulose epimerization enzyme consisting of the amino acid sequence of SEQ ID NO: 13, and the nucleotide sequence of SEQ ID NO: 14 is a polynucleotide encoding an allulose epimerization enzyme consisting 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 Nos. 10-1919713, 10-2187354, 10-1656063, 10-1695830, 10-2189458, 10-1539097, 10-1539096, 10-1455759, and 10-1318422.

[0030] According to one embodiment of the present invention, the allulose epimerization enzyme expression cassette may further comprise 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 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 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 an allulose epimerization enzyme expression cassette, which can be used in recombinant vectors, recombinant strains, and the production of allulose from fructose.

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

[0033] A recombinant strain according to one embodiment of the present invention is a recombinant Corynebacterium strain, which is obtained by transforming a host cell with the above-mentioned allulose epimerization enzyme expression cassette or a recombinant expression vector into which the expression cassette has been 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 one embodiment of the present invention includes adding a recombinant Corynebacterium strain to a fructose-containing solution and reacting the solution. The fructose-containing solution contains Ca to promote the activity of allulose epimerization enzyme. 2+ , Mn 2+ The reaction mixture may further contain metal ions such as those listed below. In the method for producing allulose from fructose, the reaction temperature is 50 to 70°C, preferably 55 to 65°C, and more preferably 60 to 65°C in consideration of smooth enzyme expression in the recombinant strain, enzyme stability, and maximum activity, and the reaction pH is 6.5 to 8, preferably 6.5 to 7.5, and more preferably 6.5 to 7. In the method for producing allulose from fructose, the fructose concentration in the fructose-containing solution is not particularly limited, but in consideration of productivity and economic efficiency, it is preferably 5 to 75% (w / w) and more preferably 10 to 55% (w / w) based on the total weight of the fructose-containing solution.

[0035] The present invention will be described in more detail with reference to the following examples, which are merely intended to clearly illustrate the technical features of the present invention and are not intended to limit the scope of protection of the present invention. The present disclosure relates, for example, to the following: [Section 1] An expression cassette comprising a polynucleotide encoding an allulose epimerization enzyme and a promoter operably linked thereto, The promoter is composed of 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. [Section 2] Item 2. The allulose epimerization enzyme expression cassette according to Item 1, wherein the allulose epimerization enzyme is derived from Flavonifractor plautii, Clostridium scindens, Treponema primitia, Ensifer adhaerens, or Ruminococcus torques. [Section 3] Item 1. The allulose epimerization enzyme expression cassette according to Item 1, wherein the allulose epimerization enzyme is composed of the amino acid sequence of SEQ ID NO: 3. [Section 4] Item 1. The allulose epimerization enzyme expression cassette according to Item 1, wherein the polynucleotide encoding the allulose epimerization enzyme is composed of the base sequence of SEQ ID NO: 2. [Section 5] Item 2. The allulose epimerization enzyme expression cassette according to Item 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. [Section 6] A recombinant expression vector into which the expression cassette according to any one of Items 1 to 5 has been inserted. [Section 7] Item 7. The recombinant expression vector according to Item 6, which has the vector map of Figure 3. [Section 8] A recombinant Corynebacterium strain, which is obtained by transforming a host strain of the genus Corynebacterium by introducing the expression cassette according to any one of Items 1 to 5 or a recombinant expression vector into which the expression cassette has been inserted. [Section 9] Item 9. The recombinant Corynebacterium strain according to Item 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. [Section 10] A method for producing allulose from fructose, comprising the step of adding the recombinant Corynebacterium strain according to Item 9 to a fructose-containing solution and reacting the mixture. [Example]

[0036] Example 1: Amplification and isolation of promoter sequences for overexpression of D-allulose 3-epimerization enzyme To obtain a polynucleotide fragment of the promoter region for superoxide dismutase (SOD) gene expression from Corynebacterium glutamicum ATCC 13032, genomic DNA was extracted from the Corynebacterium glutamicum ATCC 13032 strain. PCR was then performed using the extracted genomic DNA as a template and the primer set listed in Table 1 to obtain an amplified promoter fragment. The amplified promoter fragment was cloned into a cloning vector and sequenced. The polynucleotide fragment was confirmed to be 200 bp in length and consisted of the nucleotide sequence of SEQ ID NO: 1. [Table 1]

[0037] Generally, the exact position of the SOD promoter region relative to the superoxide dismutase (sod) gene is not specified, but the region identified using the primer set in Table 1 was confirmed to be within -200 bp of the superoxide dismutase (sod) gene. In contrast, the expression regulatory sequence described in Korean Patent Publication No. 10-1656063 is within -300 bp of the superoxide dismutase (sod) gene.

[0038] Example 2: Amplification and isolation of polynucleotide sequences encoding D-allulose 3-epimerization enzymes To clone the gene encoding the D-allulose 3-epimerase, genomic DNA was extracted from Flavonifractor plautii KCTC 5970 strain. PCR was then performed using the extracted genomic DNA as a template and the primer set listed in Table 2 to obtain an amplified D-allulose 3-epimerase gene fragment. The amplified gene fragment was cloned into a cloning vector and analyzed for its nucleotide sequence. It was confirmed to be a polynucleotide fragment measuring 885 bp in length and consisting of the nucleotide sequence of SEQ ID NO: 2, which was named the FDPE gene. The D-allulose 3-epimerase decoded by the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2 is a wild-type enzyme and consists 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 have been inserted The antibiotic resistance gene, neomycin phosphotransferase 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) was inserted to construct the recombinant plasmid vector pDS vector. Figure 1 is a schematic diagram showing the process of constructing the recombinant plasmid vector pDS vector based on the commercial shuttle vector pJC1 vector. The pDS vector contains the replication initiation protein (repA) gene and an antibiotic (kanamycin) resistance gene derived from E. coli K-12, consisting of the nucleotide sequence of SEQ ID NO: 4.

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

[0041] Example 4: Construction of a recombinant strain transformed by introducing the recombinant expression vector pDS_FDPE and confirmation of D-allulose 3-epimerization enzyme activity The recombinant expression vector pDS_FDPE constructed above was introduced into competent Corynebacterium glutamicum cells using electroporation, and the transformed recombinant Corynebacterium glutamicum strain was selected by checking for 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 recombinant Corynebacterium glutamicum 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. The recombinant Corynebacterium glutamicum cell suspension was then mixed with 50 mM Tris-HCl buffer (pH 7.0) containing 20% ​​fructose (w / w) and 1 mM MnSO4 at a volume ratio of 1:1. The enzymatic conversion reaction was carried out at 65°C for 1 hour, after which the pH was lowered to below 2 by adding 1 M hydrochloric acid. The reaction mixture was then centrifuged to remove the cells and foreign matter, and the supernatant was collected. The supernatant was then filtered through a 0.45 μm syringe filter to prepare the sample. The allulose and fructose concentrations in the samples were then measured using high-performance liquid chromatography (HPLC), and the D-allulose 3-epimerization enzyme activity of the transformed recombinant Corynebacterium glutamicum strain was confirmed. 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 Corynebacterium glutamicum strains 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 at 30°C for 16 hours. The seed culture containing the bacteria was then inoculated into 2YTG medium at a concentration of 1% (v / v). The main culture was then cultured at 30°C and samples were taken at regular intervals. The optical density at 600 nm of the sampled samples was measured to evaluate the growth rate of the strains.

[0043] Figure 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 Figure 4, there was no significant difference in the growth rates of the wild-type Corynebacterium glutamicum strain and the recombinant Corynebacterium glutamicum strain. The recombinant Corynebacterium glutamicum strain constructed in this invention did not overexpress D-allulose 3-epimerization enzyme driven by the SOD promoter in the early stage of cultivation, and showed logarithmic growth and plateau phases at levels similar to those of wild-type Corynebacterium glutamicum strains.

[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 cells. The resulting recombinant Corynebacterium glutamicum 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. The recombinant Corynebacterium glutamicum cell suspension was then mixed in a 1:1 volume ratio with 50 mM Tris-HCl buffer (pH 7.0) containing 20% ​​(w / w) fructose and 1 mM MnSO4. The enzymatic conversion reaction was carried out at 65°C, and the D-allulose 3-epimerization enzyme activity of the recombinant Corynebacterium glutamicum was measured over time. After the predetermined reaction time was reached, 1 M hydrochloric acid was added to the reaction mixture to lower the pH to below 2, terminating the reaction. The reaction mixture was then centrifuged, and the supernatant, from which the cells and foreign matter had been removed, was collected and 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] Figure 5 shows the D-allulose 3-epimerization enzyme activity of the recombinant Corynebacterium glutamicum strain, expressed as the conversion rate of fructose to allulose, over time when the recombinant Corynebacterium glutamicum strain was inoculated and cultured in a substrate solution containing fructose in Example 6. As shown in Figure 5, the recombinant Corynebacterium glutamicum strain did not show growth inhibition due to overexpression of D-allulose 3-epimerization enzyme in the early stages of culture, and after sufficient growth up to the logarithmic growth phase, it showed high expression of D-allulose 3-epimerization enzyme from the plateau phase onwards.

[0046] Example 7: Production of allulose using a recombinant Corynebacterium glutamicum strain in a commercial substrate-containing solution 500 g (corresponding to approximately 380 mL) of high fructose syrup (HFCS; sugar composition: 55% fructose by weight, 45% glucose by weight) with a solids concentration of approximately 75 Brix was mixed with 500 g of a recombinant Corynebacterium glutamicum cell suspension and 0.2 g of MnSO4·4H2O. The pH was adjusted to approximately 7 using 4% NaOH aqueous solution, and the enzymatic conversion reaction was carried out at 65°C with stirring at 200 rpm. The pH of the reaction system was maintained at 7 using 4% NaOH aqueous solution throughout the enzymatic conversion reaction. Samples were taken at regular intervals, and the reaction was terminated by adding 10% HCl aqueous solution to the sample. The sample was then diluted approximately 25-fold with distilled water and filtered through a 0.45 μm syringe filter. The filtrate was analyzed by high-performance liquid chromatography (HPLC) to determine the conversion rate. The conversion rate was calculated using the following formula: TIFF0007828399000003.tif26137

[0047] The following Table 3 summarizes the results of measuring the conversion rate of fructose to allulose over 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.

[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 are possible within the scope of 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 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.

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, wherein the expression cassette further comprises 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. A recombinant Corynebacterium strain, wherein a host strain of the genus Corynebacterium is transformed by introducing the expression cassette according to any one of claims 1 to 5 or a recombinant expression vector into which the expression cassette has been inserted.

8. 8. The recombinant Corynebacterium strain of claim 7, 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.

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

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