Psicose epimerase expression cassette and psicose preparation method using same

Abstract

One embodiment of the present invention provides a psicose epimerase expression cassette comprising a polynucleotide encoding a psicose epimerase and a promoter operably linked thereto and modulating the expression of the psicose epimerase in a Corynebacterium strain. The promoter consists of a base sequence as shown in a sequence 1, a base sequence as shown in a sequence 9, a base sequence as shown in a sequence 10 or a base sequence as shown in a sequence 11. The psicose epimerase expression cassette disclosed by the embodiment of the invention is almost not started in a normal environment for culturing a recombinant corynebacterium strain, and does not induce growth inhibition of the recombinant corynebacterium strain. Therefore, the recombinant corynebacterium strain transformed by the psicose epimerase expression cassette of one embodiment of the present invention can normally grow and proliferate to the logarithmic phase, and stably and highly express psicose epimerase after the logarithmic phase.

Description

Technical Field

[0001] The present invention relates to a psicose epimerase expression cassette, and more particularly, to a psicose epimerase expression cassette, a recombinant Corynebacterium strain comprising the same, and a method for preparing psicose using the same, which does not induce growth inhibition of a Corynebacterium host microorganism due to the low expression level of psicase in the initial stage of culture, and can highly express psicase in the stagnant period after the logarithmic growth phase of the Corynebacterium host microorganism. Background Art

[0002] D-allulose is the epimer of fructose at carbon 3, and is also called D-psicose. The sweetness of psicose is 70% of that of white sugar (Oshima, 2006), but the calorie content is only 0.3% of that of white sugar, so it is a functional monosaccharide that can be used as a low-calorie sweetener for diet foods (Matsuo et al., 2002). In addition, psicose has the function of inhibiting blood sugar suppression by inhibiting glucose absorption, so it can be used in foods for diabetic patients, diet foods, etc., and has the function of inhibiting the activity of enzymes involved in lipid synthesis in the liver, thereby inhibiting abdominal fat accumulation, so it can be used in various functional foods such as health foods, etc. (Matsuo et al., 2001; Iida et al., 2008; Hayashi et al., 2010; Hossain et al., 2011). Due to the above characteristics, psicose is a good raw material that can replace white sugar, but since it belongs to rare sugars as a monosaccharide that is extremely rare in nature, a method for efficiently producing psicose is needed for application in the food industry.

[0003] Representative biological methods for producing psicose include a method for converting fructose into psicose by directly reacting fructose with D-psicose 3-epimerase, and a method for converting fructose into psicose by reacting the bacterial cells of a strain that produces D-psicose 3-epimerase using an intracellular enzyme with fructose. A recombinant strain transformed by introducing a D-psicose 3-epimerase expression cassette is used as a strain producing the above-mentioned D-psicose 3-epimerase, and Escherichia coli is generally used as a host cell. However, the transformed recombinant Escherichia coli is not a generally recognized safe (GRAS) strain and is therefore not suitable for use as a strain for producing food materials. In order to solve this problem, it is necessary to develop a D-psicose 3-epimerase expression cassette suitable for use in a Corynebacterium strain that is a generally recognized safe strain.

[0004] For example, Korean Patent Gazette No. 10-1656063 discloses a D-psicose 3-epimerase expression cassette suitable for transforming Corynebacterium strains and a method for producing D-psicose using the same. The transcription promoter used in the above prior art contains a region for simultaneously expressing superoxide dismutase (sod) and peptide methionine sulfoxide reductase (MsrA) genes present in the full-length base sequence of Corynebacterium glutamicum strains. Therefore, if an expression cassette containing the above transcription promoter is used, there is a risk that unexpected reverse transcription may occur, and further there is a risk of adversely affecting cell growth due to reduced expression of the target protein or inefficient energy consumption. Summary of the invention

[0005] Technical issues

[0006] The present invention is proposed under the existing technical background. The object of the present invention is to provide a psicose epimerase expression cassette and various uses thereof, which does not induce growth inhibition of a Corynebacterium host microorganism due to the low expression amount of psicase in the initial culture stage, and can highly express psicase in the stagnant period after the logarithmic growth phase of the Corynebacterium host microorganism.

[0007] Technical Solution

[0008] The inventors of the present invention cloned a 200 bp promoter region for superoxide dismutase gene expression from the genomic DNA of Corynebacterium glutamicum ATCC13032 strain, and sequentially connected the promoter region with a psicose epimerase to prepare an expression cassette, which was then inserted into a recombinant shuttle vector to prepare a recombinant psicose epimerase expression vector. Then, the recombinant psicose epimerase expression vector was introduced into Corynebacterium glutamicum, which is generally considered a safe strain, for transformation and preparation of a recombinant Corynebacterium glutamicum strain. As a result of culturing the recombinant Corynebacterium glutamicum strain, it was confirmed that the growth rates in the logarithmic growth phase and the stagnant phase were similar to those of the wild-type Corynebacterium glutamicum strain, and that D-psicose 3-epimerase was highly expressed due to environmental stress starting from the stagnant phase after the logarithmic growth phase, thereby completing the present invention.

[0009] In order to solve the above problems, the present invention provides an expression cassette for psicose epimerase, comprising a polynucleotide encoding psicose epimerase and a promoter operably linked thereto. The promoter consists of the base sequence shown in SEQ ID NO: 1, the base sequence shown in SEQ ID NO: 9, the base sequence shown in SEQ ID NO: 10, or the base sequence shown in SEQ ID NO: 11.

[0010] In order to solve the above problems, the present invention provides a recombinant expression vector in one embodiment, into which a psicose epimerase expression cassette is inserted.

[0011] In order to solve the above problems, the present invention provides a recombinant Corynebacterium strain in one example, which is transformed into a Corynebacterium host strain by introducing a psicose epimerase expression cassette or a recombinant expression vector inserted with the expression cassette.

[0012] In order to solve the above problems, the present invention provides a method for preparing psicose from fructose, comprising the steps of adding a recombinant Corynebacterium strain to a fructose-containing solution and reacting.

[0013] Effects of the Invention

[0014] The psicose epimerase expression cassette of one example of the present invention is hardly activated under the normal environment of culturing the recombinant Corynebacterium strain, and does not induce growth inhibition of the recombinant Corynebacterium strain. Therefore, the recombinant Corynebacterium strain transformed by the psicose epimerase expression cassette of one example of the present invention can grow and proliferate normally until the logarithmic growth phase, and stably and highly express the psicose epimerase after the logarithmic growth phase. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The figure is a simplified diagram showing the process of preparing a pDS vector as a recombinant plasmid vector based on a pJC1 vector as a commercially available shuttle vector.

[0016] Figure 2 To illustrate the process of preparing pDS_FDPE as a recombinant expression vector from a pDS vector as a recombinant plasmid vector, Figure 3 This is the cleavage map of pDS_FDPE used as a recombinant expression vector.

[0017] Figure 4 The graph shows the results of comparing the growth rates of the wild-type Corynebacterium glutamicum strain (denoted as WT) and the recombinant Corynebacterium glutamicum strain (denoted as DS00001) in Example 5 of the present invention.

[0018] Figure 5 This is a graph showing the D-psicose 3-epimerase activity of the recombinant Corynebacterium glutamicum strain over culture time by the conversion rate of fructose into psicose when the recombinant Corynebacterium glutamicum strain is inoculated in a substrate solution containing fructose and cultured in Example 6 of the present invention. DETAILED DESCRIPTION

[0019] Hereinafter, the present invention will be described in detail.

[0020] The term "promoter" used in the present invention refers to a minimum nucleic acid sequence that is operably connected to a target nucleotide sequence as a transcription object and regulates the transcription of the above-mentioned target nucleotide sequence. In addition, the above-mentioned promoter may contain a promoter structure sufficient to express an adjustable promoter-dependent gene induced by cell type specificity or external signals or agents, and this structure may be located at the 5' or 3' part of the gene. The above-mentioned promoter includes conservative promoters and inducible promoters. The promoter sequence may be derived from prokaryotes, eukaryotes or viruses. The promoter in prokaryotes is usually defined as a binding site adjacent to the transcription start site (Transcription Start Site) that binds to RNA polymerase.

[0021] The term "homology" used in the present invention means the identity of the nucleic acid sequence with the wild type or a variant having the same activity, and the homology comparison can be calculated by the naked eye or using a comparison program that is easily available for purchase to calculate the homology percentage (%) between two or more sequences. In addition, "homology" is used to indicate the identity of the amino acid sequence with the wild type or a variant having the same activity.

[0022] The term "polynucleotide" used in the present invention refers to all polyribonucleotides (RNA) or polydeoxyribonucleotides (DNA) that are unmodified (non-modified) or modified (modified). The above-mentioned polynucleotides include single-stranded or double-stranded DNA, DNA that is a mixture of single-stranded regions and double-stranded regions, single-stranded and double-stranded RNA, RNA that is a mixture of single-stranded regions and double-stranded regions, or hybrid molecules thereof, but are not limited thereto.

[0023] The term "operably linked" as used in the present invention is defined as a state in which a promoter sequence and a nucleotide sequence encoding a target protein are functionally linked so that the promoter can regulate the expression of the target protein. For example, a promoter is linked to a coding sequence when the promoter can control the expression of the coding sequence (i.e., when the coding sequence is under the transcriptional regulation of the promoter), or a ribosome binding site is linked to a coding sequence if the ribosome binding site is located in a position that promotes translation. A coding sequence can be linked to a regulatory sequence in a sense direction or an antisense direction.

[0024] The term "recombinant vector" used in the present invention is defined as a recombinant DNA prepared by excising a promoter variant or a target gene using a restriction enzyme and inserting it into a vector.

[0025] The term "expression cassette" used in the present invention refers to a regulatory sequence that is functionally linked to a nucleotide sequence to be expressed, such as a polynucleotide sequence encoding a psicose epimerase. Therefore, unlike an expression unit, an expression cassette contains not only a nucleotide sequence that regulates transcription and translation, but also a nucleotide sequence that is expressed as a protein as a result of transcription and translation.

[0026] The term "expression vector" used in the present invention is defined as a DNA sequence required for transcribing and translating cloned DNA in an appropriate host, specifically referring to a gene construct comprising an essential gene regulatory element operably connected to an insert so that the insert is expressed when present in a cell. Standard recombinant DNA techniques can be used to prepare and purify expression vectors. The types of the above-mentioned expression vectors are not particularly limited, as long as they have the function of expressing the desired gene and producing the desired protein in various host cells of prokaryotic cells and eukaryotic cells. The expression vector at least includes a promoter, a start 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, non-translated regions on the 5' side and 3' side of the desired gene, a selection marker region or a replicable unit, etc. The above-mentioned selection marker region may be a selection marker gene for screening an antibiotic for a target vector.

[0027] The term "recombinant strain" used in the present invention refers to a cell transformed by introducing a polynucleotide encoding one or more target proteins or an expression vector having the polynucleotide into a host cell. The method of introducing the above-mentioned expression vector into a host cell to prepare a transformant includes 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 gene guns, etc., heat shock, etc., but are not limited thereto. In addition, the type of host cell of the recombinant strain is not particularly limited, as long as the promoter present in the expression vector can run smoothly, preferably a prokaryotic organism.

[0028] One aspect of the present invention relates to an expression cassette, which does not induce growth inhibition of host cells in the early stage of culture, and can induce high expression of a target protein after the logarithmic growth phase of the host cells.

[0029] The psicose epimerase expression cassette of one example of the present invention comprises a polynucleotide encoding the psicose epimerase and a promoter operably linked thereto. Preferably, the promoter is located upstream of the polynucleotide encoding the psicose epimerase as the target protein. Furthermore, the promoter regulates the expression of the psicose epimerase in a Corynebacterium strain.

[0030] The promoter as a component of the above-mentioned allulose epimerase expression cassette is a SOD promoter cloned from a specific part of the genome of Corynebacterium glutamicum ATCC 13032 strain, and is based on a polynucleotide corresponding to -200bp of the superoxide dismutase gene. The above-mentioned promoter consists of the base sequence shown in SEQ ID NO: 1. In addition, the above-mentioned promoter includes an analog of the SOD promoter. The analog of the above-mentioned SOD promoter may consist of the base sequence shown in SEQ ID NO: 9, the base sequence shown in SEQ ID NO: 10, or the base sequence shown in SEQ ID NO: 11. The length of the specific SOD promoter cloned in the present invention is 200bp, and the base sequence from the 191st base to the 200th base corresponds to the spacer sequence. The above-mentioned spacer sequence is an arrangement existing between genes, and it is presumed that it will not have a significant effect on the expression pattern of the target protein, so it can be partially modified. The above-mentioned base sequence as shown in SEQ ID NO. 9, the base sequence as shown in SEQ ID NO. 10 or the base sequence as shown in SEQ ID NO. 11 is formed by partially modifying the spacer sequence corresponding to the 191st base to the 200th base in the base sequence as shown in SEQ ID NO. 1. The promoter as a component of the expression cassette of the psicose epimerase in one example of the present invention is composed of the base sequence as shown in SEQ ID NO. 1, the base sequence as shown in SEQ ID NO. 9, the base sequence as shown in SEQ ID NO. 10 or the base sequence as shown in SEQ ID NO. 11, but the equivalent scope of the above-mentioned promoter is not necessarily limited thereto. For example, the equivalent scope of the SOD promoter of the present invention includes a sequence having substantial identity with the base sequence as shown in SEQ ID NO. 1, the base sequence as shown in SEQ ID NO. 9, the base sequence as shown in SEQ ID NO. 10 or the base sequence as shown in SEQ ID NO. 11. The above-mentioned substantial identity means that the above-mentioned any other sequence has a sequence homology of 70% or more, 90% or more, or 98% or more with the base sequence shown in sequence 1, the base sequence shown in sequence 9, the base sequence shown in sequence 10, or the base sequence shown in sequence 11 by arranging the base sequence shown in sequence 1, the base sequence shown in sequence 9, the base sequence shown in sequence 10, or the base sequence shown in sequence 11 as much as possible and analyzing the sequence. A person skilled in the art can easily understand that a polynucleotide having the same or similar activity within the scope of substantial homology can be prepared by replacing, adding, or deleting one or more bases in the above-mentioned SOD promoter base sequence using gene recombination techniques known in the art. Such a comparison of homology can be performed by calculating the homology percentage (%) between two or more sequences using a commercially available computer program.

[0031] The type of the polynucleotide encoding the psicose epimerase as a component of the psicose epimerase expression cassette is not particularly limited, as long as it is a polynucleotide encoding an enzyme having the activity of converting fructose into psicose. For example, the psicose epimerase may be derived from Flavonifractor plautii, Clostridiun scindens, Treponema primitia, Ensifer adhaerens, or Ruminococcus torques, and is preferably derived from Flavonifractor plautii in view of the activity of converting fructose into psicose. Specifically, the psicose epimerase may be composed of the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequence shown in SEQ ID NO: 13, or the amino acid sequence shown in SEQ ID NO: 15. The psicose epimerase composed of the amino acid sequence shown in SEQ ID NO. 3 is a wild-type enzyme derived from Flavonifractor plautii. The psicose epimerase composed of the amino acid sequence shown in SEQ ID NO. 13 is a sequence in which the tryptophan (Trp) at position 29 is replaced by lysine (Lys), the glycine (Gly) at position 216 is replaced by serine (Ser), and the methionine (Met) at position 234 is replaced by isoleucine (Ile). The psicose epimerase composed of the amino acid sequence shown in SEQ ID NO. 15 is a sequence in which the tryptophan at position 29 is replaced by lysine, the alanine (Ala) at position 77 is replaced by serine, the glycine at position 216 is replaced by serine, and the methionine at position 234 is replaced by isoleucine. The type of the polynucleotide encoding the psicose epimerase is not particularly limited, and preferably, the polynucleotide may include a base sequence consisting of the base sequence shown in SEQ ID NO: 2 or 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 shown in SEQ ID NO: 2. The base sequence shown in SEQ ID NO: 2 is a polynucleotide encoding the psicose epimerase consisting of the amino acid sequence shown in SEQ ID NO: 3. Furthermore, the polynucleotide encoding the psicose epimerase may consist of the base sequence shown in SEQ ID NO: 12 or the base sequence shown in SEQ ID NO: 14. The base sequence shown in SEQ ID NO: 12 is a polynucleotide encoding the psicose epimerase consisting of the amino acid sequence shown in SEQ ID NO: 13, and the base sequence shown in SEQ ID NO: 14 is a polynucleotide encoding the psicose epimerase consisting of the amino acid sequence shown in SEQ ID NO: 15.The present invention relates to a psicose epimerase and a polynucleotide encoding the same, and the present invention 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.

[0032] The psicose epimerase expression cassette of one example of the present invention may further comprise one or more sequences selected from the group consisting of a replication origin, a multi-cloning site (MCS) for cloning a target protein gene, a transcription termination sequence, and a selection marker. The selection marker is used to screen cells transformed with the vector, and a marker that confers selectable expression types such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins may be used. 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.

[0033] One aspect of the present invention relates to various uses of a psicose epimerase expression cassette. The psicose epimerase expression cassette can be used to prepare psicose from a recombinant vector, a recombinant strain, and fructose.

[0034] The recombinant vector of one embodiment of the present invention is a recombinant expression vector into which the above-mentioned psicose epimerase expression cassette is inserted. Preferably, the above-mentioned recombinant expression vector has the following Figure 3 The cutting diagram shown. Figure 3 The recombinant expression vector of the cleavage diagram shown has a structure in which a replication origin, a promoter (SOD) composed of a base sequence as shown in SEQ ID NO: 1, a polynucleotide (FDPE) encoding a psicose epimerase composed of a base sequence as shown in SEQ ID NO: 2, a transcription terminator, a kanamycin resistance gene marker (NeoR / KanR) composed of a base sequence as shown in SEQ ID NO: 4, etc. are sequentially connected.

[0035] The recombinant strain of one example of the present invention is a recombinant Corynebacterium strain transformed by introducing the above-mentioned D-psicose epimerase expression cassette or a recombinant expression vector inserted with the above-mentioned expression cassette. The type of host strain used to prepare the above-mentioned recombinant Corynebacterium strain is not particularly limited, as long as it is a Corynebacterium strain, for example, it can be selected from the group consisting of Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoacidophilum, Corynebacterium thermoaminogenes, Corynebacterium melassecola and Corynebacterium efficientens.

[0036] The method for preparing psicose from fructose in one embodiment of the present invention comprises the steps of adding a recombinant Corynebacterium strain to a fructose-containing solution and reacting the solution. The fructose-containing solution may further contain Ca 2+ , Mn 2+ and other metal ions to promote the activity of psicose epimerase. In addition, in the method for preparing psicose from the above fructose, the reaction temperature is 50-70°C, preferably 55-65°C, and more preferably in the range of 60-65°C in consideration of the smooth enzyme expression of the recombinant strain, the stability of the enzyme and the maximum activity, and the reaction pH is 6.5-8, preferably 6.5-7.5, and more preferably in the range of 6.5-7. In addition, in the method for preparing psicose from the above fructose, the fructose concentration of the fructose-containing solution is not particularly limited, but in consideration of productivity and economy, it is preferably 5-75% (w / w) based on the total weight of the fructose-containing solution, and more preferably 10-55% (w / w).

[0037] The present invention will be described in more detail below by way of examples. However, the following examples are only used to clearly illustrate the technical features of the present invention and are not intended to limit the scope of protection of the present invention.

[0038] Example 1: Amplification and obtaining of promoter sequence for overexpression of D-psicose 3-epimerase

[0039] In order to obtain the polynucleotide fragment of the promoter region for expressing superoxide dismutase gene from Corynebacterium glutamicum ATCC 13032 bacterial strain, genomic DNA was extracted from Corynebacterium glutamicum ATCC 13032 bacterial strain. Then, the genomic DNA extracted was used as template, and the primer set described in the following table 1 was used to carry out PCR to obtain the promoter fragment of amplification. The promoter fragment of amplification was cloned into the cloning vector and the result of base sequence analysis was confirmed to be a polynucleotide fragment of 200bp in length consisting of the base sequence shown in sequence 1.

[0040] Table 1

[0041]

[0042] It has been confirmed that the correct positional relationship from the superoxide dismutase gene to the SOD promoter region has not been determined, but the region obtained using the primer set in Table 1 is from the superoxide dismutase gene to -200 bp. In contrast, the expression regulatory sequence described in Korean Patent Gazette No. 10-1656063 is from the superoxide dismutase gene to -300 bp.

[0043] Example 2: Amplification and obtaining of polynucleotide sequences encoding D-psicose 3-epimerase

[0044] In order to clone the gene encoding D-psicose 3-epimerase, genomic DNA was extracted from Flavonifractor plautii KCTC 5970 strain. Then, the extracted genomic DNA was used as a template, and PCR was performed using the primer set described in Table 2 below to obtain an amplified D-psicose 3-epimerase gene fragment. The amplified gene fragment was cloned into a cloning vector and the base sequence was analyzed. It was confirmed that it was a polynucleotide fragment with a length of 885 bp consisting of the base sequence shown in SEQ ID NO: 2, and was named FDPE gene. The D-psicose 3-epimerase translated by the polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 was a wild-type enzyme, which consisted of the amino acid sequence shown in SEQ ID NO: 3.

[0045] Table 2

[0046]

[0047] Example 3: Preparation of an expression vector inserted with a SOD promoter fragment and a D-psicose 3-epimerase gene

[0048] The pDS vector as a recombinant plasmid vector was prepared by replacing the antibiotic resistance gene neomycin phosphotransferase I (NTPI) of the commercially available shuttle vector pJC1 with the NTPII gene derived from Escherichia coli (E. coli) K-12 and inserting a multiple cloning site. Figure 1 The figure is a simplified diagram showing the process of preparing a pDS vector as a recombinant plasmid vector based on a pJC1 vector as a commercially available shuttle vector. The pDS vector contains a replication initiation protein (repA) gene, an antibiotic (kanamycin) resistance gene derived from Escherichia coli (E. coli) K-12 consisting of a base sequence as shown in SEQ ID NO: 4, and the like.

[0049] Then, the SOD promoter DNA fragment and the FDPE gene fragment with a length of 200 bp obtained above were mixed and subjected to 10 cycles of PCR. Then, the SOD promoter forward primer described in Table 1 and the FDPE reverse primer described in Table 2 were added and subjected to 15 cycles of PCR to obtain a fusion DNA fragment combining the SOD promoter and the FDPE gene. Then, the ends of the fusion DNA fragment were cut with restriction enzymes PstI and BamHI, and the fusion DNA fragment was inserted into the PstI and BamHI restriction enzyme regions of the multiple cloning site of the pDS vector to prepare pDS_FDPE as a recombinant expression vector. Figure 2 To illustrate the process of preparing pDS_FDPE as a recombinant expression vector from a pDS vector as a recombinant plasmid vector, Figure 3 Figure 2 is the cleavage diagram of pDS_FDPE as a recombinant expression vector. Figure 3 As shown, pDS_FDPE as a recombinant expression vector contains a replication initiation protein gene, a replication origin, multiple cloning sites such as restriction enzyme PstI site, BamHI site, NdeI site, SOD promoter, target gene (FDPE), transcription terminator and antibiotic resistance gene marker.

[0050] Example 4: Preparation of recombinant strain transformed by introducing recombinant expression vector pDS_FDPE and confirmation of D-psicose 3-epimerase activity

[0051] Utilize electroporation that the recombinant expression vector pDS_FDPE of above preparation is introduced into Corynebacterium glutamicum competent cell and confirm antibiotic resistance to screen the recombinant Corynebacterium glutamicum bacterial strain of transformation.Selected recombinant Corynebacterium glutamicum bacterial strain is inoculated in 2YT substratum (comprising the tryptone (Trytone) of 16g / L, the yeast extract (Yeast Extract) of 10g / L, the NaCl of 5g / L), and under 30 ℃ temperature, cultivated 40 hours.Remove culture fluid by centrifugation and be suspended in the NaCl solution of 0.85% (w / w), then centrifuge again to obtain the thalline of recombinant Corynebacterium glutamicum bacterial strain. The bacterial suspension was prepared by adding the bacterial cells of the obtained recombinant Corynebacterium glutamicum strain to 50mM Tris-HCl buffer (pH7.0) containing MnSO4 at a concentration of 1mM in an amount of 0.06% (w / w). Then, 50mM Tris-HCl buffer (pH7.0) containing fructose at a concentration of 20% (w / w) and MnSO4 at a concentration of 1mM and the bacterial suspension of the recombinant Corynebacterium glutamicum strain were mixed in a volume ratio of 1:1, and the enzyme conversion reaction was carried out at a temperature of 65°C for 1 hour, and then a hydrochloric acid solution with a concentration of 1M was added to reduce the pH to less than 2 and terminate the reaction. Then, the supernatant from which the bacterial cells and impurities were removed was recovered by centrifuging the reaction product, and the supernatant was filtered with a 0.45μm syringe filter to prepare a sample. Then, the concentration of psicose and the concentration of fructose in the sample were measured by high performance liquid chromatography (HPLC) to confirm the D-psicose 3-epimerase activity of the transformed recombinant Corynebacterium glutamicum strain. The conditions for high performance liquid chromatography (HPLC) analysis are as follows.

[0052] * Column: 87C (BIO-RAD)

[0053] * Column temperature: 80℃

[0054] *Mobile phase: ion exchange water

[0055] *Flow rate: 0.6ml / min

[0056] * Detector: Refractive Index Detector (Agilent 1260TID)

[0057] Example 5: Comparison of the growth rate of wild-type Corynebacterium glutamicum strains and recombinant Corynebacterium glutamicum strains

[0058] In 2YTG medium (comprising 16g / L tryptone, 10g / L yeast extract, 5g / L NaCl, 20g / L glucose (dextrose); pH7), wild-type Corynebacterium glutamicum strain and recombinant Corynebacterium glutamicum strain were inoculated respectively, and seed culture was carried out at a temperature of 30°C for 16 hours. Then, the seed culture solution containing the bacterial cells was inoculated in 2YTG medium with an amount of 1% (v / v), and main culture was carried out at a temperature of 30°C and sampling was carried out at different times. The growth rate of the strain was evaluated by measuring the optical density (optical density) of the sampled sample at 600nm.

[0059] Figure 4 The results of comparing the growth rates of the wild-type Corynebacterium glutamicum strain (expressed as WT) and the recombinant Corynebacterium glutamicum strain (expressed as DS00001) in Example 5 of the present invention are shown. Figure 4 As shown, there is no significant difference in the growth rate between the wild-type Corynebacterium glutamicum strain and the recombinant Corynebacterium glutamicum strain. The recombinant Corynebacterium glutamicum strain prepared in the present invention does not overexpress D-psicose 3-epimerase caused by the SOD promoter in the initial stage of culture, and exhibits a logarithmic growth phase and lag phase at similar levels to the wild-type Corynebacterium glutamicum strain.

[0060] Example 6: D-psicose 3-epimerase activity of recombinant Corynebacterium glutamicum strains over culture time

[0061] Recombinant Corynebacterium glutamicum strain was inoculated in 2YT medium (containing 16 g / L tryptone, 10 g / L yeast extract, 5 g / L NaCl), and cultured at 30°C for 40 hours. The culture solution was removed by centrifugation and suspended in 0.85% (w / w) NaCl solution, then centrifuged again to obtain the cell of recombinant Corynebacterium glutamicum strain. Cell suspension was prepared by adding the cell of the recombinant Corynebacterium glutamicum strain to 50 mM Tris-HCl buffer (pH 7.0) containing 1 mM MnSO with an amount of 0.06% (w / w). Then, 50mM Tris-HCl buffer (pH7.0) containing 20% ​​(w / w) fructose and 1mM MnSO4 and a bacterial suspension of a recombinant Corynebacterium glutamicum strain were mixed in a volume ratio of 1:1, and the enzyme conversion reaction was carried out at a temperature of 65°C for 1 hour and the D-psicose 3-epimerase activity of the recombinant Corynebacterium glutamicum strain was measured along with the reaction time. When the preset reaction time was reached, a hydrochloric acid solution with a concentration of 1M was added to the reaction solution to reduce the pH to below 2 and terminate the reaction. Then, the supernatant from which the bacterial cells and impurities were removed was recovered by centrifuging the reaction solution, and the supernatant was filtered with a 0.45μm syringe filter to prepare a sample. Then, the concentration of psicose and the concentration of fructose in the sample were determined by high performance liquid chromatography (HPLC) and the conversion rate of fructose to psicose was calculated.

[0062] Figure 5 This is a graph showing the D-psicose 3-epimerase activity of the recombinant Corynebacterium glutamicum strain over the culture time by the conversion rate of fructose to psicose when the recombinant Corynebacterium glutamicum strain is inoculated in a substrate solution containing fructose and cultured in Example 6 of the present invention. Figure 5As shown, the recombinant Corynebacterium glutamicum strain did not show growth inhibition due to overexpression of D-psicose 3-epimerase in the early stage of culture, and after fully proliferating to the logarithmic growth phase, it showed high expression of D-psicose 3-epimerase from the stagnant phase.

[0063] Example 7: Production of Psicose Using Recombinant Corynebacterium glutamicum Strain in a Solution Containing Commercially Available Substrate

[0064] Mix 500g (equivalent to about 380mL) of high fructose syrup (HFCS; sugar composition: 55 weight percent fructose, 45 weight percent glucose) with a solid content concentration of about 75 degrees Brix, 500g of a bacterial suspension of a recombinant Corynebacterium glutamicum strain, and 0.2g of MnSO4.4H2O, and adjust the pH to about 7 using a 4% NaOH aqueous solution, and then perform an enzyme conversion reaction at a temperature of 65°C with a stirring condition of 200rpm. During the enzyme conversion reaction, the pH of the reaction system was maintained at 7 using a 4% NaOH aqueous solution. Sampling was performed at different times of the enzyme conversion reaction, and a 10% HCl aqueous solution was added to the sampled sample to terminate the enzyme conversion reaction. Then, the sampled sample was diluted to about 25 times with distilled water, and after filtering with a 0.45μm syringe filter, the conversion rate was determined by analyzing the filtrate using a high performance liquid chromatography (HPLC). The conversion rate was calculated using the following formula.

[0065]

[0066] Table 3 below shows the results of measuring the conversion rate of fructose to psicose with different reaction times when the recombinant Corynebacterium glutamicum strain of the present invention was inoculated into high fructose syrup as a commercially available substrate and an enzyme conversion reaction was performed.

[0067] Table 3

[0068] Reaction time (hr) Conversion rate of fructose to allulose (%) 0 0 1 13.21 2 21.66 4 26.53 6 28.82 8 29.94 10 30.01 12 29.89 14 29.93

[0069] As shown in the above Table 3, when psicose was prepared from high fructose syrup using the recombinant Corynebacterium glutamicum strain of the present invention, a conversion rate at the level of 30% was reached in about 12 hours.

[0070] As mentioned above, the present invention has been described by the above-mentioned embodiments, but the protection scope of the present invention is not limited thereto. As a matter of course, various modifications can be made without departing from the scope and thought of the present invention. Therefore, the protection scope of the present invention is not limited to the specific embodiments disclosed as the best form, and should be interpreted as including all embodiments falling within the scope of the invention claims attached to the present invention.

Claims

1. A psicose epimerase expression cassette, comprising a polynucleotide encoding a psicose epimerase and a promoter operably linked thereto, characterized in that: The above promoter consists of the base sequence shown in SEQ ID NO: 1, the base sequence shown in SEQ ID NO: 9, the base sequence shown in SEQ ID NO: 10 or the base sequence shown in SEQ ID NO:

11.

2. The expression cassette of psicose epimerase according to claim 1, characterized in that: The above-mentioned allulose epimerase is derived from Flavonoids perfringens, Clostridium fulgidus, Treponema bacteria, Sagittaria adhering or Ruminococcus contortus.

3. The expression cassette of psicose epimerase according to claim 1, characterized in that: The above-mentioned D-psicose isomerase consists of the amino acid sequence shown in SEQ ID NO:

3.

4. The expression cassette of psicose epimerase according to claim 1, characterized in that: The polynucleotide encoding the psicose epimerase consists of the base sequence shown in SEQ ID NO:

2.

5. The expression cassette of psicose epimerase according to claim 1, characterized in that: The expression cassette further comprises one or more sequences selected from the group consisting of a replication origin, a multiple cloning site for cloning a target protein gene, a transcription termination sequence and a selection marker.

6. A recombinant expression vector, characterized in that: An expression cassette according to any one of claims 1 to 5 is inserted.

7. A recombinant expression vector according to claim 6, characterized in that: It has a cutting diagram as shown in FIG3 .

8. A recombinant Corynebacterium strain, characterized in that A Corynebacterium host strain 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 is inserted.

9. The recombinant Corynebacterium strain according to claim 8, characterized in that The above-mentioned Corynebacterium host strain is selected from the group consisting of Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoacetogenicum, Corynebacterium thermoammonigenicum, Corynebacterium molasses and Corynebacterium efficacious.

10. A method for preparing psicose from fructose, characterized in that: The method comprises the steps of adding the recombinant Corynebacterium strain according to claim 9 to a fructose-containing solution and carrying out a reaction.

Images