Patent ID: 12203108

MODE FOR INVENTION

The present application will be explained in detail with reference to the following examples. However, these examples are provided to assist in understanding the present application and do not limit the scope of the present application.

EXAMPLES

Example 1: Production of Recombinant Expression Vectors of Inositol-Mono-Phosphatases and Transformed Microorganisms

To provide psicose-6-phosphate phosphatase necessary for the D-psicose production pathway, thermoresistant inositol-mono-phosphatase genes were screened. Specifically, inositol-mono-phosphatase genes (Rma, Tle, Mrub, Dtu, Msi, Mruf, Mta, Mch, and Mce) were screened from the genomic sequences ofRhodothermus marinus, Thermotoga lettingae, Meiothermus ruber, Dictyoglomus turgidum, Pyrobaculum ferrireducens, Thermoanaerobacter wiegelii, Thermus thermophilus, Thermococcus litoralis, Geobacillus stearothermophilus, Anaerolinea thermophila, Sulfolobus acidocaldarius, Thermosulfidibacter takai, Pyrococcus furiosus, Archaeoglobus fulgidus, Alicyclobacillus acidocaldarius, Meiothermus silvanus, Meiothermus rufus, Meiothermus taiwanensis, Meiothermus chliarophilus, andMeiothermus cerbereusregistered in GenBank.

Based on information on the nucleotide sequences (SEQ ID NOS: 21, 22, 23, 24, 36, 37, 38, 39, and 40 in the order of the genes) and the amino acid sequences (SEQ ID NOS: 1, 2, 3, 4, 16, 17, 18, 19, and 20 in the order of the genes) of the screened genes, forward primers (SEQ ID NOS: 41, 43, 45, 47, 49, 51, 53, 55, and 57) and reverse primers (SEQ ID NOS: 42, 44, 46, 48, 50, 52, 54, 56, and 58) were designed. The genes were amplified from the genomic DNAs ofRhodothermus marinus, Thermotoga lettingae, Meiothermus ruber, Dictyoglomus turgidum, Meiothermus silvanus, Meiothermus rufus, Meiothermus taiwanensis, Meiothermus chliarophilus,andMeiothermus cerbereusby polymerase chain reaction (PCR) using the synthesized primers. The amplified inositol-mono-phosphatase genes were inserted into plasmid vector pET21a (Novagen) forE. coliexpression using restriction enzymes NdeI and XhoI or SalI to construct recombinant expression vectors, which were named pET21a-CJ_Rma(Nde I/Xho I), pET21a-CJ_Tle(Nde I/Xho I), pET21a-CJ_Mrub(Nde I/Xho I), pET21a-CJ_Dtu(Nde I/Xho I), pET21a-CJ_Msi(Nde I/Sal I), pET21a-CJ_Mruf(Nde I/Sal I), pET21a-CJ_Mta(Nde I/Sal I), pET21a-CJ_Mch(Nde I/Sal I), and pET21a-CJ_Mce(Nde I/Sal I).

Additionally, inositol-mono-phosphatase genes (Pfe, Twi, Tth, Tli, Gst, Ath, Sac, Tta, Pfu, Afu, and Aac) derived fromPyrobaculum ferrireducens, Thermoanaerobacterwiegelii,Thermus thermophilus, Thermococcus litoralis, Geobacillus stearothermophilus, Anaerolinea thermophila, Sulfolobus acidocaldarius, Thermosulfidibacter takai, Pyrococcus furiosus, Archaeoglobus fulgidus, andAlicyclobacillus acidocaldariuswere screened. Based on information on the nucleotide sequences (SEQ ID NOS: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35 in the order of the genes) and the amino acid sequences (SEQ ID NOS: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 in the order of the genes) of the screened genes, DNA synthesis was requested to Bioneer (Korea). The DNAs were inserted into vector pBT7-C-His (Bioneer) to construct recombinant expression vectors, which were named pBT7-C-His-CJ_Pfe, pBT7-C-His-CJ_Twi, pBT7-C-His-CJ_Tth, pBT7-C-His-CJ_Tli, pBT7-C-His-CJ_Gst, pBT7-C-His-CJ_Ath, pBT7-C-His-CJ_Sac, pBT7-C-His-CJ_Tta, pBT7-C-His-CJ_Pfu, pBT7-C-His-CJ_Afu, and pBT7-C-His-CJ_Aac.

The expression vectors were transformed into strainE. coliBL21(DE3) by a general transformation technique (see Sambrook et al. 1989) to produce transformed microorganisms, which were namedE. coliBL21(DE3)/pET21a-CJ_Rma (E. coli_P1_CJ_Rma, KCCM12057P),E. coliBL21(DE3)/pET21a-CJ_Tle (E. coli_P2_CJ_Tle, KCCM12058P),E. coliBL21(DE3)/pET21a-CJ_Mrub (E. coli_P3_CJ_Mrub, KCCM12059P),E. coliBL21(DE3)/pET21a-CJ_Dtu (E. coli_P4_CJ_Dtu, KCCM12060P),E. coliBL21(DE3)/pBT7-C-His-CJ_Pfe (E. coli_P5_CJ_Pfe, KCCM12061P),E. coliBL21(DE3)/pBT7-C-His-CJ_Twi (E. coli_P6_CJ_Twi, KCCM12062P),E. coliBL21(DE3)/pBT7-C-His-CJ_Tth (E. coli_P7_CJ_Tth, KCCM12063P),E. coliBL21(DE3)/pBT7-C-His-CJ_Tli (E. coli_P8_CJ_Tli, KCCM12064P),E. coliBL21(DE3)/pBT7-C-His-CJ_Gst (E. coli_P9_CJ_Gst, KCCM12065P),E. coliBL21(DE3)/pBT7-C-His-CJ_Ath (E. coli_P10_CJ_Ath, KCCM12066P),E. coliBL21(DE3)/pBT7-C-His-CJ_Sac (E. coli_P11_CJ_Sac, KCCM12067P),E. coliBL21(DE3)/pBT7-C-His-CJ_Tta (E. coli_P12_CJ_Tta, KCCM12068P),E. coliBL21(DE3)/pBT7-C-His-CJ_Pfu (E. coli_P13_CJ_Pfu, KCCM12069P),E. coliBL21(DE3)/pBT7-C-His-CJ_Afu (E. coli_P14_CJ_Afu, KCCM12070P),E. coliBL21(DE3)/pBT7-C-His-CJ_Aac (E. coli_P15_CJ_Aac, KCCM12071P),E. coliBL21(DE3)/pET21a-CJ_Msi (E. coliP16_CJ_Msi, KCCM12072P),E. coliBL21(DE3)/pET21a-CJ_Mruf (E. coli_P17_CJ_Mruf, KCCM12073P),E. coliBL21(DE3)/pET21a-CJ_Mta (E. coli_P18_CJ_Mta, KCCM12074P),E. coliBL21(DE3)/pET21a-CJ_Mch (E. coli_P19_CJ_Mch, KCCM12075P), andE. coliBL21(DE3)/pET21a-CJ_Mce (E. coli_P20_CJ_Mce, KCCM12076P).

The transformed strains were deposited at the Korean Culture Center of Microorganisms (KCCM) on Jul. 10, 2017 under the Budapest Treaty (Accession Nos.: KCCM12057P to KCCM12076P).

Example 2: Production of Enzymes Necessary for D-Psicose Production Pathway

To provide an α-glucan phosphorylase, a phosphoglucomutase, a D-glucose-6-phosphate-isomerase, and a D-fructose-6-phosphate-3-epimerase derived fromThermotoga neapolitanaas thermoresistant enzymes necessary for the D-psicose production pathway, genes corresponding to the enzymes were screened (ct1, ct2, tn1 and fp3e in the order of the enzymes).

Based on the nucleotide sequences (SEQ ID NOS: 60, 62, 64, and 66 in the order of the enzymes) and the amino acid sequences (SEQ ID NOS: 59, 61, 63, and 65 in the order of the enzymes) of the screened genes, forward primers (SEQ ID NOS: 69, 71, 73 and 75) and reverse primers (SEQ ID NOS: 70, 72, 74 and 76) were designed. The enzyme genes were amplified from the genomic DNA ofThermotoga neapolitanaas a template by polymerase chain reaction (PCR) using the primers. PCR was performed for a total of 25 cycles using the following conditions: denaturization at 95° C. for 30 sec, annealing at 55° C. for 30 sec, and polymerization at 68° C. for 2 min. The amplified enzyme genes were inserted into plasmid vector pET21a (Novagen) forE. coliexpression using restriction enzymes NdeI and XhoI to construct recombinant expression vectors, which were named pET21a-CJ_ct1, pET21a-CJ_ct2, pET21a-CJ_tn1, and pET21a-CJ_fp3e. The recombinant expression vectors were transformed into strainE. coliBL21(DE3) by a general transformation technique (see Sambrook et al. 1989) to produce transformed microorganisms, which were namedE. coliBL21(DE3)/pET21a-CJ_ct1 (KCCM11990P),E. coliBL21(DE3)/pET21a-CJ_ct2 (KCCM11991P),E. coliBL21(DE3)/pET21a-CJ_tn1 (KCCM11992P), andE. coliBL21(DE3)/CJ_tn_fp3e (KCCM11848P). The strains were deposited at the Korean Culture Center of Microorganisms (KCCM) on Jun. 23, 2016 under the Budapest Treaty.

Example 3: Production of Recombinant Enzymes

In this example, recombinant enzymes were produced. First, a culture tube containing 5 ml of LB liquid medium was inoculated with each of the transformed microorganisms produced in Examples 1 and 2. The inoculum was cultured in a shaking incubator at 37° C. until an absorbance of 2.0 at 600 nm was reached. The culture broth was added to LB liquid medium in a culture flask, followed by main culture. When the absorbance of the culture at 600 nm reached 2.0, 1 mM IPTG was added to induce the expression and production of a recombinant enzyme. The culture temperature was maintained at 37° C. with stirring at 180 rpm. The culture broth was centrifuged at 8,000×g and 4° C. for 20 min to collect bacterial cells. The collected bacterial cells were washed twice with 50 mM Tris-HCl buffer (pH 8.0) and suspended in the same buffer. Then, cells were disrupted using an ultrasonic homogenizer. The cell lysate was centrifuged at 13,000×g and 4° C. for 20 min. The recombinant enzyme was purified from the supernatant by His-tag affinity chromatography. The purified recombinant enzyme was dialyzed against 50 mM Tris-HCl buffer (pH 8.0) and was then used for subsequent reaction. The molecular weight of the purified recombinant enzyme was determined by SDS-PAGE.

The names and molecular weights of the purified enzymes produced using the transformed microorganisms are as follows (FIGS.2a,2band2c):

30.3 kDa for the enzyme (RMA) produced fromE. coliBL21(DE3))/pET21a-CJ_Rma (E. coli_P1_CJ_Rma);

28.5 kDa for the enzyme (TLE) produced fromE. coliBL21(DE3)/pET21a-CJ_Tle (E. coli_P2_CJ_Tle);

28 kDa for the enzyme (MRUB) produced fromE. coliBL21(DE3)/pET21a-CJ_Mrub (E. coli_P3_CJ_Mrub);

30.2 kDa for the enzyme (DTU) produced fromE. coliBL21(DE3)/pET21a-CJ_Dtu (E. coli_P4_CJ_Dtu);

kDa for the enzyme (PFE) produced fromE. coliBL21(DE3)/pBT7-C-His-CJ_Pfe (E. coli_P5_CJ_Pfe);

28.8 kDa for the enzyme (TWI) produced fromE. coliBL21(DE3)/pBT7-C-His-CJ_Twi (E. coli_P6_CJ_Twi);

kDa for the enzyme (TTH) produced fromE. coliBL21(DE3)/pBT7-C-His-CJ_Tth (E. coliP7_CJ_Tth);

28 kDa for the enzyme (TLI) produced fromE. coliBL21(DE3)/pBT7-C-His-CJ_Tli (E. coli_P8_CJ_Tli);

kDa for the enzyme (GST) produced fromE. coliBL21(DE3)/pBT7-C-His-CJ_Gst (E. coli_P9_CJ_Gst);

28.7 kDa for the enzyme (ATH) produced fromE. coliBL21(DE3)/pBT7-C-His-CJ_Ath (E. coli_P10_CJ_Ath);

kDa for the enzyme (SAC) produced fromE. coliBL21(DE3)/pBT7-C-His-CJ_Sac (E. coli_P11_CJ_Sac);

28.6 kDa for the enzyme (TTA) produced fromE. coliBL21(DE3)/pBT7-C-His-CJ_Tta (E. coli_P12_CJ_Tta);

27.9 kDa for the enzyme (PFU) produced fromE. coliBL21(DE3)/pBT7-C-His-CJ_Pfu (E. coli_P13_CJ_Pfu);

28 kDa for the enzyme (AFU) produced fromE. coliBL21(DE3)/pBT7-C-His-CJ_Afu (E. coli_P14_CJ_Afu);

29 kDa for the enzyme (AAC) produced fromE. coliBL21(DE3)/pBT7-C-His-CJ_Aac (E. coli_P15_CJ_Aac);

28.1 kDa for the enzyme (MSI) produced fromE. coliBL21(DE3)/pET21a-CJ_Msi (E. coli_P16_CJ_Msi);

28 kDa for the enzyme (MRUF) produced fromE. coliBL21(DE3)/pET21a-CJ_Mruf (E. coli_P17_CJ_Mruf);

28.1 kDa for the enzyme (MTA) produced fromE. coliBL21(DE3))/pET21a-CJ_Mta (E. coli_P18_CJ_Mta);

28.4 kDa for the enzyme (MCH) produced fromE. coliBL21(DE3))/pET21a-CJ_Mch (E. coli_P19_CJ_Mch);

28.1 kDa for the enzyme (MCE) produced fromE. coliBL21(DE3))/pET21a-CJ_Mce (E. coli_P20_CJ_Mce);

The enzyme (CT1) produced fromE. coliBL21(DE3)/pET21a-CJ_ct1 (KCCM11990P);

The enzyme (CT2) produced fromE. coliBL21(DE3)/pET21a-CJ_ct2 (KCCM11991P);

The enzyme (TN1) produced fromE. coliBL21(DE3)/pET21a-CJ_tn1 (KCCM11992P); and

The enzyme (FP3E) produced fromE. coliBL21(DE3)/CJ_tn_fp3e (KCCM11848P).

Example 4: Analysis of Activities of the Inositol-Mono-Phosphatases

4-1. Analysis of Activities of the Psicose-6-Phosphate Phosphatases

Psicose-6-phosphate was difficult to purchase. Thus, the inventors directly produced D-psicose-6-phosphate from D-fructose-6-phosphate and investigated the activities of the inositol-mono-phosphatases for D-psicose production.

Specifically, 50 mM D-fructose-6-phosphate was suspended in 50 mM Tris-HCl (pH 7.0), and then the D-fructose-6-phosphate-3-epimerase (FP3E) produced in Example 3 and 0.1 unit/ml of each of the 20 inositol-mono-phosphatases were added thereto. The mixture was allowed to react at 70° C. for 1 h. The production of D-psicose was confirmed by HPLC (SP_0810 column (SHODEX), AMINEX HPX-87C column (BIO-RAD), 80° C., mobile phase flow rate 0.6 ml/min, refractive index detector).

The dephosphorylation potency of the all 20 inositol-mono-phosphatases for D-psicose-6-phosphate were investigated (FIGS.3aand3b).

4-2. Analysis of Activities of the Inositol-Mono-Phosphatases for Specific Dephosphorylation of D-Psicose-6-Phosphate

The specific dephosphorylation rates of D-psicose-6-phosphate in a mixture containing D-glucose-6-phosphate, D-glucose-1-phosphate, D-fructose-6-phosphate, and D-psicose-6-phosphate in the presence of the inositol-mono-phosphatases were measured.

Specifically, 0.1 unit/ml of each of the inositol-mono-phosphatases and 5 mM MgCl2were added to a mixture of 1% (w/v) D-glucose-6-phosphate, D-glucose-1-phosphate, D-fructose-6-phosphate, and D-psicose-6-phosphate. The reaction was allowed to proceed at 50° C. for 12 h. The reaction products were analyzed by HPLC (AMINEX HPX-87C column (BIO-RAD), 80° C., mobile phase flow rate 0.6 ml/min). A refractive index detector was used to detect the production of D-psicose and other saccharides (fructose and glucose).

As a result, the enzyme MRUB showed the highest specific dephosphorylation rate of D-psicose-6-phosphate (FIG.3a).

Example 5: Analysis of Activities of the Enzymes Through Multiple Enzymatic Reactions

For the production of D-psicose from maltodextrin, the enzymes CT1, CT2, TN1, FP3E and MRUB were allowed to simultaneously react with maltodextrin. 5% (w/v) maltodextrin was added to 0.1 unit/ml of each enzyme, 5 mM MgCl2, and 20 mM sodium phosphate (pH 7.0). The mixture was allowed to react at a temperature of 50° C. for 12 h. The reaction products were analyzed by HPLC (AMINEX HPX-87C column (BIO-RAD), 80° C., mobile phase flow rate 0.6 ml/min), refractive index detector).

As a result, the production of D-psicose from maltodextrin through the multiple enzymatic reactions was confirmed (FIG.4a).

While the embodiment of the present application has been described in detail, it will be understood by those skilled in the art that the application can be implemented in other specific forms without changing the spirit or essential features of the application. Therefore, it should be noted that the forgoing embodiments are merely illustrative in all aspects and are not to be construed as limiting the application. The scope of the application is defined by the appended claims rather than the detailed description of the application. All changes or modifications or their equivalents made within the meanings and scope of the claims should be construed as falling within the scope of the application.