Method for Efficient Biosynthesis of Reb D by Glycosyltransferase

Abstract

The disclosure discloses a method for efficient biosynthesis of Reb D by glycosyltransferase, belonging to the field of biocatalytic synthesis. According to the disclosure, a glycosyltransferase having an activity to catalyze synthesis of Reb D from Reb A is obtained, and a mutant YojK-I241T/G327N with high catalytic activity is obtained through directed evolution. The glycosyltransferase mutant YojK-I241T/G327N and a sucrose synthase AtSuSy derived from Arabidopsis thaliana are used for constructing a coupling reaction to realize efficient catalytic synthesis of Reb D with Reb A as a substrate. The reaction is carried out by using 19.32 g/L (20 mmol/L) of Reb A as the substrate for 15 h to efficiently synthesize 20.59 g/L of Reb D, and the yield of Reb D reaches 91.29%, which provides an efficient and green new pathway for production of Reb D.

Claims

1. A glycosyltransferase, wherein the amino acid sequence of the glycosyltransferase is set forth in SEQ ID NO. 1.

2. A recombinant strain expressing the glycosyltransferase according to claim 1.

3. The recombinant strain according to claim 2, wherein the recombinant strain also expresses a sucrose synthase.

4. The recombinant strain according to claim 3, wherein an amino acid sequence of the sucrose synthase is an amino acid sequence with sucrose synthase activity derived from any source.

5. The recombinant strain according to claim 4, wherein the amino acid sequence of the sucrose synthase is set forth in SEQ ID NO. 2.

6. The recombinant strain according to claim 2, wherein E. coli is used as a host cell.

7. A method for catalytic synthesis of Rebaudioside D (Reb D), comprising carrying out a catalytic reaction on Reb A as a substrate by using a cell lysis buffer of the recombinant strain according to claim 2.

8. The method for catalytic synthesis of Reb D according to claim 7, wherein the cell lysis buffer is a supernatant obtained by cell lysis after induced expression of the recombinant strain.

9. The method for catalytic synthesis of Reb D according to claim 7, wherein conditions of the catalytic reaction are as follows: in a reaction system containing 1-50 mmol/L of Reb A, 0-800 mmol/L of sucrose, 5-25% (v/v) of DMSO, 100 mmol/L of K.sub.2HPO.sub.4—KH.sub.2PO.sub.4 buffer and 100 mmol/L of NaCl, the reaction is carried out at 20-45° C. for 0-48 hours.

10. The method for catalytic synthesis of Reb D according to claim 9, wherein the reaction system further contains uridine diphosphate glucose.

11. The method for catalytic synthesis of Reb D according to claim 9, wherein the buffer has a pH of 5.5-9.0.

Description

BRIEF DESCRIPTION OF FIGURES

[0030] FIG. 1A shows protein expression and purification analysis of the glycosyltransferase YojK.

[0031] FIG. 1B shows protein expression and purification analysis of the glycosyltransferase mutant YojK-I241T/G327N.

[0032] FIG. 2 is a UPLC analysis of the conversion of Reb A catalyzed by YojK.

[0033] FIG. 3 shows LC-MS analysis of the new product after glycosylating Reb A by YojK. HRMS (ESI.sup.−) calculated for C.sub.50H.sub.80O.sub.28 [M−H].sup.−: 1127.4758; Found: 1127.4790.

[0034] FIG. 4 shows the 1H NMR spectrum of Reb D in Pyridine-d5 (600 MHz).

[0035] FIG. 5 shows the 13C NMR spectrum of Reb D in Pyridine-d5 (151 MHz).

[0036] FIG. 6 shows UPLC analysis of the conversion of Reb A catalyzed by YojK and YojK-241T/G327N.

[0037] FIG. 7 shows SDS-PAGE analysis of the heterologous expression of YojK and AtSuSy.

[0038] FIG. 8 shows optimization of pH.

[0039] FIG. 9 shows optimization of reaction temperature.

[0040] FIG. 10 shows optimization of the ratio of DMSO.

[0041] FIG. 11 shows optimization of sucrose concentration.

[0042] FIG. 12 shows comparison of the synthetic efficiency of Reb D at different concentrations of Reb A for YojK-I241T/G327N-AtSuSy and YojK-AtSuSy cascade reaction systems.

[0043] FIG. 13 shows optimization of the reaction time on a large-scale preparation of Reb D. Error bars mean the standard deviation of three repeats.

DETAILED DESCRIPTION

[0044] The disclosure will be described in detail below with reference to the accompanying drawings and specific examples. However, the examples are not intended to limit the disclosure in any form. Unless otherwise specified, reagents, methods and equipment used in the disclosure are conventional reagents, methods and equipment in the art.

[0045] Unless otherwise specified, the reagents and materials used in the following examples are commercially available commodities or can be prepared by known methods.

Example 1 Acquisition of Glycosyltransferase YojK Gene and Construction of Mutant and Recombinant Strain

[0046] An amino acid sequence (accession number: WP_004399256.1) and a nucleic acid sequence (accession number: CP053102.1) of B. subtilis 85168 glycosyltransferase were downloaded from Genbank, and the gene was synthesized by Yixin Biotechnology Co., Ltd. and linked to a multiple cloning site of a vector pET-21b(+) to obtain a recombinant plasmid pET-21b(+)-YojK.

[0047] With the recombinant plasmid pET-21b(+)-YojK as a template, whole plasmid PCR was carried out by using primers I241T-F/1241T-R and G327N-F/G327N-R (the primers are shown in Table 1) to obtain a recombinant plasmid pET-21b(+)-YojK-I241T/G327N carrying the mutant.

[0048] The obtained plasmids pET-21b(+)-YojK and pET-21b(+)-YojK-I241 T/G327N were sequenced for identification and respectively transformed into E. coli BL21(DE3) competent cells, and an LB solid plate (10 g/L of peptone, 5 g/L of tryptone, 10 g/L of NaCl and 20 g/L of agar powder) containing 100 μg/mL of ampicillin was used for screening to obtain recombinant strains E. coli BL21 (DE3) pET-21b(+)-YojK and E. coli BL21 (DE3) pET-21b(+)-YojK-I24/T/G327N.

[0049] With the recombinant plasmid pET-21b(+)-YojK as a template, whole plasmid PCR was carried out by using primers G327N-F/G327N-R (the primers are shown in Table 1) to obtain a recombinant plasmid pET-21b(+)-YojK-G327N carrying the mutant. The recombinant strain E. coli BL21 (DE3) pET-21b(+)-YojK-G327N was obtained by the same method.

TABLE-US-00001 TABLE 1 Primer names and primer sequences Primer Name Primer Sequence I241T-F TTCAATGGGAACCACTTTTAATAATCAAAAGC I241T-R GCTTTTGATTATTAAAAGTGGTTCCCATTGAA G327N-F CGATGGGAAATGACCAATTTGTTGTCGCAGATCAGG TA G327N-R ATTGGTCATTTCCCATCGGAATGACAACGAGC

Example 2 Induced Expression of Recombinant Strain and Purification of Target Protein

[0050] The recombinant strain E. coli BL21 (DE3) pET-21b(+)-YojK or the mutant E. coli BL21 (DE3) pET-21b(+)-YojK-I241T/G327N constructed in Example 1 was inoculated into 1 L of TB liquid medium (12 g/L of peptone, 24 g/L of tryptone, 5 g/L of glycerol, 2.32 g/L of KH.sub.2PO.sub.4 and 12.53 g/L of K.sub.2HPO.sub.4) containing 100 μg/mL of ampicillin, and cultured under the conditions of 135 rpm and 37° C. until OD.sub.600 was 0.6-0.8. Then the culture temperature was reduced to 18° C., isopropyl-β-thiogalactoside (IPTG) with a final concentration of 0.1 mmol/L was added, and induced culture was carried out for 8 h.

[0051] The bacterial solution obtained by induced expression was centrifuged (7000 rpm, 7 min, 4° C.), the supernatant was discarded, and the cells were collected. The cells were resuspended with a lysis buffer (50 mmol/L of Tris-HCl with pH 8.0, 300 mmol/L of NaCl, 10 mmol/L of imidazole, 10% of glycerol) according to 1 g cells/10 mL lysis buffer. The resulting bacterial solution was crushed with a high-pressure homogenizer. Then the crushed bacterial solution was centrifuged (40000×g, 30 min), and the supernatant was taken to obtain a crude enzyme solution.

[0052] The crude enzyme solution was purified by affinity chromatography with a Ni.sup.+ column. After loading was completed, 10 times of volume of lysis buffer was used for rinsing out miscellaneous proteins, and elution of the target protein was carried out with an elution buffer. The target protein obtained by elution was collected, and desalted through a desalting column (Histrp™ 5 mL Desalting) with a desalting buffer (25 mmol/L of Tris-HCl, 150 mmol/L of NaCl, 10% of glycerol). After the completion of desalting, the solution was concentrated to 10 mg/mL for subsequent reaction. The purified protein was detected by 10% SDS-PAGE gel electrophoresis. The result is shown in FIG. 1A and FIG. 1B. A pure enzyme with clear target band and accurate protein size was successfully obtained.

[0053] The enzyme activity was determined in a 200 μL system. The reaction system contained 4 mM of Reb A, 10 mM of UDPG, 10 mM of MnCl.sub.2, a proper amount of wild type YojK or a mutant thereof and 50 mM of Tris-HCl with pH 8.0. After 20 min of reaction at 35° C., the enzyme was inactivated by heating at 95° C. for 5 min immediately. Next, 5 times of volume of methanol was added for dilution, the solution was centrifuged at 20000×g for 5 min, and the supernatant was filtered through a 0.22 μm organic filter membrane. A resulting sample was analyzed using an ultra-high performance liquid chromatography (UPLC) system. The amount of Reb D produced was calculated according to a Reb D concentration standard curve, and enzyme activities of YojK and the mutants YojK-I241T/G327N and YojK-G327N were calculated. Triplicate experiments were carried out for each reaction. The enzyme activities of the wild type enzyme YojK and the mutant enzymes YojK-1241T/G327N and YojK-G327N were 8 mU/mg, 59.8 mU/mg and 34.4 mU/mg respectively.

Example 3 Synthesis of Reb D by YojK-Catalyzed Glycosylation from Reb A

[0054] The purified wild type enzyme YojK and the mutant YojK-1241T/G327N obtained in Example 2 was subjected to glycosylation.

[0055] Glycosylation was carried out in a 200 μL reaction system. The reaction system included 50 mmol/L of Tris-HCl with pH 8.0, 5 mmol/L of UDPG, 10 mmol/L of MnCl.sub.2 and 1 mmol/L of Reb A. The concentration of the pure enzyme YojK or YojK-1241T/G327 obtained in Example 2 was 10 μM. The reaction was carried out at 35° C. for 20 min. After the completion of the reaction, a resulting solution was diluted with equal volume of methanol, and centrifuged at 20000×g for 5 min. The supernatant was subjected to suction filtration through a 0.22 μM filter membrane, and ultra-high performance liquid chromatography (UPLC) was carried out for detection and analysis. During the UPLC, a BEH C18 1.7 μM reverse phase column from waters Corporation was used, the injection volume was 4 μL, the column temperature was 40° C., the mobile phase was acetonitrile in Tube A and a 1.38 g/L NaH.sub.2PO.sub.4 buffer (pH 2.6) in Tube B, and the flow rate was 0.3 mL/min. The specific process is shown in Table 2:

TABLE-US-00002 TABLE 2 UPLC reaction process Time (min) Acetonitrile 1.38 g/L NaH.sub.2PO.sub.4 (pH 2.6) 0 15% 85% 1 15% 85% 2 27% 73% 6 33% 67%

[0056] Liquid chromatography was performed. The result is shown in FIG. 2. Compared with Reb A and Reb D standards, there was obviously a new product with the same retention time as Reb D produced in the reaction system. In combination with mass spectrometry (MS) (FIG. 3) and nuclear magnetic resonance spectroscopy (NMR) (FIG. 4 and FIG. 5), the results indicated that the glycosyltransferase YojK could catalyze glycosylation of the Reb A substrate by using the UDPG as the glycosyl donor to produce Reb D. This example proved that the glycosyltransferase YojK had the catalytic activity to catalyze production of Reb D from Reb A.

Example 4 Catalytic Synthesis of Reb D from Reb a Using Glycosyltransferase Mutant YojK-I241T/G327N with High Catalytic Activity

[0057] The purified mutant enzyme YojK-I241T/G327N obtained in Example 2 was subjected to glycosylation.

[0058] The glycosylation was carried out in a 200 μL reaction system. The reaction system included 50 mmol/L of Tris-HCl with pH 8.0, 10 mmol/L of UDPG, 10 mmol/L of MnCl.sub.2 and 4 mmol/L of Reb A. The concentration of the pure enzyme YojK or YojK-I241T/G327N obtained in Example 2 was 0.45 mg/mL. The reaction was carried out at 35° C. for 20 min. After the completion of the reaction, 5 times of volume of methanol was added to terminate the reaction, and a resulting solution was centrifuged at 20000×g for 5 min. The supernatant was subjected to suction filtration through a 0.22 μM filter membrane, and ultra-high performance liquid chromatography (UPLC) was carried out for detection and analysis. The liquid chromatography was carried out as described in Example 3. As can be seen from the liquid chromatogram (FIG. 6), compared with the wild type YojK, the catalytic activity of the mutant YojK-G327N was 4.3 times that of the wild type YojK, and the catalytic activity of the mutant YojK-1241T/G327N was greatly improved, which was 7.35 times that of the wild type YojK.

Example 5 Construction of Recombinant Plasmid and Recombinant Strain of Glycosyltransferase and Sucrose Synthase

[0059] An amino acid sequence (accession number: NP_001031915) (SEQ ID NO. 2). and a nucleic acid sequence (accession number: NM_001036838.2) of sucrose synthase AtSuSy derived from Arabidopsis thaliana were downloaded from Genbank, and optimization of the E. coli preferred codon and synthesis of the gene were carried out by Yixin Biotechnology Co., Ltd. The gene YojK-I241T/G327N encoding the glycosyltransferase mutant and the gene AtSuSy encoding the sucrose synthase were linked to a multiple cloning site of pACYCDuet-1 to construct a mutant recombinant plasmid pACYCDuet-1-YojK-I24/T/G327N-AtSuSy. The obtained plasmid was sequenced for identification, and transformed into E. coli BL21(DE3) competent cells. An LB solid plate (10 g/L of peptone, 5 g/L of tryptone, 10 g/L of NaCl and 20 g/L of agar powder) containing 100 μg/mL of chloramphenicol was used for screening to obtain the recombinant strain E. coli BL21(DE3) pACYCDuet-1-YojK-I241T/G327N-AtSuSy.

Example 6 Preparation of Cell Lysis Buffer for Coupling Reaction of Glycosyltransferase Mutant and Sucrose Synthase

[0060] The recombinant strain E. coli BL21 (DE3) pACYCDuet-1-YojK-I24/T/G327N-AtSuSy obtained in Example 5 was used as a fermentation strain, and fermentation was carried out in a 7 L fermentor. A monoclonal antibody was inoculated to 5 mL of LB medium containing 34 μg/mL of chloramphenicol, cultured at 37° C. and 200 rpm overnight, transferred according to a proportion 1:100 to 100 mL of LB medium containing 34 μg/mL of chloramphenicol, and propagated for 12 h. In strict accordance with the aseptic operation regulations, the cells were inoculated according to a proportion 1:50 to 4 L of TB medium containing 34 μg/L of chloramphenicol, and cultured at 37° C. and 400 rpm until OD.sub.600 reached 6-8. 0.2 mmol/L of IPTG was added, and induced culture was carried out at 18° C. for 18 h. In the fermentation process, the pH was controlled to about 6.86 with phosphoric acid and ammonia water, glycerol was fed as a carbon source, and the dissolved oxygen was controlled at about 30%. The bacterial solution obtained by induced expression was centrifuged (7000 rpm, 7 min, 4° C.), the supernatant was discarded, and the cells were collected. The cells were washed with a potassium phosphate buffer (100 mmol/L of K.sub.2HPO.sub.4—KH.sub.2PO.sub.4 (KPi) with pH8.0) twice, and resuspended with a potassium phosphate buffer (100 mmol/L of KPi with pH8.0 and 100 mmol/L of NaCl). The resulting suspension was crushed with a high-pressure homogenizer. Then the crushed bacterial solution was centrifuged at 40000×g for 30 min, the supernatant was taken to obtain a cell lysis buffer for coupling reaction, and albumin glue detection was carried out.

[0061] The result is shown in FIG. 7. The result showed that both the glycosyltransferase and the sucrose synthase could be well expressed. The protein concentration in the cell lysis buffer was determined using a Nano-Drop 2000 UV-Vis spectrophotometer. The prepared cell lysis buffer was packaged and stored at −80° C., or directly used for coupling reaction.

Example 7 Effect of pH on Glycosylation Coupling Reaction of Glycosyltransferase and Sucrose Synthase

[0062] A glycosylation coupling reaction system was reacted in buffers with different pH values, and the effect of pH on glycosylation coupling reaction of glycosyltransferase and sucrose synthase was determined. The buffers selected were 100 mmol/L of Bis-Tris with pH 5.5-6.0 and 100 mmol/L of NaCl; 100 mmol/L of KPi with pH 6.0-8.0 and 100 mmol/L of NaCl; and 100 mmol/L of Tris-HCl with pH 8.0-9.0 and 100 mmol/L of NaCl.

[0063] The cell lysis buffer for coupling reaction was prepared according to Example 6. 1 mL of glycosylation coupling reaction system containing 40 mg/mL of cell lysis buffer, 20 mmol/L of Reb A, 200 mmol/L of sucrose, and 5% of DMSO (v/v) and the buffer was reacted at 35° C. for 6 h. After the completion of the reaction, 5 times of volume of methanol was added to terminate the reaction. Then methanol was added to make a 6-fold dilution. The diluted solution was centrifuged at 20000×g for 5 min, the supernatant was subjected to suction filtration through a 0.22 μM filter membrane, and UPLC was carried out for detection and analysis. The liquid chromatography was carried out according to Example 3, and the yield of Reb D was calculated. The result showed that when the buffer was 100 mmol/L of KPi with pH 8.0 and 100 mmol/L of NaCl, the yield of Reb D could reach 50% or above (FIG. 8).

Example 8 Effect of Temperature on Glycosylation Coupling Reaction of Glycosyltransferase and Sucrose Synthase

[0064] A glycosylation coupling reaction system was reacted at different temperatures (20-45° C.), and the effect of temperature on glycosylation coupling reaction of glycosyltransferase and sucrose synthase was determined.

[0065] The cell lysis buffer for coupling reaction was prepared according to Example 6. 1 mL of coupling reaction system containing 40 mg/mL of cell lysis buffer, 20 mmol/L of Reb A, 200 mmol/L of sucrose, 10% of DMSO (v/v), and a buffer (100 mmol/L of KPi with pH 8.0 and 100 mmol/L of NaCl) was reacted for 6 h. After the completion of the reaction, 5 times of volume of methanol was added to terminate the reaction. Then methanol was added to make a 6-fold dilution. The diluted solution was centrifuged at 20000×g for 5 min, the supernatant was subjected to suction filtration through a 0.22 μM filter membrane, and UPLC was carried out for detection and analysis. The liquid chromatography was carried out according to Example 3, and the yield of Reb D was calculated. The result showed that when the temperature was 30-40° C., the yield of Reb D could reach 50% or above (FIG. 9).

Example 9 Effect of DMSO Concentration on Glycosylation Coupling Reaction of Glycosyltransferase and Sucrose Synthase

[0066] Different concentrations of DMSO (5%-25% (v/v)) were added to a glycosylation coupling reaction system, and the reaction was carried out. The effect of DMSO concentration on glycosylation coupling reaction of glycosyltransferase and sucrose synthase was determined. The cell lysis buffer for coupling reaction was prepared according to Example 6. 1 mL of coupling reaction system containing 40 mg/mL of cell lysis buffer, 20 mmol/L of Reb A, 200 mmol/L of sucrose, DMSO (5%-25% (v/v)) and a buffer (100 mmol/L of KPi with pH 8.0 and 100 mmol/L of NaCl) was reacted at 35° C. for 6 h. After the completion of the reaction, 5 times of volume of methanol was added to terminate the reaction. Then a 6-fold dilution was made. The diluted solution was centrifuged at 20000×g for 5 min, the supernatant was subjected to suction filtration through a 0.22 μM filter membrane, and UPLC was carried out for detection and analysis. The liquid chromatography was carried out according to Example 3, and the yield of Reb D was calculated. The result showed that when the DMSO concentration was 10%-25% (v/v), the yield of Reb D could reach 55% or above (FIG. 10).

Example 10 Effect of Sucrose Concentration on Glycosylation Coupling Reaction of Glycosyltransferase and Sucrose Synthase

[0067] Different concentrations of sucrose (0-800 mmol/L) were added to a glycosylation coupling reaction system, and the reaction was carried out. The effect of sucrose concentration on glycosylation coupling reaction of glycosyltransferase and sucrose synthase was determined. The cell lysis buffer for coupling reaction was prepared according to Example 6. 1 mL of coupling reaction system containing 40 mg/mL of cell lysis buffer, 20 mmol/L of Reb A, sucrose (0-800 mmol/L), 10% (v/v) of DMSO and a buffer (100 mmol/L of KPi with pH 8.0 and 100 mmol/L of NaCl) was reacted at 35° C. for 6 h. After the completion of the reaction, 5 times of volume of methanol was added to terminate the reaction. Then a 5-fold dilution was made. The diluted solution was centrifuged at 20000×g for 5 min, the supernatant was subjected to suction filtration through a 0.22 μM filter membrane, and UPLC was carried out for detection and analysis. The liquid chromatography was carried out according to Example 3, and the yield of Reb D was calculated. The result showed that when the sucrose concentration was 200-500 mmol/L, the yield of Reb D could reach 60% or above (FIG. 11).

Example 11 Effect of the Substrate Reb a Concentration on Coupling Reaction of Glycosyltransferase and Sucrose Synthase

[0068] Different concentrations of Reb A (1-50 mmol/L) were added to a glycosylation coupling reaction system, and the reaction was carried out. The effect of Reb A concentration on glycosylation coupling reaction of glycosyltransferase and sucrose synthase was determined.

[0069] The cell lysis buffer for coupling reaction was prepared according to Example 6. 1 mL of coupling reaction system containing 40 mg/mL of cell lysis buffer, Reb A (1-50 mmol/L), 400 mmol/L of sucrose, 10% (v/v) of DMSO and a buffer (100 mmol/L of KPi with pH 8.0 and 100 mmol/L of NaCl) was reacted at 35° C. for 6 h. After the completion of the reaction, 5 times of volume of methanol was added to terminate the reaction. Then a 5-fold dilution was made. The diluted solution was centrifuged at 20000×g for 5 min, the supernatant was subjected to suction filtration through a 0.22 μM filter membrane, and ultra-high performance liquid chromatography (UPLC) was carried out for detection and analysis. The liquid chromatography was carried out according to Example 3, and the yield of Reb D was calculated. The result showed that when the substrate concentration was 20 mmol/L, 17.88 g/L of Reb D could be obtained with a yield of 79.27% (FIG. 12).

Example 12 Effect of Reaction Time on Coupling Reaction of Glycosyltransferase and Sucrose Synthase

[0070] A glycosylation coupling reaction system was reacted for different times (0-24 h), and the effect of reaction time on glycosylation coupling reaction of glycosyltransferase and sucrose synthase was determined.

[0071] The cell lysis buffer for coupling reaction was prepared according to Example 6. 20 mL of coupling reaction system containing 40 mg/mL of cell lysis buffer, 20 mmol/L of Reb A, 400 mmol/L of sucrose, 10% (v/v) of DMSO, a buffer (100 mmol/L of KPi with pH 8.0 and 100 mmol/L of NaCl) and 1 mmol/L of UDPG was reacted at 35° C. The reaction time was optimized in a range of 0-24 h. After the completion of the reaction, 5 times of volume of methanol was added to terminate the reaction. Then a 5-fold dilution was made. The diluted solution was centrifuged at 20000×g for 5 min, the supernatant was subjected to suction filtration through a 0.22 μM filter membrane, and ultra-high performance liquid chromatography (UPLC) was carried out for detection and analysis. The liquid chromatography was carried out according to Example 3, and the yield of Reb D was calculated. The result showed that when the final reaction time was 15 h, 20.59 g/L of Reb D could be obtained from Reb A at the concentration of 20 mmol/L with a yield of 91.29% (FIG. 13), which was higher than the yield of 74.6% in the production of 17.4 g/L Reb D from 20 g/L Reb A reported in documents.

[0072] Although the disclosure has been disclosed as above by way of the preferred examples, they are not intended to limit the disclosure. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the disclosure. Therefore, the protection scope of the disclosure should be as defined in the claims.