CN115433702A

CN115433702A - Method for efficiently biosynthesizing rebaudioside D2 by utilizing glycosyltransferase

Info

Publication number: CN115433702A
Application number: CN202211014088.3A
Authority: CN
Inventors: 饶义剑; 平千; 杨丽烽; 张艳; 袁振波; 蒋杰娟
Original assignee: Jiangnan University
Current assignee: Guilin Layn Natural Ingredients Corp
Priority date: 2022-08-23
Filing date: 2022-08-23
Publication date: 2022-12-06

Abstract

The invention discloses a method for efficiently biosynthesizing rebaudioside D2 by utilizing glycosyltransferase, belonging to the technical field of biocatalytic synthesis. According to the invention, the glycosyltransferase UGT94D1 capable of catalyzing rebaudioside A to synthesize rebaudioside D2 is obtained by mining, and the glycosyltransferase UGT94D1 and a sucrose synthase AtSuSy from Arabidopsis thaliana are constructed to perform coupling reaction, so that the rebaudioside D2 is efficiently catalytically synthesized by taking rebaudioside A as a substrate, the rebaudioside D2 is efficiently synthesized by taking 9.67g/L (10 mmol/L) of rebaudioside A as a substrate for 24h, the rebaudioside D2 is efficiently synthesized by 10.69g/L, the yield of the rebaudioside D2 reaches 94.66%, and a new efficient and green way is provided for the production of the rebaudioside D2.

Description

Method for efficiently biosynthesizing rebaudioside D2 by utilizing glycosyltransferase

Technical Field

The invention relates to a method for efficiently biosynthesizing rebaudioside D2 by utilizing glycosyltransferase, belonging to the technical field of biocatalytic synthesis.

Background

Stevioside is diterpene glycoside extracted and purified from stevia rebaudiana leaves, and has the advantages of high sweetness, low calorie, no side effect on human body and the like. Steviol glycosides have been approved by the food safety authorities in the united states, brazil, korea, japan and europe as safe food additives. To date, 64 steviol glycosides have been identified from stevia rebaudiana leaves, with stevioside content being the highest, 5-10% of the dry weight, followed by rebaudioside a, 2-4% of the dry weight. They exhibit a sweetness that is 250-300 times higher than sucrose, but even the high purity steviol glycosides retain the bitter taste attribute, which largely limits their use in the natural food additive market.

In recent years, researches show that the number and the types of glycosyl units connected with C-13 and C-19 have obvious influence on the properties of stevioside. For example, rebaudioside A has an additional glucose at the C-13 position, making it sweeter and more palatable than stevioside. Rebaudioside D and rebaudioside M, which are further separated from stevia, have one or two additional glucose linked at the C-19 position, making them exhibit better sweetness quality than rebaudioside a. Therefore, there is a great interest in new steviol glycosides with different glycosyl units at these positions to explore sweeteners with better quality.

UDP-glucosyltransferases (UGTs) are naturally evolved glycosylation enzymes that transfer sugar moieties from an activated sugar donor to an acceptor molecule. UGTs have found many applications in the synthesis of steviol glycosides. In 2014, prakash et al reported for the first time a novel steviol glycoside derivative, rebaudioside D2, which is a secondary product of the glycosyltransferase UGTSL2 catalyzing the conversion of rebaudioside a to rebaudioside D, and more recently, guoHong Mao reported that the glycosyltransferase EUGT11 catalyzes the conversion of rebaudioside E to rebaudioside D2. But because the yield is low, the product is mixed, the separation and purification cost is high or the substrate rebaudioside E is expensive, and further research on rebaudioside D2 is limited. Therefore, it is highly desirable to identify a novel glycosyltransferase with high catalytic activity and regioselectivity that utilizes a relatively common starting substrate (e.g., rebaudioside a) for the synthesis of rebaudioside D2.

Disclosure of Invention

In order to solve the above problems, the present invention excavates that glycoside transferase UGT94D1 from sesame catalyzes rebaudioside a to synthesize rebaudioside D2, and has an activity of catalyzing rebaudioside a to synthesize rebaudioside D2 in the presence of uridine diphosphate glucose (UDPG). And through constructing a uridine diphosphate glucose UDPG circulating regeneration system, the efficient biosynthesis of rebaudioside D2 is realized by utilizing an escherichia coli lysate, and the possibility is provided for the further research of rebaudioside D2.

In order to solve the technical problems, the technical scheme of the invention is as follows:

the first purpose of the invention is to provide a recombinant bacterium for expressing glycosyl transferase derived from sesame.

In one embodiment, the glycosyltransferase has an amino acid sequence having an accession number XP _011076907.1 and a nucleotide sequence having an accession number XM _011078605.1.

In one embodiment, the recombinant bacterium further expresses a sucrose synthase.

In one embodiment, the amino acid sequence of the sucrose synthase may be an amino acid sequence having sucrose synthase activity from any source.

In one embodiment, the NCBI accession number of the amino acid sequence of the sucrose synthase is NP _001031915.

In one embodiment, the recombinant bacterium uses escherichia coli as a host cell.

In one embodiment, the recombinant bacteria are constructed with glycosyltransferase into pET-21b (+) vector and sucrose synthase into pACYCDuet-1 vector.

A second object of the present invention is to provide a composition, which is one or more of a glycosyltransferase, the recombinant bacterium, or a cell lysate of the recombinant bacterium; the amino acid sequence of the glycosyltransferase has an accession number XP _011076907.1.

In one embodiment, the cell lysate is a supernatant obtained by cell lysis after the induction expression of the recombinant bacteria.

The third purpose of the invention is to provide a method for catalytically synthesizing rebaudioside D2, wherein rebaudioside A is used as a substrate, and the recombinant bacterium and/or the composition are/is used for carrying out catalytic reaction.

In one embodiment, the conditions of the catalytic reaction are: 1-50mmol/L rebaudioside A, 50-800mmol/L sucrose, 5-25% (v/v) DMSO, 100mmol/L K ₂ HPO ₄ -KH ₂ PO ₄ Buffer solution and 100mmol/LNaCl as a reaction system, and reacting for 0-48h at 20-45 ℃.

In one embodiment, the composition is added in an amount of at least 40mg/mL.

In one embodiment, the buffer has a pH of 5.5 to 11.0.

The invention also protects the application of the recombinant bacteria or the composition or the method in preparing a rebaudioside D2-containing product.

Has the beneficial effects that:

(1) The nucleotide sequence coding the glycosyltransferase UGT94D1 is used for preparing the recombinant protein capable of catalyzing rebaudioside A to generate rebaudioside D2, and the prepared recombinant protein can take UDPG as a glycosyl donor and glycosylate rebaudioside A as a substrate to synthesize rebaudioside D2.

(3) According to the invention, glycosyltransferase UGT94D1 and sucrose synthase AtSuSy are combined to construct a UDPG cyclic regeneration system. Through the optimization of the coupling reaction system conditions, the synthesis of 10.69g/L of rebaudioside D2 by utilizing 9.67g/L (10 mmol/L) rebaudioside A with the yield of 94.66 percent is realized.

(4) The constructed recombinant strain co-expresses glycosyltransferase derived from sesame and sucrose synthase derived from arabidopsis thaliana, and the rebaudioside D2 is synthesized by catalyzing rebaudioside A with cell lysate prepared after the recombinant strain is induced and expressed, so that a glycosyl donor is not required to be added, a cell penetrating agent is not required to be additionally used, the cost is remarkably reduced, and the method is green and environment-friendly.

Drawings

FIG. 1 shows the biosynthetic pathway for the catalysis of rebaudioside A by the glycosyltransferase UGT94D1 to produce rebaudioside D2.

FIG. 2 shows the expression and purification analysis of glycosyltransferase UGT94D1 protein in example 2.

FIG. 3 is a UPLC analysis chart of the catalytic synthesis of rebaudioside D2 from rebaudioside A by glycosyltransferase UGT94D1 in example 3. Lane 1: marker; lane 2: no IPTG induced expression samples; lane 3: crude enzyme solution; lane 4: supernatant of the crude enzyme solution; lane 5: precipitating the crude enzyme solution; lane 6: purifying the transudate liquid; lane 7: washing the heteroprotein sample; lane 8: the protein of interest elutes the sample.

FIG. 4 is a rebaudioside D2 mass spectrometry analysis of the rebaudioside A glycosylation reaction product of example 3.

FIG. 5 is a NMR spectroscopic hydrogen spectrum of rebaudioside D2, a product of example 4.

FIG. 6 is a carbon spectrum of NMR spectroscopy analysis of rebaudioside D2, a product of example 4.

FIG. 7 shows the analysis of the protein expression of the lysate of the UGT94D1-AtSuSy glycosylation coupling reaction in example 6. Lane 1: marker; lane 2: no IPTG induced expression samples; lane 3: crude enzyme solution; lane 4: supernatant of the crude enzyme solution; lane 5: precipitating the crude enzyme solution.

FIG. 8 is a graph showing the effect of pH on the buffer solution of the glycosylation coupling reaction in example 7.

FIG. 9 is a graph showing the effect of temperature on the glycosylation coupling reaction in example 8.

FIG. 10 is a graph showing the effect of DMSO concentration on the glycosylation coupling reaction in example 9.

FIG. 11 is a graph showing the effect of sucrose concentration on glycosylation coupling reaction in example 10.

FIG. 12 is a graph showing the effect of substrate concentration on glycosylation coupling reactions in example 11.

FIG. 13 is a graph showing the effect of reaction time on glycosylation coupling reaction in example 12.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, the reagents and materials used in the following examples are all commercially available or may be prepared by known methods.

The methods referred to in the following examples:

determination of the enzymatic Properties of glycosyltransferases: kinetic analysis of rebaudioside A by glycosyltransferase UGT94D1 was performed in a 200 μ L reaction system containing 5mM UDPG, 10mM MnCl ₂ 50mM Tris-HCl pH8.0 and 5 μ g of purified protein sample, with rebaudioside A concentrations ranging from 0-5mM. The reaction temperature is 35 ℃, the reaction time is 20min, and the reaction is quenched by heating at 95 ℃ for 10min immediately after the reaction is finished. Diluting with 5 times volume of methanol, centrifuging at 20000 × g for 5min, and removing protein precipitate. The supernatant liquid was filtered through a 0.22 μm filter and the sample was analyzed by UPLC.

Determination of rebaudioside D2 yield: preparing rebaudioside D2 standard solutions with different concentrations (0.4 mM, 0.8mM, 1.2mM, 1.6mM and 2.0 mM), analyzing the standard solutions by using UPLC to obtain a rebaudioside D2 concentration standard curve equation of y =1140530.429x +13834.2381 ² =0.99808. And (4) converting the yield of the rebaudioside D2 according to a standard curve. Yield = rebaudioside D2 actual yield/rebaudioside D2 theoretical yield.

Example 1 acquisition of glycosyltransferase UGT94D1 Gene and construction of recombinant Strain

The amino acid sequence (accession number: XP _ 011076907.1) and the nucleotide sequence (accession number: XM _ 011078605.1) of Bacillus glycosyltransferase were downloaded from Genbank, and were genetically synthesized by Hakken biosciences, inc. and ligated to the polyclonal enzyme cleavage site of the vector pET-21b (+), to obtain a recombinant plasmid pET-21b (+) -UGT94D1.

The obtained plasmid pET-21b (+) -UGT94D1 is subjected to sequencing identification and transformed into an Escherichia coli E.coli BL21 (DE 3) competent cell, and an LB solid plate (10 g/L peptone, 5g/L yeast powder, 10g/L NaCl,20g/L agar powder) containing 100. Mu.g/mL ampicillin is adopted for screening, so that a recombinant strain E.coli BL2L (DE 3) pET-21b (+) -UGT94D1 is obtained.

Example 2 inducible expression of recombinant strains and purification of proteins of interest

The recombinant strain E.coli BL2L (DE 3) pET-21b (+) -UGT94D1 constructed in example 1 was inoculated into 1L 2 XYT broth (16 g/L peptone, 10g/L yeast powder, 5 g/LNaCl) containing 100. Mu.g/mL ampicillin and cultured at 135rpm at 37 ℃ to OD ₆₀₀ After the concentration is 0.6-0.8 ℃, the culture temperature is reduced to 18 ℃, isopropyl-beta-thiogalactoside (IPTG) with the final concentration of 0.1mmol/L is added, and the induction culture is carried out for 8 hours.

The induced expression was centrifuged (7000rpm, 7min,4 ℃ C.), the supernatant was discarded, and the cells were collected. The cells were resuspended in lysis buffer (50 mmol/L Tris-HCl pH8.0, 300mmol/L NaCl,10mmol/L imidazole, 10% glycerol) per 10mL of lysis buffer per 1g of cells. Crushing by a high-pressure homogenizer, centrifuging the crushed bacterial liquid (40000 Xg, 30 min), and taking the supernatant to obtain a crude enzyme liquid.

Using the crude enzyme solution with Ni ⁺ The column was purified by affinity chromatography, after loading, the column was washed with 10 volumes of lysis buffer to elute the desired protein, using elution buffer (50 mmol/L Tris-HCl pH8.0, 300mmol/L NaCl,250mmol/L imidazole, 10% glycerol). The eluted target protein was collected and desalted by desalting column (HistrpTM 5 mLDesaltning) with desalting buffer (25 mmol/L Tris-HCl,150mmol/L NaCl,10% glycerol). After desalting, the mixture was concentrated to 10mg/mL and subjected to the subsequent reaction. The purified protein was detected by 10% SDS-PAGE gel electrophoresis, and the results are shown in FIG. 2, and the pure enzyme with the accurate size of the target distinct band protein was successfully obtained, and the K of UGT94D1 to rebaudioside A was determined _m The value was 0.76. + -. 0.06mM _cat The value was 15.01. + -. 0.31min ^-1 。

Example 3UGT94D1 glycosylation reaction for synthesizing rebaudioside D2 by catalyzing rebaudioside A reaction

The purified glycosyltransferase UGT94D1 obtained in example 2 was used in the glycosylation reaction (FIG. 1).

The glycosylation reaction was carried out in a 200. Mu.L reaction system as follows: 50mmol/L Tris-HCl pH 8.0，5mmol/LUDPG，10mmol/LMnCl ₂ 1mmol/L rebaudioside A, the concentration of the pure enzyme UGT94D1 obtained in example 2 was 10. Mu.M. The reaction was carried out at 35 ℃ for 20min. After the reaction is finished, the reaction solution is diluted by 5 times of volume of methanol, is centrifuged for 5min at 20000 Xg, is filtered by a 0.22 mu M filter membrane, and then is subjected to detection and analysis of Ultra Performance Liquid Chromatography (UPLC). UPLC uses waters BEH C181.7 μ M reverse column, sample size 2 μ L, column temperature 40 ℃, mobile phase using A pipeline: acetonitrile, line B: 1.38g/LNaH ₂ PO ₄ Buffer (pH 2.6), flow rate 0.3mL/min, specific procedure as shown in Table 1:

TABLE 1UPLC reaction procedure

As can be seen from the results of liquid phase analysis, as shown in FIG. 3, when compared with the rebaudioside A standard, a significant new product was produced in the reaction system, and Mass Spectrometry (MS) analysis was performed on the reaction mixture (FIG. 4), and the negative ion mode results of LC-MS showed that there was one [ M-H ] in M/z 1127.4790] ^- Peak of ion, formula C ₅₀ H ₈₀ O ₂₈ It shows that the product is a monosaccharide derivative of rebaudioside A, and has one [ M-Glc-H ] at M/z 803.3811] ^- Peak of ion. Since the ester bond at the C-19 position of the steviol glycoside is more easily cleaved in ESI-MS to generate fragment ions, it is speculated that the new glucosyl unit is augmented at the C-19-sugar portion of rebaudioside A.

Example 4 structural identification of novel rebaudioside a monoglycosylated derivatives

The novel derivatives were prepared by large scale (200 mL) glycosylation using glycosyltransferase UGT94D1, under conditions consistent with those in example 3. Purification was performed using a semi-preparative high performance liquid chromatography system using a Shim-pack GIST C18 column (10X 250mm,5 μm, SHIMADZU, japan) with a detection wavelength of 210nm, a mobile phase of a mixture of acetonitrile and water, a solvent flow rate of 5mL/min, a column temperature of 40 ℃ and an elution program of 25% acetonitrile isocratic. From the reaction mixture, 50mg of the product was obtained in high purity. Next, passing through the nucleusMagnetic resonance spectroscopy, data collected with a Bruker Avance III 400MHz spectrometer (Bruker BioSpin, karlsruhe, germany), ¹ the detection frequency of the H spectrum is 400MHz, ¹³ the C spectrum is 101MHz. The resulting product was consistent with rebaudioside D2 reported in the literature (fig. 5, fig. 6).

Example 5 construction of glycosyltransferase and sucrose synthase recombinant plasmids and recombinant strains

An amino acid sequence of a sucrose synthase AtSuSy derived from Arabidopsis thaliana (accession No: NP-001031915) and a nucleic acid sequence thereof (accession No: NM-001036838.2) were downloaded from Genbank, and codon optimization and gene synthesis preferred by Escherichia coli were carried out by also Xin Biotech Ltd. The gene AtSuSy for coding sucrose synthase is connected to a polyclonal enzyme cutting site of pACYCDuet-1 to construct a recombinant plasmid pACYCDuet-1-AtSuSy, the obtained plasmid is sequenced and identified, and is co-transformed with pET-21b (+) -UGT94D1 into a competent cell of Escherichia coli E.coli BL21 (DE 3), and an LB solid plate (10 g/L peptone, 5g/L yeast powder, 10g/LNaCl and 20g/L agar powder) containing 100 mu g/mL ampicillin and 34 mu g/mL chloramphenicol is adopted for screening to obtain a recombinant strain E.coli BL2L (DE 3) UGT94D1-AtSuSy.

EXAMPLE 6 preparation of cell lysate from glycosyltransferase and sucrose synthase coupling reaction

The recombinant strain E.coli BL2l (DE 3) UGT94D1-AtSuSy constructed in example 5 was inoculated in 5L 2 XYT medium supplemented with 100. Mu.g/mL ampicillin and 34. Mu.g/mL chloramphenicol, and cultured with shaking at 37 ℃ and 135 rpm. After the thallus grows to OD ₆₀₀ Cooling to 18 deg.C when the value is 0.6-0.8, adding isopropyl-beta-D-thiogalactoside (IPTG) with final concentration of 0.2mM, inducing protein expression at 18 deg.C for 8 hr, centrifuging at 7000 Xg for 7min, collecting thallus, and treating with lysis buffer (100 mM K) ₂ HPO ₄ -KH ₂ PO ₄ (KPi) pH8.0, 100mM NaCl) three times and then resuspended. Cells were disrupted with a high pressure homogenizer. Immediately, the cells were centrifuged at 40000 Xg for 30min to remove cell debris. And taking the supernatant to obtain the coupling reaction cell lysate. And performing protein gel detection.

The results are shown in FIG. 7, which shows that both glycosyltransferase and sucrose synthase are well expressed. The protein concentration in the cell lysate was determined using a Nano-Drop 2000UV-Vis spectrophotometer. The prepared cell lysate is subpackaged and stored at-80 ℃, or is directly used for coupling reaction.

Example 7 Effect of pH on glycosyltransferase and sucrose synthase glycosylation coupled reactions

Placing the glycosylation coupling reaction system in buffer solutions with different pH values for reaction, and determining the influence of the pH on the glycosylation coupling reaction of glycosyltransferase and sucrose synthase, wherein the selected buffer solution is 100mmol/L KPi, pH 5.5-8.0, and 100mmol/L NaCl;100mmol/L Tris-HCl pH 8.0-9.0, 100mmol/L NaCl and 100mmol/L Glycine-NaOH pH9.0-11.0, 100mmol/L NaCl.

Preparing a conjugation reaction cell lysate as described in example 6, with a glycosylation conjugation reaction system of 1mL containing 40mg/mL of conjugation reaction cell lysate, 10mmol/L of rebaudioside A, 200mmol/L of sucrose, and 5% DMSO (v/v), buffer; reacting for 6 hours at 35 ℃. After the reaction is finished, 5 times of methanol is added to stop the reaction, then methanol is added to dilute the reaction by 6 times, 20000 Xg is centrifuged for 5min, a 0.22 mu M filter membrane is filtered, and then UPLC is used for detection and analysis. The liquid phase assay was performed as described in example 3 and the rebaudioside D2 yield was calculated, showing that the rebaudioside D2 yield can reach more than 50% when the buffer was 100mmol/LKPi pH8.0, 100mmol/LNaCl (fig. 8).

Example 8 Effect of temperature on glycosyltransferase and sucrose synthase glycosylation coupling reactions

The glycosylation coupling reaction system is placed in different temperatures (20-45 ℃) for reaction, and the influence of the temperature on the glycosylation coupling reaction of the glycosyl transferase and the sucrose synthase is measured.

A coupled reaction cell lysate was prepared as described in example 6, with a coupling reaction system of 1mL containing 40mg/mL protein lysate, 10mmol/L rebaudioside A, 200mmol/L sucrose, 10% DMSO (v/v), and 100mmol/LKPI pH8.0, 100mmol/LNaCl, and reacted for 6h. After the reaction is finished, 5 times of methanol is added to stop the reaction, then methanol is added to dilute the reaction by 6 times, 20000 Xg is centrifuged for 5min, a 0.22 mu M filter membrane is used for suction filtration, and UPLC is used for detection and analysis. The liquid phase assay was performed as described in example 3 and the rebaudioside D2 yield was calculated, showing that the rebaudioside D2 yield can reach 50% or more when the temperature was 35 ℃ (fig. 9).

Example 9 Effect of DMSO concentration on glycosyltransferase and sucrose synthase glycosylation coupling reactions

Adding DMSO (5-25% (v/v)) with different concentrations into a glycosylation coupling reaction system for reaction, and determining the influence of the DMSO concentration on glycosylation coupling reaction of glycosyltransferase and sucrose synthase.

A coupled reaction cell lysate was prepared as described in example 6, with a coupling reaction system of 1mL containing 40mg/mL protein lysate, 10mmol/L rebaudioside A, 200mmol/L sucrose, DMSO (5% -25% (v/v)), and 100mmol/L KPi pH8.0, 100mmol/L NaCl, and reacted at 35 ℃ for 6h. After the reaction is finished, 5 times of methanol is added to stop the reaction, then the reaction is diluted by 6 times, 20000 Xg is centrifuged for 5min, a filter membrane with the thickness of 0.22 mu M is filtered, and detection and analysis are carried out by UPLC. The liquid phase assay was performed as described in example 3, and the rebaudioside D2 yield was calculated, and the results showed that the rebaudioside D2 yield could reach more than 50% when the DMSO concentration was 10% (v/v) (fig. 10).

Example 10 Effect of sucrose concentration on the coupling reaction of glycosyltransferase and sucrose synthase

Adding sucrose (50-800 mmol/L) with different concentrations into a glycosylation coupling reaction system for reaction, and determining the influence of the sucrose concentration on glycosylation coupling reaction of glycosyl transferase and sucrose synthase.

A coupled reaction cell lysate was prepared as described in example 6, with a coupling reaction system of 1mL containing 40mg/mL protein lysate, 10mmol/L rebaudioside A, sucrose (50-800 mmol/L), 10% (v/v) DMSO, and 100mmol/L KPi pH8.0, 100mmol/L NaCl, and reacted at 35 ℃ for 6h. After the reaction is finished, 5 times of methanol is added to stop the reaction, then the reaction is diluted by 5 times, 20000 Xg is centrifuged for 5min, a 0.22 mu M filter membrane is used for suction filtration, and detection and analysis are carried out by UPLC. The liquid phase assay was performed as described in example 3, and the rebaudioside D2 yield was calculated, and the results showed that the rebaudioside D2 yield could reach 50% or more when the sucrose concentration was 200mmol/L (fig. 11).

Example 11 Effect of rebaudioside A concentration on glycosyltransferase and sucrose synthase coupling reaction substrates

Adding rebaudioside-A (1-50 mmol/L) with different concentrations into a glycosylation coupling reaction system for reaction, and determining the influence of sucrose concentration on glycosylation coupling reaction of glycosyltransferase and sucrose synthase.

A coupled reaction cell lysate was prepared as described in example 6, with a coupling reaction system of 1mL containing 40mg/mL protein lysate, rebaudioside A (1-50 mmol/L), 200mmol/L sucrose, 10% (v/v) DMSO, and 100mmol/L KPi pH8.0, 100mmol/L NaCl, and reacted at 35 ℃ for 6h. After the reaction is finished, 5 times of methanol is added to stop the reaction, then the reaction is diluted by 5 times, 20000 Xg is centrifuged for 5min, and the filtration is carried out by using a 0.22 mu M filter membrane, and the detection and analysis are carried out by UPLC. The liquid phase detection method was performed as described in example 3, and the yield of rebaudioside D2 was calculated, and the results showed that 5.67g/L of rebaudioside D2 was obtained at a yield of 50.66% at a substrate concentration of 10mmol/L (fig. 12).

EXAMPLE 12 Effect of reaction time on glycosyltransferase and sucrose synthase coupling reactions

The glycosylation coupling reaction system is reacted for different time (0-24 h), and the influence of the sucrose concentration on the glycosylation coupling reaction of the glycosyl transferase and the sucrose synthase is measured.

The coupled reaction cell lysate is prepared as described in example 6, the coupled reaction system is 20mL, and the coupled reaction system contains 40mg/mL protein lysate, 10mmol/L rebaudioside A, 400mmol/L sucrose, 10% (v/v) DMSO, 100mmol/LKPI pH8.0, 100mmol/LNaCl, and the reaction is carried out at 35 ℃ for an optimal range of 0-24h. After the reaction is finished, 5 times of methanol is added to stop the reaction, then the reaction is diluted by 5 times, 20000 Xg is centrifuged for 5min, and the filtration membrane with the volume of 0.22 mu M is filtered and then the detection and analysis are carried out by Ultra Performance Liquid Chromatography (UPLC). The liquid phase assay method was performed as described in example 3 and the rebaudioside D2 yield was calculated and the results showed that finally 10.69g/L rebaudioside D2 was obtained at a yield of 94.66% at a rebaudioside a concentration of 10mmol/L at 24h of the reaction (fig. 13).

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A recombinant bacterium which expresses a glycosyltransferase derived from sesame, wherein the glycosyltransferase has an amino acid sequence having an NCBI accession number XP _011076907.1.

2. The recombinant bacterium of claim 1, wherein the recombinant bacterium further expresses a sucrose synthase.

3. The recombinant bacterium according to claim 2, wherein the amino acid sequence of sucrose synthase is an amino acid sequence having sucrose synthase activity from any source.

4. The recombinant bacterium according to claim 2 or 3, wherein the NCBI accession number of the amino acid sequence of the sucrose synthase is NP 001031915.

5. The recombinant bacterium according to any one of claims 1 to 4, wherein the recombinant bacterium uses Escherichia coli as a host cell.

6. The recombinant bacterium according to any one of claims 1 to 4, wherein the recombinant bacterium is a plasmid that constructs glycosyltransferase into pET-21b (+) vector and sucrose synthase into pACYCDuet-1 vector.

7. A composition comprising one or more of a glycosyltransferase, a recombinant bacterium according to any one of claims 1 to 6, or a cell lysate of a recombinant bacterium according to any one of claims 1 to 6; the amino acid sequence of the glycosyltransferase has an accession number XP _011076907.1.

8. The composition of claim 7, wherein the cell lysate is a supernatant obtained by cell lysis after the induction expression of the recombinant bacterium of any one of claims 1 to 6.

9. A method for catalytically synthesizing rebaudioside D2, wherein rebaudioside A is used as a substrate, and the recombinant bacterium of any one of claims 1 to 6 and/or the composition of claim 7 or 8 are used for catalytic reaction.

10. Use of the recombinant bacterium of any one of claims 1-6, or the composition of claim 7 or 8, or the method of claim 9, for preparing a rebaudioside D2-containing product.