Efficient biosynthesis of rebaudioside M2 ​​using glycosyltransferases

Abstract

The present invention provides an efficient method for biosynthesis of rebaudioside M2 ​​using glycosyltransferase, which belongs to the field of biocatalytic synthesis. By obtaining glycosyltransferase UGT94D1, which catalyzes the synthesis of rebaudioside M2 ​​from rebaudioside D, a new, highly efficient and environmentally friendly route for the production of rebaudioside M2 ​​is provided. No by-products are produced throughout the catalytic reaction, which is advantageous for downstream purification processes and further reduces production costs.

Description

[Technical Field]

[0001] The present invention relates to a method for efficiently biosynthesizing rebaudioside M2 ​​using glycosyltransferase, which belongs to the field of biocatalytic synthesis technology. [Background technology]

[0002] Steviol glycosides are diterpene glycosides extracted and purified from stevia leaves. They offer advantages such as high sweetness, low calories, and no side effects to the human body. Their safety as food additives has already been confirmed by the United States, Brazil, South Korea, Japan, and the European Food Safety Authority. To date, 64 steviol glycosides have been identified in stevia leaves. Of these, stevia sugar has the highest content, accounting for 5-10% of the dry weight, followed by rebaudioside A, accounting for 2-4% of the dry weight. These substances are 250-300 times sweeter than sucrose, and even highly purified steviol glycosides retain a bitter taste, significantly limiting their application in the natural food additive market.

[0003] Recent studies have shown that the number and type of glycosyl units linked at C-13 and C-19 significantly affect the properties of steviol glycosides. For example, rebaudioside A contains an additional glucose at the C-13 position, which is sweeter and more umami-tasting than stevia sugar. Rebaudioside D and rebaudioside M, obtained by further isolating stevia, contain one and two additional glucose units at the C-19 position, respectively, which significantly enhance their sweetness compared to rebaudioside A. Therefore, new steviol glycosides with different glycosyl units at these positions are attracting considerable attention in order to develop higher-quality sweeteners.

[0004] UDP-glycosyltransferases (UGTs) are enzymes that catalyze the transfer of sugar moieties from activated sugar donors to acceptor molecules, a naturally evolved glycosylation reaction. UGTs have been widely applied in the synthesis of steviol glycosides. In 2014, Prakash et al. first reported a novel steviol glycoside derivative, rebaudioside M2, as a by-product of the glycosyltransferase UGSL2 catalyzing the conversion of rebaudioside A to rebaudioside D. However, the low yield and product contamination led to high isolation and purification costs, limiting further research into rebaudioside M2. Therefore, there is a great need to identify novel glycosyltransferases with high catalytic activity and regioselectivity for the catalytic synthesis of rebaudioside M2. Summary of the Invention [Problem to be solved by the invention]

[0005] To solve the above problems, the present invention aims to discover glycoside transferase UGT94D1 derived from sesame and to confer the activity of catalyzing the synthesis of rebaudioside M2 ​​from rebaudioside D in the presence of uridine diphosphate glucose (UGPG), thereby enabling further research into rebaudioside M2. [Means for solving the problem]

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A first object of the present invention is to provide a recombinant bacterium, which expresses glycosyltransferase UGT94D1 derived from sesame.

[0008] In one embodiment, the glycosyltransferase amino acid sequence has the accession number XP_011076907.1 and the nucleotide sequence has the accession number XM_011078605.1.

[0009] In one embodiment, the recombinant bacteria are host cells that are prokaryotic or eukaryotic.

[0010] In one embodiment, the prokaryotic host cell can be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Pacific Bacillus, Staphylococcus, Streptococcus, and Streptococcus. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Clostridium, Helicobacter, Iriobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma. The eukaryotic cell is a fungal cell.

[0011] In one embodiment, the recombinant bacteria is host cell E. coli.

[0012] In one embodiment, the recombinant bacteria use a pET series expression vector.

[0013] In one embodiment, the recombinant bacterium uses pET-21b(+) as an expression vector.

[0014] A second object of the present invention is to provide a composition comprising one or more of glycosyltransferase UGT94D1, the recombinant bacterium, or a cell lysate of the recombinant bacterium, wherein the registration number of the amino acid sequence of the glycosyltransferase is XP_011076907.1.

[0015] In one embodiment, the cell lysate is a supernatant obtained by lysing cells after induction and expression of the recombinant bacterium.

[0016] A third object of the present invention is to provide a method for catalytically synthesizing rebaudioside M2, which uses rebaudioside D as a substrate and performs a catalytic reaction using glycosyltransferase UGT94D1, the recombinant bacterium, or the composition.

[0017] In one embodiment, the method uses UDP-glucose as the glycosyl donor.

[0018] In one embodiment, the catalytic system is 0.1-3.0 mM RebD, 1-10 μM glycosyltransferase UGT94D1, 1-10 mM UDPG, 5-20 mM MnCl2, 20-80 mM Tris.

[0019] In one embodiment, the catalytic reaction conditions are a pH of 5.5 to 9.0, a temperature of 30 to 50° C., and a time of 2 to 10 hours.

[0020] The present invention further provides use of glycosyltransferase UGT94D1, the recombinant bacterium, the composition, or the method in producing rebaudioside M2 ​​or a product containing rebaudioside M2.

[0021] In one embodiment, the accession number for the amino acid sequence of the glycosyltransferase UGT94D1 is XP_011076907.1 and the accession number for the nucleotide sequence is XM_011078605.1. [Effects of the Invention]

[0022] The present invention uses a nucleic acid sequence encoding glycosyltransferase UGT94D1 to prepare a recombinant protein capable of catalyzing rebaudioside D to produce rebaudioside M2. The prepared recombinant protein can glycosylate rebaudioside D as a substrate with UGPG as the glycosyl donor to produce rebaudioside M2. Furthermore, no by-products are produced during the catalytic reaction, which is advantageous for downstream purification processes and reduces production costs. The yield of rebaudioside M2 ​​is 90%, at 2.32 g / L. [Brief explanation of the drawings]

[0023] [Figure 1]FIG. 1 shows the biosynthetic pathway that catalyzes rebaudioside D to produce rebaudioside M2 ​​using glycosyltransferase UGT94D1. [Figure 2] Figure 2 shows the analysis of glycosyltransferase UGT94D1 protein expression and purification in Example 2. Lane 1: Marker, Lane 2: No IPTG-induced expression sample, Lane 3: Crude enzyme solution, Lane 4: Crude enzyme solution supernatant, Lane 5: Precipitate from crude enzyme solution, Lane 6: Purified permeate, Lane 7: Washing of heterologous protein sample, Lane 8: Eluted sample of target protein. [Figure 3] FIG. 3 is a UPLC analysis of the catalysis of rebaudioside D to produce rebaudioside M2 ​​using glycosyltransferase UGT94D1 in Example 3. [Figure 4] FIG. 4 is a mass spectrometry analysis of rebaudioside M2, a glycosylation reaction product of rebaudioside D in Example 3. [Figure 5] FIG. 5 is a hydrogen spectrum of nuclear magnetic resonance spectroscopy of rebaudioside M2, the product in Example 4. [Figure 6] FIG. 6 is a carbon spectrum of nuclear magnetic resonance spectroscopy of rebaudioside M2, the product in Example 4. [Figure 7] FIG. 7 is a COSY spectrum of nuclear magnetic resonance spectroscopy of rebaudioside M2, the product in Example 4. [Figure 8] FIG. 8 is a TOCSY spectrum of nuclear magnetic resonance spectroscopy of rebaudioside M2, the product in Example 4. [Figure 9] FIG. 9 is an HSQC spectrum of the nuclear magnetic resonance spectroscopy of rebaudioside M2, the product in Example 4. [Figure 10] FIG. 10 is a HMBC spectrum of nuclear magnetic resonance spectroscopy of rebaudioside M2, the product in Example 4. [Figure 11]FIG. 11 is a ROESY spectrum of nuclear magnetic resonance spectroscopy of rebaudioside M2, the product in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention will be described in detail below with reference to the drawings and specific examples in the specification. However, the examples are not intended to limit the scope of the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are common reagents, methods and equipment in the art.

[0025] Unless otherwise stated, all reagents and materials used in the following examples are either commercially available or prepared by known methods.

[0026] The media used in the following examples are as follows: LB solid medium: 10 g / L protein peptone, 5 g / L yeast powder, 10 g / L NaCl, 20 g / L agar powder. 2xYT liquid medium: 16g / L protein peptone, 10g / L yeast powder, 5g / L NaCl.

[0027] The methods employed in the following examples are as follows. Measurement of glycosyltransferase enzymatic properties: Kinetic analysis of rebaudioside D production by glycosyltransferase UGT94D1 was performed in a 200 μL reaction system containing 5 mM UDPG, 10 mM MnCl2, 50 mM Tris-HCl (pH 8.0), and 5 μg purified protein sample (glycosyltransferase UGT94D1). The rebaudioside D concentration ranged from 0 to 5 mM. The reaction temperature was 35°C for 2 hours. The reaction was then terminated by heating to 95°C for 5 minutes and then rapidly cooling. The reaction mixture was diluted with 2 volumes of methanol and centrifuged at 20,000 × g for 5 minutes to remove the precipitate. The supernatant was filtered through a 0.22 μm filter membrane and used for UPLC analysis.

[0028] Enzyme activity definition: The amount of enzyme required to synthesize 1 μM rebaudioside M2 ​​in 1 hour.

[0029] Measurement of the yield of rebaudioside M2: Prepare rebaudioside M2 ​​standard solutions with different concentrations (0 mM, 0.05 mM, 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM) and analyze the standard solutions using UPLC. The formula for obtaining the rebaudioside M2 ​​concentration standard curve is y = 2383564.28571x + 1556.96429, R 2 = 0.99664, and the yield of rebaudioside M2 ​​is obtained by standard curve conversion. Yield = actual yield of rebaudioside M2 ​​ / theoretical yield of rebaudioside M2.

[0030] Waters Acquity UPLC system: BEHC18 1.7 μM column (2.1 × 50 mm) was used. Liquid phase conditions: organic phase - acetonitrile, aqueous phase - ultrapure water, flow rate 0.3 mL / min, column temperature 40 °C, UV detection wavelength 210 nm. Test procedure: 0-1 min 15% organic phase, 6 min 40% organic phase, 7-8 min 15% organic phase.

[0031] Example 1: Obtaining the glycosyltransferase UGT94D1 gene and constructing a recombinant strain The amino acid sequence (accession number XP_011076907.1) and nucleotide sequence (accession number: XM_011078605.1) of Bacillus glycosyltransferase were downloaded from Genbank, and gene synthesis was carried out by Yixin Biotechnology Co., Ltd., which then ligated the sequence to the polyclonal enzyme cleavage site of the vector pET-21b(+) to obtain the recombinant plasmid pET-21b(+)-UGT94D1.

[0032] The resulting plasmid pET-21b(+)-UGT94D1 was identified by sequencing, transformed into E. coli BL21(DE3) sensitive cells, and screened on LB solid plates containing 100 μg / mL ampicillin to obtain the recombinant strain E. coli BL21(DE3)pET-21b(+)-UGT94D1.

[0033] Example 2 Induction of recombinant strain expression and purification of target protein The recombinant strain E. coli BL21(DE3)pET-21b(+)-UGT94D1 constructed in Example 1 was inoculated into 1 L of 2xYT liquid medium containing 100 μg / mL of ampicillin, and the OD 600 After culturing to pH 0.6-0.8, the culture temperature is lowered to 18°C, and isopropyl-β-thiogalactopyranoside (IPTG) is added to a final concentration of 0.1 mmol / L for 8 hours of induction culture.

[0034] The induced expression bacterial solution was centrifuged (7000 rpm, 7 minutes, 4°C), the supernatant was discarded, and the bacterial cells were collected. The cells were resuspended in 10 mL of lysis buffer (50 mmol / L Tris-HCl pH 8.0, 300 mmol / L NaCl, 10 mmol / L imidazole, 10% glycerol) per 1 g of bacterial cells, disrupted using a high-pressure homogenizer, and the disrupted bacterial solution was centrifuged (40,000 × g, 30 minutes). The supernatant was removed to obtain a crude enzyme solution.

[0035] The crude enzyme solution was + After affinity chromatography on the column, the heteroprotein was washed with 10 volumes of digestion buffer. The target protein was then eluted with elution buffer (50 mmol / L Tris-HCl pH 8.0, 300 mmol / L NaCl, 250 mmol / L imidazole, 10% glycerin). The eluted target protein was collected and desalted using a Histrp™ 5 mL desalting column. The desalting buffer (25 mmol / L Tris-HCl, 150 mmol / L NaCl, 10% glycerin) was used. After desalting, the protein was concentrated to 10 mg / mL and the subsequent reaction was observed. The purified protein was analyzed by 10% SDS-PAGE gel electrophoresis, and the results are shown in Figure 2. A pure enzyme with a clear target strip and accurate protein size was successfully obtained. The Km value of UGT94D1 for rebaudioside D was 0.89 ± 0.05 mM, and k cat The value is 0.33±0.08min -1The enzymatic activity of the purified enzyme UGT94D1 was determined to be 2.12 U / mg.

[0036] Example 3: UGT94D1 catalyzes the glycosylation reaction of rebaudioside D to synthesize rebaudioside M2 The purified glycosyltransferase UGT94D1 obtained in Example 2 is used in the glycosylation reaction (Figure 1).

[0037] The glycosylation reaction was carried out in a 200 μL reaction system containing 50 mmol / L Tris-HCl (pH 8.0), 5 mmol / L UDPG, 10 mmol / L MnCl2, 2 mmol / L rebaudioside D, and 5 μM of the purified enzyme UGT94D1 obtained in Example 2. The reaction was carried out at 35°C for 4 hours, then heated to 95°C for 5 minutes and quenched to terminate the reaction. The reaction mixture was then diluted with 2 volumes of methanol. The precipitate was removed by centrifugation at 20,000 × g for 5 minutes, and the supernatant was filtered through a 0.22 μm filter membrane before being used for UPLC and LC-MS analysis. The Waters Acquity UPLC system used a BEHC18 1.7 μM column (2.1 × 50 mm), and the liquid phase conditions were organic phase - acetonitrile, aqueous phase - ultrapure water, flow rate 0.3 mL / min, column temperature 40°C, ultraviolet detection wavelength 210 nm, and the test procedure was 0-1 min 15% organic phase, 6 min 40% organic phase, and 7-8 min 15% organic phase.

[0038] The liquid phase analysis results, shown in Figure 3, clearly show that new products and no by-products were generated in the reaction system compared with the authentic rebaudioside D. Mass spectrometry (MS) of the reaction mixture (Figure 4) was performed, and the negative ion mode results of LC-MS showed [M-H] at m / z 1289.5421. - There is an ion peak, and the molecular formula is C 56 H 90 O 33 This clearly indicated that the product was rebaudioside M2, a monosaccharide derivative of rebaudioside D. HPLC quantitative analysis showed that the yield of rebaudioside M2 ​​was 90%, with a yield of 2.32 g / L.

[0039] Example 4: Structural identification of novel rebaudioside D monosaccharide derivatives A large-scale (100 mL) glycosylation reaction was performed using glycosyltransferase UGT94D1 to prepare novel derivatives. The reaction system consisted of 2 mM RebD, 10 μM glycosyltransferase, 5 mM UDPPG, 10 mM MnCl2, and 50 mM Tris pH 8.0. The reaction mixture was incubated at 35°C for 24 hours and then quenched by heating at 95°C for 5 minutes. The reaction was then centrifuged at 20,000 × g for 5 minutes to remove the precipitate. The supernatant was then filtered through a 0.22 μm membrane and purified using a semi-preparative high-performance liquid chromatography system. The system used a Shim-pack GIST C18 column (10 × 250 mm, 5 μm, SHIMADZU, Japan). The liquid phase conditions were organic phase - acetonitrile, aqueous phase - ultrapure water, with a flow rate of 5 mL / min. The time sequence was 0-28 min 23% organic phase, 28.5-30.5 min 60% organic phase, and 31-35 min 23% organic phase. The column temperature was 40°C, and the ultraviolet detection wavelength was 210 nm. The obtained sample was dissolved in heavy water and 1D ( 1 H and 13 The complete structure of the product was analyzed by C) and 2D NMR (COSY, TCOSY, HSQC, HMBC, and ROESY) spectroscopy. Data were collected on a Bruker Avance III 600 MHz spectrometer (Bruker BioSpin, Karlsruhe, Germany). 1 The detection frequency of the H spectrum is 600 MHz. 13 The C spectrum is 151 MHz.

[0040] Detailed assignments of H and C chemical shifts for the new derivatives synthesized by 1D and 2DHMR are shown in Table 1. The structural formula of the product synthesized by UGT94D1 catalyzed by Reb D is 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid-[(2-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl)ester], which is similar to the structure of the literature-reported Reb M2 and rebaudioside M2 ​​(Figures 5-11). [Table 1]

[0041] Although the preferred embodiments of the present invention have been disclosed above, they are not intended to limit the present invention, and those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and the scope of protection of the present invention is defined by the claims.

Claims

1. A recombinant bacterium that expresses glycosyltransferase UGT94D1 derived from sesame, wherein the amino acid sequence of the glycosyltransferase UGT94D1 has the NCBI registration number XP_011076907.

1.

2. The recombinant bacterium according to claim 1, wherein the recombinant bacterium uses Escherichia coli as a host cell.

3. The recombinant bacterium according to claim 1, wherein the recombinant bacterium uses a pET series expression vector.

4. The recombinant bacterium according to claim 3, wherein the recombinant bacterium uses pET-21b(+) as an expression vector.

5. A composition comprising one or more of glycosyltransferase UGT94D1, the recombinant bacterium according to any one of claims 1 to 4, or a cell lysate of the recombinant bacterium according to any one of claims 1 to 4, wherein the registration number of the amino acid sequence of glycosyltransferase UGT94D1 is XP_011076907.

1.

6. The composition according to claim 5, wherein the cell lysate is a supernatant obtained by lysing cells after induction and expression of the recombinant bacterium according to any one of claims 1 to 4.

7. 10. A method for catalytically synthesizing rebaudioside M2, comprising: using rebaudioside D as a substrate; and performing a catalytic reaction using glycosyltransferase UGT94D1; the recombinant bacterium according to any one of claims 1 to 4; and / or the composition according to claim 5 or 6, wherein the registration number of the amino acid sequence of glycosyltransferase UGT94D1 is XP_011076907.

1.

8. 8. The method for catalytically synthesizing rebaudioside M2 ​​according to claim 7, wherein the method uses UDP-glucose as a glycosyl donor.

9. 9. The method for catalytically synthesizing rebaudioside M2 ​​according to claim 8, wherein the catalytic system comprises 0.1-3.0 mM RebD, 1-10 μM glycosyltransferase UGT94D1, 1-10 mM UDP-glucose, 5-20 mM MnCl2, and 20-80 mM Tris.

10. 10. Use of glycosyltransferase UGT94D1, the recombinant bacterium of any one of claims 1 to 4, the composition of claim 5 or 6, or the method of any one of claims 7 to 9, in the production of rebaudioside M2 ​​or a product containing rebaudioside M2, wherein the accession number of the amino acid sequence of glycosyltransferase UGT94D1 is XP_011076907.1.

Images