Method for efficient biosynthesis of rebaudioside m2 using glycosyltransferase

Abstract

The present invention belongs to the technical field of biocatalytic synthesis. Provided is a method for efficient biosynthesis of rebaudioside M2 using glycosyltransferase. Glycosyltransferase UGT94D1 that catalyzes the synthesis of rebaudioside M2 from rebaudioside D is obtained by means of excavation, which provides an efficient, green and new route for the production of rebaudioside M2. During the entire process of catalytic reaction, no byproduct is generated, which facilitates downstream purification and further reduces the production cost.

Description

A method for efficiently biosynthesizing rebaudioside M2 ​​using glycosyltransferase Technical Field

[0001] The invention relates to a method for efficiently biosynthesizing rebaudioside M2 ​​by utilizing glycosyltransferase, and belongs to the technical field of biocatalytic synthesis. Background Art

[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 on the human body. Steviol glycosides have been approved as safe food additives by the United States, Brazil, South Korea, Japan, and the European Food Safety Authority. To date, 64 steviol glycosides have been identified from stevia leaves. Stevioside has the highest content, accounting for 5-10% of the dry weight, followed by rebaudioside A, which accounts for 2-4% of the dry weight. While these glycosides exhibit a sweetness 250-300 times greater than sucrose, even highly purified steviol glycosides retain a bitter taste, significantly limiting their application in the natural food additive market.

[0003] Recent studies have revealed that the number and type of glycosyl units attached at C-13 and C-19 significantly influence the properties of steviol glycosides. For example, rebaudioside A has an additional glucose at the C-13 position, making it sweeter and more palatable than stevia. Rebaudioside D and rebaudioside M, further isolated from stevia, have one and two additional glucose units attached at the C-19 position, respectively, resulting in even better sweetness than rebaudioside A. Consequently, there is considerable interest in developing new steviol glycosides with different glycosyl units at these positions in an effort to discover higher-quality sweeteners.

[0004] UDP-glucosyltransferases (UGTs) are naturally evolved enzymes that catalyze glycosylation reactions, transferring sugar moieties from activated sugar donors to acceptor molecules. UGTs have been widely used in the synthesis of steviol glycosides. In 2014, Prakash et al. first reported a novel steviol glycoside derivative, rebaudioside M2, as a minor product of the conversion of rebaudioside A to rebaudioside D catalyzed by the glycosyltransferase UGTSL2. However, low yields, mixed products, and high isolation and purification costs have limited further research on rebaudioside M2. Therefore, it is highly desirable to identify a novel glycosyltransferase with high catalytic activity and regioselectivity to catalyze the synthesis of rebaudioside M2.

[0005] Summary of the Invention

[0006] To solve the above problems, the present invention explores the glycosyltransferase UGT94D1 from sesame, which catalyzes the synthesis of rebaudioside D into rebaudioside M2. It has the activity of catalyzing the synthesis of rebaudioside D into rebaudioside M2 ​​in the presence of uridine diphosphate glucose (UDPG), providing possibilities for further research on rebaudioside M2.

[0007] In order to solve the above technical problems, the technical solutions of the present invention are as follows:

[0008] The first object of the present invention is to provide a recombinant bacterium that overexpresses sesame-derived glycosyltransferase UGT94D1.

[0009] In one embodiment, the accession number of the amino acid sequence of the glycosyltransferase is XP_011076907.1, and the accession number of the nucleotide sequence is XM_011078605.1.

[0010] In one embodiment, the recombinant bacteria uses prokaryotic cells or eukaryotic cells as host cells.

[0011] In one embodiment, the prokaryotic host cell can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include but are not limited to: Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus and Streptomyces. Gram-negative bacteria include but are not limited to Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Thuringia, Neisseria, Pseudomonas, Salmonella, and Ureaplasma; the eukaryotic cell is a fungal cell.

[0012] In one embodiment, the recombinant bacteria uses Escherichia coli as a host cell.

[0013] In one embodiment, the recombinant bacteria uses the pET series as an expression vector.

[0014] In one embodiment, the recombinant bacteria uses pET-21b(+) as an expression vector.

[0015] The second object of the present invention is to provide a composition comprising one or more of the glycosyltransferase UGT94D1, the recombinant bacteria, or the cell lysate of the recombinant bacteria; the accession number of the amino acid sequence of the glycosyltransferase is XP_011076907.1.

[0016] In one embodiment, the cell lysate is the supernatant obtained by lysing cells of the recombinant bacteria after induction of expression.

[0017] The third object of the present invention is to provide a method for catalytically synthesizing rebaudioside M2, wherein the method uses rebaudioside D as a substrate and utilizes glycosyltransferase UGT94D1, the recombinant bacteria or the composition to carry out a catalytic reaction.

[0018] In one embodiment, the method uses UDP-glucose as the glycosyl donor.

[0019] In one embodiment, the catalytic system is 0.1-3.0 mM Reb D, 1-10 μM glycosyltransferase UGT94D1, 1-10 mM UDPG, 5-20 mM MnCl2, and 20-80 mM Tris.

[0020] In one embodiment, the catalytic reaction is carried out at a pH of 5.5-9.0 and a temperature of 30-50° C. for 2-10 hours.

[0021] The present invention also provides use of glycosyltransferase UGT94D1, the recombinant bacteria, the composition, or the method in preparing rebaudioside M2 ​​or a product containing rebaudioside M2.

[0022] In one embodiment, the accession number of the amino acid sequence of the glycosyltransferase UGT94D1 is XP_011076907.1, and the accession number of the nucleotide sequence is XM_011078605.1. Beneficial effects:

[0023] The present invention uses a nucleic acid sequence encoding the glycosyltransferase UGT94D1 to prepare a recombinant protein capable of catalyzing the conversion of rebaudioside D to rebaudioside M2. The prepared recombinant protein uses UDPG as a glycosyl donor to glycosylate rebaudioside D into rebaudioside M2. No byproducts are produced during the catalytic reaction, facilitating downstream purification and reducing production costs. The yield of rebaudioside M2 ​​is 90%, with a yield of 2.32 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG1 shows the biosynthetic pathway of rebaudioside D to rebaudioside M2 ​​catalyzed by glycosyltransferase UGT94D1.

[0025] Figure 2 shows the expression and purification analysis of the glycosyltransferase UGT94D1 protein in Example 2. Lane 1: Marker; Lane 2: Expression sample without IPTG induction; Lane 3: Crude enzyme solution; Lane 4: Crude enzyme solution supernatant; Lane 5: Crude enzyme solution precipitate; Lane 6: Purification flowthrough; Lane 7: Washed impurity protein sample; Lane 8: Eluted target protein sample.

[0026] FIG3 is a UPLC analysis chart of the synthesis of rebaudioside M2 ​​by rebaudioside D catalyzed by glycosyltransferase UGT94D1 in Example 3.

[0027] Figure 4 is the mass spectrometric analysis of rebaudioside M2, the glycosylation product of rebaudioside D in Example 3.

[0028] Figure 5 is the hydrogen nuclear magnetic resonance spectrum analysis of the product rebaudioside M2 ​​in Example 4.

[0029] Figure 6 is the carbon NMR spectrum of the product rebaudioside M2 ​​in Example 4.

[0030] FIG7 is a COSY spectrum of nuclear magnetic resonance spectroscopy analysis of rebaudioside M2, the product in Example 4.

[0031] FIG8 is a TOCSY spectrum of the nuclear magnetic resonance spectrum analysis of the product rebaudioside M2 ​​in Example 4.

[0032] FIG9 is a HSQC spectrum of the nuclear magnetic resonance spectrum analysis of the product rebaudioside M2 ​​in Example 4.

[0033] Figure 10 is the HMBC spectrum of the nuclear magnetic resonance spectrum analysis of the product rebaudioside M2 ​​in Example 4.

[0034] Figure 11 is the ROESY spectrum of the nuclear magnetic resonance spectrum analysis of the product rebaudioside M2 ​​in Example 4. DETAILED DESCRIPTION

[0035] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0036] Unless otherwise specified, the reagents and materials used in the following examples are commercially available or can be prepared by known methods.

[0037] The culture medium involved in the following examples is:

[0038] LB solid medium: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, and 20 g / L agar powder.

[0039] 2×YT liquid medium: 16 g / L peptone, 10 g / L yeast powder, 5 g / L NaCl.

[0040] The methods involved in the following embodiments are:

[0041] Glycosyltransferase Enzymatic Properties: Kinetic analysis of rebaudioside D 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 of purified protein sample (glycosyltransferase UGT94D1). Rebaudioside D concentrations ranged from 0 to 5 mM. The reaction temperature was 35°C for 2 h. The reaction was then quenched by heating at 95°C for 5 min, and the reaction mixture was diluted with 2 volumes of methanol. The precipitate was removed by centrifugation at 20,000 × g for 5 min. The supernatant was filtered through a 0.22 μm filter and used for UPLC analysis.

[0042] Definition of enzyme activity: the amount of enzyme required to synthesize 1 μM rebaudioside M2 ​​within 1 h.

[0043] Determination of the yield of rebaudioside M2: Rebaudioside M2 ​​standard solutions of different concentrations (0 mM, 0.05 mM, 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM) were prepared and analyzed by UPLC. The equation of the rebaudioside M2 ​​concentration standard curve was y = 2383564.28571x + 1556.96429, R 2 = 0.99664. The yield of rebaudioside M2 ​​was calculated based on the standard curve. Yield = actual yield of rebaudioside M2 ​​ / theoretical yield of rebaudioside M2.

[0044] Waters Acquity UPLC system: A BEH C18 1.7 μM column (2.1 × 50 mm) was used. The liquid phase conditions were: acetonitrile as the organic phase, ultrapure water as the aqueous phase; flow rate, 0.3 mL / min; column temperature, 40°C; UV detection wavelength, 210 nm; detection procedure: 15% organic phase from 0 to 1 min, 40% organic phase from 6 min, and 15% organic phase from 7 to 8 min.

[0045] Example 1 Acquisition of glycosyltransferase UGT94D1 gene and construction of recombinant strain

[0046] The amino acid sequence (accession number: XP_011076907.1) and nucleotide sequence (accession number: XM_011078605.1) of Bacillus glycosyltransferase were downloaded from Genbank, synthesized by Yixin Biotechnology Co., Ltd., and ligated into the multiple cloning restriction site of the pET-21b(+) vector to obtain the recombinant plasmid pET-21b(+)-UGT94D1.

[0047] The obtained plasmid pET-21b(+)-UGT94D1 was sequenced and transformed into E. coli BL21(DE3) competent cells. The cells were screened using LB solid plates containing 100 μg / mL ampicillin to obtain the recombinant strain E. coli BL21(DE3)pET-21b(+)-UGT94D1.

[0048] Example 2 Induced expression of recombinant strains and purification of target protein

[0049] The recombinant strain E. coli BL21(DE3)pET-21b(+)-UGT94D1 constructed in Example 1 was inoculated into 1 L 2×YT liquid culture medium containing 100 μg / mL ampicillin and cultured at 135 rpm and 37°C until OD 600 After the pH value was 0.6-0.8, the culture temperature was lowered to 18°C, and isopropyl-β-thiogalactoside (IPTG) was added at a final concentration of 0.1 mmol / L, and the culture was induced for 8 h.

[0050] The induced expression bacterial solution was centrifuged (7000 rpm, 7 min, 4°C), the supernatant discarded, and the cells collected. The cells were resuspended in lysis buffer (50 mmol / L Tris-HCl pH 8.0, 300 mmol / L NaCl, 10 mmol / L imidazole, 10% glycerol) at a ratio of 1 g of cells per 10 mL of lysis buffer. The cells were disrupted using a high-pressure homogenizer, and the disrupted cells were centrifuged (40,000 × g, 30 min). The supernatant was collected to obtain the crude enzyme solution.

[0051] The crude enzyme solution was treated with Ni + The column was purified by affinity chromatography. After the sample was loaded, 10 volumes of lysis buffer were used to wash the impurities and the target protein was eluted with elution buffer (50 mmol / L Tris-HCl pH 8.0, 300 mmol / L NaCl, 250 mmol / L imidazole, 10% glycerol). The eluted target protein was collected and desalted through a desalting column (HistrpTM 5mL Desalting) with desalting buffer (25 mmol / L Tris-HCl, 150 mmol / L NaCl, 10% glycerol). After desalting, it was concentrated to 10 mg / mL for subsequent reactions. The purified protein was detected by 10% SDS-PAGE gel electrophoresis. The results are shown in Figure 2. The pure enzyme with clear target bands and accurate protein size was successfully obtained, and the K of UGT94D1 for rebaudioside D was measured. m The value is 0.89±0.05mM, k cat The value is 0.33±0.08min -1The enzyme activity of pure enzyme UGT94D1 was measured to be 2.12 U / mg.

[0052] Example 3: UGT94D1 catalyzes the glycosylation reaction of rebaudioside D to synthesize rebaudioside M2

[0053] The purified glycosyltransferase UGT94D1 obtained in Example 2 was used for the glycosylation reaction ( FIG. 1 ).

[0054] The glycosylation reaction was performed in a 200 μL reaction system containing the following: 50 mmol / L Tris pH 8.0, 5 mmol / L UDPG, 10 mmol / L MnCl2, and 2 mmol / L rebaudioside D. The purified UGT94D1 enzyme obtained in Example 2 was used at a 5 μM concentration. The reaction was incubated at 35°C for 4 h. The reaction was then quenched by heating at 95°C for 5 min, and the reaction mixture was diluted with 2 volumes of methanol. The precipitate was removed by centrifugation at 20,000 × g for 5 min. The supernatant was filtered through a 0.22 μm filter and used for UPLC and LC-MS analysis. The Waters Acquity UPLC system used a BEH C18 1.7 μM column (2.1 × 50 mm). The liquid phase conditions were: organic phase—acetonitrile, aqueous phase—ultrapure water; flow rate 0.3 mL / min; column temperature 40°C; UV detection wavelength 210 nm; detection program: 0-1 min 15% organic phase, 6 min 40% organic phase, 7-8 min 15% organic phase.

[0055] Liquid chromatography analysis revealed that, as shown in Figure 3, new products were generated in the reaction system, and no by-products were produced, compared with the standard of rebaudioside D. The reaction mixture was analyzed by mass spectrometry (MS) (Figure 4). The negative ion mode results of LC-MS showed that there was a [MH] at m / z 1289.5421. - The peak of the ion corresponds to the molecular formula C 56 H 90 O 33 , indicating that the product is rebaudioside M2, a monosaccharide derivative of rebaudioside D. HPLC quantitative analysis showed that the yield of rebaudioside M2 ​​was 90% and the yield was 2.32 g / L.

[0056] Example 4 Structural Identification of Novel Monoglycosylated Derivatives of Rebaudioside D

[0057] The novel derivatives were prepared by large-scale (100 mL) glycosylation reaction using glycosyltransferase UGT94D1. The reaction system was as follows: 2 mM Reb D, 10 μM glycosyltransferase, 5 mM UDPG, 10 mM MnCl2, and 50 mM Tris pH 8.0. The reaction mixture was reacted at 35°C for 24 hours, then heated at 95°C for 5 minutes to quench the reaction. The precipitate was removed by centrifugation at 20,000×g for 5 minutes. The supernatant was filtered through a 0.22μm filter membrane and purified using a semi-preparative HPLC system using a Shim-pack GIST C18 column (10×250mm, 5μm, SHIMADZU, Japan). The liquid phase conditions were: organic phase - acetonitrile, aqueous phase - ultrapure water; flow rate 5mL / min; time program: 0-28min 23% organic phase; 28.5-30.5min 60% organic phase; 31-35min 23% organic phase. Column temperature: 40°C; UV detection wavelength 210nm. The obtained sample was dissolved in heavy water and purified by 1D( 1 H and 13 The complete structure of the product was analyzed by 2D NMR (COSY, TCOSY, HSQC, HMBC and ROESY) spectra. Data were collected using a Bruker Avance III 600 MHz spectrometer (Bruker BioSpin, Karlsruhe, Germany). 1 The H spectrum detection frequency is 600MHz, 13 The C spectrum is 151 MHz.

[0058] The H and C chemical shifts of the new derivatives were assigned in detail by 1D and 2D HMR, as shown in Table 1. The structural formula of the product synthesized by UGT94D1-catalyzed 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]. The structure of this product is consistent with that of Reb M2 reported in the literature. The generated product is consistent with rebaudioside M2 ​​reported in the literature (Figures 5-11).

[0059] Table 1 1 H and 13 C chemical shift assignment table (D2O)

[0060] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant bacterium, characterized in that: The glycosyltransferase UGT94D1 derived from sesame is expressed, and the NCBI accession number of the amino acid sequence of the glycosyltransferase UGT94D1 is XP_011076907.

1.

2. The recombinant bacterium according to claim 1, characterized in that The recombinant bacteria uses Escherichia coli as a host cell.

3. The recombinant bacterium according to claim 1, characterized in that The recombinant bacteria uses pET series as expression vectors.

4. The recombinant bacterium according to claim 3, characterized in that The recombinant bacteria uses pET-21b(+) as an expression vector.

5. A composition, characterized in that The composition is one or more of glycosyltransferase UGT94D1, the recombinant bacteria according to any one of claims 1 to 4, or the cell lysate of the recombinant bacteria according to any one of claims 1 to 4; the accession number of the amino acid sequence of the glycosyltransferase UGT94D1 is XP_011076907.

1.

6. The composition according to claim 5, characterized in that The cell lysate is the supernatant obtained by lysing cells after the recombinant bacteria according to any one of claims 1 to 4 are induced to express.

7. A method for catalytic synthesis of rebaudioside M2, characterized in that: The method uses rebaudioside D as a substrate and utilizes glycosyltransferase UGT94D1, the recombinant bacteria described in any one of claims 1 to 4, and / or the composition described in claim 5 or 6 to carry out a catalytic reaction; the accession number of the amino acid sequence of the glycosyltransferase UGT94D1 is XP_011076907.

1.

8. The method according to claim 7, characterized in that The method uses UDP-glucose as the glycosyl donor.

9. The method according to claim 8, characterized in that The catalytic system comprises 0.1-3.0 mM Reb D, 1-10 μM glycosyltransferase UGT94D1, 1-10 mM UDP-glucose, 5-20 mM MnCl2, and 20-80 mM Tris.

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

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