A method for efficiently biosynthesizing rebaudioside M2 ​​using glycosyltransferase.

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

[0002] Steviol glycosides are diterpene glycosides extracted and purified from stevia leaves, offering advantages such as high sweetness, low calories, and no side effects on the human body. The safety of steviol glycosides as food additives has already been confirmed by food safety authorities in the United States, Brazil, South Korea, Japan, and Europe. To date, 64 types of steviol glycosides have been identified from stevia leaves, with stevia sugar having 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 high-purity steviol glycosides retain a bitter taste, severely 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 influence the properties of steviol glycosides. For example, rebaudioside A has an additional glucose unit at the C-13 position that is sweeter and more flavorful than stevia sugar. Rebaudioside D and rebaudioside M, obtained by further isolating stevia, have one and two additional glucose units linked at the C-19 position, respectively, resulting in significantly superior sweetness compared to rebaudioside A. Therefore, novel 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 glycosylation reactions, which are naturally evolved reactions that transfer the sugar moiety from an activated sugar donor to an acceptor molecule. UGTs are widely used in the synthesis of steviol glycosides. In 2014, Prakash et al. first reported rebaudioside M2, a novel steviol glycoside derivative and byproduct of the conversion of rebaudioside A to rebaudioside D catalyzed by the glycosyltransferase UGSL2. However, low yields and mixed products led to high separation and purification costs, limiting further research on rebaudioside M2. Therefore, there is a very high need to identify a novel glycosyltransferase with high catalytic activity and domain selectivity to catalytically synthesize rebaudioside M2. [Overview of the project] [Problems that the invention aims to solve]

[0005] To solve the above problems, the present invention aims to discover the sesame-derived glycoside transferase UGT94D1, catalyze rebaudioside D to synthesize rebaudioside M2, and give it the activity to catalyze rebaudioside D to synthesize rebaudioside M2 ​​in the presence of uridine diphosphate glucose (UGPG), thereby enabling further research on rebaudioside M2. [Means for solving the problem]

[0006] To solve the above technical problems, the present invention employs the following technical solutions.

[0007] The first object of the present invention is to provide a recombinant organism that expresses the sesame-derived glycosyltransferase UGT94D1.

[0008] In one embodiment, the registration number for the amino acid sequence of the glycosyltransferase is XP_011076907.1, and the registration number for the nucleotide sequence is XM_011078605.1.

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

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

[0011] In one embodiment, the recombinant bacteria use Escherichia coli as a host cell.

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

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

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

[0015] In one embodiment, the cell degradation solution is the supernatant obtained by degrading the cells after the recombinant bacteria have been induced to express themselves.

[0016] A third object of the present invention is to provide a catalytic synthesis method for rebaudioside M2, the method using rebaudioside D as a substrate and carrying out a catalytic reaction using glycosyltransferase UGT94D1, the recombinant bacteria, or the composition.

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

[0018] In one embodiment, the catalyst system comprises 0.1 to 3.0 mM of RebD, 1 to 10 μM of glycosyltransferase UGT94D1, 1 to 10 mM of UDPG, 5 to 20 mM of MnCl₂, and 20 to 80 mM of Tris.

[0019] In one embodiment, the catalytic reaction conditions are reacting at pH 5.5 to 9.0 and 30 to 50°C for 2 to 10 hours.

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

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

[0022] In the present invention, a nucleic acid sequence encoding glycosyltransferase UGT94D1 is used 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 UDPG as a glycosyl donor to produce rebaudioside M2. In addition, no by-products are generated during the catalytic reaction, which is advantageous for downstream purification processes and can reduce production costs. The yield of rebaudioside M2 is 90%, and the titer is 2.32 g / L. Brief Description of the Drawings

[0023] [Figure 1]Figure 1 shows the biosynthetic pathway for producing rebaudioside M2 by catalyzing rebaudioside D using glycosyltransferase UGT94D1. [Figure 2] Figure 2 shows the expression and purification analysis of glycosyltransferase UGT94D1 protein in Example 2. Lane 1: Marker, Lane 2: Sample without IPTG-induced expression, Lane 3: Crude enzyme solution, Lane 4: Supernatant of crude enzyme solution, Lane 5: Precipitate of crude enzyme solution, Lane 6: Purification of permeate, Lane 7: Washing of heteroprotein sample, Lane 8: Elution sample of target protein. [Figure 3] Figure 3 is a UPLC analysis chart for producing rebaudioside M2 by catalyzing rebaudioside D using glycosyltransferase UGT94D1 in Example 3. [Figure 4] Figure 4 is mass spectrometry analysis of rebaudioside M2, which is the glycosylation product of rebaudioside D in Example 3. [Figure 5] Figure 5 is the hydrogen spectrum obtained from nuclear magnetic resonance spectrum analysis of the product rebaudioside M2 in Example 4. [Figure 6] Figure 6 is the carbon spectrum obtained from nuclear magnetic resonance spectrum analysis of the product rebaudioside M2 in Example 4. [Figure 7] Figure 7 is the COSY spectrum obtained from nuclear magnetic resonance spectrum analysis of the product rebaudioside M2 in Example 4. [Figure 8] Figure 8 is the TOCSY spectrum obtained from nuclear magnetic resonance spectrum analysis of the product rebaudioside M2 in Example 4. [Figure 9] Figure 9 is the HSQC spectrum obtained from nuclear magnetic resonance spectrum analysis of the product rebaudioside M2 in Example 4. [Figure 10] Figure 10 is the HMBC spectrum obtained from nuclear magnetic resonance spectrum analysis of the product rebaudioside M2 in Example 4. [Figure 11]Figure 11 shows the ROESY spectrum of the nuclear magnetic resonance spectroscopy analysis of rebaudioside M2, the product in Example 4. [Modes for carrying out 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 present invention. Unless otherwise stated, the reagents, methods, and equipment used in the present invention are general 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 culture medium used in the following examples is as follows: LB solid medium: 10 g / L protein peptone, 5 g / L yeast powder, 10 g / L NaCl, 20 g / L agar powder. 2×YT liquid medium: 16 g / L protein peptone, 10 g / L yeast powder, 5 g / L NaCl.

[0027] The method used in the following examples is as follows: Measurement of the enzymatic properties of glycosyltransferase: The kinetic analysis of rebaudioside D using glycosyltransferase UGT94D1 is performed in a 200 μL reaction system. The reaction system contains 5 mM UDPG, 10 mM MnCl2, 50 mM Tris-HCl pH8.0, and 5 μg of purified protein sample (glycosyltransferase UGT94D1), with a rebaudioside D concentration of 0–5 mM. The reaction temperature is 35°C, the reaction time is 2 hours, then the reaction is terminated by heating at 95°C for 5 minutes followed by rapid cooling. The reaction mixture is diluted with twice the volume of methanol, centrifuged at 20000 × g for 5 minutes to remove precipitate, and the upper layer is filtered through a 0.22 μm filtration membrane before being used for ULC analysis.

[0028] Definition of enzyme activity: The amount of enzyme required to synthesize 1 μM rebaudioside M2 ​​within one hour.

[0029] Measurement of rebaudioside M2 ​​yield: Standard solutions of rebaudioside M2 ​​at different concentrations (0 mM, 0.05 mM, 0.1 mM, 0.15 mM, 0.2 mM, 0.25 mM, 0.3 mM) are prepared and analyzed using UPLC. The formula for obtaining the standard concentration curve of rebaudioside M2 ​​is y = 2383564.28571x + 1556.96429, R 2 Set the value to 0.99664 and obtain the yield of rebaudioside M2 ​​by converting to the standard curve. Yield = Actual yield of rebaudioside M2 ​​ / Theoretical yield of rebaudioside M2.

[0030] WatersAcquityUPLC system: Using a BEHC18 1.7μM column (2.1×50mm), liquid phase conditions: organic phase - acetonitrile, aqueous phase - ultrapure water, flow rate 0.3mL / min, column temperature 40℃, UV detection wavelength 210nm, inspection 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 recombinant strains. The amino acid sequence (registration number XP_011076907.1) and nucleotide sequence (registration number XM_011078605.1) of Bacillus glycosyltransferase were downloaded from Genbank, and Yixin Biotechnology Co., Ltd. performed gene synthesis to bind it to the polyclonal enzyme cut site of the vector pET-21b(+) to obtain recombinant plasmid pET-21b(+)-UGT94D1.

[0032] The obtained plasmid pET-21b(+)-UGT94D1 was identified by sequence analysis, converted into E. coli BL 21(DE3)-sensitive cells, and screened using LB solid plates containing 100 μg / mL ampicillin to obtain recombinant strain E. coli BL21(DE3)pET-21b(+)-UGT94D1.

[0033] Example 2: Induced expression of recombinant bacterial strain 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 2×YT liquid medium containing 100 μg / mL of ampicillin, and OD was performed under conditions of 135 rpm and 37°C. 600 After culturing at 0.6-0.8°C, the culture temperature is lowered to 18°C, isopropyl-β-thiogalactopyranoside (IPTG) with a final concentration of 0.1 mmol / L is added, and induction culture is performed for 8 hours.

[0034] The induced bacterial suspension is centrifuged (7000 rpm, 7 min, 4°C), the supernatant is discarded, and the bacterial cells are collected. The bacterial cells are resuspended in a cell degradation buffer (50 mmol / L Tris-HCl pH 8.0, 300 mmol / L NaCl, 10 mmol / L imidazole, 10% glycerin) at a rate of 10 mL per 1 g of bacterial cells. After lysing using a high-pressure homogenizer, the lysed bacterial suspension is centrifuged (40000 × g, 30 min), and the supernatant is taken to obtain the crude enzyme solution.

[0035] Crude enzyme solution Ni + The sample was purified by affinity chromatography using a column. After sample loading, the heteroprotein was washed with 10 times the volume of degradation buffer, and the target protein was eluted using 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 desalting column (Histrp™ 5 mL D Desalting). Desalting buffer (25 mmol / L Tris-HCl, 150 mmol / L NaCl, 10% glycerin). After desalting, the solution was concentrated to 10 mg / mL to observe subsequent reactions. The purified protein was measured by 10% SDS-PAGE gel electrophoresis, and the results are shown in Figure 2. Pure enzyme was successfully obtained with a clear target strip and accurate protein size, and the Km value of UGT94D1 for rebaudioside D was 0.89 ± 0.05 mM, k cat The value is 0.33 ± 0.08 min -1The enzymatic activity of the purified enzyme UGT94D1 was measured to be 2.12 U / mg.

[0036] Example 3 Glycosylation reaction in which UGT94D1 catalyzes 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 is carried out in a 200 μL reaction system, and the composition of the reaction system is as follows: 50 mmol / L Tris-HCl pH 8.0, 5 mmol / L UDPG, 10 mmol / L MnCl₂, 2 mmol / L rebaudioside D, and the concentration of the purified enzyme UGT94D1 obtained in Example 2 is 5 μM. The reaction is carried out at 35°C for 4 hours, then terminated by heating at 95°C for 5 minutes followed by quenching, and the reaction mixture is diluted with twice the volume of methanol. The precipitate is removed by centrifugation at 20000×g for 5 minutes, and the supernatant is collected, filtered through a 0.22 μm filter membrane, and then used for UPLC and LC-MS analysis. The Waters Acquity UPLC system uses a BEH C18 1.7 μM column (2.1×50 mm), the liquid phase conditions are: organic phase - acetonitrile, aqueous phase - ultrapure water, flow rate 0.3 mL / min, column temperature 40°C, ultraviolet detection wavelength 210 nm, and the detection procedure is: 0-1 min 15% organic phase, 6 min 40% organic phase, 7-8 min 15% organic phase.

[0038] As shown in Figure 3 from the liquid phase analysis results, compared with the rebaudioside D standard, a distinct new product is obviously produced in the reaction system, and no by-product is generated. Mass spectrometry (MS) was performed on the reaction mixture (Figure 4). According to the result of LC-MS in negative ion mode, [M-H at m / z 1289.5421 - ⁻ there is an ion peak, corresponding to the molecular formula C 56 ₅₄H 90 ₈₀O 33 ₂₉, which clearly demonstrates that the product is rebaudioside M2, a monosaccharide derivative of rebaudioside D. According to HPLC quantitative analysis, the yield of rebaudioside M2 is 90%, and the titer is 2.32 g / L.

[0039] Example 4: Structural identification of a novel rebaudioside D monosaccharide derivative Novel derivatives were prepared by a large-scale (100 mL) glycosylation reaction using glycosyltransferase UGT94D1. The reaction system was as follows: 2 mM RBebD, 10 μM glycosyltransferase, 5 mM MUDPG, 10 mM MnCl2, 50 mM Tris pH 8.0. The reaction mixture was reacted at 35°C for 24 hours, then heated to 95°C for 5 minutes and rapidly cooled to terminate the reaction. The precipitate was removed by centrifugation at 20000 × g for 5 minutes, the upper liquid was taken and filtered through a 0.22 μm filtration membrane, and purified using a semi-prepared high-efficiency 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, flow rate 5 mL / min. The time procedure was 0-28 mins 23% organic phase, 28.5-30.5 mins 60% organic phase, and 31-35 mins 23% organic phase. Column temperature: 40°C, UV detection wavelength 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, and data were collected using a BrukerAvance III 600MHz spectrometer (Bruker BioSpin, Karlsruhe, Germany). 1 The detection frequency for the H spectrum is 600 MHz. 13 The C spectrum is 151 MHz.

[0040] Table 1 shows the detailed assignment of the H and C chemical shifts of the new derivatives catalyzed by 1D and 2DHMR. The structural formula of the product synthesized by catalyzing Reb D of UGT94D1 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], and the structure of this product is similar to that of Reb M2 in the literature, and the product is also similar to rebaudioside M2 ​​reported in the literature (Figure 5-11). [Table 1]

[0041] While preferred embodiments of the present invention have been disclosed above, this does not limit the invention, and those skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention, and the scope of protection of the present invention is defined by the claims.

Claims

1. Using rebaudioside D as a substrate, a catalytic reaction is carried out using glycosyltransferase UGT94D1, recombinant bacteria, and / or compositions. The NCBI registry number for the amino acid sequence of the glycosyltransferase UGT94D1 is XP_011076907.1 (Sequence ID 1). The recombinant bacteria express the sesame-derived glycosyltransferase UGT94D1, and the NCBI registry number for the amino acid sequence of the glycosyltransferase UGT94D1 is XP_011076907.1 (SEQ ID NO: 1). The composition comprises one or more of the glycosyltransferase UGT94D1, the recombinant bacteria, or the cell degradation solution of the recombinant bacteria, and the NCBI registration number of the amino acid sequence of the glycosyltransferase UGT94D1 is XP_011076907.1 (SEQ ID NO: 1), characterized in that it is a rebaudioside M2 ​​catalytic synthesis method.

2. The method described above is characterized by using UDP-glucose as a glycosyl donor, as described in claim 1, for the catalyst synthesis of rebaudioside M2.

3. The catalytic system contains 0.1–3.0 mM RebD, 1–10 μM glycosyltransferase UGT94D1, 1–10 mM UDP-glucose, and 5–20 mM MnCl. ２ The method for synthesizing a rebaudioside M2 ​​catalyst according to claim 2, characterized by containing 20 to 80 mM Tris.

4. The use of glycosyltransferase UGT94D1, recombinant bacteria, or compositions in the manufacture of rebaudioside M2 ​​or products containing rebaudioside M2, The NCBI registry number for the amino acid sequence of the glycosyltransferase UGT94D1 is XP_011076907.1 (Sequence ID 1). The recombinant bacteria express the sesame-derived glycosyltransferase UGT94D1, and the NCBI registry number for the amino acid sequence of the glycosyltransferase UGT94D1 is XP_011076907.1 (SEQ ID NO: 1). The composition comprises glycosyltransferase UGT94D1, the recombinant bacteria, or one or more of the cytolytic solutions of the recombinant bacteria, wherein the NCBI registry number for the amino acid sequence of glycosyltransferase UGT94D1 is XP_011076907.1 (SEQ ID NO: 1).

5. Use of the method according to claim 1 in the manufacture of rebaudioside M2 ​​or a product containing rebaudioside M2.

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