Glycosyl transferase mutant and application thereof in catalytic synthesis of rebaudioside M
By using a multi-enzyme cascade catalysis of glycosyltransferase mutants G1-Mu5 and C1-Mu4, the problem of low catalytic efficiency of Reb M in existing technologies has been solved, and the efficient synthesis of Rebdi glycoside M has been achieved with high space-time yield and high purity, making it suitable for industrial production.
Patent Information
- Application Number
- CN202511604583.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-05
- Publication Date
- 2026-01-13
AI Technical Summary
Existing technologies have low catalytic efficiency of glycosyltransferases and large differences in parameters of multi-enzyme cascade reactions, making it difficult to meet the needs of industrial production. Traditional plant extraction processes have low yields and high costs, while microbial fermentation methods have insufficient output, making it difficult to realize the industrial application of Reb M.
Reb glycoside M was synthesized via a multi-enzyme cascade catalysis using glycosyltransferase mutants G1-Mu5 and C1-Mu4. These enzymes were expressed in Escherichia coli using a recombinant expression vector and co-expressing engineered bacteria. Reb A was used as the substrate, sucrose as the glycoside donor, and UDP coenzyme was used as an auxiliary agent to synthesize Reb M under mild conditions.
The synthesis of Reb M with high space-time yield and high purity was achieved, with a space-time yield of up to 3.33 g/L/h, purity >98%, and yield of 84%. The purification process was simplified, and it has industrial application value.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biocatalysis technology, specifically relating to glycosyltransferase mutants and their application in the multi-enzyme-level synthesis of the natural sweetener Reb M (also known as Reb M). Background Technology
[0002] With rapid socio-economic development and continuous improvement in civilization, people's quality of life has undergone a qualitative leap. This change is not only reflected in the significant improvement in material conditions, but also in people's higher pursuit of a better quality of life. Pursuing scientific diets and healthy living concepts has become an important life goal for modern people. However, excessive intake of high-calorie sweeteners such as sucrose and fructose may induce obesity, diabetes, and other metabolic diseases. Against this backdrop, developing natural sweeteners that are both high in sweetness and low in calories, while also being safe and reliable, has become an important research direction in the field of food health.
[0003] Steviol glycosides are a type of glycoside derived from stevia (stevia) Stevia rebaudiana Steviosides, a natural sweetener extracted from sucrose, are characterized by high sweetness, low calories, and good safety. Their sweetness can reach 200-300 times that of sucrose, but they contain almost no calories and do not cause blood sugar fluctuations, making them an ideal alternative to sucrose and widely used in food, beverages, and health products. Studies have shown that steviol glycosides not only satisfy people's craving for sweetness but also help control weight and regulate blood sugar, making them suitable for diabetics and obese individuals. Furthermore, they possess potential health benefits such as antioxidant and anti-inflammatory properties and are recognized as a safe food additive by authoritative organizations such as the US FDA and the EU EFSA.
[0004] Although steviol glycosides, as a high-intensity sweetener, possess significant sweetness, they are often accompanied by unpleasant flavor characteristics such as noticeable bitterness and a licorice-like aftertaste, limiting their application in beverages, dairy products, and other fields with stringent taste requirements. As a new generation of high-intensity sweetener, Reb M (also known as Reb A) significantly outperforms traditional steviol glycosides (such as Reb A) in both sensory characteristics and functional performance. Its core advantages are: 1) In terms of sensory characteristics, it eliminates the characteristic bitterness, metallic taste, and licorice-like taste of Reb A, presenting a pure sweetness with a flavor highly similar to sucrose; 2) In terms of functional parameters, it has a sweetness 350 times that of sucrose with zero calories, while also possessing excellent thermal stability, demonstrating outstanding application adaptability in food systems such as beverages, baked goods, and dairy products. However, Reb M is present in very low amounts in stevia, accounting for only 0.4-0.5% of the dry weight of the leaves. Traditional plant extraction processes not only result in low yields but also in high production costs, which severely restricts its industrial application.
[0005] This technological bottleneck has been effectively addressed in recent years through breakthroughs in biomanufacturing technology. Currently, the industry mainly employs two innovative preparation pathways: enzymatic conversion and microbial fermentation. Enzymatic conversion is a relatively mature production process, primarily using glycosyltransferases to convert relatively inexpensive and readily available Reb A or Reb D into Reb M. However, the catalytic efficiency of natural glycosyltransferases is generally low, and the reaction parameters of different enzyme systems in multi-enzyme cascade reactions vary significantly, resulting in poor system adaptability, low conversion rates, and difficulty in meeting the needs of industrial production. Microbial fermentation utilizes synthetic biology techniques to construct genetically engineered strains (such as yeast or bacteria) and achieves de novo biosynthesis of Reb M by optimizing the fermentation process. However, currently reported Reb M yields via fermentation are mostly at the gram level, still far from industrial-scale production. Summary of the Invention
[0006] To overcome the shortcomings of low catalytic efficiency and slow conversion rate in the existing enzymatic conversion method for preparing Reb M, this invention provides a glycosyltransferase mutant and its application in the multi-enzyme cascade catalytic synthesis of the natural sweetener Reb M.
[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a glycosyltransferase G1 mutant, wherein the glycosyltransferase G1 mutant is formed by mutation of any one, two, three, four, or five amino acid residues at any of the following positions in the amino acid sequence shown in SEQ ID NO. 1: T284A, T284S, M88I, M88L, M88V, L200A, L200S, L200T, L200Q, W197Y, W197F, I199F, I199A, I199L, L379G.
[0008] The amino acid residue mutation mode is represented in the form XnY, where X represents the original amino acid, n represents the mutation site, and Y represents the mutated amino acid; for example, T284A means that the amino acid T at position 284 in the amino acid sequence shown in SEQ ID NO. 1 is mutated to A.
[0009] In a preferred embodiment, the glycosyltransferase G1 mutant is a five-point mutant G1-Mu5, obtained by mutating the amino acid residues M88I / W197Y / L200A / I199F / L379G in the amino acid sequence shown in SEQ ID NO. 1. The amino acid sequence of the mutant G1-Mu5 is shown in SEQ ID NO. 2, and the gene sequence encoding the mutant G1-Mu5 is shown in SEQ ID NO. 3.
[0010] In a second aspect, the present invention provides a glycosyltransferase C1 mutant, wherein the glycosyltransferase C1 mutant is formed by mutation of any one, two, three, or four amino acid residues at any of the following positions in the amino acid sequence shown in SEQ ID NO. 4: L358C, R91F, R91M, L181I, L181V, N118A, N118F, N118L, N118M, N178D, N178E, Y263W.
[0011] In a preferred embodiment, the glycosyltransferase C1 mutant is a four-point mutant C1-Mu4, obtained by mutating the amino acid residues L358C / R91M / N178E / N263P in the amino acid sequence shown in SEQ ID NO. 4. The amino acid sequence of the mutant C1-Mu4 is shown in SEQ ID NO. 5, and the gene sequence encoding the mutant C1-Mu4 is shown in SEQ ID NO. 6.
[0012] The glycosyltransferase G1 mutant and glycosyltransferase C1 mutant provided above can be used in the catalytic synthesis of rebaudioside M.
[0013] In a third aspect, the present invention provides a recombinant expression vector containing the coding gene of the above-mentioned glycosyltransferase G1 mutant and the coding gene of the glycosyltransferase C1 mutant.
[0014] As one possible implementation, the mutant genes of the two glycosyltransferases are further recombined on the pET-30a plasmid; the expression vector of the mutant is obtained by circular PCR using the pET-30a-G1-C1 plasmid as a template and mutant primers.
[0015] In a fourth aspect, the present invention provides a co-expression engineered bacterium, which is obtained by transforming the above-mentioned recombinant expression vector and a recombinant expression vector containing the SUS gene encoding sucrose synthase into host bacteria.
[0016] As one possible implementation, the sucrose synthase SUS encoding gene is further recombined on the pCDFDuet-1 plasmid; the amino acid sequence of the sucrose synthase SUS is shown in SEQ ID NO. 7, and the DNA sequence of its encoding gene is shown in SEQ ID NO. 8.
[0017] As one possible implementation, the host bacteria is Escherichia coli, more preferably Escherichia coli BL21(DE3).
[0018] In a fifth aspect, the present invention also provides a method for synthesizing rabodiin M, using Reb A as a substrate, sucrose as a glycoside donor, and utilizing the glycosyltransferase G1 mutant as described in claim 1, the glycosyltransferase C1 mutant as described in claim 3, and sucrose synthase SUS as catalysts, to synthesize rabodiin M through a multi-enzyme cascade catalysis.
[0019] As one possible implementation, further, during the enzyme reaction, the coenzyme UDP is added to activate the reaction; The glycosyltransferase G1 mutant, glycosyltransferase C1 mutant, and sucrose synthase SUS are derived from whole cells or cell lysates of recombinant cells co-expressing the glycosyltransferase G1 mutant, glycosyltransferase C1 mutant, and sucrose synthase SUS using genetic engineering methods, or from whole cells or cell lysates of recombinant cells expressing the glycosyltransferase G1 mutant, glycosyltransferase C1 mutant, and sucrose synthase SUS individually.
[0020] As one possible implementation, Reb A is further synthesized into Reb M using wet cells or cell lysate of recombinant cells co-expressing glycosyltransferase G1 mutant, glycosyltransferase C1 mutant, and sucrose synthase SUS; the reaction system contains substrate Reb A, glycoside donor sucrose, coenzyme UDP, and wet cells or lysate of the above-mentioned engineered bacteria; the reaction is stirred at 35-40°C and pH 7.0-7.5 until the reaction is complete.
[0021] Furthermore, the method for synthesizing Reb M also includes: purifying the synthesized Reb M, with the following steps: The reaction solution was subjected to steps including heating to dissolve, centrifugation, membrane filtration, vacuum concentration, cooling, rinsing and drying to obtain Reb M white powder, with a purity >98% as determined by HPLC.
[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention has advantages such as high spacetime yield, easy purification, and being environmentally friendly.
[0023] This invention utilizes whole cells or lysed enzyme solutions of engineered bacteria co-expressing glycosyltransferase G1 mutant, glycosyltransferase C1 mutant, and sucrose synthase SUS as catalysts to synthesize Reb M under mild conditions, using Reb A as substrate and sucrose as glycoside donor. The engineered bacteria whole cells or lysed enzyme solutions can completely convert 30 g / L of Reb A within 12 h, achieving a space-time yield of 3.33 g / L / h for Reb M. After conversion, the reaction system is simple in composition, containing only the product Reb M, sucrose, fructose, whole-cell or crude enzyme protein, and trace amounts of the coenzyme UDP(G), with no accumulation of substrate or intermediate products, facilitating downstream purification. After dissolution and concentration, the target product Reb M with a purity >98% and a yield of 84% can be obtained, demonstrating extremely high industrial application value. Attached Figure Description
[0024] Figure 1 This describes the reaction route for the synthesis of Reb M from Reb A via a multi-enzyme cascade catalysis. Figure 2 Synthesis time curves of Reb M at a scale of 3 L; Figure 3 Images of Reb M products; Figure 4 This is a high-performance liquid chromatography (HPLC) chromatogram of the Reb M product. Detailed Implementation
[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0026] This invention provides a glycosyltransferase G1 mutant, wherein the glycosyltransferase G1 mutant is formed by mutation of any one, two, three, four, or five amino acid residues at any of the following positions in the amino acid sequence shown in SEQ ID NO. 1: T284A, T284S, M88I, M88L, M88V, L200A, L200S, L200T, L200Q, W197Y, W197F, I199F, I199A, I199L, L379G.
[0027] The present invention also provides a glycosyltransferase C1 mutant, wherein the glycosyltransferase C1 mutant is formed by mutation of any one, two, three or four amino acid residues at any of the following positions in the amino acid sequence shown in SEQ ID NO. 4: L358C, R91F, R91M, L181I, L181V, N118A, N118F, N118L, N118M, N178D, N178E, Y263W.
[0028] Furthermore, this invention also provides a method for synthesizing rebodiin M, using Reb A as a substrate, sucrose as a glycoside donor, and utilizing the aforementioned glycosyltransferase G1 mutant, glycosyltransferase C1 mutant, and sucrose synthase SUS as catalysts, to synthesize rebodiin M through a multi-enzyme cascade catalysis. The catalytic reaction route is as follows: Figure 1 As shown.
[0029] The amino acid sequence of sucrose synthase SUS is shown in SEQ ID NO. 7, and the DNA sequence of its encoding gene is shown in SEQ ID NO. 8.
[0030] Example 1: Construction of expression vector pET-30a-G1-C1 The genes of two glycosyltransferases, G1 and C1, were recombined into the pET-30a plasmid using In-Fusion seamless cloning technology. The primer information used is shown in Table 1.
[0031] First, the G1 and C1 coding genes (1431 bp and 1366 bp, respectively) were amplified using primer pairs F1 / R1 and F2 / R2. Then, using the amplified G1 and C1 gene fragments as templates, a fusion PCR was performed using primer pairs F1 / R2 to amplify the large G1-C1 fragment (2778 bp). Simultaneously, the linearized pET-30a backbone (5355 bp) was amplified using primer pairs F3 / R3. The large G1-C1 fragment and pET-30a backbone were purified using a DNA purification kit. Then, the large G1-C1 fragment and pET-30a backbone were recombined using recombinase. The recombinant product (10 μL) was completely transformed into E. coli BL21(DE3) competent cells, plated on LB agar plates containing 50 mg / L kanamycin, and incubated at 37°C. Transformants were selected and colony PCR was performed using the F1 / R2 primer pair. Positive clones (transformants that could amplify a band of about 2700 bp) were sequenced for verification. Once the sequence was confirmed to be correct, it was identified as the pET-30a-G1-C1 vector.
[0032] Example 2: Construction of expression vector pCDFDuet-SUS The gene for sucrose synthase SUS was recombined into the pCDFDuet-1 plasmid using In-Fusion seamless cloning technology. Primer information used is shown in Table 1.
[0033] First, the SUS coding gene (2448 bp) was amplified using the F4 / R4 primer pair. Simultaneously, the linearized pCDFDuet-1 backbone (3727 bp) was amplified using the F5 / R5 primer pair. The SUS gene fragment and pCDFDuet-1 backbone were purified using a DNA purification kit. Then, the SUS gene fragment and pCDFDuet-1 backbone were recombined using recombinase. The recombinant product (10 μL) was completely transformed into *E. coli* BL21(DE3) competent cells, plated on LB agar plates containing 50 mg / L streptomycin, and incubated at 37°C. Transformants were picked and colony PCR was performed using the F4 / R4 primer pair for verification. Positive clones (transformants that amplified a band of approximately 2400 bp) were sequenced for verification. Once the sequence was confirmed to be correct, it was identified as the pCDFDuet-SUS vector.
[0034] Example 3: Construction of co-expressed engineered bacteria The pET-30a-G1-C1 vector and the pCDFDuet-SUS vector were co-transformed into Escherichia coli BL21(DE3) competent cells, spread on LB plates containing 50 mg / L streptomycin and 50 mg / L kanamycin, and incubated at 37°C. The resulting transformants were engineered bacteria that co-expressed G1, C1 and SUS.
[0035] Example 4: Construction of mutants E. coli BL21(DE3) containing the pCDFDuet-SUS vector were prepared into competent cells and frozen at -80℃. Using the recombinant plasmid pET-30a-G1-C1 as a template, circular plasmid PCR was performed using KOD one DNA polymerase to amplify the expression plasmid of the mutant. The primers used are shown in Table 1. The amplification program was: 98℃, 3 min; 98℃, 10 s, 60℃, 20 s, 68℃, 40 s, 30 cycles; 68℃, 2 min. After PCR, 0.3 U DMT was added to the reaction system, and the reaction was carried out at 37℃ for 1 h to digest the template. After digestion, 3 µL of the digestion product was transferred into E. coli BL21(DE3) competent cells containing the pCDFDuet-SUS vector and plated on LB agar plates containing 50 mg / L streptomycin and 50 mg / L kanamycin.
[0036] Table 1. Primer Information
[0037] Example 5: Mutant Screening Single colonies obtained from site-directed mutagenesis were picked and inoculated into 1 mL of LB liquid medium containing 50 mg / L streptomycin and 50 mg / L kanamycin. The culture was incubated at 37°C for 8 h at 200 rpm to obtain the primary seed culture. 0.5 mL of the primary seed culture was inoculated into 25 mL of fresh resistant LB liquid medium and incubated at 37°C for 2–3 h at 200 rpm to obtain the secondary culture. IPTG was added to the secondary culture to a final concentration of 0.1 mM, and the culture was transferred to 25°C at 200 rpm for 12 h. After incubation, the fermentation broth was centrifuged (12000 rpm, 3 min), the supernatant was discarded, and the bacterial resuspended in phosphate buffer (100 mM, pH 7.5) to a concentration of 100 mg / mL. 250 μL of whole-cell resuspension, 20 mg Reb A, 200 mg sucrose, and 1 mM UDP were added to an EP tube, and water was added to bring the volume to 1 mL. After reacting the mixture at 40℃ and 200 rpm for 4 h, a sample was diluted 20-fold with 30% acetonitrile, filtered through a 0.22 μm filter, and then analyzed by HPLC. The chromatographic column was a Supersil ODS2 C18 column, the column oven temperature was 40℃, the detection wavelength was 196 nm, and the mobile phase and ratio were acetonitrile:phosphate buffer (10 mM, pH 2.6) = 28:72 (v / v), with an injection volume of 10 μL. The Reb M generation rate was calculated based on the peak areas of the substrate and product. The Reb M generation rates of each mutant are shown in Table 2. After multiple rounds of mutagenesis, the optimal G1 five-point mutant and C1 four-point mutant were finally selected. The engineered bacterium was *Escherichia coli* BL21(DE3) (pET-30a-G1-Mu5+C1-Mu4, pCDFDuet-SUS), which could generate 8.1 g / L of Reb M within 4 h.
[0038] Table 2. Yields of RebM synthesized from RebA catalyzed by mutants
[0039] Example 6: Fermentation preparation of engineered Escherichia coli The engineered *E. coli* strain BL21(DE3) (pET30a-G1-Mu5+C1-Mu4, pCDFDuet-SUS) was activated on LB agar plates containing 50 mg / L kanamycin and 50 mg / L streptomycin. After single colonies grew, single colonies of the engineered strain were picked and inoculated into 150 mL of resistant LB medium. The medium was incubated at 37°C and 200 rpm for 12 h to obtain the seed culture. The seed culture was then completely inoculated into a 5 L fermenter (containing 3 L of liquid, 10 g / L glycerol, 15 g / L yeast extract, 4 g / L K₂HPO₄·3H₂O, 2.24 g / L NaH₂PO₄·2H₂O, 3 g / L NaCl, 2.5 g / L (NH₄)₂SO₄, 2.1 g / L citric acid, 2 g / L glucose, 0.49 g / L MgSO₄·7H₂O, and 0.3 g / L FeSO₄). Fermentation was initiated with a concentration of 110 g / L yeast extract and 600 g / L glycerol mixture. The stirring speed was set to 300 rpm, and the temperature to 37℃. During fermentation, the pH was maintained at approximately 7.0 by automatically adding NH4OH (25%, v / v). In the early stages of fed-batch fermentation, dissolved oxygen (DO) slowly decreased as the cells proliferated. At this point, the stirring speed was increased to maintain DO at approximately 30%, with an upper limit of 600 rpm. When nutrients were depleted, dissolved oxygen and pH spiked. Feeding was then initiated (a mixture of 110 g / L yeast extract and 600 g / L glycerol), and the feeding speed was adjusted to maintain DO at approximately 15%. When OD600 reached approximately 60, IPTG at a final concentration of 0.2 mM was added to induce the expression of the target gene. After 26 h of fermentation, the fermentation broth was centrifuged (6000 rpm, 10 min) to obtain 560 g of wet cells.
[0040] Example 7: Synthesis of Reb M from Reb A catalyzed by whole-cell or lysed enzyme solution in 3 L scale Add 90 g RebA, 900 g sucrose, 1.3 g UDP, and 2.3 L water to a 5 L reactor. Start stirring at 300 rpm and maintain the temperature at 35-40 °C. Continue stirring until the materials are completely dissolved, then adjust the pH to approximately 7.0 with 5 M sodium hydroxide solution. Add 150 g of wet cells or lysed enzyme solution of engineered bacteria to initiate the reaction. Perform HPLC analysis every 2 h. The time curve for RebM synthesis is shown below. Figure 2 As shown in the figure, the conversion rate reached 99% when the reaction continued for 12 hours.
[0041] Example 8: Isolation and purification of Reb M The 3 L reaction solution from Example 7 was heated to 80°C, and water was added with continuous stirring to dissolve Reb M. The solution was centrifuged (7000 rpm, 10 min), and the supernatant was collected. The supernatant was filtered through a membrane to remove the protein. The solution was concentrated under reduced pressure, cooled, and crystallized to obtain a pale yellow solid. The solid was washed with water, filtered, and dried to obtain 95 g of white powder (e.g., ...). Figure 3 As shown), this is Reb M, with a purity >98% (e.g. Figure 4 As shown in the figure, the yield reached 84%.
[0042] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A glycosyltransferase G1 mutant, characterized in that, The glycosyltransferase G1 mutant is formed by any one, two, three, four, or five amino acid residue mutations at the following positions in the amino acid sequence shown in SEQ ID NO.1: T284A, T284S, M88I, M88L, M88V, L200A, L200S, L200T, L200Q, W197Y, W197F, I199F, I199A, I199L, L379G.
2. The mutant of glycosyltransferase G1 according to claim 1, characterized in that, The glycosyltransferase G1 mutant is a five-point mutant G1-Mu5, which is obtained by mutating the amino acid residues M88I / W197Y / L200A / I199F / L379G in the amino acid sequence shown in SEQ ID NO.
1.
3. A glycosyltransferase C1 mutant, characterized in that, The glycosyltransferase C1 mutant is formed by mutating any one, two, three, or four amino acid residues at the following positions in the amino acid sequence shown in SEQ ID NO.4: L358C, R91F, R91M, L181I, L181V, N118A, N118F, N118L, N118M, N178D, N178E, Y263W.
4. The mutant of glycosyltransferase C1 according to claim 3, characterized in that, The glycosyltransferase C1 mutant is a four-point mutant C1-Mu4, which is obtained by mutating the amino acid residues L358C / R91M / N178E / N263P in the amino acid sequence shown in SEQ ID NO.
4.
5. The application of the glycosyltransferase G1 mutant as described in claim 1 or 2, or the glycosyltransferase C1 mutant as described in claim 3 or 4, in the catalytic synthesis of rebodiin M.
6. A recombinant expression vector containing the coding gene for the glycosyltransferase G1 mutant as described in claim 1 or 2 and the coding gene for the glycosyltransferase C1 mutant as described in claim 3 or 4.
7. A co-expression engineered bacterium, characterized in that, The co-expression engineered bacteria are obtained by transforming the recombinant expression vector as described in claim 6 and the recombinant expression vector containing the SUS gene encoding sucrose synthase into host bacteria; The amino acid sequence of the sucrose synthase SUS is shown in SEQ ID NO. 7, and the DNA sequence of its encoding gene is shown in SEQ ID NO.
8.
8. The co-expression engineered bacteria according to claim 7, characterized in that, The host bacteria is Escherichia coli BL21(DE3).
9. A method for synthesizing rabodiin M, characterized in that, Using RebA as a substrate and sucrose as a glycoside donor, a multi-enzyme cascade catalytic synthesis of Rebadi glycoside M was achieved using the glycosyltransferase G1 mutant as described in claim 1, the glycosyltransferase C1 mutant as described in claim 3, and sucrose synthase SUS as catalysts.
10. The method for synthesizing rabodiin M according to claim 9, characterized in that, During the enzyme reaction, the coenzyme UDP is added to activate the reaction; The glycosyltransferase G1 mutant, glycosyltransferase C1 mutant, and sucrose synthase SUS are derived from whole cells or cell lysates of recombinant cells co-expressing the glycosyltransferase G1 mutant, glycosyltransferase C1 mutant, and sucrose synthase SUS using genetic engineering methods, or from whole cells or cell lysates of recombinant cells expressing the glycosyltransferase G1 mutant, glycosyltransferase C1 mutant, and sucrose synthase SUS individually.