A group of glycosyltransferase genes and their application in the asiaticoside / madecassoside biosynthesis pathway

Abstract

The present invention discloses a group of glycosyltransferase encoding genes and their applications. The glycosyltransferases UGT94M2, UGT94BE1 and their derived polypeptides provided by the present invention catalyze the first sugar group at the C-28 position of a pentacyclic triterpenoid compound substrate to extend the sugar chain and generate asiatic acid disaccharide, madecassoside, asiaticoside and madecassoside. The nucleotide sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; the amino acid sequences encoded by the glycosyltransferase genes are also disclosed, as shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The present invention also discloses an expression vector containing the above-mentioned gene, and a host cell transformed by the expression vector. The present invention screens and identifies key glycosyltransferases UGT94M2 and UGT94BE1 based on relevant data of genome and transcriptome, completely resolves the biosynthetic pathway of asiaticoside / madecassoside, and lays a theoretical foundation for producing asiaticoside / madecassoside using synthetic biology methods.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a group of glycosyltransferase genes and applications thereof in an asiaticoside / madecassoside synthesis pathway. Background Art

[0002] Centella asiatica, also known as Gotu Kola, Horseshoe Grass, etc., is the dried whole herb of Centella asiatica (L.) Urb. of the Umbelliferae family. It has the effects of clearing away heat and dampness, detoxifying and reducing swelling. It is mainly distributed in South my country, East China, Central South and Southwest China south of the Yangtze River. Centella asiatica mainly contains triterpenes and their glycosides, polyacetylenes, volatile oils, flavonoids, alkaloids and other compounds. Among them, the triterpenoid asiaticoside has been studied the most and is considered to be the main active ingredient of Centella asiatica to exert related effects. As one of the main pharmacologically active ingredients of Centella asiatica, asiaticoside has multiple pharmacological effects such as anti-tumor, anti-inflammatory, anti-fibrosis, anti-Alzheimer's disease, improving learning and memory ability, anti-depression, inhibiting scar hyperplasia and repairing skin damage. In addition, asiaticoside can also inhibit proinflammatory cytokines, promote skin hydration, promote hyaluronic acid secretion, maintain skin homeostasis, and has potential cosmetic application value. In addition, hydroxymadecassoside is also one of the effective components in Centella asiatica. Studies have shown that the pharmacological effects of hydroxymadecassoside are also very extensive. It is clinically used for burn scars, skin whitening, anti-inflammatory and antioxidant, anti-depression, anti-tumor, and prevention and treatment of coronary heart disease.

[0003] At present, the main way to obtain Centella asiatica and madecassoside is to extract them from Centella asiatica by decoction, reflux, ultrasound, etc., while the extraction from plants has problems such as complex process, high energy consumption, long production cycle, and requires a large amount of plant resources, which may even seriously affect the ecological environment. In addition, due to the complexity of their molecular structure, chemical synthesis of Centella asiatica and madecassoside is also difficult to achieve.

[0004] The development of synthetic biology has provided new opportunities for the heterologous synthesis of natural products from plants. Using microorganisms such as yeast as a chassis, through the assembly and optimization of metabolic pathways, it has great potential in the drug synthesis of natural products. Using yeast chassis cells to heterologously synthesize Centella asiatica / madecassoside through synthetic biology methods, the raw materials are cheap, and the fermentation preparation process is safe and controllable. Therefore, the preparation of Centella asiatica by synthetic biology technology not only has cost advantages, but also can ensure the quality and safety of the finished product. Using synthetic biology technology to prepare sufficient amounts of various high-purity Centella asiatica and madecassoside monomers can be used for commercial purposes or scientific experiments.

[0005] Although asiatica / madecassoside has been shown to have multiple effects in pharmacology and cosmetics, the biosynthetic pathway of asiatica / madecassoside has not yet been elucidated. In Centella asiatica, multiple genes involved in the formation of triterpenoids have been found, including farnesyl pyrophosphate synthase (CaFPS), squalene synthase (CaSQS), β-amyrin synthase (CaβAS) and dammarane synthase (CaDDS). Modification of the triterpene skeleton includes oxidation, substitution or glycosylation by P450 monooxygenase, UDP-Glc glycosyltransferase and other enzymes. Glycosyltransferase is a key enzyme in the glycosylation process of plants and is essential for the biological activity of saponins. Asiaticoside / madecassoside is obtained by continuous three-step glycosylation of asiatic acid / madecassoside. At present, the glycosyltransferases (UGT73AD1, UGT73AH1 and CaUGT1) that catalyze the production of asiatic acid / madecassoside monoglycosides have been identified, but the remaining two glycosylation steps have not yet been resolved. In the present invention, screening analysis combined with genome and transcriptome and in vitro enzyme activity experimental verification are used to identify the key glycosyltransferases in Centella asiatica that catalyze the production of asiatic acid / madecassoside monoglycoside disaccharides and further catalyze and generate asiaticoside / madecassoside, completely resolve the biosynthetic pathway of asiaticoside / madecassoside, and also lay a theoretical foundation for the production of asiaticoside / madecassoside by synthetic biology. Summary of the invention

[0006] Aiming at the unresolved biosynthetic pathway of asiaticoside / madecassoside in Centella asiatica, the present invention provides two Centella asiatica glycosyltransferases and their encoding genes, which can catalyze the synthesis of asiaticoside / madecassoside from asiatic acid monosaccharide / madecassoside.

[0007] The present invention integrates the technology and method of functional genomics and transcriptomics to mine and obtain two glycosyltransferase genes in the Centella asiatica (L.) Urban genome, which are named UGT94M2 and UGT94BE1, respectively, corresponding to the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0008] The present invention also protects nucleotide sequences having a homology of ≥95%, preferably ≥98%, such as 98%, 99%, 99.8%, etc., to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0009] The present invention also protects a nucleotide sequence that is complementary (preferably completely complementary) to any of the aforementioned nucleotide sequences.

[0010] The present invention also provides a glycosyltransferase encoded by the above glycosyltransferase gene, the amino acid sequence of which is shown in SEQ ID NO: 3 and SEQ ID NO: 4, wherein:

[0011] The amino acid sequence of SEQ ID NO: 3 is encoded by the nucleotide sequence of SEQ ID NO: 1;

[0012] The amino acid sequence of SEQ ID NO:4 is encoded by the nucleotide sequence of SEQ ID NO:2.

[0013] In a specific embodiment, the present invention also protects a derivative polypeptide having glycosyltransferase activity formed by adding a tag sequence, a signal sequence or a secretion signal sequence to the sequence SEQ ID NO: 3 or the sequence SEQ ID NO: 4.

[0014] In a specific embodiment, the present invention also protects a derivative polypeptide having an amino acid sequence with a homology of ≥95% with the amino acid sequence shown in either SEQ ID NO: 3 or SEQ ID NO: 4 and having glycosyltransferase activity.

[0015] In order to facilitate the purification or detection of the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4, a tag sequence may be connected to the amino terminus or carboxyl terminus of the polypeptide.

[0016] A person skilled in the art can easily adopt known methods to mutate the nucleotide sequence of the polypeptide of the present invention. Those artificially modified nucleotide sequences that have more than 95% identity with the nucleotide sequence of the present invention and have the same function as the polypeptide of the present invention are all derivative polypeptides of the present invention.

[0017] The polypeptide of the present invention and its derivative polypeptide catalyze the first sugar group at the C-28 position of the pentacyclic triterpenoid compound substrate to extend the sugar chain and generate asiatic acid disaccharide, hydroxy-madecassoside, asiaticoside and hydroxy-madecassoside.

[0018] The present invention also protects a biological material, which is any one of the following (C1)-(C4):

[0019] (C1) an expression cassette containing the polynucleotide described above;

[0020] (C2) a recombinant vector containing the polynucleotide described above, or a recombinant vector containing the expression cassette described in (C1);

[0021] (C3) a recombinant microorganism containing the polynucleotide described above, a recombinant microorganism containing the expression cassette described in (C1), or a recombinant microorganism containing the recombinant vector described in (C2);

[0022] (C4) A genetically engineered host cell containing the polynucleotide described above, a genetically engineered host cell containing the expression cassette described in (C1), or a genetically engineered host cell containing the recombinant vector described in (C2).

[0023] The vectors of the present invention are well known to those skilled in the art, including but not limited to plasmids, bacteriophages, viral vectors, etc. In one embodiment of the present invention, the vectors are pSJ8 vectors and pGEX-6P vectors.

[0024] The present invention also protects the use of the polypeptide described above in catalyzing one or more of the following reactions:

[0025] (D1) catalyzes asiatic acid monosaccharide to produce asiatic acid disaccharide;

[0026] (D2) catalyzes madecassic acid monosaccharide to produce madecassic acid disaccharide;

[0027] (D3) catalyzes the production of asiatic acid disaccharide to asiaticoside;

[0028] (D4) catalyzes the production of madecassoside from madecassoside.

[0029] The present invention also protects the use of the above-mentioned polypeptide, the above-mentioned polynucleotide, and the above-mentioned biological material as an enzyme catalyst in the preparation of one or more of the following reactions:

[0030] (D1) catalyzes asiatic acid monosaccharide to produce asiatic acid disaccharide;

[0031] (D2) catalyzes madecassic acid monosaccharide to produce madecassic acid disaccharide;

[0032] (D3) catalyzes the production of asiatic acid disaccharide to asiaticoside;

[0033] (D4) catalyzes the production of madecassoside from madecassoside.

[0034] The present invention also protects the use of the aforementioned polypeptide, the aforementioned polynucleotide, and the aforementioned biological material in the preparation of asiatic acid disaccharide, madecassoside, asiaticoside, or madecassoside.

[0035] The present invention also protects a method for producing the polypeptide described above, which comprises using the coding gene of the polypeptide described above to express in an organism to obtain the polypeptide, wherein the organism is a microorganism or a non-human animal.

[0036] The present invention also protects an in vitro glycosylation method, comprising the steps of: transferring the glycosyl of a glycosyl donor to the C28 site of a pentacyclic triterpenoid compound in the presence of a glycosyltransferase, thereby forming a further glycosylated pentacyclic triterpenoid compound; wherein the glycosyltransferase is selected from: the glycosyltransferases shown in SEQ ID NO: 3 and SEQ ID NO: 4 or their derivative polypeptides.

[0037] Based on the relevant results of the Centella asiatica genome and transcriptome and in vitro enzyme activity analysis, the present invention identified the key glycosyltransferases UGT94M2 and UGT94BE1 in the biosynthesis of asiaticoside, completely analyzed the biosynthetic pathway of asiaticoside / madecassoside, and laid a theoretical foundation for the production of asiaticoside / madecassoside using synthetic biology. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Diagram of the synthetic pathway from asiatic acid / madecassic acid to asiaticoside / madecassososide.

[0039] Figure 2 This is the agarose gel electrophoresis diagram of UGT73AD1, UGT94M2 and UGT94BE1 gene cloning, where lane 1 is UGT73AD1, lane 2 is UGT94M2, and lane 3 is UGT94BE1.

[0040] Figure 3 This is the SDS-PAGE run of the recombinant proteins of UGT73AD1, UGT94M2 and UGT94BE1, where lane 1 is PSJ8-UGT73AD1, lane 2 is PSJ8-UGT94M2, and lane 3 is pGEX-6P-UGT94BE1.

[0041] Figure 4 This is the HPLC chart of in vitro enzymatic activity analysis of recombinant protein UGT on Centella asiatica monosaccharide.

[0042] Figure 5 This is the HPLC chart of in vitro enzymatic activity analysis of recombinant protein UGT on hydroxy-madecassic acid monosaccharide.

[0043] Figure 6 This is the MS spectrum of compound 1 (Asiatic acid standard).

[0044] Figure 7 This is the MS spectrum of compound 2 (Centella asiaticacid monosaccharide).

[0045] Figure 8 This is the MS spectrum of compound 3 (Centella asiatica disaccharide).

[0046] Fig. 9 This is the MS spectrum of compound 4 (Asiaticoside).

[0047] Fig.10 This is the MS spectrum of compound 5 (madecassoic acid standard).

[0048] Fig.11 This is the MS spectrum of compound 6 (madecassoside).

[0049] Fig.12 This is the MS spectrum of compound 7 (madecassoside).

[0050] Fig.13 This is the MS spectrum of compound 8 (madecassoside).

[0051] Fig.14 This is the MS / MS spectrum of compound 4.

[0052] Fig.15 This is the MS / MS spectrum of compound 8. DETAILED DESCRIPTION

[0053] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources. The following examples are convenient for a better understanding of the present invention, and these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0054] Example 1: Acquisition of glycosyltransferase genes UGT94M2 and UGT94BE1

[0055] (1) HMMER3.0 was used to annotate the published genome data of Centella asiatica. 134 glycosyltransferases were annotated from all proteins in the published genome of Centella asiatica. A phylogenetic tree was constructed with all the pentacyclic triterpenoid glycosyltransferases identified so far for analysis.

[0056] (2) Combined with the genome data, the published transcriptome information of Centella asiatica was analyzed, and the expression of different glycosyltransferases in roots, stems and leaves was analyzed. The glycosyltransferases with high transcriptional expression levels in leaves were selected for functional verification. Finally, two new glycosyltransferase genes, UGT94M2 and UGT94BE1, were mined in the Centella asiatica genome.

[0057] The nucleotide and amino acid sequences of the obtained glycosyltransferase UGT94M2 are shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively;

[0058] The nucleotide and amino acid sequences of the obtained glycosyltransferase UGT94BE1 are shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

[0059] Example 2: Gene cloning and vector construction

[0060] Centella asiatica (L.) Urban plant leaves were collected, and total RNA was extracted from the leaves using the Aidlab EASYspin Universal Plant RNA Rapid Extraction Kit, and reverse transcribed using the Novozymes reverse transcription kit to synthesize cDNA.

[0061] Primer sequences were designed as follows: sequences SEQ ID NO: 5 and SEQ ID NO: 6 were used to amplify UGT94M2, sequences SEQ ID NO: 7 and SEQ ID NO: 8 were used to amplify UGT94BE1, and sequences SEQ ID NO: 9 and SEQ ID NO: 10 were used to amplify UGT73AD1. Using leaf cDNA as template, TOYOBO KOD plus high-fidelity enzyme was used to amplify UGT94M2, UGT94BE1 and UGT73AD1 (GenBank: KP195716.1), respectively. The reaction system was 50 μL: 1.0 μL primer F (10 ng / μL), 1.0 μL primer R (10 ng / μL), 5 μL dNTP mixture, 3.5 μL MgSO4, 5 μL KOD Buffer, 0.5 μL KOD plus polymerase, 1 μL template and 33 μL water. The reaction procedure was as follows: 95°C for 3 min; 95°C for 30 s, 60°C for 30 s, 68°C for 2 min and 30 s, for a total of 35 cycles; 72°C for 5 min. Detection was performed by 1% agarose gel electrophoresis. The results are shown in the table. Figure 2Lane 1 is the UGT94M2 PCR amplification product, lane 2 is the UGT94BE1 PCR amplification product, and lane 3 is the UGT73AD1 PCR amplification product. The PCR amplification product was recovered using the Axygen gel recovery kit. The UGT94M2 gene PCR amplification product obtained in the previous step was digested with restriction endonucleases EcoRI and NotI, the UGT94BE1 gene PCR amplification product was digested with restriction endonucleases BamHI and XhoI, and the UGT73AD1 amplification product obtained in the previous step was digested with EcoRV and NotI. The digested genes were recovered using the Axygen column recovery kit. The PSJ8 vector was double-digested with restriction endonucleases EcoRV, NotI and EcoRI, NotI, respectively, and the pGEX-6P vector was digested with restriction endonucleases BamHI and XhoI to recover the vector backbone. The double-digested gene and vector were connected using T4 ligase at 16°C overnight. The ligation product was transformed into DH5α Escherichia coli competent cells. The transformed bacterial solution was spread on an LB solid plate containing Amp resistance and incubated inverted at 37°C for 12-16 hours. Single colonies were picked for PCR screening of positive clones. The corresponding positive colonies were sent to Jinweizhi for sequencing verification. The sequencing results were compared with the original sequences of the UGT94M2 and UGT73AD1 genes, and the positive bacteria were expanded and cultured for recombinant plasmid extraction. The plasmid extraction experiment was carried out according to the steps of the plasmid extraction kit of Axygen. Finally, the recombinant plasmids PSJ8-UGT94M2, pGEX-6P-UGT94BE1 and PSJ8-UGT73AD1 were obtained.

[0062] Example 3: Expression and purification of glycosyltransferase genes in Escherichia coli

[0063] The extracted plasmids PSJ8-UGT94M2, pGEX-6P-UGT94BE1 and PSJ8-UGT73AD1 were transformed into competent E. coli BL21 (DE3). Similarly, a single clone was picked and positive verification was performed using bacterial solution PCR. The positive bacterial solution was streaked and the single clone grown after streaking was picked and cultured in 1 mL of LB liquid medium containing Amp antibiotics at 37°C overnight with shaking. 3 mL of turbid bacterial solution was transferred to 300 mL of LB liquid medium containing Amp antibiotics and shaken at 37°C until the bacterial solution OD reached 0. 600 When the value reaches 0.6-0.8, add the prepared IPTG (final concentration reaches 0.5mM) for induction. After culturing at 16℃, 160rpm for 20h, collect the bacterial liquid by centrifugation and store it at -80℃.

[0064] Take out the Ni-NTA agarose and gently invert it several times, pipette 1 mL of the mixed Ni-NTA into a 15 mL centrifuge tube, centrifuge at 500 g for 5 min, and gently aspirate the supernatant; continue to add 5 mL of sterile distilled water, gently invert the column at 4°C for 3 min, centrifuge at 500 g for 5 min, and gently aspirate the supernatant; add 5 mL of Binding buffer, gently invert the column at 4°C for 3 min, centrifuge at 500 g for 5 min, and gently aspirate the supernatant; add an equal volume of Binding buffer (1 mL) to the Ni column to make a 50% slurry.

[0065] Take out the bacteria stored at -80℃, add Lysis buffer (10mL), and thaw on ice for half an hour; ultrasonic on ice for 4min (10 times, 10s each time, power not exceeding 200W) until the bacterial solution is no longer viscous. Centrifuge at 4℃, 4000r / min for 40min; add 10mL supernatant after centrifugation to the treated 1mL Ni column, and incubate the mixture at 4℃ for 1-2 hours with low-speed shaking; wash the column with pre-cooled 10mL (2-3 times) 1×Wash Buffer; wash the column with 5mL (2-3 times) 1×Elution Buffer, and collect the effluent (operate on ice). The eluate is further concentrated to protein and impurities, and the purified protein is filtered and concentrated using a 50KDa ultrafiltration tube. Determine the concentration of the purified protein, add an equal volume of 50% glycerol to the successfully purified, concentrated and impurity-removed protein, mix well, and dispense (100μL per tube), and store at -80℃ for later use. Take out a small amount of purified protein and run SDS-PAGE electrophoresis gel detection. The detection picture is as follows Figure 3 As shown, Marker: protein molecular mass standard; Lane 1: PSJ8-UGT73AD1; Lane 2: PSJ8-UGT94M2; Lane 3: pGEX-6P-UGT94BE1.

[0066] Example 4: In vitro enzyme activity reaction and product detection

[0067] After the concentration of purified proteins of PSJ8-UGT94M2, pGEX-6P-UGT94BE1 and PSJ8-UGT73AD1 was determined by BCA method, in vitro enzyme activity experiments were performed. The in vitro enzyme activity reaction system was as follows (200 μL): 500 μM substrate (asiatic acid / madecassic acid), 50 μM sugar donor (UDP-glucose / UDP-Rha), 1 mM DTT and 20 mM Bicine (pH 8.5), 100 μL purified protein, and water was added to 200 μL. The reaction conditions were 37 ° C, 300 rpm, incubation overnight, adding 200 μL methanol to terminate the reaction, centrifugation at 12,000 rpm for 10 min, removing the supernatant, nitrogen blowing, evaporation, adding 200 μL methanol to dissolve, and LC-MS detection and analysis.

[0068] The product components were detected by LC / MS coupling method, using Agilent 1290 high performance liquid chromatography system, chromatographic column Agilent Zorbax Extend C-18 column (250mm×4.6mm, 5μm); column oven temperature was 30℃; mobile phase: pure water (A)-pure acetonitrile (B). The flow rate was 1mL / min; the injection volume was 10μL; the post-run time was 5 minutes. Mass spectrometry analysis conditions: Agilent quadrupole-time of flight mass spectrometer Agilent 6545QTOF MS, set the operating parameters, use positive ion mode to detect samples, set the gradient elution conditions: 0-6min, 10%-30% B; 6-10min, 30%-37% B; 10-20min, 37%-63% B; 20-25min, 63%-90% B; 25-30min, 90% B.

[0069] According to literature reports, asiatic acid / madecassic acid can be catalyzed by UGT73AD1 to produce asiatic acid monoglycoside / madecassic acid monoglycoside. Since asiatic acid monoglycoside / madecassic acid monoglycoside is currently unavailable for purchase and difficult to extract or synthesize, we used asiatic acid monoglycoside / madecassic acid monoglycoside produced by UGT73AD1 and substrate asiatic acid / madecassic acid to react with the next one or two steps of enzymes and perform detection. UGT94M2 and UGT94BE1 glycosyltransferase catalyzed steps such as Figure 1 The liquid phase test results are shown in Figure 4 and Figure 5As shown, UGT73AD1 catalyzes compound 1 (asiatic acid) and compound 5 (madecassoside) to generate compound 2 (asiatic acid monosaccharide) and compound 6 (madecassoside monosaccharide). UGT94M2 can further catalyze compound 2 and compound 6 to generate compound 3 and compound 7. The molecular weight of compound 3 is consistent with that of asiatic acid disaccharide, and the molecular weight of compound 7 is consistent with that of madecassoside disaccharide. UGT94BE1 can catalyze compound 3 and compound 7 to generate compound 4 and compound 8, respectively. The retention time and molecular weight of compound 4 are consistent with those of asiaticoside, and the retention time and molecular weight of compound 8 are consistent with those of madecassoside. Through Q-TOF analysis, the fragment ion peaks of compound 4 and compound 8 are consistent with those of asiaticoside standard and madecassoside standard, respectively. The MS data of compounds 1-8 are shown as follows: Figure 6-Figure 13 shown.

Claims

1. A polypeptide, characterized in that The polypeptide is a polypeptide with an amino acid sequence shown in SEQ ID NO:

4.

2. A polynucleotide, characterized in that The polynucleotide is selected from the group consisting of: (B1) a nucleotide sequence encoding the polypeptide of claim 1; (B2) the sequence shown in SEQ ID NO: 2; (B3) A nucleotide sequence complementary to the nucleotide sequence described in any one of (B1) to (B2).

3. A biological material, wherein the biological material is any one of the following (C1) to (C4): (C1) an expression cassette comprising the polynucleotide of claim 2; (C2) a recombinant vector containing the polynucleotide of claim 2, and a recombinant vector containing the expression cassette of (C1); (C3) a recombinant microorganism containing the polynucleotide of claim 2, a recombinant microorganism containing the expression cassette of (C1), or a recombinant microorganism containing the recombinant vector of (C2); (C4) A genetically engineered host cell containing the polynucleotide of claim 2, a genetically engineered host cell containing the expression cassette of (C1), or a genetically engineered host cell containing the recombinant vector of (C2).

4. Use of the polypeptide according to claim 1 in catalyzing one or more of the following reactions: (D1) catalyzes the production of asiaticoside from asiatic acid disaccharide; (D2) catalyzes the production of madecassoside from madecassoside.

5. Use of the polypeptide according to claim 1, the polynucleotide according to claim 2, or the biomaterial according to claim 3 in preparing an enzyme catalyst in one or more of the following reactions: (D1) catalyzes the production of asiaticoside from asiatic acid disaccharide; (D2) catalyzes the production of madecassoside from madecassoside.

6. Use of the polypeptide according to claim 1, the polynucleotide according to claim 2, or the biomaterial according to claim 3 in the preparation of asiaticaoside or madecassoside.

7. A method for producing the polypeptide according to claim 1, characterized in that The method comprises using the coding gene of the polypeptide according to claim 1 to express in an organism to obtain the polypeptide, and the organism is a microorganism.

8. An in vitro glycosylation method, characterized in that The method comprises the following steps: in the presence of a glycosyltransferase, transferring the glycosyl of a glycosyl donor to the C28 site of asiatic acid disaccharide or madecassoside, thereby forming asiaticoside or madecassoside; wherein the glycosyltransferase is selected from the glycosyltransferase shown in SEQ ID NO: 4.

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