A mutant of udp-glycosyltransferase for the exclusive synthesis of rhodioloside or icariside d2

Abstract

The application discloses a UDP-glycosyltransferase mutant for specifically synthesizing rhodiolin or icariside D2, and belongs to the technical field of enzyme engineering. BL 1-A235E, UGT BL 1-A235N, UGT BL The regional selectivity of the 1-A235W crude enzyme solution within 10 hours is 100% phenolic hydroxyl group regional selectivity, and the WT is only 50.87%. BL The alcohol hydroxyl group regional selectivity of the 1-G233V is 100%. Compared with the WT, the mutants have a significant improvement in the regional selectivity, can realize precise tyrosol glycosylation, can be used for synthesizing glycoside compounds with specific structures and functions, and further promote the development of related research and application.

Description

Technical Field

[0001] This invention relates to a UDP-glycosyltransferase mutant for the specific synthesis of rhodioloside or icariin D2, and belongs to the field of enzyme engineering technology. Background Technology

[0002] Salidroside, chemical name: 2-[4-hydroxyphenyl]ethyl β-D-glucopyranoside, molecular formula C 14 H 20 O7, CAS No. 10338-51-9, as a rare glycoside compound, has shown significant efficacy in areas such as nervous system regulation, anti-fatigue, prevention and treatment of altitude sickness, and cardiovascular protection. Its anti-tumor and radiation protection properties have made it a hot topic in modern pharmaceutical research and development. According to a MarketWatch report, the global market size of rhodioloside has an annual growth rate of 8.7%, exceeding $320 million in 2023. Icariside D2, chemical name: 4-β-D-glucosyloxyphenylethanol, molecular formula C 14 H 20 O7, CAS No. 38954-02-8, is a key component for bone metabolism regulation and has unique advantages in the treatment of osteoporosis and immunomodulation. Its global clinical demand is increasing at an average annual rate of 12%.

[0003] Current industrial production of rhodioloside relies on extraction from plants of the genus *Rhodiola rosea* L., but this species has a long growth cycle of 4-5 years and an effective component content of less than 0.3%, resulting in raw material costs accounting for over 60% of the product's cost. Furthermore, due to the similar polarity of polyphenols to rhodioloside, polyphenols may also be extracted simultaneously during the extraction process, complicating subsequent purification. HPLC analysis shows 7-9 impurity peaks in the product. Current industrial production of icariin D2 mainly relies on chemical synthesis, which requires eight reaction steps. The yield of the key glycosidic bond construction step is only 28%-35%, and the use of heavy metal catalysts (such as Pd / C) results in metal residues in the final product reaching 50-80 ppm, far exceeding the ICH Q3D standard.

[0004] Microbial synthesis systems have emerged as novel preparation methods due to their environmental friendliness and process controllability. UDP-glycosyltransferase (UGT) is the core glycosylation enzyme in the biosynthesis of rhodioloside and icariin D2. It catalyzes the formation of rhodioloside by transferring glucose residues from UDP-glucose to the hydroxyl position of tyrosol, and catalyzes the formation of icariin D2 by transferring glucose residues to the phenolic hydroxyl position of tyrosol.

[0005] However, the performance of UDP-glycosyltransferases still restricts the industrialization process of rhodioloside and icariin D2. Specifically, (1) low catalytic efficiency: Glycosylation is the rate-limiting step in the biosynthesis of rhodioloside and icariin D2, and its catalytic efficiency directly affects the product yield. Existing enzyme libraries (such as UGT76G1, UGT78K1) have limited K-type catalytic efficiency for tyrosol. m Values ​​are generally higher than 5 mM, catalytic efficiency (kJ / mM) cat / K m <50s -1 ·M -1 .

[0006] (2) Poor substrate specificity: The synthesis of icariin D2 using UDP-glycosyltransferase requires precise C-7 glycosylation, but conventional UDP-glycosyltransferases have insufficient regioselectivity for tyrosine hydroxyl groups. In addition, since rhodioloside and icariin D2 have similar polarity, the cost of separating and purifying the two using existing technologies (chromatographic separation or supercritical fluid extraction) to obtain a single product is relatively high.

[0007] In existing research, the enzymes that catalyze the in vitro tyrosol glycosylation are mainly UGT1 family proteins: UGT85A1, UGT73B6, UGT72B14, RrUGT17, and UGT. BL 1, of which wild-type UGT BL The 1-peptide tyrosol exhibits the highest conversion rate (86.1%) and has significant application potential. The catalytic processes of members of the UGT1 (EC 2.4.1) family typically follow a double substitution mechanism (SN2-like), exhibiting a distinct transition state (UDP-G-tyrosol complex) compared to traditional SN2 catalysis. This family of proteins possesses a highly conserved catalytic binary, such as His-Asp or His-Glu, which interact to form an acid-base pair. His then increases nucleophilicity by deprotonating specific hydroxyl groups on the acceptor molecule. The deprotonated hydroxyl group (O-) then acts as a nucleophile, attacking the C- group on the glycogen (UDP-G), thereby achieving glycosyl group transfer. However, when the substrate has multiple hydroxyl functional groups, it can lead to inconsistent products. For example, the two nucleophilic attack sites of tyrosol correspond to rhodioloside and icariin D2, respectively, and the two products have similar polarities, increasing the cost of separation and purification.

[0008] Existing technologies for modifying UGT1 primarily focus on improving the regioselectivity of the alcohol hydroxyl groups in the synthesis of rhodioloside, often at the expense of the yield of its phenolic hydroxyl glycosylation product, icariin D2. Compared to rhodioloside, icariin D2 is currently difficult to produce on a large scale, with a market price as high as ¥1060 / mg, far exceeding that of rhodioloside. Furthermore, although the regioselectivity of tyrosol can be altered through the combined effects of channel modification and multiple sequence alignment mutations, achieving selectivity of 48%-99% (rhodioloside) or 52%-99% (icariin D2), complete synthesis of a single product remains difficult, and the yield of icariin D2 is only increased to 1.4 times that of the wild type. Summary of the Invention

[0009] [Technical Issues]

[0010] The technical problem to be solved by this invention is to directionally switch the regioselectivity of UDP-glycosyltransferase, so that UDP-glycosyltransferase specifically catalyzes the synthesis of rhodioloside or icariin D2, thereby improving the catalytic efficiency of rhodioloside and icariin D2 and saving industrial production costs.

[0011] [Technical Solution]

[0012] This invention, starting from the different polarities of the two nucleophilic attack sites of tyrosol, utilizes molecular docking combined with virtual saturation mutagenesis to assist in designing intermolecular interaction forces, providing a UDP-glycosyltransferase mutant with directional switching of regioselectivity. The amino acid sequence of the UDP-glycosyltransferase mutant is mutated to V and E / N / W at positions 233 and 235 respectively, relative to SEQ ID NO.1 (i.e., glycine at position 233 is mutated to valine; alanine at position 235 is mutated to glutamic acid, asparagine, or tryptophan, respectively, and named UGT). BL 1-G233V, UGT BL 1-A235E, UGT BL 1-A235N, UGT BL 1-A235W.

[0013] In one embodiment of the present invention, the parent of the UDP-glycosyltransferase mutant is derived from Bacillus licheniformis ZSP01.

[0014] The present invention also provides a gene sequence encoding the mutant.

[0015] The present invention also provides an expression vector carrying the gene.

[0016] In one embodiment of the present invention, the expression vector includes the pET 28a(+) vector.

[0017] The present invention also provides an engineered Escherichia coli strain expressing the mutant.

[0018] In one embodiment of the invention, Escherichia coli BL 21 is the host cell and pET 28a(+) is the expression vector.

[0019] This invention also provides a method for preparing UDP-glycosyltransferase mutants, comprising the following steps:

[0020] (1) The gene sequence is ligated into an expression vector to obtain a recombinant expression vector.

[0021] (2) The recombinant expression vector was transferred into the host, and the host was cultured to express the UDP-glycosyltransferase mutant.

[0022] (3) The UDP-glycosyltransferase mutant was isolated and purified from the host culture medium.

[0023] This invention also provides a method for preparing salidroside or icariside D2, using a mutant as a catalyst and uridine diphosphate-glucose (UDPG) and tyrosol as substrates, and carrying out the reaction under suitable conditions. These suitable conditions refer to conditions conducive to the enzyme's catalytic activity, such as the optimal pH, temperature, and substrate concentration for the enzyme's reaction.

[0024] In one embodiment of the present invention, the reaction is carried out at 30°C for 10 minutes.

[0025] The present invention also provides the application of the mutant, the gene, the expression vector, or the engineered Escherichia coli in the preparation of rhodioloside or icariin D2.

[0026] [Beneficial Effects]

[0027] This invention utilizes UGT BL A semi-rational design and single-point mutagenesis yielded a regioselective mutant, enabling directional switching of the complete catalytic synthesis of rhodioloside and icariin D2 as single products, and significantly increasing the yield of icariin D2 to twice that of the wild type. This breakthrough addresses the shortcomings of existing technologies, provides a practical solution for industrial production, greatly reduces separation and purification costs, and enhances market competitiveness.

[0028] UGT BL 1-G233V exhibits 100% regioselectivity of the alcohol hydroxyl group, with a relative conversion of rhodioloside of 167% to WT within 10 min; UGT BL 1-A235E, UGTBL 1-A235N, UGT BL The phenolic hydroxyl group regioselectivity of 1-A235W was 100%, and the relative conversion rates of icariin D2 were 130%, 120%, and 156% of that of WT. The above mutants achieved the transformation synthesis of a single product.

[0029] Existing modifications of UDP-glycosyltransferases to target the tyrosine hydroxyl region often sacrifice enzyme activity to improve the selectivity of icariin D2. The variant UGT provided in this invention... BL 1-A235E, UGT BL 1-A235N, UGT BL 1-A235W further achieved a catalytic efficiency increase of up to 56% based on the regioselectivity of phenolic hydroxyl groups.

[0030] The cost of enzymatic production of rhodioloside and icariin D2 is reduced by 30%-50% compared to traditional methods, demonstrating a significant price advantage. Furthermore, the enzyme modified in this invention, when used to synthesize rhodioloside and icariin D2, achieves a product purity of over 99%, further reducing industrial production costs. Attached Figure Description

[0031] Figure 1 Relative conversion rate bar chart;

[0032] Figure 2 Line graph of icariin D2 yield versus reaction time;

[0033] Figure 3 Rhodioloside yield-reaction time line graph;

[0034] Figure 4 Figure 1 shows the liquid phase detection results of products catalyzed by different mutant enzymes. Detailed Implementation

[0035] Regarding terminology

[0036] In this invention, the term "directional switching" refers to the precise control of the catalytic site of an enzyme substrate through specific enzyme modification technology, enabling the reaction to switch from bidirectional to unidirectional, thereby improving the selectivity and yield of a single target product.

[0037] In this invention, the term "regioselectivity" refers to the selective action of an enzyme on a specific region of the substrate during a catalytic reaction, which preferentially acts on a specific location or region of the substrate, thereby improving the purity and quality of a single product.

[0038] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0039] The DNA polymerase (2×Super Pfu PCR Mix) used in this embodiment of the invention was purchased from Hangzhou Baosai Biotechnology Co., Ltd.; Dpn I enzyme, recombinant cloning kit and plasmid extraction kit were purchased from Nanjing Novizan Biotechnology Co., Ltd.; gene and primer synthesis and gene sequencing were performed by General Shanghai Sangon Biotech Co., Ltd., and the usage of the above reagents is as described in the product manual.

[0040] All tyrosol, rhodioloside standards, and UDP-glucose used in this invention were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0041] The LB medium used in the following examples consists of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride (NaCl), pH 7.4; LB liquid medium with 2% agar added is LB solid medium.

[0042] The PB buffer used in the following examples: 2.30g Na2HPO4, 0.46g NaH2PO4, adjusted to 1L.

[0043] The tris-HCl (50mM) buffer used in the following examples: 6.06g of tris(hydroxymethyl)aminomethane, brought to a final volume of 1L, and pH adjusted to 8.0.

[0044] Solution A (equilibration solution) used in the following examples: 0.34 g / L imidazole (5 mM) and 29.22 g / L NaCl (0.5 M) were added to the above PB buffer solution to adjust the pH to 8.0.

[0045] The B solution (elution buffer) used in the following examples is as follows: 34.08 g / L imidazole (500 mM) and 29.22 g / L NaCl (0.5 M) are added to the above PB buffer to adjust the pH to 8.0.

[0046] The enzyme activity assay method used in the following examples was as follows: The enzyme was incubated at 30°C for 10 min in 100 μL of 50 mM tris-HCl buffer (pH 8.0) containing 5 mM tyrosol, 10 mM UDP-glucose (UDP-G).

[0047] Enzyme activity is defined as the amount of enzyme required to convert 1 μmol of tyrosol within 1 minute, which is one unit of enzyme activity (U).

[0048] Enzyme activity calculation: Enzyme activity (U / mg) = Enzyme activity (U) / Protein content (mg)

[0049] The determination method of the product in the following examples: The determination of tyrosol glucoside was performed using a C18 column, with water (A) and acetonitrile (B) as the mobile phase. The elution was linear: 0-2 min 30% B, 2-3 min 30%-70% B, 3-6 min 30% B, 6-7 min 70%-30% B, and 7-12 min 30% B. The flow rate was 1 ml / min, and the detector was a UV detector with a detection wavelength of 272 nm.

[0050] The formula for calculating the "relative conversion rate" in the following examples is:

[0051] Relative conversion rate (%) = (Rhodioloside or Icariin D2 yield of mutant (mM) / (Rhodioloside or Icariin D2 yield of wild type (mM)) * 100%

[0052] The formula for calculating the "crude enzyme conversion rate of tyrosol" in the following examples is:

[0053] Crude tyrosol conversion rate (%) = [Initial tyrosol catalytic concentration (mM) - Residual tyrosol concentration after catalysis (mM)] / Initial tyrosol catalytic concentration (mM) * 100%

[0054] The formula for calculating the "regioselectivity of alcohol or phenolic hydroxyl groups" in the following examples is:

[0055] Alcohol or phenolic hydroxyl regioselectivity (%) = Rhodioloside (alcohol) or Icariin D2 (phenol) yield (mM) / Tyrosol consumption (mM) * 100%

[0056] Example 1: UDP-glycosyltransferase UGT BL Construction of 1 and its mutants

[0057] (1) Construction of recombinant plasmids containing genes encoding UDP-glycosyltransferase

[0058] The UDP-glycosyltransferase derived from Bacillus licheniformis ZSP01 was selected as the wild type. The amino acid sequence of the wild type enzyme is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the wild type is shown in SEQ ID NO.2.

[0059] SEQ ID NO.1:

[0060] MGHKHIAIFNIPAHGHINPTLALTASLVKRGYRVTYPVTDEFVKAVEETGAEPLNYRSTLNIDPQQIRELMKNKKDMTQAPMMFMKEMEEVLPQLEALYENDKPDLILFDFMAMAGKMLAEKFGIEAVRLCSTYAQNEHFSFKSMSEEFKIELTPEQEAALKNANLPSFNFEEMFEPAKLNIVFMPRAFQPYGETFDERFSFVGPSLAKRKFQEKDTPVISDSGRPVMLISLGTAFNAWPEFYHMCIEAFRDTKWQVIMAVGTTIDPESFDDIPDNFSIHQRVPQLEILKKAELFITHGGMNSTMEGLNAGVPLVAVPQMPEQEITARRVEELGLGKHLQPEDTTVASLREAVSQTDGNLDVLKRVKDMQEHIKQAGGAEKAADEIESFLAPAGVK

[0061] SEQ ID NO.2:

[0062]

[0063] EcoRI and XhoI on pET28a were selected as insertion sites for the target gene, and the target gene was amplified using the following primers: upstream primer (SEQ ID NO.3): GCGGATCCGAATTCATGGGACATAAACATAT, downstream primer (SEQ ID NO.4): GTGGTGGTGGTGCTCGATTT TACTCCTGCGGGTGC, thus imbuing the target gene with restriction enzyme sites. pET28a was double-digested and purified to obtain a linear plasmid fragment. The target gene with restriction sites was then ligated to the linear plasmid fragment via homologous recombination. The system used for homologous recombination contained: 1 μL of digested linear plasmid, 1 μL of the target fragment, 2 μL of 5×Reaction Buffer, and 1 μL of... Add recombinase and 5 μL H2O. Homologous recombination was performed at 37 °C for 30 min. Then, the ligation product was stored at -20 °C.

[0064] The ligation product was converted into Escherichia coli DE3 ( BL 21) Competent cells were cultured at 37°C for 1 hour, then spread onto LB agar plates containing kanamycin-resistant bacteria. After overnight culture, single colonies were picked and colony PCR was performed using universal primers. Plasmids with positive PCR results were sequenced for verification, ultimately yielding the correct UDP-glycosyltransferase gene recombinant plasmid WT-UGT. BL 1-pET28a.

[0065] (2) Construction of recombinant plasmids of UDP-glycosyltransferase mutants

[0066] With WT-UGT BL Using the 1-pET28a plasmid as a template, full-plasmid PCR was performed using the primers listed in Table 1 to introduce the six mutation sites. The recombinant plasmid was digested with DpnI enzyme at 37°C for 2 hours and then transformed into *E. coli* using the heat shock method. BL Single colonies were selected from 21(DE3) competent cells and cultured overnight in LB medium at 37°C and 200 rpm. Plasmids were extracted from the single colony cultures and sent to a sequencing company for sequencing. The mutants with correct sequencing results were the successfully mutated mutants.

[0067] Table 1 Primers for mutant construction

[0068]

[0069] (3) Induction and purification of the target protein

[0070] The correctly sequenced single-clone strains were activated by shaking in LB medium for 12 h, then inoculated into fresh LB medium and cultured at 37°C and 200 rpm until the OD reached 0.5. IPTG inducer was added to a final concentration of 1 mM, and the culture was continued at 16°C and 200 rpm for 24 h. After culture, the precipitate was collected by centrifugation, and the supernatant was obtained after cell disruption by centrifugation to obtain the crude enzyme solution. The specific enzyme activity of the wild-type crude enzyme solution was 1380 U / mg, and the specific enzyme activities of the crude enzyme solutions of each mutant were UGT. BL 1-G233V: 1376U / mg, UGT BL 1-A235E: 1352 U / mg, UGT BL 1-A235N: 1298 U / mg, UGT BL 1-A235W: 1400U / mg, UGT BL 1-M320Y: 1831 U / mg. Pure enzyme was obtained using nickel column purification.

[0071] The method for nickel column purification is as follows:

[0072] First, equilibrate the nickel column with equilibration solution A, then accelerate the reaction at a rate of 1 mL / min. -1 The sample was injected at a flow rate of [flow rate missing]. After injection, the column was washed again with solution A for 10 min. Then, the mixing ratio of solution B and solution A was adjusted to achieve an imidazole concentration of 100 mM in the eluent. Impurities were then eluted with the eluent. The imidazole concentration in the eluent was then adjusted to 200 mM, and the target protein was eluted and collected. The collected eluent containing the target protein (wild-type, UDP-glycosyltransferase mutant) was desalted and concentrated using an ultrafiltration tube. The concentrate was stored at 4°C for subsequent experiments.

[0073] Example 2: Comparison of tyrosol regioselectivity

[0074] Add 10 mM UDP-G and 5 mM tyrosol to 100 μL tris-HCl buffer (pH 8.0), followed by the addition of wild-type UDP-glycosyltransferase UGT purified by a 0.5 g / L nickel column. BL 1. The concentrated solution of the above mutant was reacted at 30°C for 10 min, and the reaction was terminated by adding 100 μL of acetonitrile. The contents of icariin D2, rhodioloside, and tyrosol were determined. The relative conversion rates of icariin D2 and rhodioloside during the reaction are shown in the figure. Figure 1 As shown.

[0075] Among them, UGT BL 1-A235E, UGT BL 1-A235N, UGT BL 1-A235W and UGT BLThe phenolic hydroxyl region selectivity of the four enzyme elements of 1-M320Y was 100%, 100%, 100%, and 74.36% respectively within 10 min.

[0076] UGT BL 1-G233V exhibits 100% regioselectivity of alcohol hydroxyl groups, and its pure enzyme solution yields 1.67 times that of the wild type.

[0077] Example 3: Synthesis of Tyrosol Glucoside using UDP-glycosyltransferase

[0078] (1) Wild type: 10 mM UDP-G and 5 mM tyrosol were added to a 10 mL phosphate buffer (pH 6.5) system, and 1 g / L wild type UDP-glycosyltransferase UGT was added to each. BL 1. Crude enzyme solution was reacted at 30℃ for 10 h. 100 μl samples were taken at different reaction time points to determine the contents of icariin D2, rhodioloside, and tyrosol. The synthesis amounts of icariin D2 and rhodioloside during the reaction are shown below. Figure 2 and Figure 3 As shown. After 10 h of reaction, the synthesis amounts of icariin D2 and rhodioloside were 0.70 mM and 0.57 mM, respectively, the crude enzyme conversion rate of tyrosol was 27.6%, and the regioselectivity of alcoholic and phenolic hydroxyl groups were 49.13% and 50.87%, respectively.

[0079] (2)UGT BL 1-G233V: using the mutant UGT BL The 1-G233V mutant was used to replace the wild type in the experiment of Example (1). The results showed that the mutant synthesized icariin D2 and rhodioloside in amounts of 0 mM and 1.38 mM, respectively, with a crude enzyme conversion rate of tyrosol of 27.52% and a regioselectivity of hydroxyl groups of 100%. Figure 4 ).

[0080] (3) UGT BL 1-A235E: Using the mutant UGT BL The 1-A235E mutant was used to replace the wild type in the experiment of Example (1). The results showed that the mutant synthesized 1.35 mM and 0 mM of icariin D2 and rhodioloside, respectively, the crude enzyme conversion rate of tyrosol was 27.04%, and the regioselectivity of phenolic hydroxyl groups was 100%. Figure 4 ).

[0081] (4) UGT BL 1-A235N: Using the mutant UGT BLThe 1-A235N mutant was used to replace the wild type in the experiment of Example (1). The results showed that the mutant synthesized 1.30 mM and 0 mM of icariin D2 and rhodioloside, respectively, the crude enzyme conversion rate of tyrosol was 25.96%, and the regioselectivity of phenolic hydroxyl groups was 100%. Figure 4 ).

[0082] (5) UGT BL 1-A235W: Using the mutant UGT BL The 1-A235W mutant was used to replace the wild type in the experiment of Example (1). The results showed that the mutant synthesized 1.40 mM and 0 mM of icariin D2 and rhodioloside, respectively, the crude enzyme conversion rate of tyrosol was 28%, and the regioselectivity of phenolic hydroxyl groups was 100%. Figure 4 ).

[0083] The results showed that several variants at site A235 exhibited absolute phenolic hydroxyl regioselectivity in the regioselectivity for catalyzing tyrosol glucose glycosylation, and had little impact on tyrosol conversion compared to the wild type. In fact, the yield of icariin D2 in A235W was twice that of the wild type, the highest reported to date. Variant UGT BL 1-G233V exhibits absolute regioselectivity of alcohol hydroxyl groups, and its final rhodioloside yield is 1.38 mM, which is 2.42 times that of the wild type.

[0084] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A UDP-glycosyltransferase mutant, characterized in that, Rhodioloside or icariin D2 can be specifically catalyzed and synthesized using uridine diphosphate-glucose and tyrosol as substrates. in, The UDP-glycosyltransferase mutant capable of specifically catalyzing the synthesis of rhodioloside has an amino acid sequence obtained by mutating glycine at position 233 of the amino acid sequence shown in SEQ ID NO.1 to valine. A UDP-glycosyltransferase mutant capable of specifically catalyzing the synthesis of icariin D2: its amino acid sequence is obtained by mutating alanine at position 235 of the amino acid sequence shown in SEQ ID NO. 1 to glutamic acid, asparagine, or tryptophan.

2. The gene encoding the UDP-glycosyltransferase mutant of claim 1.

3. An expression vector carrying the gene of claim 2.

4. Engineered bacteria expressing the UDP-glycosyltransferase mutant of claim 1.

5. The engineered bacteria according to claim 4, characterized in that, Escherichia coli is the host.

6. The engineered bacteria according to claim 5, characterized in that, by Escherichia coli BL 21 is the host cell, and pET28a(+) is the expression vector.

7. A method for preparing rhodioloside or icariin D2, characterized in that, Using the mutant described in claim 1 as a catalyst, and uridine diphosphate-glucose and tyrosol as substrates, the reaction is carried out under suitable conditions.

8. The method according to claim 7, characterized in that, React at 30℃ for 10 min.

9. The use of the UDP-glycosyltransferase mutant of claim 1, or the gene of claim 2, or the expression vector of claim 3, or the engineered bacteria of claim 4 in the preparation of rhodioloside or icariin D2.

10. The application according to claim 9, characterized in that, Using the UDP-glycosyltransferase mutant of claim 1 as a catalyst, and uridine diphosphate-glucose and tyrosol as substrates, the reaction is carried out under suitable conditions.

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