Screening and identification of a key enzyme involved in glycosylation modification of triterpenoid saponins and application thereof

Abstract

The application provides a key enzyme involved in glycosylation modification of triterpene saponins, and an application thereof, and belongs to the technical field of genetic engineering. The glycosyltransferase gene is screened based on the omics research of the base plant of traditional Chinese medicine Duanxueliu, i.e. clinopodium gracile, and a pET28a-MBP-CcGT-g51295 prokaryotic expression vector is constructed to successfully obtain the glycosyltransferase. It is found that the glycosyltransferase can catalyze Saikogenin A and Saikogenin F to generate Prosaikogenin A and Prosaikogenin F respectively by using a glycosyl donor UDP-fucose; can catalyze Saikogenin A to generate clinopodiside I and clinopodiside X by using UDP-glucose; can catalyze Prosaikogenin A to generate Saikosaponin B1, Saikosaponin c1 and Saikosaponin X by using UDP-glucose; and can catalyze Prosaikogenin F to generate Saikosaponin A and Buddlejasaponin IV by using UDP-glucose, which reveals the catalytic function of the glycosyltransferase and provides a gene component for the biological research of triterpene saponins, and solves the problem of biological resources of rare saponins.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically to the screening, identification and application of a key enzyme involved in the glycosylation modification of triterpenoid saponins. Background Technology

[0002] Triterpenoid saponins are widely found in medicinal plants and are the main active ingredients of many precious traditional Chinese medicines. However, the content of triterpenoid saponins in natural medicinal materials is relatively low, and the high cost of chemical extraction and severe pollution during purification processes limit their potential for new drug development and clinical application. Therefore, the biosynthesis of triterpenoid saponins has become a research hotspot. *Duanxueliu*, one of the "Ten Great Anhui Medicines," possesses significant pharmacological activity and has been developed into a drug; triterpenoid saponins (such as bupleurum saponin and Buddleja saponin) are its important active ingredients.

[0003] Triterpenoid saponins are a class of natural compounds whose basic structure includes a triterpenoid skeleton and a sugar chain. The sugar chain is linked to the triterpenoid skeleton by glycosidic bonds. The type, number, and position of the sugar chain determine the diversity and function of triterpenoid saponins. Glycosyltransferases are enzymes that catalyze glycosylation reactions, responsible for transferring activated sugar donors, such as UDP-fucose, UDP-glucose, and UDP-rhamnose, to the triterpenoid skeleton to form glycosidic bonds. Different glycosyltransferases can recognize different sugar donors and acceptors, resulting in structurally diverse triterpenoid saponins. Glycosylation not only increases the structural complexity of triterpenoid saponins but also significantly affects their physicochemical properties and biological activities. Therefore, glycosyltransferases are crucial for the synthesis of triterpenoid saponins.

[0004] However, research on glycosyltransferases that catalyze the C-3 position fucosylation and glucosylation modification of triterpenoids has not yet yielded breakthroughs, and systematic research is urgently needed.

[0005] Therefore, how to provide a glycosyltransferase that can catalyze the C-3 position fucosylation and glucosylation modification of triterpenoid compounds is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a key enzyme screening, identification and application for participating in the glycosylation modification of triterpenoid saponins. It successfully discovered a glycosyltransferase that can catalyze fucosylation and glucosylation modification at the C-3 position, providing genetic components for the synthetic biology research of triterpenoid saponins and solving the problem of rare saponins in terms of biological resources.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A glycosyltransferase involved in the glycosylation modification of triterpenoid saponins, wherein the glycosyltransferase is composed of the amino acid sequence shown in SEQ ID NO. 6 or an amino acid sequence shown in SEQ ID NO. 6 that retains the same enzymatic catalytic performance after substitution, deletion and / or addition of one or more amino acids.

[0009] Another object of the present invention is to provide a gene encoding the above-mentioned glycosyltransferase, said gene being composed of a nucleotide sequence of SEQ ID No. 3 or a nucleotide sequence of SEQ ID No. 3 that, after substitution, deletion and / or addition of one or more bases, still has the same enzymatic catalytic performance.

[0010] Another object of the present invention is to provide a primer set for amplifying the above-mentioned glycosyltransferase gene, said primer set comprising CcGT-g51295-F and CcGT-g51295-R, the specific sequences of which are as follows:

[0011] CcGT-g51295-F: 5'-ATGGCGATCAATGGAAAACCGCCTC-3', SEQ IDNo.1;

[0012] CcGT-g51295-R: 5'-TCACTGGACTAGGATTTGAGAAGAAATGTC-3', SEQ ID No. 2.

[0013] Another object of the present invention is to provide a biological material containing the above-mentioned glycosyltransferase gene, wherein the biological material is a recombinant vector, expression cassette, transgenic cell line or recombinant bacteria.

[0014] Another object of the present invention is to provide the application of the above-mentioned glycosyltransferase or the above-mentioned gene encoding the glycosyltransferase or the above-mentioned biological material, wherein the application is in any of the following directions:

[0015] (1) Application in the preparation of glycosyltransferases and / or products containing glycosyltransferases;

[0016] (2) Application in the preparation of glycosyltransferase mutants and / or products containing glycosyltransferase mutants;

[0017] (3) Application in the preparation of recombinant glycosyltransferases and / or related products containing recombinant glycosyltransferases;

[0018] (4) Application in the C-3 position fucosylation and glucosylation modification of triterpenoid saponins;

[0019] (5) Application in the glycosylation modification of triterpenoid saponins;

[0020] (6) Application in the biosynthesis of triterpenoid saponins.

[0021] Preferably, in the application of steps (4)-(6), the glycosyltransferase utilizes the glycosyl donor UDP-fucose to catalyze the generation of Prosaikogenin A and Prosaikogenin F from Saikogenin A and Saikogenin F, respectively;

[0022] The synthesis of clinopodiside I and clinopodiside X from Saikogenin A was catalyzed by UDP-glucose.

[0023] Prosaikogenin A was converted into Saikosaponin B1, Saikosaponin c1 and Saikosaponin X using UDP-glucose catalysis;

[0024] Prosaikogenin F was converted to Saikosaponin A and Buddlejasaponin IV using UDP-glucose catalysis.

[0025] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0026] This invention utilizes liquid chromatography-mass spectrometry (LC-MS) to detect the differences in the content of eight target components in the roots, stems, and leaves of *Gnaphalium affine*. Then, based on the *Gnaphalium affine* genome, members of the *Gnaphalium affine* glycosyltransferase (GT) gene family were identified, and a phylogenetic tree was constructed to analyze the evolutionary relationship between *Gnaphalium affine* and GT genes in other species, initially screening for key enzymes involved in the glycosylation modification of triterpenoid saponins. Finally, based on transcriptome data from different tissues of *Gnaphalium affine*, differential gene expression was analyzed to further screen for key enzyme genes involved in the glycosylation modification of triterpenoid saponins. The above method successfully screened glycosyltransferase genes.

[0027] Using the above-mentioned gene, the prokaryotic expression vector pET28a-MBP-CcGT-g51295 was constructed, and glycosyltransferases were successfully obtained. The catalytic function of the enzymes was verified in vitro using Saikogenin A, Saikogenin F, Prosaikogenin A, and Prosaikogenin F as substrates. The study found that the glycosyltransferases of this invention can utilize the glycosyl donor UDP-fucose to catalyze the generation of Prosaikogenin A and Prosaikogenin F from Saikogenin A and Saikogenin F, respectively; utilize UDP-glucose to catalyze the generation of clinopodiside I and clinopodiside X from Saikogenin A; utilize UDP-glucose to catalyze the generation of Saikosaponin B1, Saikosaponin c1, and Saikosaponin X from Prosaikogenin A; and utilize UDP-glucose to catalyze the generation of Saikosaponin A and Buddlejasaponin IV from Prosaikogenin F. In summary, the glycosyltransferase of this invention catalyzes the C-3 fucosylation and glucosylation modification of triterpenoid compounds, providing genetic components for the synthetic biology research of triterpenoid saponins and solving the problem of rare saponins in terms of biological resources. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0029] Figure 1 Metabolomics study of triterpenoid saponins in different tissues of *Gnaphalium affine*; 1A-1F represent the contents of triterpenoid saponins in different tissues of *Gnaphalium affine*.

[0030] Figure 2 The results are as follows: Screening and phylogenetic analysis of key glycosyltransferases for the biosynthesis of triterpenoid saponins in *Gnaphalium affine*; 2A shows 11 candidate genes screened in Example 2; 2B is the ML phylogenetic tree.

[0031] Figure 3The following are the in vitro functional verification results of CcGT-g51295 catalyzing the conversion of Saikogenin A and Saikogenin F from Saikogenin A and Saikogenin F, respectively; 3A and 3B are the circuit diagrams of CcGT-g51295 catalyzing the conversion of Saikogenin A and Saikogenin F from Saikogenin A and Saikogenin F, respectively; 3C and 3D are the mass spectrometry detection results of CcGT-g51295 catalyzing the conversion of Saikogenin A and Saikogenin F from Saikogenin A and Saikogenin F, respectively; 3E and 3F are the mass spectrometry fragmentation patterns of the catalytic products Prosaikogenin A and Prosaikogenin F, respectively.

[0032] Figure 4 For: In vitro functional verification of CcGT-g51295 catalysis for the formation of clinopodiside I and clinopodiside X from Saikogenin A; 4A, catalytic roadmap; 4B, liquid phase detection results.

[0033] Figure 5 For: clinopodiside I 1 H NMR and 13 10⁻⁵ C NMR mass spectrometry results; 5A, clinopodiside I 1 1H NMR mass spectrometry results (600MHz, measured inpyridine-d5); 5B, clinopodiside I 13 CNMR mass spectrometry results (150 MHz, measured in pyridine-d5).

[0034] Figure 6 The results are as follows: HSQC and HMBC mass spectrometry detection results of clinopodiside I; 6A, HSQC mass spectrometry detection results of clinopodiside I (measured inpyridine-d5); 6B, HMBC mass spectrometry detection results of clinopodiside I (measured inpyridine-d5).

[0035] Figure 7 For: clinopodiside I's NOESY and 1 H- 17A, NOESY mass spectrometry results of clinopodiside I (measured in pyridine-d5); 7B, NOESY mass spectrometry results of clinopodiside I. 1 H- 1 H COSY mass spectrometry results (measured inpyridine-d5).

[0036] Figure 8 For: clinopodiside X 1 H NMR and 13 12C NMR mass spectrometry results; 8A, clinopodiside I 1 1H NMR mass spectrometry results (600MHz, measured inpyridine-d5); 8B, clinopodiside I 13 CNMR mass spectrometry results (150 MHz, measured in pyridine-d5).

[0037] Figure 9 9A, HSQC and HMBC mass spectrometry results of clinopodiside X; 9B, HSQC mass spectrometry results of clinopodiside I (measured in pyridine-d5); 9C, HMBC mass spectrometry results of clinopodiside I (measured in pyridine-d5).

[0038] Figure 10 For: clinopodiside X's NOESY and 1 H- 1 10A, NOESY mass spectrometry results for clinopodiside I (measured in pyridine-d5); 10B, NOESY mass spectrometry results for clinopodiside I. 1 H- 1 H COSY mass spectrometry results (measured inpyridine-d5).

[0039] Figure 11 For: In vitro functional verification of the formation of Saikosaponin B1, Saikosaponinc1 and SaikosaponinX from Prosaikogenin A catalyzed by CcGT-g51295; 11A, catalytic roadmap; 11B, liquid phase detection results; C, mass spectrometry fragmentation pattern of catalytic products.

[0040] Figure 12 The figures are: 12A and 12B, which are circuit diagrams of CcGT-g51295 catalyzing the formation of Saikosaponin A and Buddlejasaponin IV from Prosaikogenin F; 12C and 12D, which are mass spectrometry results of CcGT-g51295 catalyzing the formation of Saikosaponin A and Buddlejasaponin IV from Prosaikogenin F; and 12E and 12F, which are mass spectrometry fragmentation patterns of the catalytic products Saikosaponin A and Buddlejasaponin IV. Detailed Implementation

[0041] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0042] Example 1

[0043] Metabolomics study of triterpenoid saponins from different tissues of *Gnaphalium affine*

[0044] Samples of roots, stems, and leaves of *Saikosaponin* stored at -80℃ were removed and placed in liquid nitrogen, ground into a fine powder, and 1.0 g was quickly weighed into a centrifuge tube. 10 mL of methanol was added for ultrasonic extraction. After concentration, 1.0 mL of methanol was added to redissolve the extract. The relative contents of eight compounds (Saikogenin A, Saikogenin F, Prosaikogenin A, Prosaikogenin F, Saikosaponin B1, Saikosaponin A, Buddlejasaponin IV, and Buddlejasaponin IVb) in various tissues of *Saikosaponin* were determined and analyzed using liquid chromatography-mass spectrometry (LC-MS).

[0045] The measurement conditions were as follows: the instruments were an Agilent Technologies 1290 Infinity II and a 6545Q-TOF liquid chromatography-mass spectrometry system; the measurement was performed using Waters Acquity... BEH C18 column (1.7μm, 100×2.1mm) chromatographic column, column temperature 40℃;

[0046] Chromatographic conditions: Mobile phase: A-water (containing 0.1% formic acid), B-acetonitrile (containing 0.1% formic acid), flow rate: 0.2 mL / min, elution program: 0-5 min (25% B to 40% B), 5-13 min (40% B to 42.5% B), 13-14 min (42.5% B to 100% B), 14-20 min (100% B), 20-21 min (100% B to 25% B), 21-26 min (25% B);

[0047] Mass spectrometry conditions: dry gas temperature 350℃ (flow rate 8.0 L / min), sheath gas temperature 250℃ (flow rate 11.0 L / min), nebulizer 45 psig, VCap 4000 V;

[0048] The relative contents of eight compounds in different tissues were compared and analyzed using Agilent MassHunter Qualitative Analysis 10.0.

[0049] Results analysis: The results showed that Buddlejasaponin IVb was found in high concentrations in the leaves of *Buddlejasaponin var. fasciata*, and its biosynthetic precursors Prosaikogenin A and Saikosaponin B1 were present in the roots of *Buddlejasaponin var. fasciata*. Figure 1 A-1C); Buddlejasaponin IV is found in higher concentrations in the leaves of *Sedum aizoon*, while its direct biosynthetic precursor Saikosaponin A mainly accumulates in the leaves, and its indirect biosynthetic precursor Prosaikogenin F is found in higher concentrations in the roots of *Sedum aizoon*. Figure 1 D-1F); Saikogenin A and Saikogenin F were not detected.

[0050] Example 2

[0051] Based on multi-omics research on *Gnaphalium affine*, the gene encoding the key CcGT enzyme in the biosynthesis of triterpenoid saponins was screened.

[0052] Based on the genome data of *Arabidopsis thaliana*, UGT protein sequences from the TAIR database were extracted and BLASTP sequences were used to identify homologous genes in *Arabidopsis thaliana*. Protein sequences were aligned using Mega software to construct an ML phylogenetic tree, with 1000 bootstrap replicates. RNA-Seq transcriptome data from *Arabidopsis thaliana* roots, stems, and leaves were aligned to the *Arabidopsis thaliana* genome using HiSAT2. Gene expression FPKM values ​​were calculated using Cufflinks to further screen for UGT genes specifically highly expressed in *Arabidopsis thaliana*.

[0053] Results analysis: A total of 214 GT-coding genes were annotated in the genome using bioinformatics techniques, and 11 candidate genes were selected based on differential gene expression analysis. Figure 2 A), among which g51295 was significantly highly expressed in plant leaves; phylogenetic analysis showed that g51295 belongs to the UGT73 subfamily, suggesting that it has the activity of C-3 glycosylation modification of blood flow interrupted triterpenoid saponins (A). Figure 2 B).

[0054] Example 3

[0055] Synthesis of CcGT-g51295, a glycosyltransferase involved in the glycosylation modification of triterpenoid saponins.

[0056] (1) Cloning of the target gene

[0057] RNA was extracted from the leaves and roots of *Gnaphalium affine* using the Tiangen Polysaccharide and Polyphenol Plant Total RNA Extraction Kit. cDNA templates were synthesized using Takara's PrimeScript™ II 1st Strand cDNA Synthesis Kit for cloning the CcGT target gene.

[0058] Based on the bioinformatics analysis results and the sequence characteristics of CcGT-g51295, cloning primers for CcGT-g51295 were designed, and the target gene was cloned using Novizan 2×Phanta Flash MasterMix.

[0059] The amplification primer sequences are as follows:

[0060] CcGT-g51295-F: 5'-ATGGCGATCAATGGAAAACCGCCTC-3', SEQ ID No. 1;

[0061] CcGT-g51295-R: 5'-TCACTGGACTAGGATTTGAGAAGAAATGTC-3', SEQ ID No. 2.

[0062] The cloning system is as follows: Primers: 2×Phanta Flash MasterMix: 25μL; CcGT-g51295-F (10μM): 2μL; Primers: CcGT-g51295-R (10μM): 2μL; cDNA: 2μL; ddH2O: 19μL.

[0063] PCR reaction parameters: denaturation at 98℃ for 30 s; denaturation at 98℃ for 10 s; annealing at 54℃ for 10 s; extension at 72℃ for 20 s; 40 cycles; final extension at 72℃ for 1 min.

[0064] DNA electrophoresis was used to obtain the target gene band. The target gene band was then recovered using the Tiangen agarose gel DNA recovery kit. The target gene fragment was then A-tailed using the Takara DNA A-Tailing Kit. Takara's pMD... TM The target gene was ligated into a T vector using the 18-T Vector Cloning Kit; the sequence accuracy of the amplified gene was verified by first-generation sequencing; and the pMD18-T-CcGT-g51295 sequenced without errors was used to extract plasmids using the Tiangen Plasmid Mini-Prep Kit.

[0065] The nucleotide sequence of the target gene CcGT-g51295 is as follows:

[0066]

[0067] (2) Construction of expression carrier

[0068] The pET28a (Cat. No. 69864-3) expression vector was purchased from Novagen. MBP·Tag was synthesized using gene chemical synthesis and inserted between the NcoI and NheI restriction enzyme sites of the pET28a vector to form the pET28a-MBP vector. The pET28a-MBP vector was then cleaved using Takara's EcoRI and XhoI restriction enzymes to obtain a linearized vector fragment. The pET28a-MBP-CcGT-g51295 prokaryotic expression vector was constructed using the Novizan ClonExpress II One Step Cloning Kit based on a homologous recombination strategy.

[0069] The accuracy of the expression vector construction was verified by first-generation sequencing technology. The pET28a-MBP-CcGT-g51295, which was sequenced without error, was used to extract plasmids using the Tiangen Plasmid Mini-Prep Kit.

[0070] The amplification primer sequences for the expression vector are as follows:

[0071] pET28a-MBP-CcGT-g51295-F: 5'-atgggtcgcggatccgaattcATGGCGATCAATGGAAAACCG-3', SEQ ID No. 4;

[0072] pET28a-MBP-CcGT-g51295-R: 5'-gtggtggtggtggtgctcgagTCACTGGACTAGGATTTGAGAAGAAA-3', SEQ ID No. 5.

[0073] (3) Synthesis of glycosyltransferase CcGT-g51295

[0074] The recombinant expression vector pET28a-MBP-CcGT-g51295 was transformed into the BL21(DE3) *Escherichia coli* expression strain. 2 mL of the overnight culture was added to 40 mL of LB broth (containing 50 μg / mL kanamycin resistance), and activated at 37°C and 200 rpm for 3 h. IPTG was then added to a final concentration of 0.3 mM, and the culture was induced at 16°C and 160 rpm for 24 h. The cells were collected by centrifugation and sonicated to obtain the crude protein enzyme. The amino acid sequence of the obtained glycosyltransferase CcGT-g51295 was determined as follows:

[0075] MAINGKPPHFALLPFLAQGHLIPMVDIAKLLAKRGAAVSFLVTPQNFNRVKKVLDRAVDSGLSIRVCLLTLPGAEAGLPEDCENFDMLPSMNYVLNFFKATAMMGGQVEDLLVELNPTCLIADMCFP WATDVAMKLRIPRLVFHGTSCFSLVCMNVLKNSKLLEGVNGDLEYFVVPDLPDRIEITKAQLRGSAEDISPEWMEIRQQIFSSEEDAAGTVANTFHELEPEYVRAYIKLRNKNLWCIGPVSLCNVDDS DKAERGNTAAIDGHDCLEWLDSHAPESVVYACLGSISRVVTTQLKEMGLGLEASDRPFIWVIREAPDEFNAWLTSEKFEERVKGKGLLIRGWAPQVLILSHPSVGGFVTHCGWNSTLEGVSAGLPMI TWPVFAEQFCNEKFIVNVIKTGVRVGVEVPVLLGMDEGAQVQVKSDDVKMAIDRLMEGGEEGEERRERARKLGEAAKRAVEEGGSSHLNMGQLIQDMVALKAKYGEKSVDGAKLDDISSQILVQ, SEQ ID NO.6.

[0076] Example 4

[0077] Functional verification of CcGT-g51295, a glycosyltransferase involved in the glycosylation modification of triterpenoid saponins

[0078] The crude protein enzyme obtained in Example 3 was used to perform an in vitro reaction (200 μL reaction system: 100 mM Tris-HCl at pH 8.5, 100 μM substrate (Saikogenin A, Saikogenin F, Prosaikogenin A, Prosaikogenin F), 5 mM glycosyl donor (UDP-fucose or UDP-glucose), 50 μL crude protein; reaction at 30°C for 3 h) to verify its catalytic activity. For substrates and products with available standards, qualitative analysis was performed using liquid chromatography-mass spectrometry (LC-MS / MS) (as described in the metabolomics detection methods above). For unknown products, product preparation was performed, and structural characterization was conducted using nuclear magnetic resonance spectroscopy.

[0079] Results analysis: CcGT-g51295 has the following function: It utilizes the glycosyl donor UDP-fucose to catalyze the generation of Prosaikogenin A and Prosaikogenin F from Saikogenin A and Saikogenin F, respectively. Figure 3 );

[0080] The synthesis of clinopodiside I and clinopodiside X from Saikogenin A using UDP-glucose catalysis. Figures 4-10 );

[0081] The synthesis of Saikosaponin B1, Saikosaponin C1, and Saikosaponin X from Prosaikogenin A using UDP-glucose catalysis. Figure 11 );

[0082] The synthesis of Saikosaponin A and Buddlejasaponin IV from Prosaikogenin F using UDP-glucose catalysis. Figure 12 ).

[0083] The various embodiments in the specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0084] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A glycosyltransferase involved in the glycosylation modification of triterpenoid saponins, characterized in that, The amino acid sequence of the glycosyltransferase is shown in SEQ ID NO.

6.

2. A gene encoding the glycosyltransferase of claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

3. A primer set for amplifying the glycosyltransferase gene of claim 2, characterized in that, The primer set includes CcGT-g51295-F and CcGT-g51295-R, with the following specific sequences: CcGT-g51295-F: 5'-ATGGCGATCAATGGAAAACCGCCTC-3', SEQ ID No. 1; CcGT-g51295-R: 5'-TCACTGGACTAGGATTTGAGAAGAAATGTC-3', SEQ ID No.

2.

4. A biomaterial containing the glycosyltransferase gene of claim 2, characterized in that, The biological material is a recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria.

5. The application of the glycosyltransferase of claim 1, the gene encoding the glycosyltransferase of claim 2, or the biomaterial of claim 4, characterized in that, The application is in any of the following directions: (1) Application in the preparation of glycosyltransferases and / or products containing glycosyltransferases; (2) Application in the preparation of recombinant glycosyltransferases and / or related products containing recombinant glycosyltransferases; (3) Application in the C-3 position fucosylation and glucosylation modification of triterpenoid saponins; (4) Application in the glycosylation modification of triterpenoid saponins; (5) Application in the biosynthesis of triterpenoid saponins; In steps (3)-(5), the glycosyltransferases utilize the glycosyl donor UDP-fucose to catalyze the generation of Prosaikogenin A and Prosaikogenin F from Saikogenin A and Saikogenin F, respectively; utilize UDP-glucose to catalyze the generation of clinopodiside I and clinopodiside X from Saikogenin A; utilize UDP-glucose to catalyze the generation of Saikosaponin B1, Saikosaponin c1 and Saikosaponin X from Prosaikogenin A; and utilize UDP-glucose to catalyze the generation of Saikosaponin A and Buddlejasaponin IV from Prosaikogenin F.

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