Dipsacus asper glycosyltransferase and application thereof in production of alpha-hederasaponin and dipsacus asper saponin B

Abstract

The invention discloses teasel glycosyl transferase and application thereof in production of alpha-hederaoside and teasel saponin B. The amino acid sequence of the teasel glycosyl transferase is shown as SEQ ID NO.1, and the nucleotide sequence of the coding gene of the teasel glycosyl transferase is shown as SEQ ID NO.2. The invention further discloses a preparation method of the teasel glycosyl transferase. After the glycosyl transferase is subjected to prokaryotic expression, a series of triterpenoid saponins can be catalyzed to be converted into triterpenoid saponin-3-(2-O)-glycoside products in a glycosylation manner, and the triterpenoid saponin-3-(2-O)-glycoside products comprise alpha-hederin and dipsacus asper saponin B. The discovery of the dipsacus asper triterpenoid saponin glycosyltransferase provides more optional biological elements for the biosynthesis of natural products, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant molecular biology, and particularly relates to a glycosyltransferase DaUGT103 derived from Dipsacus asperoides and application of the glycosyltransferase DaUGT103 in production of alpha-hederin and Dipsacoside B. BACKGROUND

[0002] Dipsacus asperoides is a traditional Chinese medicine, also known as "Heshangtou", which is the dried root of Dipsacus asperoides, mainly distributed in Sichuan, Hunan, Hubei, Yunnan, Guizhou and other places. As a multi-purpose traditional Chinese medicine, the dried root of Dipsacus asperoides is used to treat various diseases, including liver and kidney deficiency, sprains, fractures and metrorrhagia. The chemical composition of Dipsacus asperoides is complex, and triterpenoid saponins are the main components, among which oleanane-type saponins are particularly prominent. The structures of these saponins are mainly oleanane-type, which usually undergo glycosylation modification at C-3 or C-28. Modern pharmacological studies have shown that the saponin extract of Dipsacus asperoides exhibits multiple biological activities such as anti-inflammatory, antioxidant, anti-Alzheimer's disease, antifungal, anti-apoptosis and anti-tumor. Hederagenin, Dipsacoside VI and alpha-hederin are the main active components of Dipsacus asperoides. For alpha-hederin, there have been a large number of modern pharmacological activity studies, mainly for anti-cancer and anti-tumor. However, the biosynthetic pathway of these compounds has not been fully elucidated.

[0003] With the progress of synthetic biology, it is now possible to synthesize these triterpenoid saponins through biotechnology in microorganisms and improve their content, reducing the direct dependence on natural plant resources. In-depth understanding of the biosynthetic pathway of oleanolic acid type triterpenoid saponins and the discovery of key genes are the basis for realizing microbial production of these compounds. In addition, the application of genetic engineering and metabolic engineering opens up new ways for the de novo synthesis of these compounds, improves the sustainable use of rare resources and provides new strategies for microbial manufacturing of natural products.

[0004] Studies have identified a variety of enzymes closely related to triterpenoid saponin biosynthesis, such as OSC, CYP450 and UGTs, which play a key role in saponin production. For example, UGT74AG5 can glycosylate at C-28 of hederagenin, while UGT73F3 specifically transfers glucose to C-3.

[0005] However, so far there has been no report of a related glycosyltransferase that can catalyze the synthesis of alpha-hederin. In response to this problem, a rhamnose glycosyltransferase that can catalyze the synthesis of alpha-hederin in Dipsacus asperoides has been successfully identified and cloned. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application aims to provide a glycosyltransferase DaUGT103 derived from Radix Dipsaci, which has the function of 1,2-rhamnose glycosylation modification of various triterpene saponins.

[0007] Another object of the present application is to provide the use of the above-mentioned glycosyltransferase DaUGT103 in the production of α-hederin and Dipsacoside B.

[0008] The object of the present application can be achieved by the following technical solutions.

[0009] In a first aspect, the present application claims a Radix Dipsaci glycosyltransferase DaUGT103 having the function of catalyzing 1,2-rhamnose glycosylation modification of triterpene saponins, which is at least one of the following (a1) to (a4):

[0010] (a1) a protein having the amino acid residue sequence shown in SEQ ID NO. 1;

[0011] (a2) a derivative protein having the function of catalyzing 1,2-rhamnose glycosylation modification of triterpene saponins, which is obtained by substitution and / or deletion and / or addition of amino acid residues to the amino acid sequence shown in SEQ ID NO. 1;

[0012] (a3) a protein derived from the amino acid sequence described in (a1), which has 80% or more homology with the amino acid sequence described in (a1) or (a2) and has the same enzyme function;

[0013] (a4) a protein having a tag sequence or a signal peptide sequence at the N or C terminus of the amino acid sequence described in (a1) or (a2).

[0014] In a second aspect, the present application claims a gene encoding the above-mentioned Radix Dipsaci glycosyltransferase, the nucleotide sequence of which is as follows (b1) or (b2):

[0015] (b1) the nucleotide sequence shown in SEQ ID NO. 2;

[0016] (b2) a nucleotide sequence having 80% or more homology with the nucleotide sequence shown in SEQ ID NO. 2 and encoding a glycosyltransferase having the function of catalyzing 1,2-rhamnose glycosylation modification of triterpene saponins.

[0017] In a third aspect, the present application claims a biological material containing the above-mentioned gene, which is at least one of the following (cl) to (c4):

[0018] (cl) an expression cassette containing the above-mentioned gene;

[0019] (c2) a recombinant vector containing the gene, or a recombinant vector containing the expression cassette of (cl);

[0020] (c3) a transgenic non-plant cell line containing the gene, or a transgenic non-plant cell line containing the expression cassette of (cl), or a transgenic non-plant cell line containing the recombinant vector of (c2);

[0021] (c4) a genetically engineered bacterium containing the gene, or a genetically engineered bacterium containing the expression cassette of (cl), or a genetically engineered bacterium containing the recombinant vector of (c2).

[0022] Further, the recombinant vector is a recombinant expression vector, which is a recombinant expression vector obtained by inserting the gene into a prokaryotic or eukaryotic expression vector to express the glycosyltransferase. Further, the recombinant expression vector is obtained by inserting the nucleotide sequence shown in SEQ ID NO. 2 into the BamHI and EcoRI enzyme cutting sites of the pET28a(+) vector.

[0023] Further, the genetically engineered bacterium is obtained by inserting the gene or the recombinant expression vector into an engineered bacterium; the engineered bacterium is a fungus or other bacteria, including but not limited to Escherichia coli, Saccharomyces cerevisiae or Pichia pastoris; the engineered bacterium in the specific embodiment of the present application is BL21(DE3), but is not limited thereto.

[0024] In a fourth aspect, the present application claims the use of the above-mentioned Radix Dipsaci glycosyltransferase, the above-mentioned gene, or the above-mentioned biological material in catalyzing the 1,2-rhamnose glycosylation modification of triterpenoid saponins to generate triterpenoid saponin-3-(2-O)-glycoside products.

[0025] Further, the method for catalyzing the 1,2-rhamnose glycosylation modification of triterpenoid saponins to generate triterpenoid saponin-3-(2-O)-glycoside products in the above-mentioned use is as follows (d1) or (d2) or (d3):

[0026] (d1) using the Radix Dipsaci glycosyltransferase to catalyze the generation of triterpenoid saponin-3-(2-O)-glycoside products in vitro;

[0027] (d2) adding the genetically engineered bacterium in the biological material to a reaction system containing triterpenoid saponin substrates to generate corresponding triterpenoid saponin-3-(2-O)-glycoside products;

[0028] (d3) using the genetically engineered bacterium in the biological material to ferment triterpenoid saponin-3-(2-O)-glycoside products, and directly feeding triterpenoid saponin substrates to the fermentation broth to generate corresponding triterpenoid saponin-3-(2-O)-glycoside products.

[0029] In a fifth aspect, the present application claims a method for producing triterpene saponin-3-(2-O)-glycoside products, which is as follows (d1) or (d2) or (d3):

[0030] (d1) using the said Dipsacus fullonum glycosyltransferase to catalyze the production of triterpene saponin-3-(2-O)-glycoside products in vitro;

[0031] (d2) adding the genetically engineered bacteria in the said biological material to a reaction system containing triterpene saponin substrates to generate corresponding triterpene saponin-3-(2-O)-glycoside products;

[0032] (d3) using the genetically engineered bacteria in the said biological material to ferment and produce triterpene saponin-3-(2-O)-glycoside products, and directly feeding triterpene saponin substrates to the fermentation broth to generate corresponding triterpene saponin-3-(2-O)-glycoside products.

[0033] Further, the enzymatic reaction temperature of the above-mentioned in vitro catalysis is 30-70℃, preferably 30-40℃; the pH is 5.0-10.0, preferably pH 6.0-10.0, further preferably 7.0-9.0.

[0034] In the specific embodiments of the present application, the triterpene saponin substrates are Wulingxian saponin A or / and Dipsacus fullonum saponin VI; and the triterpene saponin-3-(2-O)-glycoside products are α-hederin or / and Dipsacoside B.

[0035] In the specific embodiments of the present application, the method for catalyzing the production of α-hederin and Dipsacoside B and other triterpene saponin-3-(2-O)-glycosides by using Dipsacus fullonum glycosyltransferase DaUGT103 includes the following steps:

[0036] (1) fermenting and culturing the said genetically engineered bacteria, collecting the fermentation broth, centrifuging to collect the bacterial bodies, breaking the bacterial bodies to collect the supernatant, and separating and purifying the proteins;

[0037] (2) reacting the said supernatant or the separated and purified proteins with triterpene saponin substrates in a buffer to catalyze the production of corresponding triterpene saponin-3-(2-O)-glycosides; the enzymatic reaction temperature is 30-70℃, and the reaction is carried out in a pH 5.0-10.0 buffer.

[0038] In specific embodiments of the present application, the Rhizoma Dipsaci glycosyltransferase DaUGT103 is used in a method for in vitro catalytic production of triterpenoid saponin-3-(2-O)-glycosides such as alpha-hederin and Rhizoma Dipsaci saponin B. The genetically engineered bacteria can be added to a reaction system containing substrate triterpenoid saponin, and the triterpenoid saponin-3-(2-O)-glycoside product can be prepared by reaction; or the substrate can be directly fed to produce triterpenoid saponin-3-(2-O)-glycoside by in vivo fermentation.

[0039] Compared with the prior art, the glycosyltransferase DaUGT103 disclosed in the present application has the following advantages:

[0040] The present application discloses a Rhizoma Dipsaci-derived glycosyltransferase. Research has found that the amino acid sequence of the glycosyltransferase is shown as SEQ ID NO. 1, and the nucleotide sequence encoded by the glycosyltransferase is shown as SEQ ID NO. 2. After prokaryotic expression of the glycosyltransferase, a series of triterpenoid saponin glycosylation can be catalyzed to convert into triterpenoid saponin-3-(2-O)-glycoside product, including alpha-hederin and Rhizoma Dipsaci saponin B.

[0041] The discovery of the Rhizoma Dipsaci triterpenoid saponin glycosyltransferase provides more biological elements for the biosynthesis of natural products, provides certain guidance and theoretical basis for the molecular modification of this type of enzyme, and has good application prospects. The present application improves the understanding of triterpenoid saponin biosynthesis.

[0042] The concept, specific experimental scheme and effects of the present application will be further described below in combination with specific implementation methods. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 It is an agarose gel electrophoresis map of the DaUGT103 gene.

[0044] Figure 2 It is a sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis map of pET28a-DaUGT103.

[0045] Figure 3 It is an HPLC map of the DaUGT103 catalyzed rhiociguat rhamnose glycosylation to generate alpha-hederin.

[0046] Figure 4 It is an UPLC-MS analysis map of the DaUGT103 catalyzed rhiociguat rhamnose glycosylation to generate alpha-hederin. A is the DaUGT103 catalyzed rhiociguat to generate alpha-hederin; B is the extracted ion chromatogram of the glycosylation reaction; and C is the TOF / MS spectrum.

[0047] Figure 5The UPLC-MS chart of DaUGT103 catalyzing the rhamnose glycosylation of Asperosaponin VI to generate Asperosaponin B; wherein, A is DaUGT103 catalyzing Asperosaponin VI to generate Asperosaponin B; B is the extracted ion chromatogram of the glycosylation reaction; and C is the TOF / MS spectrum.

[0048] Figure 6 The SDS-PAGE chart of the purified recombinant glycosyltransferase DaUGT103.

[0049] Figure 7 The temperature stability investigation chart of the recombinant glycosyltransferase DaUGT103.

[0050] Figure 8 The pH stability investigation chart of the recombinant glycosyltransferase DaUGT103. DETAILED DESCRIPTION

[0051] The following examples are used to illustrate the present application, making its technical content more clear and convenient to understand. The present application can be embodied by different examples, and the following is an exemplary description, and the modification or replacement of the method, step or condition of the present application without departing from the spirit and essence of the present application, all belong to the scope of the present application.

[0052] The experimental methods used in the following examples are conventional methods unless otherwise specified; and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0053] Example 1: Cloning of glycosyltransferase gene

[0054] (I) Extraction of total RNA of Asiasarum and synthesis of first strand of cDNA:

[0055] (1) Take an appropriate amount of fresh or frozen Asiasarum plant samples stored at -80℃, and transfer them to a pre-sterilized and pre-cooled mortar for grinding. Add liquid nitrogen during the grinding process until the material is ground into a white fine powder. Use the total RNA rapid extraction kit (centrifugal column type) of Jetser to extract the total RNA according to the instructions. Take 3 μL of the newly extracted total RNA for agarose gel electrophoresis analysis.

[0056] (2) Use the PrimerScript TM IVStrand cDNA Synthesis Mix reverse transcription kit of Takara company to reverse transcribe the total RNA of Asiasarum as a template according to the instructions to obtain the cDNA template.

[0057] (II) Cloning of DaUGT103 gene:

[0058] Design specific primers, and the specific primer sequences are as follows:

[0059] DaUGT103-F: 5'-cgcGGATCCATGACAACTGATGACTTGAAGCTCCATGTTGTAGT-3'

[0060] DaUGT103-R: 5'-ccgGAATTCTCATTTGCTTAAGCTGAAGAAATTTTTTGT-3'

[0061] Using cDNA from *Dendrobium nobile* as a template, PCR amplification was performed using a designed pair of specific primers. A high-fidelity DNA polymerase was used. MAX DNA Polymerase amplification yields the amplified target gene, such as... Figure 1 As shown.

[0062] PCR reaction program: 98℃ pre-denaturation for 3 min; 98℃ for 10 s, 60℃ for 5 s, 72℃ for 1.5 min, 32 cycles; 72℃ extension for 10 min.

[0063] After the reaction was completed, a complete gold product recovery kit was used. The PCR Purification Kit recovers the target gene fragment.

[0064] PCR products were double-digested with Thermo Fisher Scientific's Fast Digest restriction endonucleases BamHI and EcoRI, and the pET28a vector was also double-digested. The digestion system was then analyzed using a full-length gold gel recovery kit. The agarose gel was extracted using the Quick Gel Extraction Kit, and the DNA was ligated using T4 DNA ligase to obtain the recombinant expression vector pET28a-DaUGT103.

[0065] The recombinant expression vector was transformed into E. coli BL21(DE3) competent cells, plated on LB agar plates containing kanamycin and incubated overnight at 37°C. Single colonies were picked from the plates and subjected to PCR amplification and gel electrophoresis. Single clones were picked and PCR was confirmed to be positive, thus obtaining the recombinant strain BL21-pET28a-DaUGT103.

[0066] Example 2: Prokaryotic expression to verify the function of DaUGT103:

[0067] (1) Prokaryotic expression of DaUGT103

[0068] In the blank liquid LB medium, 50mg / ml Kana was added at 1‰ concentration (v / v), mixed well. In the super-clean bench, 4ml LB+Kana (0.1‰, v / v) liquid medium was added in a 15ml centrifuge tube, a single colony was taken, and mixed well. It was cultured at 37℃, 220rpm for 24h. In the super-clean bench, 100ml LB+Kana (0.1‰, v / v) liquid medium was added in a 500ml conical flask, 1ml bacterial solution was added, mixed well. It was cultured at 37℃, 220rpm for about 2.5h, and the bacterial solution concentration reached OD 600 0.6-1, the medium was cooled, 12μL IPTG (1M) was added to the medium, and the final concentration of IPTG was 0.12mM. It was cultured at 120rpm, 13℃ for 16h. The harvested bacterial solution was centrifuged at 4000rpm, the supernatant was removed, and the bacterial pellet was resuspended with 50mM Tris-HCl (pH 7.4). After ultrasonic disruption, the supernatant was obtained by centrifugation at 4℃ to obtain the crude protein.

[0069] The obtained protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as shown in Figure 2 The results showed that the solubility of DaUGT103 recombinant protein was good, the calculated protein size was about 54.5kDa, Figure 2 The size was consistent with the expected value, which confirmed that the soluble protein was correctly expressed.

[0070] (2) Verification of the enzyme activity of DaUGT103

[0071] The supernatant obtained in (1) was used as DaUGT103 crude enzyme for enzymatic reaction, and the following reaction system (100μL) was configured:

[0072]

[0073] The reaction was carried out at 37℃ overnight, the reaction system was added with twice the volume of methanol to terminate the reaction, vortexed and centrifuged to take the supernatant, and then nitrogen was blown. After drying, 200μL of methanol was added for resuspension, centrifuged to take the supernatant, and HPLC and UPLC-Q-TOF-MS / MS were used for detection.

[0074] The reaction product of enzyme activity was detected by HPLC, and the product was analyzed by HPLC-Agilent 1260 detector. The chromatographic column was ZORBAX Eclipse Plus C18 chromatographic column (4.6 mm x 250 mm, 5 μm); the mobile phase was 0.05% phosphoric acid aqueous solution-acetonitrile (v / v), linear gradient elution: 0-10 min, 8%-30% acetonitrile; 10-30 min, 30%-50% acetonitrile; 30-40 min, 50%-80% acetonitrile; 40-50 min, 80%-95% acetonitrile; the column temperature was 25°C, the volume flow rate was 1.0 mL / min; the injection volume was 10 μL; the post-equilibration time was 10 min. The HPLC-1260 results are shown in Figure 3 . The product appeared in the experimental group and basically corresponded to the peak time of the standard.

[0075] The reaction product of enzyme activity was detected by LC-MS, and the UPLC-Q-TOF-MS / MS chromatographic mass spectrometry conditions were as follows: the mobile phase consisted of acetonitrile (A) and 0.1% formic acid water (v / v) (B), the ACQUITY HSS C18 (2.1 x 100 mm, 1.8 μm) chromatographic column. The mass spectrometry parameters included: ion source: Turbo V TM ; negative ion scanning mode; TOF MS ~ Product Ion ~ IDA mode for collecting mass spectrometry data; scanning range 100-1500 m / z; atomizing gas (GS1): 344.738 kPa (50 psi); atomizing gas (GS2) 344.738 kPa (50 psi); curtain gas (CUR): 241.317 kPa (35 psi); ion source temperature (TEM) 550°C; ion source voltage (IS) -4500 V; primary scanning: de-clustering voltage (DP) -100 V; focusing voltage (CE) -10 V; secondary scanning: de-clustering voltage (DP) -100 V; focusing voltage (CE) -40 V. The mass spectrometry data were collected using Analyst software, the data were analyzed using PeakView software, and the rapid matching identification of compounds was performed using MarkerView software.

[0076] Based on the HPLC and LC-MS results, Figures 3 to 5 it was shown that the glycosyltransferase DaUGT103 effectively catalyzed the conversion of triterpene saponins to the corresponding triterpene saponin-3-(2-O)-glycoside using UDP-rhamnose as the glycosyl donor. HPLC analysis detected a product peak at 28 minutes that matched a- hederin (see Figure 3 ), and UPLC-Q-TOF-MS analysis further verified the molecular structure, confirming that the secondary fragment ion spectrum was consistent with that of the standard (see Figure 4). These results collectively demonstrate the unique function of DaUGT103 protein in catalyzing the addition of rhamnose group at 3-OH position of Disanguisorin A. As shown in Fig. Figure 5 ), when Disanguisorin VI, UDP-Rha and DaUGT103 were reacted, a new peak was detected at 9.65 min. After comparison with the standard sample, it was confirmed that the new peak was Disanguisorin B. Through secondary fragment analysis, it was further confirmed that DaUGT103 could catalyze the transfer of rhamnose sugar group at 3-OH position of Disanguisorin VI. Detailed analysis of HPLC and LC-MS revealed the specific glycosyltransferase activity of DaUGT103 on various triterpene saponin substrates, especially the ability to add rhamnose on Disanguisorin A and Disanguisorin VI. DaUGT103 efficiently utilized UDP-rhamnose to catalyze the production of triterpene saponin-3-(2-O)-glycosides such as α-Hederin and Disanguisorin B in vitro, showing a wide substrate adaptability. These experimental results are crucial for understanding and utilizing the role of DaUGT103 glycosyltransferase in triterpene saponin biosynthesis.

[0077] Example 3: Optimum temperature of recombinant glycosyltransferase DaUGT103

[0078] (1) Purification of glycosyltransferase DaUGT103

[0079] Using the crude enzyme solution of recombinant glycosyltransferase DaUGT103 obtained in Example 2 as the original material, 5 mL HisTrap pre-packed nickel column was installed on a peristaltic pump. First, 10 column volumes of pre-washing were performed at a flow rate of 5 mL per minute using 20 mM pH 7.4 sodium phosphate buffer A containing 20 mM imidazole and 500 mM NaCl, and then the crude protein was loaded into the nickel column at the same flow rate. Subsequently, elution was performed with 5 column volumes of buffer containing 10 to 500 mM imidazole to achieve complete elution of the protein, and the corresponding 25 mL eluate was collected for subsequent analysis.

[0080] After analyzing the eluate by SDS-PAGE protein electrophoresis technique, the eluate containing the target protein was selected for further purification. The eluate was transferred to a 50 kDa ultrafiltration tube and centrifuged at 2300 rpm at 4°C until the protein solution was reduced to about 1 mL. At this time, 20 mL of D buffer (20 mM pH 7.4 sodium phosphate buffer containing 500 mM NaCl) was added and ultrafiltration was continued. This process was repeated 3 to 5 times to ensure that the target protein was highly purified. As shown in Figure 6 The SDS-PAGE results showed that the purified protein had clear and single bands.

[0081] (2) Optimum temperature of recombinant glycosyltransferase DaUGT103

[0082] The protein was quantitatively analyzed by BCA protein quantitative kit, 10 μg of recombinant glycosyltransferase DaUGT103 and 100 μM of wilging saponin A and 500 μM of UDP-rhamnose were used as the components of the reaction system. These components were mixed with 50 mM Tris-HCl buffer solution with pH 7.0, and the total volume was adjusted to 100 μL, and the reaction was carried out at 30-70 °C for 12 hours. In order to terminate the reaction, 200 μL of methanol was added to the reaction mixture, and the supernatant was collected by vortex and centrifugation. Then, it was treated by nitrogen blowing dry, and 200 μL of methanol was used for re-dissolution. After re-dissolution, the supernatant was collected by twice centrifugation, and was used for LC-MS detection analysis.

[0083] According to the relative activity measurement results defined as 100% of the highest activity, it is revealed that the enzyme exhibits the optimal activity at the reaction temperature of 35 °C. This result shows that the reaction temperature has a significant influence on the catalytic efficiency of DaUGT103, and 35 °C is the temperature point at which the enzyme exhibits the best activity.

[0084] Table 1: Optimum temperature of glycosyltransferase DaUGT103

[0085] Temperature (°C) Relative activity (%) 30 88.2±2.2 35 96.5±3.1 40 85.5±0.7 45 71.5±5.7 50 61.5±2.8 55 53.7±5.8 60 52.5±3.8 65 49.5±2.2 70 49.7±2.3

[0086] Example 4: Optimum pH of recombinant glycosyltransferase DaUGT103

[0087] 2-3 ng of purified recombinant glycosyltransferase DaUGT103 and 100 μM of wilging saponin A and 500 μM of UDP-rhamnose were added to 50 mM citric acid-sodium citrate buffer (pH 5.0-6.0), sodium phosphate dibasic-sodium phosphate monobasic buffer (pH 6.0-8.0), Tris-hydrochloric acid buffer (pH 8.0-9.0) and sodium carbonate-sodium bicarbonate buffer (pH 9.0-11.0) to make up the system to 100 μL, and incubated at the optimum temperature of 35 °C for 12 hours. 200 μL of methanol was added to terminate the reaction, vortexed and centrifuged to collect the supernatant, and then blown dry with nitrogen. After blowing dry, 200 μL of methanol was added for re-dissolution, and the supernatant was collected by centrifugation, and was used for detection by LC-MS. With the highest activity as 100%, the relative activity results are shown in Table 2, and the enzyme has the highest reaction activity in sodium phosphate dibasic-sodium phosphate monobasic buffer (pH = 8.0).

[0088] Table 2: Optimum pH of glycosyltransferase DaUGT103

[0089] pH Relative activity (%) 5.0 (Citric acid-sodium citrate buffer) 23.7±4.4 6.0 (Citric acid-sodium citrate buffer) 46.1±5.0 6.0 (Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer) 59.5±6.0 7.0 (Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer) 82.9±5.3 8.0 (Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer) 96.2±3.8 8.0 (Tris-hydrochloric acid buffer) 81.7±5.0 9.0 (Tris-hydrochloric acid buffer) 95.9±3.5 9.0 (Sodium carbonate-sodium bicarbonate buffer) 76.2±4.2 10.0 (Sodium carbonate-sodium bicarbonate buffer) 90.8±9.5 11.0 (Sodium carbonate-sodium bicarbonate buffer) 58.3±6.6

[0090] Example 5: Temperature stability of recombinant glycosyltransferase DaUGT103

[0091] In the study, 2-3 ng of purified recombinant glycosyltransferase DaUGT103 was reacted with the final concentration of 100 mM of wilsonia saponin A and 500 mM of UDP-rhamnose in 50 mM of sodium phosphate dibasic-sodium phosphate monobasic buffer at pH 8.0 at different temperatures. At the end of the experiment, the reaction was terminated by adding 200 μL of methanol, and the supernatant was collected after vortex centrifugation. Subsequently, the supernatant obtained after centrifugation was analyzed by LC-MS after nitrogen blowing and re-dissolving with 200 μL of methanol. The relative residual activity of DaUGT103 was determined by setting the highest value of enzyme activity as 100%. The results show that the stability of DaUGT103 significantly decreased above 40°C. Between 30°C and 40°C, DaUGT103 exhibited a high level of activity, indicating its characteristics as a mesophilic enzyme. Further evaluation of its temperature stability showed that DaUGT103 could maintain at least 50% of the initial activity after incubation at 45°C for 30 minutes or 60 minutes. However, when the incubation temperature reached above 50°C, the enzyme activity was almost completely lost after 30 minutes, which clearly demonstrated the insufficient stability of DaUGT103 in high temperature environment. The experimental results revealed that DaUGT103 has good activity and stability in a moderate temperature range, but the activity decreases sharply at higher temperatures. Figure 7

[0092] Example 6: pH stability of recombinant glycosyltransferase DaUGT103

[0093] At different pH, 2-3 ng of purified recombinant glycosyltransferase DaUGT103 and the final concentration of 100 mM of wilsonia saponin A and 500 mM of UDP-rhamnose were reacted at 35°C. At the end of the reaction, 200 μL of methanol was added to terminate the reaction, and the supernatant was collected after vortex centrifugation and nitrogen blowing. After blowing dry, 200 μL of methanol was added for re-dissolution, and the supernatant was collected after centrifugation and detected by LC-MS. The highest activity was set as 100%, and the relative residual activity results are shown in Figure 8 DaUGT103 exhibited the highest catalytic activity at pH 9. It showed better stability in an alkaline environment. After incubation at pH 9 for 60 min, the activity of DaUGT103 could still maintain about 90% of the residual activity, while the enzyme activity decreased sharply after incubation at pH 10 for 60 min. In the extremely alkaline (pH 10.0) environment, a significant decrease in enzyme activity was observed. In addition, the stability of DaUGT103 was evaluated in the pH range of 6 to 10, and it was found that the stability was best in the moderately alkaline condition.

[0094] ​In exploring the activity and stability of recombinant glycosyltransferase DaUGT103 under different pH conditions, the experiment used 2-3 ng of purified enzyme and 100 μM of wilson saponin A and 500 μM of UDP-rhamnose as the reaction system, and the reaction was carried out at 35°C. The reaction was terminated by adding 200 μL of methanol and vortexing to extract the supernatant, and then the supernatant was treated with nitrogen blowing dry, resuspended in 200 μL of methanol, and centrifuged to obtain the final supernatant for LC-MS analysis. Based on the definition of the maximum enzyme activity as 100%, the determination results of the relative residual activity showed that Figure 8 ), DaUGT103 showed the highest catalytic activity at pH 9. The enzyme showed better stability in alkaline environment, especially at pH 9, it could maintain about 90% of the residual activity after 60 minutes of incubation. In contrast, the enzyme activity showed a significant decrease under the same incubation time at pH 10. The significant decrease in enzyme activity in the extremely alkaline (pH 10) environment was confirmed. In addition, the evaluation of the stability of DaUGT103 in the pH range of 6 to 10 showed that its stability was best under moderate alkaline conditions. These experimental results revealed that the activity and stability of DaUGT103 were significantly affected by pH, especially under alkaline conditions, it showed higher activity and stability, but the activity decreased rapidly in the over-alkaline environment. This indicates that the catalytic action of DaUGT103 is sensitive to pH conditions, and it has the best activity performance in a specific pH range.

[0095] The above detailed the preferred embodiments of the present application. It should be understood that those of ordinary skill in the art can make many modifications and changes without creative labor based on the concept of the present application. Therefore, any technical solutions obtained by logical analysis, reasoning or limited experiments based on the prior art according to the concept of the present application shall be within the protection scope defined by the claims.

[0096] SEQ ID NO. 1 304

[0098] Dipsacus asper Wall. ex Henry

[0099] MTTDDLKLHVVVLPYFTPSHMIPLIDVARLLAARGVIITIISTPFNARLFQDSVEAEVADGHQITIRTLQLPSAEVGLPEGIENFNTITSPDMPAKVYQGFAMLQQPMEQLIRDLNPDCI FSDLSYPWTVDLADELHVPRLLFDPSSFLAHCVFDSRDKYKPHLNIKSDDETFLVPNLPHKVEMKGSMLEDYVKAETQYGQIVKMIEASKLRSFGYVHNTFYELEPSFADHYRKIKGNKTFHVGPVYRFSNRERFQLQHHKCLTWLDTQKPNSVIYIAFGTLVRFPDSQLIEIAHGIELSGHKFIWVVRNREKKSDKENWLPEGFEERMSKANKGMIVRDWVPQSKILTHDAIGGFMTHCGWNSILEASTMGVPLITWPLFAEQFYNEMVITINGTGVRVGSDIWHPWFEITEPTVGREKIKTAIIRLMGDTEEAEQIRKRAKEMSVKAKNTVAEGGSSYNQLTTLIEELKACALAKKQKNFFSLSK*

[0100] SEQ ID NO. 2 1464

[0102] Dipsacus asper Wall. ex Henry

[0103]

Claims

1. A glycosyltransferase of Dipsacus asperoides, characterized in that, The glycosyltransferase is at least one of the following (a1) to (a4): (a1) A protein having the amino acid residue sequence shown in SEQ ID NO.1; (a2) A derivative protein with catalytic 1,2-rhamnosyl glycosylation modification function of triterpenoid saponins obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID NO.

1. (a3) A protein derived from the amino acid sequence described in (a1) that has more than 80% homology with the amino acid sequence described in (a1) or (a2) and has the same enzymatic function; (a4) A protein having a tag sequence or signal peptide sequence at the N or C terminus of the amino acid sequence described in (a1) or (a2).

2. The gene encoding the dipysmosyltransferase of claim 1, characterized in that, The nucleotide sequence of this gene is as follows (b1) or (b2): (b1) The nucleotide sequence as shown in SEQ ID NO.2; (b2) A nucleotide sequence having more than 80% homology with the nucleotide sequence shown in SEQ ID NO.2 and encoding a glycosyltransferase that catalyzes the 1,2-rhamnose glycosylation modification of triterpenoid saponins.

3. A biomaterial containing the gene of claim 2, characterized in that, The biomaterial is at least one of the following (c1) to (c4): (cl) An expression cassette containing the gene described in claim 2; (c2) A recombinant vector containing the gene described in claim 2, or a recombinant vector containing the expression cassette described in (c1); (c3) A transgenic non-plant cell line containing the gene described in claim 2, or a transgenic non-plant cell line containing the expression cassette described in (c1), or a transgenic non-plant cell line containing the recombinant vector described in (c2); (c4) A transgenic engineered bacterium containing the gene described in claim 2, or a transgenic engineered bacterium containing the expression cassette described in (c1), or a transgenic engineered bacterium containing the recombinant vector described in (c2).

4. The biomaterial according to claim 3, characterized in that, The recombinant vector described in (c2) is a recombinant expression vector, which is a recombinant expression vector that expresses glycosyltransferase by inserting the gene described in claim 2 into a prokaryotic or eukaryotic expression vector; the transgenic engineered bacteria described in (c4) are obtained by transferring the gene described in claim 2 or the recombinant vector described in (c2) into engineered bacteria.

5. The use of the glutamyl transferase of claim 1, the gene of claim 2, or the biomaterial of claim 3 or 4 in catalyzing the 1,2-rhamnose glycosylation modification of triterpenoid saponins to generate triterpenoid saponin-3-(2-O)-glycoside products.

6. The application according to claim 5, characterized in that, The method for catalytic 1,2-rhamnose glycosylation modification of triterpenoid saponins to generate triterpenoid saponin-3-(2-O)-glycoside products is as follows (d1) or (d2) or (d3): (d1) Triterpenoid saponin-3-(2-O)-glycoside products were generated in vitro using the aforementioned Dipsacus glycosyltransferase catalysis; (d2) The transgenic engineered bacteria in the biomaterial are added to a reaction system containing triterpenoid saponin substrates to carry out the reaction and generate the corresponding triterpenoid saponin-3-(2-O)-glycoside products; (d3) The transgenic engineered bacteria in the biomaterial are used to ferment and produce triterpenoid saponin-3-(2-O)-glycoside products. The triterpenoid saponin substrate is directly fed into the fermentation broth to generate the corresponding triterpenoid saponin-3-(2-O)-glycoside products.

7. A method for producing triterpenoid saponin-3-(2-O)-glycoside products, characterized in that, The method is as follows (d1) or (d2) or (d3): (d1) Using the Dipsacus asperoides glycosyltransferase described in claim 1 to catalyze the in vitro generation of triterpenoid saponin-3-(2-O)-glycoside products; (d2) The transgenic engineered bacteria in the biomaterial described in claim 3 are added to a reaction system containing a triterpenoid saponin substrate, and the reaction is carried out to generate the corresponding triterpenoid saponin-3-(2-O)-glycoside product; (d3) Using the transgenic engineered bacteria in the biomaterial described in claim 3 to produce triterpenoid saponin-3-(2-O)-glycoside products by fermentation, and directly feeding triterpenoid saponin substrates into the fermentation broth to generate the corresponding triterpenoid saponin-3-(2-O)-glycoside products.

8. The method according to claim 7, characterized in that, The in vitro catalytic enzyme reaction temperature described in (d1) is 30-70℃, preferably 30-40℃; pH 5.0-10.0, preferably pH 6.0-10.0, and more preferably pH 7.0-9.

0.

9. The application as described in claim 5 or 6, the method as described in claim 7, characterized in that, The triterpenoid saponin substrate is Clematis chinensis saponin A or / and Dipsacus asperoidin VI; the triterpenoid saponin-3-(2-O)-glycoside product is α-hederone or / and Dipsacus asperoidin B.

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