Dipsacus asper glycosyltransferase and application thereof in production of clematis saponin A

Abstract

The invention discloses teasel glycosyl transferase and application thereof in production of clematis saponin A. The amino acid sequence of the teasel glycosyl transferase is shown as SEQ ID NO.1, and the nucleotide sequence of a 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-O-glycoside products through glycosylation, and the triterpenoid saponin-3-O-glycoside products comprise radix clematidis saponin A. 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 DaUGT121 derived from Dipsacus asper and application of the glycosyltransferase DaUGT121 in production of Cauloside A. BACKGROUND

[0002] Dipsacus asper Wall.ex Henry is a dry root of Dipsacus asper Wall.ex Henry, and the earliest medicinal record appears in Shennong Bencao Jing. Dipsacus asper has the effects of tonifying liver and kidney, strengthening muscles and bones, and stopping bleeding. Because of its remarkable curative effect, it is widely used in China and is one of the commonly used bulk medicinal materials. Research has found that the main active ingredient in Dipsacus asper is triterpene saponins. Triterpene saponins are a class of natural products with a wide range of pharmacological activities, showing quite diverse biological and pharmacological activities, such as hemolysis, cytotoxicity, anti-tumor, anti-inflammatory, anti-hypercholesterolemia and other pharmacological effects, and have been widely used in clinical research and drug development.

[0003] Oleanane-type triterpene saponins are compounds with oleanolic acid as the aglycone, which are widely found in nature, especially in dicotyledonous plants such as ginseng, American ginseng and notoginseng. The extraction and purification of these compounds are challenging, which limits the in-depth study of their structure, properties and pharmacological effects. Existing research has proved that they have a wide range of biological activities in anti-inflammatory, antibacterial and anti-tumor aspects, and may have potential for the prevention and treatment of cardiovascular diseases, neurodegenerative diseases and some cancers. Not only are they applied in the medical field, but also show wide potential in the development of health products, perfumes, condiments and cosmetics industry. Especially in cosmetics, due to their anti-inflammatory and antibacterial properties, they have been used as effective ingredients to improve skin health.

[0004] The rapid progress of synthetic biology has made it possible to synthesize and enhance the content of triterpene saponins in microorganisms through biotechnological means, realizing the microbial production of medicinal natural products, rather than relying entirely on traditional plant sources. Detailed analysis of the oleanolic acid-type triterpene saponin biosynthesis pathway and exploration of biosynthesis genes are key steps for the production of these precious ingredients in microorganisms through metabolic engineering. In addition, the exploration of biosynthetic pathways for the de novo synthesis of target compounds through genetic engineering and metabolic engineering opens up an innovative way to obtain natural compounds that are difficult to extract and have limited sources. These advances not only improve the efficiency of sustainable use of rare resources, but also provide new strategies for microbial manufacturing of natural products.

[0005] A number of studies have successfully isolated and identified various UGTs from various plants, and revealed the biosynthetic pathways of oleanolic acid type triterpenoid saponins. A triterpenoid saponin 2"-O-rhamnosyltransferase GuRhaGT with high site specificity and donor selectivity was identified from Glycyrrhiza uralensis, which can effectively promote the 2"-O-rhamnosylation of 33 saponins. The UGT73 family modifies pentacyclic triterpenoids in plants through glycosylation, and performs glycosyltransfer at the C3, C23 and C28 positions of oleanolic acid and hederasaponin. In particular, 10 glycosyltransferases found in Brassica rapa are involved in this process, UGT73C10 and UGT73C11 mainly perform glucosylation at the C3 position of oleanolic acid, and UGT73C12 and UGT73C13 can add two glucose groups at the position to generate a double glycosylation product. Meanwhile, a number of enzymes capable of glucosylation at the C3-OH and C28-COOH positions of oleanolic acid type triterpenoid saponins have been reported. However, there is a lack of reports on the arabinosylation function gene of hederagenin, which limits the further application of oleanolic acid type triterpenoid saponins. In view of this problem, a glycosyltransferase capable of adding arabinose and xylose at the C3-OH position of oleanolic acid type saponin was successfully identified and cloned in Dipsacus asper. SUMMARY

[0006] In view of the deficiencies in the prior art, the present application aims to provide a glycosyltransferase DaUGT121 derived from Dipsacus asper, which has the function of glycosylating and modifying the 3-hydroxyl group of various triterpenoid saponins.

[0007] Another object of the present application is to provide the application of the above-mentioned glycosyltransferase DaUGT121 in the production of Cauloside A.

[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 Dipsacus asper glycosyltransferase DaUGT121 having the function of catalyzing the glycosylation and modification of the 3-hydroxyl group of triterpenoid 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 the glycosylation and modification of the 3-hydroxyl group of triterpenoid saponins, which is obtained by substitution, and / or deletion, and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID NO. 1;

[0012] (a3) a protein derived from the amino acid sequence described in (a1) having 80% or more homology with the amino acid sequence described in (a1) or (a2) and having 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 to protect a gene encoding the above-mentioned Dioscorea opposita Thunb. glycosyltransferase DaUGT121, the nucleotide sequence of the gene being as follows (b1) or (b2):

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

[0016] (b2) a nucleotide sequence having 80% or more homology with the nucleotide sequence shown as SEQ ID NO. 2 and encoding a glycosyltransferase having the function of catalyzing the 3-hydroxyl glycosylation modification of triterpene saponins.

[0017] In a third aspect, the present application claims to protect a biological material containing the above-mentioned gene, the biological material being at least one of the following (c1) to (c4):

[0018] (c1) an expression cassette containing the above-mentioned gene;

[0019] (c2) a recombinant vector containing the above-mentioned gene or a recombinant vector containing the expression cassette described in (c1);

[0020] (c3) a transgenic non-plant cell line containing the above-mentioned gene 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);

[0021] (c4) a genetically engineered bacterium containing the above-mentioned gene or a genetically engineered bacterium containing the expression cassette described in (c1) or a genetically engineered bacterium containing the recombinant vector described in (c2).

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

[0023] Further, the genetically engineered bacteria is obtained by transforming the gene or the recombinant expression vector into the engineered bacteria; further, the engineered bacteria is fungi or other bacteria, including but not limited to Escherichia coli, Saccharomyces cerevisiae or Pichia pastoris; the engineered bacteria in the specific embodiments 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 Dioscorea panthaica glycosyltransferase, the gene or the biological material in the catalysis of 3-hydroxyl glycosylation modification of triterpene saponins to generate triterpene saponin 3-hydroxyl glycoside products.

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

[0026] (d1) using the Dioscorea panthaica glycosyltransferase to catalyze the generation of triterpene saponin 3-hydroxyl glycoside products in vitro;

[0027] (d2) adding the genetically engineered bacteria in the biological material to a reaction system containing triterpene saponin substrates to generate corresponding triterpene saponin 3-hydroxyl glycoside products;

[0028] (d3) using the genetically engineered bacteria in the biological material to ferment to produce triterpene saponin 3-hydroxyl glycoside products, and directly feeding triterpene saponin substrates to the fermentation broth to generate corresponding triterpene saponin 3-hydroxyl glycoside products.

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

[0030] (d1) using the Dioscorea panthaica glycosyltransferase to catalyze the generation of triterpene saponin 3-hydroxyl glycoside products in vitro;

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

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

[0033] Further, the enzymatic reaction temperature for the in vitro catalysis is 10-70℃, preferably 35-55℃, and further preferably 35-45℃; and the pH is 6.0-10.0, preferably pH 7.0-8.0.

[0034] In the embodiment of the present application, the triterpene saponin substrate is hederagenin, oleanolic acid, ursolic acid, asiatic acid and betulinic acid; and the triterpene saponin 3-hydroxyl glycoside product is saponin A.

[0035] In the embodiment of the present application, the method for producing triterpene saponin 3-hydroxyl glycoside products such as saponin A catalyzed by the Rabdosia rubescens glycosyltransferase DaUGT121 comprises the following steps:

[0036] (1) The genetically engineered bacteria are subjected to fermentation culture, the fermentation broth is collected, the bacteria are collected by centrifugation, the bacteria are broken, and the supernatant is collected after the supernatant is separated and purified to obtain the protein;

[0037] (2) The supernatant or the separated and purified protein is reacted with the triterpene saponin substrate in a buffer solution to catalyze the generation of the corresponding triterpene saponin-3-O-glycoside; the enzymatic reaction temperature is 10-70 DEG C, and the reaction is carried out in a buffer solution with pH 6.0-10.0.

[0038] In the embodiment of the present application, the Rabdosia rubescens glycosyltransferase DaUGT121 is used for in vitro catalytic production of triterpene saponin 3-hydroxyl glycosides such as saponin A, the genetically engineered bacteria can be added to a reaction system containing the substrate triterpene saponin to carry out the reaction, and the triterpene saponin-3-O-glycoside product is prepared; or the substrate is directly fed to produce the triterpene saponin-3-O-glycoside in an in vivo fermentation mode.

[0039] Compared with the prior art, the glycosyltransferase DaUGT121 disclosed in the present application has the following advantages:

[0040] The present application discloses a Rabdosia rubescens-derived glycosyltransferase, and research shows 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 the glycosyltransferase is expressed in prokaryotes, it can catalyze a series of triterpene saponin glycosylation to be converted into triterpene saponin-3-O-glycoside products.

[0041] The discovery of the Rabdosia rubescens triterpene 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 prospect. The present application improves the understanding of the biosynthesis of triterpene saponins.

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

[0043] Figure 1 It is an agarose gel electrophoresis map of the DaUGT121 gene.

[0044] Figure 2 The sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoretogram of pET28a-DaUGT121.

[0045] Figure 3 The HPLC chart of DaUGT121 catalyzing the arabinose glycosylation of the saponin genin of Hederae to generate Anemarrhenae saponin A.

[0046] Figure 4 The UPLC-MS analysis chart of DaUGT121 catalyzing the arabinose glycosylation of the saponin genin of Hederae to generate Anemarrhenae saponin A;

[0047] Wherein, A is DaUGT121 catalyzing the saponin genin of Hederae to generate Anemarrhenae saponin A; B is the extracted ion chromatogram of the glycosylation reaction; C is the TOF / MS spectrum.

[0048] Figure 5 The HPLC chart of DaUGT121 catalyzing the xylose glycosylation of the saponin genin of Hederae to generate Colchiside A.

[0049] Figure 6 The UPLC-MS chart of DaUGT121 catalyzing the arabinose glycosylation of oleanolic acid to generate oleanolic acid-3-O-α-L-arabinoside;

[0050] Wherein, A is DaUGT121 catalyzing the oleanolic acid to generate oleanolic acid-3-O-α-L-arabinoside; B is the extracted ion chromatogram of the glycosylation reaction; C is the TOF / MS spectrum.

[0051] Figure 7 The UPLC-MS chart of DaUGT121 catalyzing the arabinose glycosylation of ursolic acid to generate ursolic acid-3-O-α-L-arabinoside;

[0052] Wherein, A is DaUGT121 catalyzing the ursolic acid to generate ursolic acid-3-O-α-L-arabinoside; B is the extracted ion chromatogram of the glycosylation reaction; C is the TOF / MS spectrum.

[0053] Figure 8 The UPLC-MS chart of DaUGT121 catalyzing the arabinose glycosylation of asiatic acid to generate asiatic acid-3-O-α-L-arabinoside;

[0054] Wherein, A is DaUGT121 catalyzing the asiatic acid to generate asiatic acid-3-O-α-L-arabinoside; B is the extracted ion chromatogram of the glycosylation reaction; C is the TOF / MS spectrum.

[0055] Figure 9UPLC-MS image of betulinic acid arabinose glycosylation to betulinic acid-3-O-α-L-arabinoside catalyzed by DaUGT121;

[0056] In this spectrum, A represents the DaUGT121-catalyzed formation of betulinic acid 3-O-α-L-arabinoside from betulinic acid; B is the extracted ion chromatogram of the glycosylation reaction; and C is the TOF / MS spectrum.

[0057] Figure 10 This is an SDS-PAGE image of the purified recombinant glycosyltransferase DaUGT121.

[0058] Figure 11 Figure showing the temperature stability of recombinant glycosyltransferase DaUGT121.

[0059] Figure 12 Figure showing the pH stability of recombinant glycosyltransferase DaUGT121.

[0060] Figure 13 The effect of metal ions on the activity of DaUGT121 enzyme. Detailed Implementation

[0061] The following embodiments are provided to illustrate the present invention, making its technical content clearer and easier to understand. The present invention can be embodied through different embodiments. The following are exemplary descriptions. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the present invention.

[0062] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; and the materials and reagents used are commercially available unless otherwise specified.

[0063] Example 1: Cloning of the glycosyltransferase gene

[0064] (I) Extraction of total RNA from Dipsacus asperoides and synthesis of cDNA first strand:

[0065] (1) Take an appropriate amount of fresh or frozen plant sample of Dipsacus asperoides at -80℃, transfer it to a pre-sterilized and pre-cooled mortar for grinding, and continuously add liquid nitrogen during the grinding process until the material is ground into a white fine powder; use the Jereh Total RNA Rapid Extraction Kit (centrifuge column type) to extract total RNA according to the instructions; take 3μL of freshly extracted total RNA for agarose gel electrophoresis analysis.

[0066] (2) Using TaKaRa's PrimerScript TMThe IVStrand cDNASynthesisMix reverse transcription kit was used to reverse transcribe total RNA from Dipsacus asperoides as a template to obtain a cDNA template, according to the instructions.

[0067] (II) Cloning of the DaUGT121 gene:

[0068] Design specific primers, with the specific primer sequences as follows:

[0069] DaUGT121-F: 5'-cgcGGATCCATGGAAGAAATCAGTTCAAAGCCGCAT-3'

[0070] DaUGT121-R: 5'-cccAAGCTTTTTAGGATTTTGCGAGCAAAACTTCA-3'

[0071] 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.

[0072] 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.

[0073] After the reaction was completed, a complete gold product recovery kit was used. The PCR Purification Kit recovers the target gene fragment.

[0074] PCR products were double-digested with Thermo Fisher Scientific's Fast Digest restriction endonucleases BamHI and HindIII. 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 recovered using the Quick Gel Extraction Kit, and the DNA was ligated using T4 DNA ligase to obtain the recombinant expression vector pET28a-DaUGT121.

[0075] 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-DaUGT121.

[0076] Example 2: Prokaryotic expression to verify the function of DaUGT121

[0077] (1) Prokaryotic expression of DaUGT121

[0078] In the blank liquid LB medium, 50 mg / ml Kana was added at a concentration of 1‰ (v / v), and mixed. In the clean bench, 4 ml of LB+Kana (0.1‰, v / v) liquid medium was added to a 15 ml centrifuge tube, and a single colony was taken and mixed. It was cultured at 37°C, 220 rpm on a shaker for 24 h. In the clean bench, 100 ml of LB+Kana (0.1‰, v / v) liquid medium was added to a 500 ml conical flask, 1 ml of bacterial solution was added, and mixed. It was cultured at 37°C, 220 rpm on a shaker for about 2.5 h, and the bacterial solution concentration reached OD 600 0.6-1, the medium was cooled, 12 μL of IPTG (1M) was added to the medium, and the final concentration of IPTG was 0.12 mM. It was cultured at 120 rpm, 13°C for 16 h. The harvested bacterial solution was centrifuged at 4000 rpm, the supernatant was removed, and the bacterial pellet was resuspended with 50 mM Tris-HCl (pH 7.4). After ultrasonic disruption, the supernatant was obtained by centrifugation at 4°C to obtain the crude protein.

[0079] 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 DaUGT121 recombinant protein was good, the calculated protein size was about 54.5 kDa, Figure 2 , which was consistent with the expected value, confirming that the soluble protein was correctly expressed.

[0080] (2) Verification of the enzyme activity of DaUGT121

[0081] The supernatant obtained in (1) was used as DaUGT121 crude enzyme for enzymatic reaction, and the following reaction system (100 μL) was configured:

[0082]

[0083] The reaction was carried out at 37°C overnight, the reaction system was added with two times the volume of methanol to terminate the reaction, vortexed and centrifuged to take the supernatant, and then nitrogen was blown. After drying, 200 μL of methanol was added for resuspension, centrifugation was performed to take the supernatant, and HPLC and UPLC-Q-TOF-MS / MS were used for detection.

[0084] The enzyme activity reaction products were analyzed by HPLC using an Agilent 1260 detector. The chromatographic column was a ZORBAX Eclipse Plus C18 column (4.6 mm × 250 mm, 5 μm); the mobile phase was 0.05% phosphoric acid aqueous solution-acetonitrile (v / v), with 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℃; the flow rate was 1.0 mL / min; the injection volume was 10 μL; and equilibration was performed for 10 min. HPLC-1260 results are shown below. Figure 3 As shown, products appeared in the experimental group, and their peak times basically corresponded to those of the standard.

[0085] The enzyme activity reaction products were detected by LC-MS. The UPLC-Q-TOF-MS / MS chromatographic and mass spectrometric conditions were as follows: the mobile phase consisted of acetonitrile (A) and 0.1% formic acid water (v / v) (B). HSS C18 (2.1×100mm, 1.8μm) column. Mass spectrometry parameters included: ion source: Turbo V. TM The mass spectrometry data was acquired using negative ion scanning mode and TOF MS-Product Ion-IDA mode. The scan range was 100–1500 m / z. Nebulized gas (GS1): 344.738 kPa (50 psi); Nebulized gas (GS2): 344.738 kPa (50 psi); Curtain gas (CUR): 241.317 kPa (35 psi); Ion source temperature (TEM): 550℃; Ion source voltage (IS): -4500 V; First-stage scan: Declustering voltage (DP): -100 V; Focusing voltage (CE): -10 V; Second-stage scan: Declustering voltage (DP): -100 V; Focusing voltage (CE): -40 V. Mass spectrometry data acquisition was performed using Analyst software, data analysis using PeakView software, and rapid compound matching and identification using MarkerView software.

[0086] Combining HPLC and LC-MS results, Figures 3-9 This indicates that the glycosyltransferase DaUGT121 uses UDP-arabinose and UDP-xylose as glycosyl donors to catalyze the formation of corresponding triterpenoid saponins from triterpenoid saponins – 3-O-glycosides. HPLC analysis showed a product peak matching that of Clematis chinensis saponin A at 28 minutes. Figure 3 ), while UPLC-Q-TOF-MS analysis further confirmed the molecular structure of the product, and its secondary fragment ion spectrum was completely consistent with the standard. Figure 4). The results collectively demonstrate the unique function of DaUGT121 in catalyzing the addition of an arabinose group at the 3-OH position of hederagenin. As shown in Fig. Figure 5 ), a new peak was detected at 29 min when hederagenin, UDP-Xyl, and DaUGT121 were reacted. From the previous experiment, it was known that Cauloside A could be produced when hederagenin, UDP-Ara, and DaUGT121 were reacted. It was thus determined that DaUGT121 has the function of catalyzing the transfer of a xylose group at the C-3 position of hederagenin. When reacted with oleanolic acid and UDP-Ara, a new peak was detected at 13.5 min, with a parent ion of 587.3376 m / z. By comparison with the parent ion of oleanolic acid-3-O-α-L-arabinoside, the consistency of the parent ion supports the hypothesis that DaUGT121 has the ability to transfer an arabinose group at the 3-OH position of oleanolic acid Figure 6 ). In the reaction system of ursolic acid, UDP-Ara, and DaUGT121 Figure 7 ), a new peak was detected at 13.2 min, with a parent ion of 587.3379 m / z. By comparison with the parent ion of ursolic acid-3-O-α-L-arabinoside, the same parent ion data further indicates the arabinose group transfer function of DaUGT121 at the 3-OH position of ursolic acid. In the glycosylation reaction of asiatic acid, UDP-Ara, and DaUGT121 Figure 8 ), a new peak was detected at 20.5 min, with a parent ion of 619.3833 m / z. Comparison of this parent ion with that of asiatic acid-3-O-α-L-arabinoside confirms the arabinose group transfer activity of DaUGT121 at the C-3 position of asiatic acid. When betulinic acid, UDP-Ara, and DaUGT121 were reacted, a new peak was detected at 17 min, with a parent ion of 587.3951 m / z. Then, by comparing the secondary fragment ions of this peak with those of betulinic acid-3-O-α-L-arabinoside, it was found that the parent ions were the same, and it was thus determined that DaUGT121 has the function of transferring an arabinose group at the 3-OH position of betulinic acid Figure 9

[0087] Through detailed HPLC and LC-MS analysis, the research reveals the specific glycosyltransferase activity of DaUGT121 on various triterpenoid saponin substrates, especially emphasizing its ability to add arabinose and xylose to hederagenin, oleanolic acid, ursolic acid, and asiatic acid. DaUGT121 exhibits high utilization ability for UDP-arabinose and UDP-xylose as glycosyl donors, which indicates its broad substrate adaptability. These experimental results are of great significance for understanding and utilizing the role of DaUGT121 glycosyltransferase in triterpenoid saponin biosynthesis. ​

[0088] Example 3: Optimum temperature of recombinant glycosyltransferase DaUGT121

[0089] (1) Purification of glycosyltransferase DaUGT121

[0090] Using the crude enzyme solution of recombinant glycosyltransferase DaUGT121 obtained in Example 2 as the original material, 5 mL of HisTrap pre-packed nickel column was mounted on a peristaltic pump. The crude protein was loaded into the nickel column at a flow rate of 5 mL / min using 10 column volumes of A solution (20 mM sodium phosphate buffer at pH 7.4 containing 20 mM imidazole and 500 mM NaCl). The protein was eluted with 5 column volumes of eluent containing 10-500 mM imidazole at the same flow rate, and the corresponding eluent (25 mL / tube) was collected.

[0091] The eluent was analyzed by SDS-PAGE protein electrophoresis, and the eluent containing the target protein was placed in a 50 kD ultrafiltration tube and centrifuged at 2300 rpm at 4°C. When the protein solution was about 1 mL, 20 mL of D solution (20 mM sodium phosphate buffer at pH 7.4 containing 500 mM NaCl) was added for further ultrafiltration, and the process was repeated 3-5 times. The purified target protein was obtained, as shown in Figure 10 by SDS-PAGE detection, the protein band was relatively single.

[0092] (2) Optimum temperature of recombinant glycosyltransferase DaUGT121

[0093] The protein was quantitatively analyzed by BCA protein quantitative kit, 10 μg of recombinant glycosyltransferase DaUGT121, 100 μM of hederagenin and 500 μM of UDP-arabinose were used. These reactants were mixed with 50 mM Tris-HCl buffer at pH 7.0, and the total volume was adjusted to 100 μL. The mixture was reacted at 30-70°C for 12 hours. After the reaction was completed, 200 μL of methanol was added to terminate the reaction, and the supernatant was collected after vortex centrifugation. Then, 200 μL of methanol was added for re-dissolution after nitrogen blowing. After re-centrifugation, the supernatant was collected and detected by LC-MS. Based on the standard of setting the highest activity as 100%, the relative activity determination results are shown in Table 1, which shows that the enzyme exhibits the highest reaction activity at 45°C.

[0094] Table 1: Optimum temperature of glycosyltransferase DaUGT121

[0095]

[0096]

[0097] Example 4: Optimum pH of recombinant glycosyltransferase DaUGT121

[0098] Take 2-3 ng of purified recombinant glycosyltransferase DaUGT121 and final concentration of 100 μM of hederagenin and 500 μM of UDP-arabinose, respectively, add 50 mM of citric acid-sodium citrate buffer (pH 3.0-6.0), sodium phosphate dibasic-sodium phosphate 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, incubate at the optimum temperature of 45 ℃ for 12 hours. Add 200 μL of methanol to terminate the reaction, vortex centrifugation to take the supernatant, nitrogen blow, add 200 μL of methanol after drying, centrifugation to take the supernatant, and use LC-MS for detection. With the highest activity as 100%, the relative activity results are shown in Table 2, and the enzyme has the highest reaction activity in Tris-hydrochloric acid buffer (pH = 9.0).

[0099] Table 2: Optimum pH of glycosyltransferase DaUGT121

[0100] pH Relative viability (%) 3.0 (Citric acid-sodium citrate buffer) 0.3±0.1 4.0 (Citric acid-sodium citrate buffer) 0.2±0.0 5.0 (Citric acid-sodium citrate buffer) 0.8±0.7 6.0 (Citric acid-sodium citrate buffer) 28.1±4.5 6.0 (Disodium hydrogen phosphate-monosodium dihydrogen phosphate buffer) 34.8±7.9 7.0 (Disodium hydrogen phosphate-monosodium dihydrogen phosphate buffer) 54.4±1.2 8.0 (Disodium hydrogen phosphate-monosodium dihydrogen phosphate buffer) 81.8±2.8 8.0 (Tris-hydrochloric acid buffer) 92.3±4.4 9.0 (Tris-hydrochloric acid buffer) 95.7±0.2 9.0 (Sodium carbonate-sodium bicarbonate buffer) 95.4±4.4 10.0 (Sodium carbonate-sodium bicarbonate buffer) 37.2±3.7 11.0 (Sodium carbonate-sodium bicarbonate buffer) 1.2±0.2

[0101] Example 5: Temperature stability of recombinant glycosyltransferase DaUGT121

[0102] At different temperatures, take 2-3 ng of purified recombinant glycosyltransferase DaUGT121 and final concentration of 100 μM of hederagenin and 500 μM of UDP-arabinose in 50 mM, pH 9.0 Tris-hydrochloric acid buffer. Reaction was terminated by adding 200 μL of methanol, vortex centrifugation to take the supernatant, nitrogen blow, add 200 μL of methanol after drying, centrifugation to take the supernatant, and use LC-MS for detection. With the highest activity as 100%, the relative residual activity results are shown in Table 3, and it is found that DaUGT121 has a significant decrease in stability when the temperature exceeds 40 ℃. Between 30 ℃ and 35 ℃, DaUGT121 exhibits relatively high catalytic activity. After incubation at 35 ℃ for 40 min, the relative enzyme activity can still remain more than 60%. In addition, the activity can be maintained at more than 40% of the initial level after incubation below 40 ℃ for 20 minutes. However, when the temperature rises above 45 ℃, the activity of the enzyme is almost lost within 20 minutes. Figure 11

[0103] Example 6: pH stability of recombinant glycosyltransferase DaUGT121

[0104] ​At different pH, 2-3 ng of purified recombinant glycosyltransferase DaUGT121 and 100 μΜ of hederagenin and 500 μΜ of UDP-arabinose were reacted at 45 °C. After reaction, 200 μL of methanol was added to terminate the reaction, and the supernatant was taken after vortex centrifugation and nitrogen blowing. After drying, 200 μL of methanol was added for redissolution, and the supernatant was taken after centrifugation. LC-MS was used for detection. With the highest activity as 100%, the relative residual activity results are shown in Figure 12 . DaUGT121 showed the highest catalytic activity at pH 9. When the reaction pH value was more than 9, the enzyme activity began to decrease, which was probably caused by enzyme structure denaturation and active center damage in strong alkaline environment. In extremely acidic (pH 3.0 to 5.0) and extremely alkaline (pH 10.0) environments, a significant decrease in enzyme activity was observed. In addition, the stability of DaUGT121 in the pH 6 to 10 range was also evaluated, and it was found that the stability was best in the medium alkaline condition. After 40 minutes of incubation at pH 7, the enzyme could still maintain more than 80% activity.

[0105] Example 7: Effect of metal ions on recombinant glycosyltransferase DaUGT121

[0106] 2-3 ng of purified recombinant glycosyltransferase DaUGT121 was reacted in 50 mM Tris-hydrochloric acid buffer at pH 9.0, and 5 mM of Co 2+ , MO 6+ , Ca 2+ , Mg 2+ , Zn 2+ , EDTA 2+ , Cu 2+ , Fe 3+ , Ba 2+ , Mn 2+ and NH4 -1 metal ions were added, and 100 μΜ of hederagenin and 500 μΜ of UDP-arabinose were reacted at 45 °C for 12 h. After reaction, 200 μL of methanol was added to terminate the reaction, and the supernatant was taken after vortex centrifugation and nitrogen blowing. After drying, 200 μL of methanol was added for redissolution, and the supernatant was taken after centrifugation. LC-MS was used for detection. With the activity of the blank control group without metal ions as 100%, the relative residual activity results are shown in Figure 13 . Fe 3+ was the only one that could significantly enhance the enzyme activity to 120%, while the addition of Mn 2+ had no significant effect on the enzyme activity, which was flat with the control group. EDTA 2+ had the most significant inhibition on the activity of DaUGT121, which reduced the activity to 38% of the control group.

[0107] The foregoing describes in detail preferred embodiments of this application. It is understood to those of ordinary skill in the art that modifications and changes can be made thereto without departing from the spirit and scope of the application as set forth in the following claims. It is therefore desired that what is claimed should be interpreted as being the broadest interpretation feasible under the prior art to allow the public to fully utilize the benefits of the application.

[0108] SEQ ID NO. 1 299

[0110] Dipsacus asper Wall. ex Henry

[0111] MEEISSKPHAVFAPYPLLSHINPSLKLAKLIHQKGFHITFVIPDYNYNSILSSLGPHALEGLPDFQFKSMALDVPPANSLPPPDYFGVLMKSIWANFLPLFRELVNKLNKDESWPKVSCIISDGVMTQSTVVAEELGVPIALSWITGANGFTNIARHRDLVRKCVAGLRDGSIDIKSEELDKPIIGFLPGRKAMSMREFLNFMQTTHTKEYMNESVVGEVQRTSNSSALIFETFEGLEKEALDEIRKKVPHVYSVAPLPLLLKNEVNDERVVKLVGDDIWKEDIECSRWLDSKAPNSVLYVSFGTFAITSQQHLIEIAWGLAASKKNFLWIIREDMVVGDVVTTLPEGFVEETKDRGLIKSWCKQEQVLNHPAIGGFFSHCGWNSVLESITAGIPMICRPYFGEHIISSKMICEEWEFGIEVNDNIDRRQVENAVKEILEGEKGKIIREKAKEWMRIAKEATYLDGTSSQNLDKLVNEVLLAKS

[0112] SEQ ID NO. 2 1455

[0114] Dipsacus asper Wall. ex Henry

[0115]

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 3-hydroxyglycosylation 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 that has more than 80% homology with the nucleotide sequence shown in SEQ ID NO.2 and encodes a glycosyltransferase that catalyzes the 3-hydroxy 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 of claim 2, or a recombinant vector containing the expression cassette of (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 described in claim 1, the gene described in claim 2, or the biomaterial described in claim 3 or 4 in catalyzing the 3-hydroxyglycosylation modification of triterpenoid saponins to generate triterpenoid saponin 3-hydroxyglycoside products.

6. The application according to claim 5, characterized in that, The method for catalytically modifying triterpenoid saponins by 3-hydroxyglycosylation to generate triterpenoid saponin 3-hydroxyglycoside products is as follows (d1) or (d2) or (d3): (d1) Triterpenoid saponin 3-hydroxy glycoside products were generated in vitro using the aforementioned Dipsacus asperoides 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-hydroxy glycoside products; (d3) The transgenic engineered bacteria in the biomaterial are used to ferment and produce triterpenoid saponin 3-hydroxy glycoside products. The triterpenoid saponin substrate is directly fed into the fermentation broth to generate the corresponding triterpenoid saponin 3-hydroxy glycoside products.

7. A method for producing triterpenoid saponin 3-hydroxyglycoside 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-hydroxy glycoside products; (d2) The transgenic engineered bacteria in the biomaterial described in claim 3 are added to a reaction system containing triterpenoid saponin substrates to carry out the reaction and generate the corresponding triterpenoid saponin 3-hydroxy glycoside product; (d3) Using the transgenic engineered bacteria in the biomaterial described in claim 3 to produce triterpenoid saponin 3-hydroxy glycoside products by fermentation, and directly feeding triterpenoid saponin substrates into the fermentation broth to generate the corresponding triterpenoid saponin 3-hydroxy glycoside products.

8. The method according to claim 7, characterized in that, The in vitro catalytic enzyme reaction temperature described in (d1) is 10-70℃, preferably 35-55℃, more preferably 35-45℃; pH 6.0-10.0, preferably pH 7.0-8.

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 at least one of hederaponin, oleanolic acid, ursolic acid, asiatic acid and betulinic acid; the triterpenoid saponin 3-hydroxyglycoside product is Clematis chinensis saponin A.

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