Gynostemma pentaphyllum glycosyl transferase GpUGT74A1 and application thereof
Gynostemma pentaphyllum glycosyltransferase GpUGT74A1 catalyzes the formation of glycosidic bonds between protopanaxatriol PPT and protopanaxadiol PPD and glucose, which solves the problem that existing technology cannot meet the large-scale demand for ginsenosides and achieves the efficient preparation of ginsenosides F1 and CK. .
Patent Information
- Application Number
- CN202411567604.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-05
- Publication Date
- 2025-01-21
AI Technical Summary
The existing gynostemma cyanobacter glycosyltransferase cannot meet the large demand for ginseng saponins, and finding other ways to prepare ginseng saponins is an urgent problem.
Provide a twisted blue sugar -based metastase GPUGT74A1. By catalyzing the original ginseng triam PPT and the ginseng glycol PPD to form glucoside bonds with glucose, the rare human ginsenoside F1 and CK are generated.
The efficient preparation of ginsenosides F1 and CK was achieved with a simple method, high yield, low consumption, and enriched the glycosyltransferase database.
Smart Images
Figure CN119331848A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, and in particular to a gynostemma pentaphyllum glycosyltransferase GpUGT74A1 and an application thereof. Background Art
[0002] Gynostemma pentaphyllum (Thunb.) Makino is a perennial climbing herb of the genus Gynostemma in the Cucurbitaceae family. It contains more than 10 ingredients, including saponins, polysaccharides, flavonoids, amino acids, vitamins, etc., among which gypenosides are one of the main active ingredients. At present, 445 types of gypenosides have been separated from Gynostemma pentaphyllum, and it has been found that the total saponin content of Gynostemma pentaphyllum is about 3 times that of ginseng. Among them, saponins Re, Rg2, Rc, Fl, Rd, Rf, etc. are common components of ginseng and Gynostemma pentaphyllum. The structures of gypenosides Gyp-III, Gyp-IV, Gyp-VIII, and Gyp-XII are the same as those of ginsenosides Rbl, Rb3, Rd, and F2, respectively.
[0003] Ginsenosides, as the main component of the precious Chinese medicine ginseng, have multiple pharmacological effects. At present, the main method for producing ginsenosides is to extract them from cultivated ginseng. However, the chemical extraction of saponins has low efficiency, low yield, and high production cost, which limits the development of ginsenosides as medicine. Ginseng has a long growth cycle, and the process of extracting ginsenosides is complex, low in yield, and easy to cause environmental pollution. The glycosidase method has the advantages of stereoselectivity, high yield, few by-products, and can obtain rare ginsenosides. It is considered to be the most promising method for producing rare ginsenosides.
[0004] In order to alleviate the pressure on medicinal material resources, the heterologous biosynthesis and directional preparation of ginsenosides, especially rare saponins, have attracted much attention. The prior art has disclosed the functions of 8 kinds of Gynostemma pentaphyllum glycosyltransferases, which can catalyze the production of ginsenosides CK, F1, F2, Rd, Rh2 and other secondary saponins from protopanaxadiol or protopanaxatriol ginsenosides. However, the existing Gynostemma pentaphyllum glycosyltransferases still cannot meet the current large demand for ginsenosides, and finding other methods for preparing other ginsenosides is an urgent problem to be solved in this field. Summary of the invention
[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a gynostemma pentaphyllum glycosyltransferase GpUGT74A1 and its application, which can catalyze the formation of glycosidic bonds between the 20th hydroxyl group of protopanaxatriol PPT and protopanaxadiol PPD and glucose molecules, thereby generating rare ginsenosides F1 and CK.
[0006] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:
[0007] Provided is a gynostemma pentaphyllum glycosyltransferase GpUGT74A1, the amino acid sequence of the gynostemma pentaphyllum glycosyltransferase GpUGT74A1 is shown in SEQ ID NO.3.
[0008] The present invention also provides the use of the above-mentioned Gynostemma pentaphyllum glycosyltransferase GpUGT74A1 in the preparation of ginsenoside F1 and ginsenoside CK.
[0009] Furthermore, the specific method for preparing ginsenoside F1 and ginsenoside CK by Gynostemma pentaphyllum glycosyltransferase GpUGT74A1 is as follows:
[0010] A1: preparing a reaction solution, the reaction solution comprising uridine diphosphate glucose disodium salt, protopanaxatriol PPT or protopanaxadiol PPD, Gynostemma pentaphyllum glycosyltransferase GpUGT74A1 enzyme solution and tris(hydroxymethyl)aminomethane hydrochloride;
[0011] A2: After reacting at 37°C for 12 h, add the same volume of ethyl acetate to terminate the reaction;
[0012] A3: Take the upper organic phase, rotary evaporate and dry it, and purify it with a silica gel column to obtain ginsenoside F1 or ginsenoside CK; when protopanaxatriol PPT is added to the reaction solution, ginsenoside F1 is obtained; when protopanaxadiol PPD is added to the reaction solution, ginsenoside CK is obtained.
[0013] Further, the concentration of uridine diphosphate glucose disodium salt is 50mM; the concentration of protopanaxatriol PPT or protopanaxadiol PPD is 10mM; the concentration of Gynostemma pentaphyllum glycosyltransferase GpUGT74A1 enzyme solution is 60mg / ml; the concentration of tris(hydroxymethyl)aminomethane hydrochloride is 100mM, and the pH is 8.0;
[0014] The volume ratio of UDP-glucose disodium salt, protopanaxatriol PPT or protopanaxadiol PPD, Gynostemma pentaphyllum glycosyltransferase GpUGT74A1 enzyme solution and tris(hydroxymethyl)aminomethane hydrochloride is 3:3:1:23.
[0015] Furthermore, in step A3, the eluent used in the silica gel column purification is chloroform:methanol=85:15.
[0016] The invention also provides a genetically engineered Escherichia coli bacterium expressing the gynostemma pentaphyllum glycosyltransferase GpUGT74A1.
[0017] The present invention also provides a method for constructing the above-mentioned genetically engineered Escherichia coli, comprising the following steps:
[0018] B1: extract total RNA from Gynostemma pentaphyllum and obtain Gynostemma pentaphyllum cDNA library by reverse transcription;
[0019] B2: Design specific primers to amplify the gene of GpUGT74A1, a gypenoside glycosyltransferase, from the Gynostemma cDNA library;
[0020] B3: Prepare a plasmid for constructing a metabolic pathway based on the gypenoside glycosyltransferase gene GpUGT74A1;
[0021] B4: The host bacteria E. coli BL21 (DE3) is subjected to lysogeny treatment, and the plasmid for constructing the metabolic pathway is transformed into the lysogeny bacteria, thereby obtaining the genetically engineered E. coli bacteria expressing the Gynostemma pentaphyllum glycosyltransferase GpUGT74A1.
[0022] Furthermore, the specific primers include a forward primer and a reverse primer. The sequence of the forward primer is shown in the sequence listing as SEQ ID NO.1; the sequence of the reverse primer is shown in the sequence listing as SEQ ID NO.2.
[0023] The present invention also provides the use of the above-mentioned genetically engineered Escherichia coli in synthesizing ginsenoside F1 and ginsenoside CK in vitro.
[0024] The present invention also provides a gene encoding the gynostemma pentaphyllum glycosyltransferase GpUGT74A1, and the gene sequence is shown in SEQ ID NO.4.
[0025] The beneficial effects of the present invention are:
[0026] The present invention proposes a novel gynostemma glycosyltransferase, which catalyzes the formation of glycosidic bonds between the 20th hydroxyl group of protopanaxatriol PPT and protopanaxadiol PPD and glucose molecules to generate ginsenoside F1 and ginsenoside CK. The gynostemma glycosyltransferase is compared with several known gynostemma glycosyltransferases, and the highest homology is UGTGp5, which is 36.32%.
[0027] When the gynostemma pentaphyllum glycosyltransferase is used to prepare ginsenoside F1 and ginsenoside CK, the method and process are simple, the yield is high, and the consumption is low, thus enriching the current glycosyltransferase database. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 is the plasmid map of the recombinant plasmid pSJ8-MBP-GpUGT74A1;
[0029] Figure 2 The mass spectra of the product in Example 3 and the standard are compared;
[0030] Figure 3 HPLC comparison spectra of the product in Example 3 and the standard product. DETAILED DESCRIPTION
[0031] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.
[0032] Unless otherwise specified, the materials, reagents, instruments and methods used in the following examples of the present invention are conventional materials, reagents, instruments and methods in the art and can be obtained through commercial channels; for example, Tryptone, Yeast Extract, Agar; ampicillin, E. coli DH5α, etc. In the present invention, the plasmid extraction adopts the high-purity plasmid small-scale rapid extraction kit (Catalog NO.: hdp201-02) of Huiling Biotechnology (Shanghai) Co., Ltd., the gel excision recovery adopts the quick agarose gel DNA recovery kit (Catalog NO.: dp1702) of Baitai Biotechnology (Fuzhou) Co., Ltd., the connection of DNA fragments uses the DNA Assembly Mix Plus (Catalog NO.: D0204P) of Rambolide Biotechnology Co., Ltd., the amplification of DNA fragments uses the T8 High-Fidelity Master Mix (Catalog NO.: TSE111) of Qingke Biotechnology (Beijing) Co., Ltd., the digestion of PCR plasmid template uses Xho I (Calalog NO.: F5583S) and Not I (Calalog NO.: F5553S) of Rambolide Biotechnology Co., Ltd., and the E. coli heat shock transformation experiment uses the constant temperature water bath (Catalog NO.: HH-2) of Guohua Electric Co., Ltd.
[0033] Example 1 Cloning of Glycosyltransferase GpUGT74A1 from Gynostemma pentaphyllum
[0034] With the help of transcriptomics data analysis, combined with the resources of databases such as NCBT, and the principles of plant PSPG boxes, a new coding gene was screened and obtained, and the sequence is shown in SEQ ID NO.4. According to SEQ ID NO.4, the forward primer sequence is designed as shown in the sequence list SEQ ID NO.1; the reverse primer sequence is shown in the sequence list SEQ ID NO.2; the gene is amplified from the Gynostemma pentaphyllum cDNA library by PCR using a polymerase. The amplified fragment was gel-cut and purified, and seamlessly homologously recombined with the pSJ8-MBP plasmid linearized after reverse PCR. According to the instructions of the homologous recombination reagent, the linearized vector and the target gene fragment with homology arms were mixed in a certain amount, and an appropriate amount of DNA Assembly Mix reagent was added and reacted at 50°C for 15 minutes. The recombinant product was transformed into E.coli DH5α, and cultured at 37°C overnight on an LB plate containing ampicillin, and positive clones were screened and sequenced for verification. The recombinant plasmid pSJ8-MBP-GpUGT74A1 was obtained, and the plasmid map is shown as follows Figure 1 shown.
[0035] Example 2 Establishment of Escherichia coli expression strain and purification of Gynostemma pentaphyllum glycosyltransferase GpUGT74A1
[0036] The recombinant plasmid pSJ8-MBP-GpUGT74A1 obtained in Example 1 was transformed into the host bacteria E. coli BL21 (DE3) competent cells to obtain the recombinant expression strain of Gynostemma pentaphyllum glycosyltransferase. The recombinant bacteria were cultured to an OD of 0.6 to 0.8, 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) was added, and induced at 16°C for 16 hours. The cells were collected by centrifugation at 4°C and 5000 rpm, and the cells were ultrasonically disrupted. The enzyme solution of Gynostemma pentaphyllum glycosyltransferase GpUGT74A1 was purified by Ni-NTA His-binding resin, and the amino acid sequence shown in SEQ ID NO.3 was obtained by sequencing.
[0037] Example 3 Preparation of Ginsenoside F1 and Ginsenoside CK by Gynostemma Pentaphyllum Glycosyltransferase
[0038] Take the Gynostemma pentaphyllum glycosyltransferase GpUGT74A1 enzyme solution prepared in Example 2, and prepare the reaction solution according to Table 1;
[0039] Table 1
[0040] Components Dosage UDP-Glucose diphosphate disodium salt (50 mM) 30μL Protopanaxatriol PPT or protopanaxadiol PPD (10 mM) 30μL Gynostemma pentaphyllum glycosyltransferase GpUGT74A1 enzyme solution (60mg / ml) 10μL Tris-HCL (100mM, pH 8.0) 230μL Overall system 300μL
[0041] 300 μL of reaction solution 1 containing protopanaxatriol PPT and 300 μL of reaction solution 2 containing protopanaxadiol PPD were prepared respectively; reaction solution 1 and reaction solution 2 were reacted at 37°C for 12 hours, and then the same volume of ethyl acetate was added to terminate the reaction; the upper organic phase after the reaction was completed was rotary evaporated and dried, and purified by silica gel column; the eluent used in the purification was chloroform: methanol = 85: 15. Product 1 was purified by reaction solution 1 containing protopanaxatriol PPT, and product 2 was purified by reaction solution 2 containing protopanaxadiol PPD.
[0042] For each 5 ml portion of the purified product, aliquots were collected, and the collected samples were subjected to EST and HPLC analysis (conditions were the same as described above) to obtain product 1 or product 2 with a purity of <90%;
[0043] In the specific implementation, for further purification, the collected portion is purified using a Sep-Pak tC18 column (Waters), water (A) and acetonitrile (B) are used as eluents, and gradient elution (20%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%) is adopted. When eluting with 70% and 75% acetonitrile, product 1 or product 2 is obtained, and the purity reaches 98%. White powder is obtained by rotary evaporation or freeze drying at 40°C.
[0044] The reaction system was extracted three times with 3 volumes of ethyl acetate, the upper layers were combined, dried in vacuo, re-dissolved with 50% mass spectrometry grade methanol, filtered through a 0.22 μm filter membrane, and then detected by electrospray ionization mass spectrometry (ESI) using a Finnigan LCQ Advantage MAX ion trap massspectromcter (Thermo Eloctron, CA). The results are shown in Figure 2 As shown. Among them, Figure 2 a is the ion peak of ginsenoside F1 standard; Figure 2 b is the ion peak of product 1; Figure 2 c is the ion peak of ginsenoside CK standard; Figure 2 b is the ion peak of product 2;
[0045] Depend on Figure 2 It can be seen that the sodium addition peak (m / z) 661.43 of product 1 is consistent with the standard ginsenoside F1 (Na + The molecular weight of product 2 is consistent with that of ginsenoside CK (Na + The molecular weight of )(645) is consistent.
[0046] The protopanaxatriol PPT, protopanaxadiol PPD, standard ginsenoside F1, standard ginsenoside CK, as well as the normal reaction solution and the inactivated reaction solution were subjected to liquid phase detection and analysis, specifically: using Shimadzu LC-20A liquid chromatograph (Shimadzu, Japan), using Ultimate XB-C18 (5μm 4.6mm×250mm), A: ultrapure water, mobile phase B: 100% acetonitrile, flow rate 1.0mL·min-1, column temperature: 30℃, detection wavelength: 203nm, sample volume: 10μL. The gradient elution program was: 0-5 min, 10% B; 5-10 min, 10%-30% B; 10-25 min, 30%-70% B; 25-30 min, 70% B; 30-35 min, 70%-76% B; 35-40 min, 76%-95% B; 40-50 min, 95% B; 50-55 min, 95%-60% B; 55-60 min, 60%-30% B; 60-65 min, 30%-10% B; 65-70 min, 10% B.
[0047] Test results such as Figure 3 As shown by Figure 3 It can be seen that standard ginsenoside F1 and standard ginsenoside CK peaked at around 22min and 33min respectively; the negative control reaction solution (inactivated) had no product at around 22min and 33min; the reaction solution had product peaks at around 22min and 33min respectively, and the peak values were relatively high. It can be seen that the conversion of protopanaxatriol PPT or protopanaxadiol PPD to ginsenoside F1 or ginsenoside CK requires the participation of active Gynostemma pentaphyllum glycosyltransferase GpUGT74A1.
Claims
1. A Gynostemma pentaphyllum glycosyltransferase GpUGT74A1, characterized in that The amino acid sequence of the Gynostemma pentaphyllum glycosyltransferase GpUGT74A1 is shown in SEQ ID NO.
3.
2. Use of the Gynostemma pentaphyllum glycosyltransferase GpUGT74A1 according to claim 1 in the preparation of ginsenoside F1 and ginsenoside CK.
3. The use according to claim 2, characterized in that: The specific method for preparing ginsenoside F1 and ginsenoside CK by using Gynostemma pentaphyllum glycosyltransferase GpUGT74A1 is as follows: A1: preparing a reaction solution, wherein the reaction solution includes uridine diphosphate glucose disodium salt, protopanaxatriol PPT or protopanaxadiol PPD, Gynostemma pentaphyllum glycosyltransferase GpUGT74A1 enzyme solution and tris(hydroxymethyl)aminomethane hydrochloride; A2: After reacting at 37°C for 12 h, add the same volume of ethyl acetate to terminate the reaction; A3: Take the upper organic phase, rotary evaporate and dry it, and purify it with a silica gel column to obtain ginsenoside F1 or ginsenoside CK; when protopanaxatriol PPT is added to the reaction solution, ginsenoside F1 is obtained; when protopanaxadiol PPD is added to the reaction solution, ginsenoside CK is obtained.
4. The use according to claim 3, characterized in that: The concentration of uridine diphosphate glucose disodium salt is 50mM; the concentration of protopanaxatriol PPT or protopanaxadiol PPD is 10mM; the concentration of Gynostemma pentaphyllum glycosyltransferase GpUGT74A1 enzyme solution is 60mg / ml; the concentration of tris(hydroxymethyl)aminomethane hydrochloride is 100mM, and the pH is 8.0; The volume ratio of UDP-glucose disodium salt, protopanaxatriol PPT or protopanaxadiol PPD, Gynostemma pentaphyllum glycosyltransferase GpUGT74A1 enzyme solution and tris(hydroxymethyl)aminomethane hydrochloride is 3:3:1:
23.
5. The use according to claim 3, characterized in that: In the step A3, the eluent used in the silica gel column purification is chloroform:methanol=85:
15.
6. A genetically engineered Escherichia coli bacterium expressing GpUGT74A1, a glycosyltransferase of Gynostemma pentaphyllum.
7. A method for constructing the genetically engineered Escherichia coli according to claim 6, characterized in that: The steps include: B1: extract total RNA from Gynostemma pentaphyllum and obtain Gynostemma pentaphyllum cDNA library by reverse transcription; B2: Design specific primers to amplify the gene of GpUGT74A1, a gypenoside glycosyltransferase, from the Gynostemma cDNA library; B3: Prepare a plasmid for constructing a metabolic pathway based on the gypenoside glycosyltransferase gene GpUGT74A1; B4: The host bacteria E. coli BL21 (DE3) is subjected to lysogeny treatment, and the plasmid for constructing the metabolic pathway is transformed into the lysogeny bacteria, thereby obtaining the genetically engineered E. coli bacteria expressing the Gynostemma pentaphyllum glycosyltransferase GpUGT74A1.
8. The construction method according to claim 7, characterized in that: The specific primers include a forward primer and a reverse primer. The sequence of the forward primer is shown in the sequence list as SEQ ID NO.1; the sequence of the reverse primer is shown in the sequence list as SEQ ID NO.
2.
9. Use of the genetically engineered Escherichia coli bacteria according to claim 6 in the in vitro synthesis of ginsenoside F1 and ginsenoside CK.
10. A gene encoding Gynostemma pentaphyllum glycosyltransferase GpUGT74A1, characterized in that: The gene sequence is shown as SEQ ID NO.4.