Glycosyl transferase gene for Gypensaposide LVI biosynthesis and application of glycosyl transferase gene

Abstract

The invention discloses a glycosyl transferase gene used for biosynthesis of Gypensaposide LVI and an application of the glycosyl transferase gene used for biosynthesis of Gypensaposide LVI. According to the invention, a coding gene and an expression vector containing the coding gene are found in gynostemma pentaphylla, and the protein coded by the coding gene can catalyze Gypenoside XLVI to generate Gypenoside LVI. Therefore, the coding gene, the expression vector containing the coding gene and the protein coded by the coding gene can be used for synthesizing and preparing the Gypensaposide LVI, the yield of the Gypensaposide LVI can be increased, and the economic value is relatively high.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biology, and relates to a glycosyltransferase gene for Gypenoside LVI biosynthesis and use thereof. BACKGROUND

[0002] Dammarane-type triterpenoid saponins are a class of natural compounds with important pharmacological activities such as anti-inflammatory and anti-tumor, and widely exist in plants such as ginseng and gynostemma. They have significant application value in the fields of medicine and functional food. Among them, gynostemma is an ideal alternative resource for obtaining this type of saponins due to its strong environmental adaptability, low planting cost, and higher total content of gypenosides (dammarane type) than ginseng.

[0003] Among various monomers of gypenosides, Gypenoside LVI has become the research core due to its excellent biological activity. Modern pharmacology has confirmed that Gypenoside LVI can exert a strong anti-tumor effect through mechanisms such as regulation of tumor cell signaling pathways and induction of apoptosis, and has low toxicity to normal cells and high safety, making it a highly potential anti-tumor lead drug. With the rising incidence of cancer worldwide and the growing demand for high-efficiency and low-toxicity anti-tumor drugs, the market demand for Gypenoside LVI continues to surge.

[0004] However, the current acquisition of Gypenoside LVI still relies on extraction and separation from gynostemma plants, which has significant bottlenecks. On the one hand, the content of Gypenoside LVI in gynostemma is low, and its structure is similar to that of other saponin monomers, resulting in a complex and costly extraction and separation process that cannot meet the needs of industrial production. On the other hand, the growth of plants is greatly affected by natural environment and planting conditions, and the quality and yield fluctuate significantly, further reducing the stability and efficiency of Gypenoside LVI acquisition. Therefore, how to break through the limitations of existing technology, improve the yield of Gypenoside LVI and reduce the cost, has become the key to promoting its industrialization.

[0005] The synthesis of plant natural products depends on the catalytic regulation of enzymes, and the function of enzymes is determined by coding genes. In the biosynthesis pathway of Gypenoside LVI, glycosyltransferase is a core modification enzyme that can catalyze the transfer of a sugar group to a saponin precursor, directly determining the synthesis efficiency and yield of Gypenoside LVI. Therefore, identifying the glycosyltransferase involved in the synthesis of Gypenoside LVI in gynostemma and its coding gene, and clarifying its function and mechanism, not only can perfect the molecular regulation network of Gypenoside LVI biosynthesis, but also can provide core resources for optimizing the synthesis process through gene engineering technology (such as overexpression of key genes and construction of microbial synthesis system), fundamentally solving the problems of yield and cost.

[0006] At present, the research on the biosynthesis related genes of Gypenoside LVI in Gynostemma pentaphyllum is still in the initial stage, especially the glycosyltransferase gene specifically catalyzing the synthesis of Gypenoside LVI has not been identified and cloned. This technical blank seriously limits the regulation and optimization of the biosynthesis pathway of Gypenoside LVI, and directly restricts the industrialization process. Therefore, mining and verifying the glycosyltransferase gene for the biosynthesis of Gypenoside LVI, and clarifying its application have important scientific significance and high economic value for breaking the production bottleneck and improving the industrialization level. SUMMARY

[0007] The present application aims at the deficiencies of the prior art, and provides a kind of encoding

[0008] Gypenoside LVI biosynthesis glycosyltransferase gene, in particular, the application of the glycosyltransferase in

[0009] Gypenoside LVI biosynthesis.

[0010] The object of the present application can be achieved by the following technical solutions:

[0011] The present application provides a kind of g107610 gene, the gene encodes a kind of glycosyltransferase, can catalyze Gypenoside XLVI to be converted into Gypenoside LVI. The nucleotide sequence of the gene is as shown in the sequence table SEQ ID NO:1.

[0012] The amino acid sequence of the glycosyltransferase encoded by the gene is as shown in the sequence table SEQ ID NO:2. Through the verification of two experimental systems, the catalytic activity of the enzyme is proved, and technical support is provided for the efficient synthesis of Gypenoside LVI.

[0013] The present application also provides a recombinant expression vector containing the glycosyltransferase gene g107610.

[0014] Preferably, pHB-g107610 vector and pGEX-6P-g107610 vector are provided for expressing the glycosyltransferase in Agrobacterium GV3101 and Escherichia coli BL21, respectively.

[0015] Further preferably, the pGEX-6P-g107610 vector is obtained by inserting the glycosyltransferase gene of claim 1 into the BamH I and Not I enzyme cutting sites of the pGEX-6P-1 vector.

[0016] Genetically engineered bacteria containing the glycosyltransferase gene g107610.

[0017] Using these vectors and genetically engineered bacteria, this invention successfully expressed glycosyltransferases and verified their catalytic activity in in vitro enzyme activity experiments. Specifically, in a tobacco transient expression system, Agrobacterium GV3101 was transformed into the pHB-g107610 vector, successfully expressing the g107610 protein in tobacco leaves, and the formation of Gypenoside LVI was detected by LC-MS. In an Escherichia coli expression system, the pGEX-6P-g107610 vector was transformed into strain BL21, and the protein expressed via IPTG induction was purified, further verifying its catalytic activity in vitro.

[0018] The application of the glycosyltransferase gene, the recombinant expression vector, and the genetically engineered bacteria is selected from any one of the following:

[0019] (1) Prepare an enzyme for the biosynthesis of Gypenoside LVI;

[0020] (2) Catalyze the conversion of Gypenoside XLVI to Gypenoside LVI.

[0021] The application of the described gynostemma pentaphyllum glycosyltransferase in the biosynthesis of Gypenoside LVI.

[0022] This invention provides a method for preparing Gypenoside LVI, which uses Gypenoside XLVI as a substrate, UDP-xylose as a glycosyl donor, and completes the glycosylation reaction under the catalysis of g107610 glycosyltransferase to generate Gypenoside LVI.

[0023] Beneficial effects

[0024] This invention identifies a coding gene in Gynostemma pentaphyllum, and an expression vector containing this gene, along with the protein encoded by this gene, can catalyze the formation of Gypenoside XLVI from Gypenoside LVI. Therefore, this coding gene, the expression vector containing this gene, and the protein encoded by this gene can be used for…

[0025] The synthesis and preparation of Gypenoside LVI can help increase the yield of Gypenoside LVI and has high economic value. Attached Figure Description

[0026] Figure 1 This is an agarose gel electrophoresis image of g107610 amplification; where M is the marker and 1 is the target gene g107610;

[0027] Figure 2 This is an LC / MS chromatogram of the transient expression product of pHB-g107610 tobacco.

[0028] Figure 3 This is an SDS-PAGE gel electrophoresis image of the purified pGEX-6P-g107610 expressed protein; where M is the marker; 1 is the elution buffer; 2 is the eluent; and 3 is the lysate supernatant.

[0029] Figure 4 This is an LC / MS detection image of the pGEX-6P-g107610 prokaryotic expression product. Detailed Implementation

[0030] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. The substantive content of the present invention is described in detail below with reference to embodiments, but this does not limit the scope of protection of the present invention.

[0031] Example 1: Cloning of g107610

[0032] Step 1: Extraction and reverse transcription of total RNA from Gynostemma pentaphyllum to synthesize cDNA

[0033] The experimental material used in this experiment was Gynostemma pentaphyllum from Hunan Province, transplanted and cultivated in the Medicinal Botanical Garden of China Pharmaceutical University. The roots, stems, and leaves of Gynostemma pentaphyllum were used for DNA and RNA extraction. Total RNA was extracted according to the instructions of the Plant Tissue Total RNA Extraction Kit from Novizumi Biotechnology Co., Ltd. The concentration and purity of the product were determined using a Nano-100 micro spectrophotometer and stored at -80℃ for later use. The prepared RNA solution was then taken out and reagents were added according to the following system.

[0034] Table 1-1 Reverse Transcription System

[0035]

[0036] The above reaction system was placed in a 42°C water bath and incubated for 2 minutes. Then, 5 μL of 4×Hifair II-I SuperMixplus was added. The reaction products were then subjected to reverse transcription in a PCR instrument according to the following temperature settings.

[0037] Table 1-2 Reverse Transcription Temperature System

[0038]

[0039] The reverse transcription product, cDNA, is stored at -20°C for later use.

[0040] Step 2: Full-length amplification of g107610

[0041] Based on the full-length CDS sequence (Sequence NO.1) of g107610, specific primers were designed. The primer list is shown in the table below:

[0042] Table 1-3 Primer Information

[0043]

[0044] The full-length g107610 was amplified using Gynostemma pentaphyllum cDNA as a template. The PCR amplification system is as follows:

[0045] Table 1-4 PCR Amplification System

[0046]

[0047] The amplification program was as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 15 s; annealing at 60℃ for 15 s; extension at 72℃ for 44 s, for 35 cycles; final extension at 72℃ for 5 min; and storage at 4℃ for later use. 10× DNA Loading Buffer was added to the PCR amplification products, and the amplification products were detected using an agarose gel electrophoresis system. After gel excision, the DNA gel was recovered according to the Axygen DNA gel recovery kit. The concentration and purity of the recovered products were determined, and then stored at -20℃ for later use. The gene sequence is approximately 1350 bp in length, and the sequence is shown in SEQ ID NO.1. Figure 1 Agarose gel electrophoresis image for amplifying the full length of g107610.

[0048] Example 2: Construction of g107610 tobacco transient expression vector

[0049] Step 1: Double digestion of the g107610 tobacco transient expression vector.

[0050] The selected tobacco transient expression vector pHB plasmid was subjected to a double enzyme digestion reaction according to the two restriction enzyme sites added by the designed primers. The double enzyme digestion reaction system is as follows:

[0051] Table 2-1 pHB plasmid digestion system

[0052]

[0053] The double enzyme digestion reaction was performed at 37°C for 40 min. The digested plasmid was then subjected to agarose gel electrophoresis. The target band was excised and recovered using the Axygen DNA gel recovery kit. After determining the concentration and purity of the recovered product, it was stored at -20°C for later use.

[0054] Step 2: Recombination and transformation of the g107610 tobacco transient expression vector into E. coli DH5α competent cells

[0055] The target gene was recombined with the enzyme-digested plasmid. The recombination reaction system is as follows:

[0056] Table 2-2 Carrier Linkage System

[0057]

[0058] The recombination conditions were a metal bath at 37°C for 30 minutes. The product was then transformed into *E. coli* DH5α competent cells, following the steps outlined below:

[0059] (1) Take the DH5α Escherichia coli competent cells out of the -80℃ freezer and place them on ice to thaw.

[0060] (2) When it becomes an ice-water mixture, add the product from the previous step to the competent state and let it stand on ice for 30 minutes.

[0061] (3) Heat shock in a 42℃ metal bath for 90 seconds, then quickly place back on ice and let stand for 2 minutes.

[0062] (4) Add 800 μL of antibiotic-free LB liquid medium and revive in a shaker at 37°C for 60 min.

[0063] (5) Centrifuge at 8000 rpm for 1 min using a room temperature centrifuge, retain approximately 50 μL of supernatant, resuspend the bacterial cells, and spread them onto LB solid medium containing Kans using sterile glass beads. Invert the medium and incubate overnight at 37°C. Once colonies have grown, pick several single colonies and incubate them separately in LB liquid medium containing Kans until the culture becomes turbid. Then, colony PCR can be performed. The reaction system is as follows:

[0064] Table 2-3 Bacterial PCR System

[0065]

[0066] The PCR amplification procedure was the same as the gene cloning procedure described above. After the procedure, the products were detected by agarose gel electrophoresis, and one positive colony was sent to General Electric for sequencing. 600 μL of the successfully sequenced colony was taken and added to 400 μL of 50% glycerol, and incubated at -80°C. Another 60 μL of the colony was added to 6 mL of LB broth containing Kans, and incubated overnight at 37°C with shaking.

[0067] Following the instructions of the Axygen plasmid extraction kit, plasmids were extracted from the overnight culture to obtain plasmid pHB-g107610. Its concentration and purity were determined using Nano, and it was stored at -20°C for later use.

[0068] Example 3: Tobacco Expression Verification Experiment

[0069] Step 1: Transform GV3101 Agrobacterium competent cells

[0070] (1) Take GV3101 Agrobacterium competent cells out of the -80℃ freezer and place them on ice to thaw.

[0071] (2) When it becomes an ice-water mixture, add 1 μL of plasmid to the competent state and let it stand on ice for 5 min. (3) Quick freeze in liquid nitrogen for 5 min, and then quickly place it in a 37℃ digital display constant temperature water bath for 5 min heat shock.

[0072] (4) Gently place it back on the ice and let it stand for 5 minutes.

[0073] (5) Add 800 μL of antibiotic-free LB liquid medium and revive in a shaker at 28°C for 2 h.

[0074] (6) Centrifuge at 8000 rpm for 1 min using a room temperature centrifuge, retain approximately 50 μL of supernatant, resuspend the bacterial cells, and spread them onto LB solid medium containing Kan-Rif using sterile glass beads. Invert the medium and incubate overnight at 30°C. Once colonies have grown, pick several single colonies and place them separately in LB liquid medium containing Kan-Rif until the culture becomes turbid. Add 10 μL of 20 mM sodium hydroxide solution to 20 μL of the bacterial culture and heat in a 99°C metal bath for 20 min. Colony PCR can then be performed. The colony PCR system and procedure are the same as above.

[0075] Add an equal proportion of 50% sterile glycerol to the positive strain and store it in a -80°C refrigerator for later use.

[0076] Step 2: Instantaneous conversion of tobacco

[0077] (1) Streak positive bacterial suspension onto LB agar plates containing Kan-Rif. Pick a single Agrobacterium GV3101-pHB-g107610 colony from the plate, add it to LB liquid medium containing Kan-Rif, and incubate at 28°C and 220 rpm with shaking for 2 days until the suspension becomes turbid. Transfer 200 μL of the turbid suspension to 20 mL of LB liquid medium containing Kan-Rif, and incubate at 28°C and 220 rpm with shaking until the OD600 value reaches approximately 1.0. Transfer the Agrobacterium to a 50 mL centrifuge tube, centrifuge at 4000 rpm for 10 min, discard the supernatant, resuspend the suspension in MES buffer, and adjust the OD600 value to approximately 0.6. Incubate in the dark for 2 h.

[0078] (2) Select 3 tobacco plants in good condition, and use a 1mL syringe (without a needle) to...

[0079] GV3101-pHB-g107610 bacterial suspension was injected onto the underside of tobacco leaves. A blank pHB Agrobacterium suspension was injected into a tobacco plant using the same method.

[0080] (3) Place the injected tobacco in the dark at room temperature. After about 3 days, take it out and dissolve the Gypenoside XLVI standard in 1% DMSO (100 μM). Use a 1 mL syringe without a needle to inject the substrate Gypenoside XLVI onto the back of the tobacco leaf.

[0081] (4) After the injected tobacco leaves are left at room temperature for 2 days, they are taken out, the leaves of the injected tobacco are cut off, and dried in an oven at 37°C until the weight no longer changes.

[0082] (5) The dried tobacco leaves were placed in a mortar and ground into powder using liquid nitrogen. The powder was dissolved in methanol, extracted by ultrasonication for 1 hour, centrifuged at 12,000 rpm for 15 minutes, and the supernatant was collected, filtered, and analyzed by LC / MS. An Agilent LC-QTOF 6545 quadrupole-time-of-flight mass spectrometer was used with the system's default operating parameters.

[0083] The operating parameters were as follows: drying gas was N2, drying gas flow rate was 8 L / min, and temperature was 320℃. The spray needle nozzle pressure was 35 psig, capillary voltage was 3500 V, skimmer voltage was 65 V, octapole voltage was 750 V, and fragmentor voltage was 120 V. Negative ion mode was used for sample detection (50-1000 m / z). The chromatographic column was an Agilent Infinity Lab Poroshell 120EC-C18 (100 mm × 2.1 mm, 1.9 μm). Mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was acetonitrile solution, the column oven was set to 40℃, the injection volume was 5 μL, the flow rate was 0.3 mL / min, and the run time was 2 min.

[0084] The mobile phase elution gradient is as follows:

[0085] Table 3-1 Elution gradient

[0086]

[0087] Data acquisition and processing were performed using Agilent UPLC-QTOF-MS Mass Hunter Acquisition Software Version A.01.00 (Agilent Technologies) and Mass Hunter Workstation Software Version B.05.00 (Agilent Technologies), respectively.

[0088] In this embodiment, the pHB empty-vector Agrobacterium group was used as a negative control, and the pHB-g107610 group was cultured in parallel. Tobacco leaves were harvested, extracted with methanol, and the extract was analyzed by LC / MS. The results are as follows: Figure 2 As shown, the retention time was 4.842 min, and a fragment ion peak with [MH]-MS / MS ratio of 1093.58 appeared, which is consistent with...

[0089] The results showed that g107610 was consistent with the standard Gypenoside LVI. Therefore, we confirmed that g107610 does indeed have significant catalytic activity, capable of catalyzing the conversion of Gypenoside XLVI to Gypenoside LVI. This has a significant impact on the exploration of the biosynthetic pathway of Gypenoside LVI and has important economic value for its synthesis.

[0090] Example 4: In vitro functional verification of g107610 The specific steps for implementing the in vitro functional verification of g107610 are as follows:

[0091] Step 1: Construction of the g107610 prokaryotic expression vector

[0092] The selected prokaryotic expression vector pGEX-6P-1 plasmid was subjected to double digestion according to the two restriction enzyme sites added by the designed primers. The reaction system is as follows:

[0093] Table 4-1 pGEX-6P-1 plasmid digestion system

[0094]

[0095] The double enzyme digestion reaction was performed at 37°C for 40 min. The digested plasmid was then subjected to agarose gel electrophoresis. The target band was excised and recovered using the Axygen DNA gel recovery kit. After determining the concentration and purity of the recovered product, it was stored at -20°C for later use.

[0096] Following the method in Example 1, the target gene g107610 was amplified by PCR using pGEX-6P-g107610-F and pGEX-6P-g107610-R as primers. The target gene was then recombined with the enzyme-digested plasmid. The recombination system is as follows:

[0097] Table 4-2 Carrier Linkage System

[0098]

[0099]

[0100] The recombination conditions were 37°C in a metal bath for 30 minutes. The product was temporarily stored at 4°C and promptly transformed into *E. coli* DH5α competent cells. The specific steps for *E. coli* DH5α transformation with the pHB recombinant plasmid were the same as those for pHB recombinant plasmid, and positive colonies were verified by bacterial culture PCR. The pGEX-6P-g107610 plasmid of positive bacteria was extracted and transformed into *E. coli* BL21. The specific steps for *E. coli* DH5α transformation with the pHB recombinant plasmid were the same as those for pHB recombinant plasmid, and positive colonies were verified by bacterial culture PCR. Positive bacterial cultures were selected and stored at -80°C for subsequent *E. coli* heterologous protein expression experiments.

[0101] Step 2: Protein Induction and Purification

[0102] (1) Protein induction

[0103] Take the preserved positive *E. coli* BL21 bacterial culture and culture it in 5 mL of LB liquid medium containing Amp until turbid. Add the entire culture to 500 mL of LB liquid medium containing Amp and shake vigorously. Incubate at 37°C and 180 rpm for approximately 6 hours until the OD600 reaches 0.6–0.8. Add 500 μL of 0.5 mol / L IPTG and induce culture at 16°C and 160 rpm for 20 hours. Centrifuge at 4000 rpm for 10 min to collect the bacterial pellet in a 50 mL centrifuge tube and store at -80°C.

[0104] (2) Protein purification

[0105] 1) Ni column pretreatment

[0106] Gently invert and mix the Ni-NTA agarose. Take 1 mL and place it into a 12 mL empty affinity chromatography column tube. Open the bottom outlet of the empty column tube to allow the protective solution of the Ni column to flow out. Add pre-cooled sterile distilled water and rinse the column 2-3 times. Add 5 mL of pre-cooled 1×Ni-NTA Binding Buffer and rinse. Close the bottom outlet of the empty column tube and add an equal volume of pre-cooled 1×Ni-NTA Binding Buffer to prepare a 50% slurry.

[0107] 2) Enzymatic hydrolysis and ultrasonic disruption

[0108] Take the bacterial precipitate stored at -80℃, add 10 mL of 1×Ni-NTA Lysis Buffer, and thaw on ice. Add 100 μL of benzyl thiosulfate fluoride (PMSF), mix well, and then use an ultrasonic homogenizer to homogenize the bacterial cells on ice (sonicate for 10 seconds, pause for 10 seconds) for 10 minutes until the bacterial solution is no longer viscous.

[0109] 3) Binding and elution with Ni column

[0110] Centrifuge the completely disrupted bacterial culture at 4000 rpm for 40 min at 4°C. Collect the supernatant and add it to an empty column containing a pretreated Ni column. Seal both ends and incubate at 4°C with low-speed shaking for 2 h. Allow the column to stand vertically until the liquid separates into layers. Open the bottom outlet and allow the liquid to filter completely. Add 10 mL of pre-cooled 1×Ni-NTA Wash Buffer, allow to stand, and repeat twice. Add 5 mL of 1×Ni-NTA Elution Buffer, allow to stand, collect the eluent, and repeat once more.

[0111] 4) Protein purification

[0112] The collected effluent was concentrated by filtration using a 50 kDa ultrafiltration tube, and an equal volume of 50% glycerol was added. After determining the protein concentration, the effluent was aliquoted and stored at -80°C.

[0113] (3) SDS-PAGE electrophoresis detection of purified protein

[0114] 1) Take 16 μL of protein sample and 4 μL of 5×SDS protein loading buffer, mix well, and load a total of 20 μL.

[0115] 2) Use 220V electrophoresis for 30 minutes.

[0116] 3) Remove the gel and stain it in protein staining solution for 30 minutes.

[0117] 4) After the bands appear, replace the staining solution with distilled water for destaining until clear protein bands are visible and the gel background is light. Scan and save the results. The predicted molecular weight of the pGEX-6P-g107610 recombinant protein is approximately 78.69 kDa. See SDS-PAGE gel staining for details. Figure 3 .

[0118] Step 3: Verify protein function using in vitro enzyme activity reaction.

[0119] The protein was purified using pGEX-6P-g107610 for in vitro experiments. The in vitro enzyme activity reaction system is as follows:

[0120] Table 4-3 In vitro enzyme activity reaction system

[0121]

[0122] Incubate overnight at 30°C, add 100 μL of methanol to terminate the reaction, centrifuge at 12000 rpm for 15 min, take the supernatant and blow it with nitrogen, add 100 μL of methanol to reconstitute, and analyze by LC-MS.

[0123] The detection method for prokaryotic expression products is the same as that for the determination of transiently converted tobacco products in Example 3.

[0124] According to the in vitro enzyme activity reaction system in Table 4-3, the pGEX-6P-g107610 recombinant protein, prepared under parallel conditions at 99℃ in a metal bath for 15 min, served as a negative control and was detected by LC / MS. The results are as follows: Figure 4 As shown, the retention time was 4.842 min, and a fragment ion peak with a [MH]-MS / MS ratio of 1093.58 appeared, consistent with the Gypenoside LVI standard. These results further validated the catalytic activity of g107610 in vitro.

[0125] SEQ ID NO: 1

[0126]

[0127] SEQ ID NO:2

[0128] MDSISKQTKSSILMLPWLAHGHISPFLELAKTLSQRNFNIFLCSTPVNLDSIKPKLPPLFTSSIHLVEIHLPSSVDLPPHLHTTTGLPSHLMSSLKEAFSMAAHNFHSILQKLQPDLVIYDCVQPWAPEVASSMDIPAILFNTTGALVMSFGLHSIQTQGLIEFPFPDAVIEKHWSAKYIADDGVFTEEAKKVSAEFMISLYSSKSVILINSFRELEGKFVDFSSSLLDKKVIPVGPLIHEPEEDDDYSSIINWLDKKEPSSAVFVSFGSEYFPSKEELEEIAYGLELSEANFIWVIRFPRGNDMKSIEESLPEGFMKRVGEKGMMVKDWAPQARILKHCNIGGFVSHCGWNSMIESIMFGVPIIAIPMQLDQPYNARLVEDVGVGVEAKRDSNGKIQRDEVGKLIREVVVDKTREDVRKKVREMGDVLKRKGEEKIDEFVAEISGLCK。

Claims

1. A glycosyltransferase gene derived from Gynostemma pentaphyllum, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The glycosyltransferase gene encoding Gynostemma pentaphyllum glycosyltransferase according to claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A recombinant expression vector containing the glycosyltransferase gene as described in claim 1.

4. The recombinant expression vector according to claim 3, characterized in that, The gene was obtained by inserting the glycosyltransferase gene described in claim 1 into the BamHI and NotHI restriction sites of the vector using the pGEX-6P-1 vector as the starting vector.

5. Genetically engineered bacteria containing the glycosyltransferase gene as described in claim 1.

6. The application of the glycosyltransferase gene according to claim 1, selected from any one of the following: (1) Prepare an enzyme for the biosynthesis of Gypenoside LVI; (2) Catalyze the conversion of Gypenoside XLVI to Gypenoside LVI.

7. The application of the Gynostemma pentaphyllum glycosyltransferase according to claim 2 in the biosynthesis of Gypenoside LVI.

8. The use of the recombinant expression vector of claim 3 or 4 and the genetically engineered bacteria of claim 5 in the preparation of enzymes for the biosynthesis of Gypenoside LVI.

9. The application of the recombinant expression vector of claim 3 or 4 and the genetically engineered bacteria of claim 5 in catalyzing the conversion of Gypenoside XLVI to Gypenoside LVI.

10. A method for preparing Gypenoside LVI, characterized in that, Using Gypenoside XLVI and UDP-xylose as substrates, Gypenoside XLVI undergoes a glycosylation reaction to generate Gypenoside LVI under the catalysis of the Gynostemma pentaphyllum glycosyltransferase described in claim 2.

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