Epimerase Epi20 gene and application thereof in preparation of cucurbitacine

Abstract

The invention relates to the field of bioengineering, in particular to an epimerase Epi20 gene, the sequence of the gene is shown as SEQ NO: 1, the epimerase Epi20 gene can effectively utilize cucurbitacine 6a to synthesize cucurbitacine 6d, and a source is provided for cucurbitacine production.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and specifically relates to an epimerase Epi20 gene and application thereof in the preparation of cucurbitacin. Background Art

[0002] Hemsleya chinensis Cogn.ex FBForbes&Hemsl. is a plant of the genus Hemsleya in the family Cucurbitaceae. About 30 species of this genus are native to the subtropical to temperate regions of Asia, and the other two species are native to eastern India and northern Vietnam. It is produced throughout my country, mainly distributed in the southwest to central and southern parts of China (Hubei, Sichuan, Guizhou, Yunnan, etc.), with a small part distributed in Zhejiang, Anhui, Fujian, etc. It likes to grow under mixed woods or along the edge of forests at an altitude of 1200-2100 meters. Hemsleya chinensis is a commonly used Chinese herbal medicine, with its tubers used as medicine. It contains saponins, bitter substances, etc., and has the functions of clearing away heat and detoxification, antibacterial and anti-inflammatory, and reducing swelling and relieving pain. Among them, cucurbitacin is a bitter substance that can be isolated from Hemsleya chinensis. Cucurbitacin can be divided into 12 categories according to the different oxygen-containing functional groups at different positions, namely cucurbitacin A, B, C, D, E, F, G and H, I and L, J and K, O and P and Q, R and S and T, and mixed categories. Studies have found that cucurbitacin has multiple effects such as anti-cancer, anti-hyperglycemia, antioxidant, anti-inflammatory, antibacterial, anti-depressant and cardioprotective. It has also been found that cucurbitacin F is cytotoxic to human tumor cells and has an antagonistic effect on insect steroid hormones.

[0003] The function of the gene HcCYP81Q58 has been verified and a patent has been applied for. The strain Cuol-04-1 constructed by the patent proves that HcCYP81Q58 can catalyze the C25 hydroxylation and C23 hydroxylation of 11-carbonyl-20β-hydroxy-Cuol to form 11-Carbonyl-cucurbita-5,23-diene-3β,22,25-triol(5),

[0004] 11-Carbonyl-cucurbita-5,24-diene-3β,22,23-triol(5b), and then the HCSDR34 gene ketolyzes the C3 position to form 11-Carbonyl-cucurbita-5,23-diene-22,25-diol-3β-one(5a),

[0005] 11-Carbonyl-cucurbita-5,24-diene-22,23-diol-3β-one(5c). The function of the HcCYP87D19 gene has been verified and a patent has been applied for. In this patent, the two genes HcCYP81Q58 and HcCYP87D19 were introduced into the yeast engineered bacteria DNHC87-03 that was constructed in the early stage to produce high levels of 11-carbonyl-20β-hydroxy-Cuol. It was found that the HcCYP87D19 gene can catalyze the production of 11-Carbonyl-cucurbita-5,23-diene-3β,22,25-triol(5) and

[0006] The C16 position of 11-Carbonyl-cucurbita-5,24-diene-3β,22,23-triol (5b) was hydroxylated to form

[0007] 11-Carbonyl-cucurbita-5,23-diene-3β,16α,20,25-tetrol(6),

[0008] 11-Carbonyl-cucurbita-5,23-diene-3β,16α,20,25-tetrol(6b), and then the HCSDR34 gene ketolylates the C3 position to form 11-Carbonyl-cucurbita-5,23-diene-16α,20,25-triol-3-one(6a) and 11-Carbonyl-cucurbita-5,24-diene-16α,20,23-triol-3-one(6c).

[0009] With the increasing number of patients with gastritis, gastric ulcers and bronchitis, the gap in the raw material of cucurbitacin, snow gall, is increasing. At present, the raw materials of cucurbitacin on the market are mainly obtained by extraction, and the annual demand for dry medicinal materials used to extract cucurbitacin is very high. The large-scale excavation in recent decades has caused a sharp decline in wild resource reserves and is on the verge of exhaustion. At the same time, snow gall cultivation conditions prefer a moist and cool environment, are not drought-resistant, and are not resistant to waterlogging. Planting in poorly drained or rainy areas will cause poor plant growth and development and easily cause root rot. Planting in arid areas has an extremely low survival rate of plants, making it difficult to cultivate them on a large scale, further expanding the market gap of cucurbitacin raw materials, and prices are rising year by year. In recent years, with the rapid development of the field of synthetic biology, the use of synthetic biological snow technology to produce natural drug monomers can effectively solve the above problems. However, to understand the biosynthetic pathways of these active ingredients, it is necessary to identify the key genes related to these pathways, and discovering these catalytic enzyme genes has become a key link in studying the biosynthetic pathways of plant metabolites. Summary of the invention

[0010] In order to solve the above problems, the present invention provides a epimerase HcEpi20 gene, which can be used as a biosynthesis regulatory gene of cucurbitacin 6d and applied to the preparation of cucurbitacin 6d.

[0011] The first aspect of the present invention provides a epimerase HcEpi20 gene, the nucleic acid sequence of the epimerase HcEpi20 gene is shown in SEQ ID NO.1.

[0012] The second aspect of the present invention provides a protein encoded by the above-mentioned epimerase HcEpi20 gene, and the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0013] The third aspect of the present invention provides a recombinant plasmid, which is obtained by homologous recombination of the above-mentioned epimerase HcEpi20 gene and the pET28a vector.

[0014] The fourth aspect of the present invention provides a genetically engineered bacterium, wherein the genetically engineered bacterium contains the recombinant plasmid, or the exogenous epimerase HcEpi20 gene is integrated into the genome of the genetically engineered bacterium.

[0015] Preferably, the genetically modified bacteria is Escherichia coli BL21 (DE3) strain.

[0016] A fifth aspect of the present invention provides an application of an epimerase HcEpi20 gene in the preparation of cucurbitacin 6d.

[0017] In one embodiment, the keto group at the C-3 position of cucurbitacin 6a is reduced to the hydroxyl group at the C-3 position by epimerase HcEpi20 to obtain cucurbitacin 6d.

[0018] In one embodiment, the present invention obtains the target protein through in vitro expression by recombinant plasmid, and further catalyzes the substrate 6a to directly generate cucurbitacin 6d.

[0019] The sixth aspect of the present invention provides a method for preparing the epimerase HcEpi20 gene, which comprises extracting RNA from snow thorn tubers using an RNA reagent, reverse transcribing it into cDNA, and then performing PCR amplification to obtain it.

[0020] The epimerase HcEpi20 gene isolated and identified from the snow gall can be used as an important marker gene for molecular-assisted breeding of snow gall, and can also be used as an important candidate gene for producing cucurbitacin in the construction of yeast chassis cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1.Schematic diagram of the derived synthetic route of cucurbitacin 6d.

[0022] Figure 2 . Schematic diagram of the construction of the recombinant expression plasmid Pet28a-HcEpi20 (used to express the epimerase HcEpi20 gene).

[0023] Figure 3 .The electrophoresis detection results of HcEpi20 after recombinant.

[0024] Figure 4 . SDS-PAGE protein electrophoresis detection diagram of epimerase HcEpi20. Wherein, M: protein molecular weight standard; lanes 1, 2, 3, 4, and 5 are respectively precipitation eluent, flow-through eluent, precipitation, 20mmol / L imidazole eluent, and 40mmol / L imidazole eluent; lanes 6-9 are all proteins in 100mmol / L imidazole eluent; lane 10 is protein in 150mmol / L imidazole eluent; lanes 12-14 are proteins in 200mmol / L imidazole eluent.

[0025] Figure 5 HPLC detection of the hydroxylation of the keto group at the C-3 position of cucurbitacin 6a by epimerase HcEpi20. The enzymatic activity reaction results of epimerase HcEpi20, the horizontal axis is time, unit min; among them, standard product: cucurbitacin 6a standard; CK: control group (cucurbitacin 6a + NADH + inactivated epimerase HcEpi20 + buffer Na 2 HPO 4 ·NaH 2 PO 4 ) enzyme inactivation reaction results; HcEpi20: experimental group (cucurbitacin 6a + NADH + epimerase HcEpi20 + buffer Na 2 HPO 4 ·NaH 2 PO 4 ) enzyme activity reaction results.

[0026] Figure 6 . Mass spectrometry analysis (LC / MS / MS) showed that epimerase HcEpi20 hydroxylated the keto group at the C-3 position of cucurbitacin 6a. The retention time of cucurbitacin 6a was 20.9 minutes, and the retention time of cucurbitacin 6d was 15.4 minutes.

[0027] Figure 7 .Fragment ion pattern of standard cucurbitacin 6a (theoretical molecular weight 486) (LC / MS / MS).

[0028] Figure 8. Reaction product cucurbitacin 6d and fragment ion pattern of cucurbitacin 6a in enzyme reaction (theoretical molecular weight 488) (LC / MS / MS). DETAILED DESCRIPTION

[0029] Example 1 Acquisition and purification of epimerase HcEpi20 gene

[0030] Based on the Unigene basic function annotation information of the snow bile transcriptome, 720 candidate genes were obtained. Then, according to the reported function of Arabidopsis thaliana to change the keto group at C-3 to the α-hydroxyl at C-3, the three most closely related Epi candidate genes were screened out in the sequencing annotation results. The enzyme activity reaction was verified to finally determine that the HcEpi20 gene has the function of changing the keto group at C-3 to the α-hydroxyl at C-3. At the same time, the epimerase (Epi) identified in the plant was analyzed by sequence local BLAST, and then the screening results were sorted and analyzed, and finally 1 epimerase (Epi) HcEpi20 gene was found. The nucleic acid sequence of the epimerase HcEpi20 gene is shown in SEQ ID NO.1, the full length of the sequence is 987bp, and the protein sequence encoded by it is shown in SEQ ID NO.2. The amplification method of this gene is as follows:

[0031] (1) Preparation of cDNA template

[0032] Fresh samples of snow dandelion tubers were taken, sliced, and then quickly frozen in liquid nitrogen for RNA extraction. Total RNA was extracted using the HiPure Plant RNAMini Kit from Magen (Guangzhou Meiji Biotechnology Co., Ltd.). RNA was extracted according to the operating procedures of the kit. After passing the test, the RNA was reverse transcribed into cDNA using the TAKARA reverse transcription kit and stored at -20°C for later use.

[0033] (2) Gene amplification and recovery

[0034] Using primer design software (CE Design) v1.04, primers with homology arms for the gene were designed (the homology arms were from Escherichia coli pET28a). The primers with homology arms are as follows:

[0035] SEQ ID NO: 3

[0036] 5'F: gtggacagcaaatgggtcgcggatccATGAGCAGCGGAGCAGGGAA

[0037] SEQ ID NO: 4

[0038] 3'R: tgtcgacggagctcgaattcggatccTCAGAAGCTGATAAATTTCTTTTCCTTCAAGC

[0039] Then, 2×Phanta Max Master Mix high-fidelity DNA polymerase was used for gene amplification. The PCR reaction system was: 95℃, 3min; 95℃, 15S, 60℃, 15S, 72℃, 30S, 35 cycles; 72℃, 5min. After the PCR was completed, the gel was run, and the target band was recovered after confirming the successful amplification. The EasyPure Quick Gel Extraction Kit of Beijing Quanshijin Biotechnology Co., Ltd. was used to recover the target gene. After recovery, the recovery concentration was measured on the NanoReady ultra-micro UV-visible spectrophotometer, and finally stored in a -20℃ refrigerator for later use.

[0040] (3) Construction and identification of gene recombination vectors

[0041] Schematic diagram of homologous recombination Figure 2 As shown. First, the vector pET28a was linearized and the linearized vector was obtained by single restriction digestion with BamH I enzyme. During homologous recombination, the assembly was performed according to the operating instructions of the homologous recombination enzyme, and then the amount of each component was calculated according to the concentration of the inserted fragment and the vector and the recombination instructions; finally, each component was added to the PCR reaction tube on ice. After assembly, the results were tested and sent to the company for sequencing. The electrophoresis test results after assembly are shown in Figure 3 , indicating that the assembly is successful. Reassemble the operation according to the following process:

[0042] Table 2-4Candidate genes Recombination System

[0043]

[0044] Where X = (0.02 × pET28a base pairs) ng / linearized pET28a concentration ng / μL; Y = (0.02 × pET28a base pairs) ng / HcEpi20 recovery concentration ng / μL;

[0045] (4) SDS-PAGE protein electrophoresis detection

[0046] After a small protein expression test, the protein induction conditions of HcEpi20 were determined to be: 16°C, 0.25mM IPTG, 160r / min, induction for 16h; then shake vigorously, collect bacteria, break the cell wall, obtain protein supernatant after high-speed centrifugation, and then use SDS-PAGE protein electrophoresis and detection. The test results are shown in Figure 4 , Figure 4 The results showed that HcEpi20 protein could be eluted and purified in 100mmol / L imidazole eluent.

[0047] Example 3 Preparation of cucurbitacin 6d using isomerase HcEpi20

[0048] The reaction was carried out in a 1.5 mL centrifuge tube. The reaction system contained: 10 μL of 100 mM NADH, 10 μL of 100 mM cucurbitacin 6a, 40 μg of the epimerase HcEpi20 protein purified in Example 1, and 50 mM Na 2 HPO 4 ·NaH 2 PO 4 Buffer (pH = 8.0) was added to a total volume of 100 μL, and the total volume of the reaction system was 100 μL. After incubation at 37°C for 8 hours, an equal volume of 1 M chromatographic methanol was added to terminate the reaction, and the mixture was centrifuged (12000 r / min, 10 min) to obtain the supernatant. Finally, the reaction product was detected by HPLC and LC-MS / MS analysis.

[0049] Control group (CK) reaction system: 10 μl of 100 mM NADH, 10 μl of 100 mM cucurbitacin 6a, 40 μg of inactivated purified epimerase HcEpi20 protein, and 50 mM Na 2 HPO 4 ·NaH 2 PO 4 Buffer (pH=8.0) was added to a total volume of 100 μL, and the total volume of the reaction system was 100 μL.

[0050] Standard products: 20 μl of 100 mM cucurbitacin 6a, add 180 μl of methanol; 20 μl of 100 mM cucurbitacin 6d, add 180 μl of methanol.

[0051] (6) Product testing

[0052] HPLC detection conditions are as follows:

[0053] The instrument used for HPLC detection is Waters ultra-high performance liquid chromatograph. The chromatographic column is Phenomenex Kinetex C18 analytical column (4.6×100 mm, 2.6 μm), and the mobile phase for the determination of cucurbitacin 6d is 0.1% formic acid aqueous solution (A)-acetonitrile (B), with gradient elution: 0-6 min, 25%-25% B; 6-16 min, 25%-42% B; 16-22 min, 42%-68% B; 22-28 min, 68%-90% B; 28-33 min, 90%-95% B; 33-38 min, 95%-95% B; 38-42 min, 95%-25% B; 42-47 min, 25%-25% B; the detection wavelength is 195 nm. The test results are shown in Figure 5 , indicating the production of cucurbitacin 6d.

[0054] LC-MS detection conditions are as follows:

[0055] In order to further confirm the reaction products detected by HPLC, Agilent 1290UPLC / 6540Q-TOF liquid chromatography-mass spectrometry (LC / MS) was used for detection. The detection method was as follows: Mass spectrometry conditions: the ion source used was in positive ion mode, voltage: 3500 V; fragmentation voltage: 135 V; cone voltage: 60 V; radio frequency voltage: 750 V, scanning range: 100-1000 m / z. Chromatographic conditions: The chromatographic column was Phenomenex Kinetex C18 analytical column (4.6×100 mm, 2.6 μm), the mobile phase for determination of cucurbitacin 6d was 0.1% formic acid aqueous solution (A)-acetonitrile (B), gradient elution: 0-6 min, 25%-25% B; 6-16 min, 25%-42% B; 16-22 min, 42%-68% B; 22-28 min, 68%-90% B; 28-33 min, 90%-95% B; 33-38 min, 95%-95% B; 38-42 min, 95%-25% B; 42-47 min, 25%-25% B; the detection wavelength was 195 nm.

[0056] The results are as follows Figure 6-8 As shown, according to the analysis of the total ion current of mass spectrometry, the enzymatic reaction of Epi20 with the cofactor NADH using 6a as substrate can produce a new product 6d, but cucurbitacin 6a is not completely consumed in the reaction.

[0057] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A epimerase HcEpi20 gene, characterized in that: The nucleic acid sequence of the epimerase HcEpi20 gene is shown in SEQ ID NO.

1.

2. A protein encoded by an epimerase HcEpi20 gene, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A recombinant plasmid, characterized in that: The recombinant plasmid is obtained by homologous recombination of the epimerase HcEpi20 gene as claimed in claim 1 and the pET28a vector.

4. A genetically modified bacteria, characterized in that: The genetically engineered bacteria contains the recombinant plasmid as claimed in claim 3, or the epimerase HcEpi20 gene as claimed in claim 1 is integrated into the genome of the genetically engineered bacteria.

5. The genetically modified bacteria according to claim 4, characterized in that: The transgenic engineering bacteria are Escherichia coli BL21 (DE3) strains.

6. Use of the gene according to claim 1, the protein according to claim 2, the recombinant plasmid according to claim 3 or the genetically modified engineering bacteria according to claim 4 in the preparation of cucurbitacin 6d.

7. The use according to claim 6, characterized in that The application comprises reducing the keto group at the C-3 position of cucurbitacin 6a to the hydroxyl group at the C-3 position by using epimerase HcEpi20 to obtain cucurbitacin 6d.

8. The use according to claim 6, characterized in that The application includes obtaining the epimerase HcEpi20 protein after in vitro expression through a recombinant plasmid, and further catalyzing the substrate 6a to directly generate cucurbitacin 6d.

9. A method for preparing an epimerase HcEpi20 gene, characterized in that: The method comprises extracting RNA from snow thorn tubers by using RNA reagent, reverse transcribing the RNA into cDNA and then performing PCR amplification to obtain the RNA. The primers for PCR amplification are shown in SEQ ID NOs. 3 and 4.

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