A epimerase Epi20 gene and its application in preparing cucurbitacin

Abstract

The present application relates to the field of bioengineering, and particularly provides a epimerase Epi20 gene, the sequence of the gene is shown as SEQ NO:1, the enzyme can effectively utilize cucurbitacin 6a to synthesize cucurbitacin 6d, and provides a source for cucurbitacin production.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering. Specifically, this invention relates to an epimerase Epi20 gene and its application in the preparation of cucurbitacin. Background Technology

[0002] *Hemsleya chinensis* Cogn. ex FBForbes & Hemsl. is a plant belonging to the genus *Hemsleya* in the family Cucurbitaceae. About 30 species of this genus are found in subtropical to temperate regions of Asia, with two more species found in eastern India and northern Vietnam. It is found throughout my country, mainly in the southwest to south-central regions (Hubei, Sichuan, Guizhou, Yunnan, etc.), with smaller distributions in Zhejiang, Anhui, and Fujian. It prefers to grow in mixed forests or along forest edges and ditches at altitudes of 1200-2100 meters. *Hemsleya chinensis* is a commonly used traditional Chinese medicine, with the tuber used medicinally. It contains saponins and bitter substances, and has the functions of clearing heat and detoxifying, antibacterial and anti-inflammatory, reducing swelling and relieving pain. Cucurbitacin is a bitter substance that can be isolated from the *Hemsleya chinensis* plant. Cucurbitacins can be classified into 12 major classes based on the different oxygen-containing functional groups at different positions: cucurbitacins A, B, C, D, E, F, G and H, I and L, J and K, O, P and Q, R, S and T, as well as mixed classes. Studies have found that cucurbitacins possess various effects, including anticancer, anti-hyperglycemic, antioxidant, anti-inflammatory, antibacterial, antidepressant, and cardioprotective properties. Cucurbitacin F has also been found to be cytotoxic to human tumor cells and antagonistic to insect steroid hormones.

[0003] The function of the HcCYP81Q58 gene has been verified and patented. The strain Cuol-04-1 constructed in this patent demonstrates that HcCYP81Q58 can catalyze the C25 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) was then ketolylated at the C3 position via the HCSDR34 gene 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 patented. In this patent, by introducing the HcCYP81Q58 and HcCYP87D19 genes into the previously constructed high-level yeast engineer DNHC87-03 for the production of 11-carbonyl-20β-hydroxy-Cuol, it was found that the HcCYP87D19 gene can catalyze 11-Carbonyl-cucurbita-5,23-diene-3β,22,25-triol(5) and

[0006] Hydroxylation at the C16 position of 11-Carbonyl-cucurbita-5,24-diene-3β,22,23-triol(5b) forms, respectively

[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) was then ketolylated at the C3 position by the HCSDR34 gene 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 suffering from gastritis, gastric ulcers, and bronchitis, the shortage of cucurbitacin raw material, *Gentiana scabra*, is widening. Currently, the market supply of cucurbitacin mainly relies on extraction, and the annual demand for dried medicinal materials used in cucurbitacin extraction is extremely high. Decades of over-harvesting have led to a sharp decline in wild resource reserves, bringing them to the brink of depletion. Simultaneously, *Gentiana scabra* thrives in moist, shady environments and is intolerant of drought and waterlogging. Planting in poorly drained or rainy areas results in poor plant growth and development, easily leading to root rot. In arid regions, plant survival rates are extremely low, making large-scale artificial cultivation difficult and further widening the market gap for cucurbitacin raw materials, causing prices to rise year by year. In recent years, with the rapid development of synthetic biology, the use of synthetic biotechnology to produce natural drug monomers can effectively solve these problems. However, to clarify the biosynthetic pathways of these active ingredients, it is necessary to identify the key genes involved in these pathways, and discovering these catalytic enzyme genes is a crucial step in studying the biosynthetic pathways of plant metabolites. Summary of the Invention

[0010] To address the aforementioned problems, this invention provides an epimerase HcEpi20 gene, which can serve as a regulatory gene for the biosynthesis of cucurbitacin 6d and be applied to the preparation of cucurbitacin 6d.

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

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

[0013] A third aspect of the present invention provides a recombinant plasmid obtained by homologous recombination of the epimerase HcEpi20 gene described above with the pET28a vector.

[0014] In a fourth aspect, the present invention provides a transgenic engineered bacterium containing the recombinant plasmid described above, or having an exogenous epimerase HcEpi20 gene integrated into the genome of the engineered bacterium.

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

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

[0017] In one embodiment, cucurbitacin 6a is obtained by reducing the ketone group at the C-3 position to the hydroxyl group at the C-3 position using the epimerase HcEpi20 to yield cucurbitacin 6d.

[0018] In one embodiment, the present invention obtains the target protein by expressing it in vitro using a recombinant plasmid, and then directly generates cucurbitacin 6d by catalyzing substrate 6a.

[0019] A sixth aspect of the present invention provides a method for preparing the epimerase HcEpi20 gene, the method comprising extracting RNA from the tuber of *Gentiana macrophylla* using an RNA reagent, reverse transcribing it into cDNA, and then performing PCR amplification.

[0020] The epimerase HcEpi20 gene isolated and identified from *Gynostemma pentaphyllum* can serve as an important marker gene for molecular-assisted breeding of *Gynostemma pentaphyllum*, and also as an important candidate gene for cucurbitacin production in yeast chassis cell construction. Attached Figure Description

[0021] Figure 1A schematic diagram of the synthetic pathway of cucurbitacin 6d.

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

[0023] Figure 3 Electrophoretic detection results of HcEpi20 after recombination.

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

[0025] Figure 5 HPLC was used to detect the hydroxylation of the keto group at the C-3 position of cucurbitacin 6a by epimerase HcEpi20. The enzyme activity results of epimerase HcEpi20 are shown on the x-axis, representing time in minutes. Standard: Cucurbitacin 6a standard; CK: Control group (cucurbitacin 6a + NADH + inactivated epimerase HcEpi20 + buffer Na2HPO4·NaH2PO4) enzyme activity results; HcEpi20: Experimental group (cucurbitacin 6a + NADH + epimerase HcEpi20 + buffer Na2HPO4·NaH2PO4) enzyme activity results.

[0026] Figure 6 Mass spectrometry (LC / MS / MS) analysis was performed to detect the hydroxylation of the ketone group at the C-3 position of cucurbitacin 6a by the epimerase HcEpi20. The retention time of cucurbitacin 6a was 20.9 min, and that of cucurbitacin 6d was 15.4 min.

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

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

[0029] Example 1: Obtaining and purifying the epimerase HcEpi20 gene

[0030] Based on the basic functional annotation information of the *Gnaphalium affine* transcriptome Unigene, 720 candidate genes were obtained. Then, based on the reported function of *Arabidopsis thaliana* involving the transformation of the keto group at the C-3 position to the α-hydroxyl group at the C-3 position, the three most closely related Epi candidate genes were screened from the sequencing annotation results. Enzymatic activity testing confirmed that the HcEpi20 gene possesses the function of transforming the keto group at the C-3 position to the α-hydroxyl group at the C-3 position. Simultaneously, using epimerases (Epi) identified in plants, sequence-local BLAST analysis was performed, and the screening results were then analyzed and compiled, ultimately identifying one epimerase (Epi) gene, HcEpi20. The nucleic acid sequence of the epimerase HcEpi20 gene is shown in SEQ ID NO.1, with a full length of 987 bp, and its encoded protein sequence is shown in SEQ ID NO.2. The amplification method for this gene is as follows:

[0031] (1) Preparation of cDNA template

[0032] Fresh samples of *Gentiana macrophylla* tubers were collected, sliced, and flash-frozen in liquid nitrogen for RNA extraction. Total RNA was extracted using the Magen (Guangzhou Meiji Biotechnology Co., Ltd.) HiPure Plant RNAMini Kit. RNA was extracted according to the kit's operating procedures, and after passing the tests, the RNA was reverse transcribed into cDNA using the TAKARA reverse transcription kit and stored at -20℃ for later use.

[0033] (2) Gene amplification and recovery

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

[0035] SEQ ID NO: 3

[0036] 5'F: gtggacagcaaatgggtcgcggatccATGAGCAGCGGAGCAGGGAA

[0037] SEQ ID NO: 4

[0038] 3'R: tgtcgacggagctcgaattcggatccTCAGAAGCTGATAAATTTCTTTTCCTTCAAGC

[0039] Gene amplification was then performed using 2×PhantaMax Master Mix high-fidelity DNA polymerase. The PCR reaction system was as follows: 95℃ for 3 min; 95℃ for 15 s, 60℃ for 15 s, 72℃ for 30 s, 35 cycles; 72℃ for 5 min. After PCR, gel electrophoresis was performed to confirm successful amplification, and the target band was then recovered. Gene digestion and recovery were performed using the EasyPure Quick Gel Extraction Kit from Beijing TransGen Biotechnology Co., Ltd. The recovered gene concentration was measured using a NanoReady ultra-micro UV-Vis spectrophotometer, and the recovered gene was stored at -20℃ for later use.

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

[0041] A schematic diagram of homologous recombination is shown below. Figure 2 As shown in the diagram. First, the vector pET28a was linearized using BamHI enzyme digestion to obtain the linearized vector. For homologous recombination, assembly was performed according to the instructions for homologous recombinase. Then, based on the concentrations of the insert fragment and vector, the amounts of each component were calculated according to the recombination instructions. Finally, each component was added to the PCR reaction tube on ice. After assembly, the results were detected and sent to the company for sequencing. The electrophoresis results after assembly are shown in the diagram. Figure 3 This indicates successful assembly. Reassemble using the following procedure:

[0042] Table 2-4 Candidate gene 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-scale protein expression experiment, the optimal induction conditions for HcEpi20 were determined to be: 16℃, 0.25mM IPTG, 160r / min, for 16h induction; followed by vigorous shaking, bacterial collection, cell disruption, and high-speed centrifugation to obtain the protein supernatant. SDS-PAGE protein electrophoresis was then performed for detection. The results are shown below. Figure 4 , Figure 4 This indicates that HcEpi20 protein can be purified by elution with 100 mmol / L imidazole elution buffer.

[0047] Example 3: Preparation of Cucurbitacin 6d using the isomerase HcEpi20

[0048] The reaction was carried out in 1.5 mL centrifuge tubes. The mixture in the reaction system contained: 10 μL of 100 mM NADH, 10 μL of 100 mM cucurbitacin 6a, 40 μg of the purified epimerase HcEpi20 protein from Example 1, and 50 mM Na2HPO4·NaH2PO4 buffer (pH = 8.0) was added to a total volume of 100 μL. The total reaction volume was 100 μL. After incubation at 37 °C for 8 hours, the reaction was terminated with an equal volume of 1 M methanol, centrifuged (12000 r / min, 10 min), and the supernatant was collected. Finally, the reaction products were detected by HPLC and LC-MS / MS analysis.

[0049] The control group (CK) reaction system consisted of 10 μL of 100 mM NADH, 10 μL of 100 mM cucurbitacin 6a, and 40 μg of inactivated purified epimerase HcEpi20 protein. 50 mM Na2HPO4·NaH2PO4 buffer (pH = 8.0) was added to a total volume of 100 μL. The total reaction volume was 100 μL.

[0050] Standard formulation: 20 μL of 100 mM cucurbitacin 6a, added to 180 μL of methanol; 20 μL of 100 Mm cucurbitacin 6d, added to 180 μL of methanol.

[0051] (6) Product testing

[0052] The HPLC detection conditions are as follows:

[0053] The instrument used for HPLC analysis was a Waters ultra-high performance liquid chromatograph. The chromatographic column was a Phenomenex Kinetex C18 analytical column (4.6 × 100 mm, 2.6 μm). The mobile phase for determining cucurbitacin for 6 days was 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 was 195 nm. The results are shown in the table below. Figure 5 This indicates the production of cucurbitacin 6d.

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

[0055] To further confirm the reaction products detected by HPLC, an Agilent 1290UPLC / 6540Q-TOF liquid chromatography-mass spectrometry (LC / MS) system was used for detection. The detection method was as follows: Mass spectrometry conditions: positive ion mode was used for the ion source, voltage: 3500V; fragmentation voltage: 135V; cone voltage: 60V; radio frequency voltage: 750V; scan range: 100-1000m / z. Chromatographic conditions: The chromatographic column was a Phenomenex Kinetex C18 analytical column (4.6 × 100 mm, 2.6 μm). The mobile phase for determining cucurbitacin for 6 days was 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; detection wavelength was 195 nm.

[0056] The results are as follows Figure 6-8 As shown, according to the total ion chromatogram analysis of mass spectrometry, the enzymatic reaction of Epi20 with 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 modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. An 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 the 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 described in claim 1 with the pET28a vector.

4. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria contain the recombinant plasmid as described in claim 3, or the genome of the genetically engineered bacteria is integrated with the epimerase HcEpi20 gene as described in claim 1.

5. The genetically engineered bacteria as described in claim 4, characterized in that, The genetically engineered bacteria is Escherichia coli BL21(DE3) strain.

6. The application of the gene as described in claim 1, the protein as described in claim 2, the recombinant plasmid as described in claim 3, or the transgenic engineered bacteria as described in claim 4 in the preparation of cucurbitacin 6d, characterized in that, The application involves reducing the ketone group at the C-3 position of cucurbitacin 6a to a hydroxyl group at the C-3 position using the epimerase HcEpi20 to obtain cucurbitacin 6d. The cucurbitacin 6a is The cucurbitacin 6d is .

7. The application as described in claim 6, characterized in that, The application involves obtaining the epimerase HcEpi20 protein through in vitro expression of a recombinant plasmid, which then catalyzes the substrate cucurbitacin 6a to directly generate cucurbitacin 6d.

8. A method for preparing the epimerase HcEpi20 gene, characterized in that, The nucleic acid sequence of the epimerase HcEpi20 gene is shown in SEQ ID NO.

1. The preparation method includes extracting RNA from the tuber of *Gentiana scabra* using RNA reagent, reverse transcribing it into cDNA, and then performing PCR amplification. The primers for the PCR amplification are shown in SEQ ID NO.3 and 4.

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