Hemsleya amabilis cytochrome oxidase HcCYP81Q59 gene and application thereof

Abstract

The invention relates to a hemsleya amabilis cytochrome oxidase HcCYP81Q59 gene and application thereof, and belongs to the technical field of biology. The nucleotide sequence of the hemsleya amabilis cytochrome oxidase HcCYP81Q59 gene disclosed by the invention is as shown in SEQ ID NO. 1. According to the invention, the cytochrome oxidase HcCYP81Q59 gene of hemsleya amabilis is utilized to synthesize 22-Deoxucurbitacin D through a bioengineering method, so that a foundation is further laid for regulation and control research on biosynthesis of hemsleya amabilis, and meanwhile, an early-stage foundation is provided for biological industrialized production of cucurbitacin in the future.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a snow gall cytochrome oxidase HcCYP81Q59 gene and an application thereof. Background Art

[0002] Cucurbitacin is a highly oxidized tetracyclic triterpenoid compound with cucurbitacin as the basic skeleton, which has high medicinal value, especially in anti-tumor. Although it has high anti-tumor activity, low therapeutic index and non-specificity, its toxicity has greatly hindered the clinical application and biological research of cucurbitacin. In addition, the structure of triterpenoid components is relatively complex, the content in plants is low and it often exists in a mixture of multiple components with similar structures, which is difficult to obtain by extraction, separation or chemical synthesis. Only a small number of cucurbitacin have been developed as drugs (CuB, snow gallbladder A, etc.). More importantly, the complete biosynthetic pathway for synthesizing cucurbitacin triterpenoids by modifying their skeleton is still unclear. The lack of this production pathway limits the effectiveness and biological research of clinical candidate drugs for this highly oxidized compound (there are mixed cucurbitacin tablets on the market, and the team of Tu Pengfei from Peking University has published an article on the use of cucurbitacin B in the treatment of liver cancer). Studies have shown that impurities in cucurbitacin preparations may be the cause of toxicity. Therefore, synthetic biology strategies based on the elucidation of biosynthetic pathways seem to be a more promising, sustainable and alternative approach.

[0003] Synthetic biology plays an important role in the production of pharmacological monomers. Using synthetic biology to achieve heterologous synthesis of cucurbitacin monomers is one of the most important strategies. In recent years, more and more active pharmaceutical ingredients have been successfully biosynthesized in microorganisms, such as paclitaxel precursor, artemisinic acid, ginsenosides and their precursors. However, cucurbitacin biosynthesis is currently limited to the precursor cucurbitadienol (Cuol), mainly because little is known about its biosynthetic pathway. At the same time, there are very few systems that use heterologous hosts to synthesize cucurbitacin monomers and intermediates. So far, there has been no report on the production of cucurbitacin monomers and intermediates by metabolic engineering. The tubers of the snow thorn plant are rich in cucurbitacin components, especially the snow thorn protein (a mixture of snow thorn protein A and snow thorn protein B) extracted from the tubers of the plants of this genus (Cucurbitaceae), which is the raw material for the production of snow thorn protein tablets (protected variety of Chinese medicine) and snow thorn protein capsules. This medicine has the functions of clearing away heat and detoxifying, and antibacterial and anti-inflammatory. It is mainly used clinically to treat bacillary dysentery, enteritis, bronchitis, acute tonsillitis and other diseases.

[0004] Cytochrome P450 (CYP450) is an ancient supergene family and the largest enzyme family in plant metabolism. CYP450 has a wide range of catalytic activities. In biochemical reactions, it can insert an oxygen atom into a hydrophobic molecule to obtain higher activity or hydrophilicity. It is also called monooxygenase (MFO). The most common reaction is the monooxygenase reaction, and the modification of the triterpene carbon skeleton catalyzed by it significantly promotes the structural diversity of triterpene components.

[0005] Studies have shown that cucurbitacin biosynthesis starts with 2,3-oxidosqualene cyclization to form cucurbitacinol (Cuol), which is catalyzed by cucurbitacinol synthase (CBS) of the oxidosqualene cyclase (OSCs) family. Under the catalysis of cytochrome P450 and SDR34, cucurbitacinol undergoes oxidation modification at specific sites by introducing hydroxyl, carboxyl or epoxy groups, thereby producing a variety of tetracyclic triterpenoids. The functions of the two genes HcCYP81Q58 and HcCYP87D19 have been verified and patents have been applied for (CN 116334106A), the strain Cuol-05-1 constructed in the patent introduced the two genes HcCYP81Q58 and HcCYP87D19 into the yeast engineering strain DNHC87-03 constructed in the previous period for high-level production of 11-carbonyl-20β-hydroxy-Cuol, and found that the HcCYP87D19 gene could catalyze the C16 hydroxylation of 11-Carbonyl-cucurbita-5,23-diene-3β,22,25-triol (5) and 11-Carbonyl-cucurbita-5,24-diene-3β,22,23-triol (5b) to form 11-Carbonyl-cucurbita-5,23-diene-3β,22,23-triol (5b), respectively. rbita-5,23-diene-3β,16α,20,25-tetrol(6), 11-Carbonyl-cucurbita-5,23-diene-3β,16α,20,25-tetrol(6b), and then ketolyzed at C3 by the HCSDR34 gene to form 11-Carbonyl-cucurbita-5,23-diene-16α,20,25-triol-3-one(6a), 11-Carbonyl-cucurbita-5,24-diene-16α,20,23-triol-3-one(6c), respectively. Figure 2 The structural formula is the same as listed in .

[0006] Compounds such as cucurbitacin are highly oxidized, with low content in plants, limited utilization, and complex and time-consuming extraction and purification processes, requiring a large amount of manpower, material resources and other resources. At present, the development of related drugs is mainly prepared by extraction from plants. There are still many challenges to achieve the chemical synthesis of cucurbitacin today, especially the key intermediate compounds of cucurbitacin.

[0007] Therefore, how to overcome the shortcomings of existing technologies is an urgent problem to be solved in the field of biotechnology. Summary of the invention

[0008] The purpose of the present invention is to solve the deficiencies of the prior art and to provide a snow gall cytochrome oxidase HcCYP81Q59 gene and its application.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] The first aspect of the present invention provides a snow gall cytochrome oxidase HcCYP81Q59 gene, and the nucleotide sequence of the snow gall cytochrome oxidase HcCYP81Q59 gene is shown in SEQ ID NO.1.

[0011] The second aspect of the present invention provides the protein encoded by the snow bile cytochrome oxidase HcCYP81Q59 gene, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0012] The third aspect of the present invention provides a recombinant plasmid containing the above-mentioned snow bile cytochrome oxidase HcCYP81Q59 gene.

[0013] Furthermore, the snow gall cytochrome oxidase HcCYP81Q59 gene was homologously recombined with the YCplac33 vector to obtain the Y33-HcCYP81Q59 recombinant plasmid.

[0014] The fourth aspect of the present invention provides a genetically modified engineered bacterium, comprising the above-mentioned recombinant plasmid, or the genome of the genetically modified bacterium is integrated with the exogenous above-mentioned snow gall cytochrome oxidase HcCYP81Q59 gene.

[0015] Furthermore, the transgenic engineered bacteria is an engineered yeast of Saccharomyces cerevisiae capable of producing 22-Deoxucucurbitacin D.

[0016] Furthermore, the method for constructing the engineered yeast of Saccharomyces cerevisiae comprises the following steps: cloning the exogenous gene HcCYP81Q59, the promoter TEF1, and the terminator ADH and fusing the fragments to construct a gene expression cassette, and integrating the gene expression cassette into the yeast chassis Coul-05-1 by lithium acetate transformation.

[0017] The fifth aspect of the present invention provides the use of the above-mentioned snow gall cytochrome oxidase HcCYP81Q59 gene in the preparation of 22-Deoxucucurbitacin D.

[0018] Furthermore, 11-Carbonyl-cucurbita-5.23-diene-3β,16a,203,25-tetrol was used as a substrate to generate 22-Deoxucucurbitacin D through C2 hydroxylation and C3 ketolation under the catalysis of HcCYP81Q59 and SDR34, respectively.

[0019] The present invention introduces the HcCYP81Q59 gene into the yeast engineering bacterium Cuol-05-1 (existing strain), and finds that the HcCYP81Q59 gene can catalyze the C2 hydroxylation of 11-Carbonyl-cucurbita-5,23-diene-3β,16α,20,25-tetrol (6), and then ketolyzes the C3 position through the HCSDR34 gene to form 22-Deoxucucurbitacin D (7a).

[0020] The synthesis path is as follows:

[0021]

[0022] The structural formula of 11-Carbonyl-cucurbita-5.23-diene-3β,16a,203,25-tetrol is as follows:

[0023]

[0024] Chinese name: De-22-oxidized cucurbitacin D, molecular formula: C 30 H 46 O6, molecular weight: 502; structural formula is as follows:

[0025]

[0026] Table 1 HcCYP81Q59 products 13 C& 1 H NMR (600 MHz, solvent: CDCl3) data (Jin Hz, δin ppm)

[0027]

[0028]

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention provides a transgenic saccharomyces cerevisiae engineering bacterium Coul-06 containing cytochrome oxidase HcCYP81Q59, and synthesizes 22-Deoxucucurbitacin D (7a) by a bioengineering method, further laying a foundation for the study of cytochrome oxidase biosynthesis regulation, and providing a preliminary foundation for the future bio-factory production of cucurbitacin. 22-Deoxucucurbitacin D (7a) is a direct precursor of cucurbitacin D and an important precursor of cucurbitacin B. At present, CuB is mainly extracted from the pedicel of melon (Cucumis melo), and its content is extremely low and extraction is difficult, resulting in limited sources. Referring to previous studies, it is found that the content of cucurbitacin in most plants is very low, accounting for only 0.000012% to 0.5% in plant raw materials; for example, Huang Sanwen's team in my country once isolated 480 mg of acetyl-deficient cucurbitacin C from 375 kg of cucumber leaves, which was used for in vitro experiments to study the key enzyme gene function of cucurbitacin biosynthesis. The huge material input and workload are daunting. Therefore, the heterologous synthesis of 22-Deoxucucurbitacin D (7a) and the clarification of the biosynthesis mechanism of cucurbitacin are of great significance for the molecular breeding of Cucurbitaceae plants and the efficient heterologous synthesis of cucurbitacin compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram for constructing transgenic Saccharomyces cerevisiae engineered strain Coul-06;

[0032] Figure 2 The synthetic route of 22-Deoxucucurbitacin D is shown in FIG.

[0033] Figure 3 The TLC chart of compound 22-Deoxucucurbitacin D (7a); A is the initial fermentation test chart, and B is the test chart after liquid phase preparation and purification; the compound marked with an asterisk is a new compound produced after the introduction of snow gall cytochrome oxidase HcCYP81Q59 (represented as Coul-05-1+HcQ59) into the engineered yeast Coul-05-1 of cerevisiae; CK-6 is the standard of compound 6 and is a precursor compound;

[0034] Figure 4 LC-MS data of compounds 7 and 7a;

[0035] Figure 5 NMR C spectrum of 22-Deoxucucurbitacin D (deuterated chloroform, 600 Hz);

[0036] Figure 6This is the NMR H spectrum of 22-Deoxucucurbitacin D (deuterated chloroform, 600HZ) DETAILED DESCRIPTION

[0037] The present invention is further described in detail below in conjunction with embodiments.

[0038] Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product specifications are used. If the manufacturer of the materials or equipment used is not specified, they are all conventional products that can be purchased.

[0039] Unless otherwise specified in the present invention, percentages are by mass.

[0040] The Y33-HcCYP81Q59 recombinant plasmid was purchased from Wuhan Jinkairui Bioengineering Co., Ltd. The recombinant plasmid was obtained by homologous recombination of the snow bile cytochrome oxidase HcCYP81Q59 gene and the YCplac33 vector.

[0041] (1) Method for constructing genetically modified bacteria

[0042] (1.1) Transfer the host bacteria of Saccharomyces cerevisiae (strain Cuol-05-1) into YPD liquid medium for activation to obtain yeast liquid;

[0043] (1.2) transferring the yeast culture to YPD liquid medium for cultivation, transferring the culture solution to a sterile 50 mL centrifuge tube, and centrifuging (8000 rpm, 5 min) to collect the yeast cells;

[0044] (1.3) resuspending the yeast cells in deionized water and centrifuging to obtain yeast precipitate;

[0045] (1.4) Add yeast transformation system to yeast precipitate, mix well, culture at 30°C, heat shock, spread plate, and culture in a constant temperature incubator at 30°C.

[0046] In step (1.1), the activation conditions are 30°C and 220 rpm for 24 h;

[0047] In step (1.2), the culture method is: the initial OD600 is 0.4, and the culture is carried out at 30°C and 220 rpm in a shaker until the OD600 reaches 0.8-0.9;

[0048] YPD liquid medium contains 10% yeast extract powder, 20% peptone, and 2% glucose;

[0049] In step (1.4), the yeast transformation system includes the following components: PEG4000 solution, LiAc solution, ssDNA, recombinant plasmid and sterilized deionized water, wherein PEG4000 solution 240μL, LiAc solution (1mol / L) 36μL, ssDNA 10μL, gene expression cassette 3NμL (N=800 / gene expression cassette concentration ng / mL, gene expression cassette includes P1 gene cassette, P2 gene cassette and P3 gene cassette, the amount of each of the three is NμL), deionized water (74-3N)μL. The static culture time is 60min; the heat shock condition is to maintain at 42°C for 40min.

[0050] (2) Construction of the recombinant Saccharomyces cerevisiae strain Coul-06

[0051] Using the strain Cuol-05-1 as the host yeast of Saccharomyces cerevisiae, the exogenous gene HcCYP81Q59, the promoter TEF1, and the terminator ADH were cloned and fragments were fused to construct a gene expression cassette. According to the method described in (4), the gene expression cassette was integrated into the existing yeast chassis Coul-05-1. The constructed strain that successfully expressed the exogenous gene HcCYP81Q59 was named Coul-06. The construction diagram is shown in Figure 1 shown.

[0052] S1. The enzymatic steps of the cucurbitacin biological pathway are long and complex, and there is a lack of intermediates. In order to characterize and screen the functions of candidate genes, this study constructed a yeast engineered bacterium that produces high levels of cucurbitacin precursors and intermediates. In the early stage, a high-level production of 11-Carbonyl-cucurbita-5,23-diene-3β,16a,20,25-tetrol (6), 11-Carbonyl-cucurbita-5,23-diene-3β,16α,20,25-tetrol (6b), 11-Carbonyl-cucurbita-5,23-diene-16α,20,25-triol-3-one (6a), 11-Carbonyl-cucurbita-5,2 The yeast engineered strain Coul-05-1, which produces 4-diene-16α,20,23-triol-3-one (6c), was constructed by combining the mevalonate pathway-related genes ERG20, ERG9, ERG1 and tHMG1 from Saccharomyces cerevisiae BY4742 and the cucurbitadienol synthase genes synHcOSC6, synHcCYP87D20 and cytochrome P450 reductase gene synHcCPR1 from the plant Saccharomyces cerevisiae to form gene expression cassettes to form modules for the production of 11-carbonyl-20β-hydroxy-Cuol synthesis pathway. At the same time, based on the same method, we also expressed two other cucurbitacin pathway key enzyme genes HcCYP81Q58 and HcCYP87D19 to construct the yeast engineered strain Coul-05-1. On this basis, we expressed another cucurbitacin pathway key enzyme gene HcCYP81Q59 to provide an enzyme catalytic reaction for the C2 hydroxylation of cucurbitacin in yeast.

[0053] S2. The gene expression cassette was co-transformed into the δDNA site of Saccharomyces cerevisiae BY4742 using the lithium acetate transformation method. The yeast homologous recombination ability was used to assemble and contain the selection marker gene HIS3 to form the Saccharomyces cerevisiae engineered strain Coul-06 containing the 22-Deoxucucurbitacin D (7a) biosynthetic pathway.

[0054] The method for constructing the gene expression cassette provided by the Saccharomyces cerevisiae engineered bacteria Coul-06 utilizes constitutive promoters UP and TEF1 and terminators ADH and Down to control the expression of HcCYP81Q59 and HIS3, namely, the module for producing the 22-DeoxucucurbitacinD (7a) pathway.

[0055] The screening marker gene in the method for constructing the gene expression cassette provided by the Saccharomyces cerevisiae engineered bacterium Coul-06 is HIS3.

[0056] Table 2 Primers for engineered bacteria Cuol-06

[0057]

[0058]

[0059] (1) Construction of P1 gene cassette:

[0060] (1.1) Using primers DN-F and TEF1-DN-R and the Saccharomyces cerevisiae genome as a template, clone fragment DN;

[0061] (1.2) Use primers DN-TEF1-F and Hc81Q59+TEF1-R to clone the fragment TEF1 using the Saccharomyces cerevisiae genome as a template;

[0062] (1.3) Using the obtained fragments DN and TEF1 as templates, primers DN-F and Hc81Q59+TEF1-R were used for fusion PCR amplification to obtain the P1 gene cassette;

[0063] (2) Construction of P2 gene cassette:

[0064] (2.1) Use primers TEF1-Hc81Q59-F and ADH-Hc81Q59-R and plasmid (synthesized by the company) as template to clone fragment Hc81Q59;

[0065] (2.2) Using primers Hc81Q59-ADH-F and HIS3-ADH-R and the Saccharomyces cerevisiae genome as a template, clone the fragment ADH;

[0066] (2.3) Using the obtained fragments HC81Q59 and ADH as templates, primers TEF1-HC81Q59-F and HIS3-ADH-R were used for fusion PCR amplification to obtain the P2 gene cassette;

[0067] (3) Construction of P3 gene cassette:

[0068] (3.1) Use primers ADH-HIS3-F and UP-HIS3-R and plasmid (synthesized by the company) as template to clone fragment HIS3;

[0069] (3.2) Use primers HIS3-UP-F and UP-R and the Saccharomyces cerevisiae genome as a template to clone fragment UP.

[0070] (3.3) Using the obtained fragments HIS3 and UP as templates, primers ADH-HIS3-F and UP-R were used for fusion PCR amplification to obtain the P3 gene cassette;

[0071] In steps (1), (2), and (3), the PCR reaction system was 50 μL: 1 μL template, 1 μL 10 mM upstream primer, 1 μL 10 mM downstream primer, 25 μL enzyme mixture (2×Phanta Max Master Mix), and deionized water to 50 μL;

[0072] The PCR reaction procedures are:

[0073] 95℃, 3min; 95℃, 15s, 58℃, 15s, 72℃, 1min20s, 35 cycles; 72℃, 5min.

[0074] (4) Obtaining an engineered strain of Saccharomyces cerevisiae for producing 22-Deoxucucurbitacin D:

[0075] The obtained P1 gene cassette, P2 gene cassette and P3 gene cassette were transferred into the cerevisiae engineering strain Cuol-05-1, and cultured in the nutrient-deficient solid plate CM-LH at 30°C until transformants grew out, and the positive transformants were picked for fermentation and production measurement to obtain the cerevisiae engineering strain (i.e., cerevisiae engineering strain Coul-06) producing 22-Deoxucucurbitacin D. The formula of the nutrient-deficient solid plate CM-LH: YNB: 0.67% (6.7 g / L), glucose: 2% (20 g / L), Dropmix Powder: 0.083% (0.83 g / L); Dropmix Powder contains (mg / L): threonine (Thr) 150mg / L, tyrosine (Try) 30mg / L, valine (Val) 150mg / L, lysine (Lys) 30mg / L, glutamate (Glu) 100mg / L, serine (Ser) 150mg / L, aspartic acid (Asp) 100mg / L, methionine (Met) 20mg / L, phenylalanine (Phe) 50mg / L, isoleucine (He) 30mg / L, arginine (Arg) 20mg / L; on this basis, uracil (Ura) 50mg / L, adenine (Ade) 50mg / L, tryptophan (Trp) 100mg / L are added.

[0076] The specific method for preparing 22-Deoxucucurbitacin D using engineered bacteria and detecting the prepared 22-Deoxucucurbitacin D is as follows:

[0077] (1) Activate strain Cuol-06 on YPD solid medium plates by streaking;

[0078] (2) Prepare seed solution: shake 3 mL of the solution on a microplate and then expand to 50 mL;

[0079] (3) Expanding fermentation;

[0080] (4) Collecting bacteria;

[0081] (5) Product extraction;

[0082] (6) Liquid phase detection.

[0083] In step (1), the activation condition is culturing in a constant temperature incubator at 30° C. for 24 h; the YPD liquid culture medium formula is 10% yeast extract powder, 20% peptone, and 2% glucose, and the solid culture medium needs to add 2% agar powder on the basis of the liquid culture medium;

[0084] In step (2), a single colony is picked up and placed in 3 mL YPD liquid culture medium and cultured overnight at 30°C and 220 rpm in a shaker, generally for 12-24 h; during expansion shaking, 1 mL is added to 50 mL YPD liquid culture medium and cultured at 30°C and 220 rpm in a shaker for 6-12 h;

[0085] In step (3), the expanded fermentation is generally carried out by adding 3% of the volume of the culture medium to the seed solution; the fermentation conditions are 30°C shaker at 220 rpm for 150 to 160 hours;

[0086] In step (4), the bacteria were collected by centrifugation at 5000 rpm for 10 min to separate the bacteria and the culture medium;

[0087] In step (5), the product in the culture medium is extracted twice with ethyl acetate and culture medium in a ratio of 1:1, the aqueous phase is discarded, and the compound is collected by rotary evaporation. The product in the cell body is extracted with ethyl acetate and bacteria in a ratio of 1:1 and ultrasonicated for 40 minutes. After the ultrasonication, the upper organic phase is taken and rotary evaporated. Generally, the ultrasonication step is repeated twice.

[0088] In step (6), the liquid phase detection solvent is phase A: water and phase B: acetonitrile; gradient elution is adopted, the elution program is shown in Table 3, and the gradient change is a linear change.

[0089] Table 3

[0090] Time (min) Flow rate (mL / min) ％A ％B 0 1.5 70 30 5 1.5 70 30 16 1.5 55 45 22 1.5 32 68 28 1.5 10 90 34 1.5 10 90

[0091] Compound 7 peaked at 22.08-22.52 min, and compound 7a peaked at 23.19-23.65 min.

[0092] Test results such as Figure 3 to Figure 6 shown.

[0093] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A snow gall cytochrome oxidase HcCYP81Q59 gene, characterized in that: The nucleotide sequence of the snow gall cytochrome oxidase HcCYP81Q59 gene is shown in SEQ ID NO.

1.

2. The protein encoded by the snow gall cytochrome oxidase HcCYP81Q59 gene according to claim 1, characterized in that: The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

3. A recombinant plasmid containing the snow gall cytochrome oxidase HcCYP81Q59 gene according to claim 1.

4. The recombinant plasmid of the snow bile cytochrome oxidase HcCYP81Q59 gene according to claim 3, characterized in that: The snow bile cytochrome oxidase HcCYP81Q59 gene was homologously recombined with the YCplac33 vector to obtain the Y33-HcCYP81Q59 recombinant plasmid.

5. A genetically modified engineered bacterium, comprising the recombinant plasmid of claim 3, or the genome of the genetically modified bacterium is integrated with the exogenous snow gall cytochrome oxidase HcCYP81Q59 gene of claim 1.

6. The genetically modified bacteria according to claim 5, characterized in that: The transgenic engineering bacteria are engineering yeasts capable of producing 22-Deoxucucurbitacin D.

7. The method for constructing an engineered strain of Saccharomyces cerevisiae according to claim 6, characterized in that: The method comprises the following steps: cloning the exogenous gene HcCYP81Q59, the promoter TEF1 and the terminator ADH, fusing the fragments to construct a gene expression cassette, and integrating the gene expression cassette into the yeast chassis Coul-05-1 by lithium acetate transformation method.

8. Use of the snow bile cytochrome oxidase HcCYP81Q59 gene according to claim 1 in the preparation of 22-Deoxucucurbitacin D.

9. Use of the snow bile cytochrome oxidase HcCYP81Q59 gene in the preparation of 22-Deoxucucurbitacin D according to claim 8, characterized in that: Using 11-Carbonyl-cucurbita-5.23-diene-3β,16a,203,25-tetrol as substrate, 22-Deoxucucurbitacin D was generated by C2 hydroxylation and C3 ketolation catalyzed by HcCYP81Q59 and SDR34, respectively.

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