Dioxygenase from taxus chinensis as well as preparation method and application of dioxygenase

Abstract

The invention discloses taxus chinensis-sourced dioxygenase as well as a preparation method and application thereof, and belongs to the technical field of natural product biosynthesis. The dioxygenase TcOGD1 and the dioxygenase TcOGD2 are identified and obtained from taxus chinensis var mairei, and both the dioxygenase TcOGD1 and the dioxygenase TcOGD2 can participate in oxidation modification and skeleton rearrangement reaction of taxane natural products. The TcOGD1 shows strong catalytic activity and wider substrate adaptability, in-vitro synthesis of the 5 / 7 / 6 type taxane taxupine J with the potential of reversing multidrug resistance of tumors is realized, and a new class of 5 / 7 / 6 type taxane compound is obtained. The TcOGD2 can produce selective oxidation products for specific substrates. The dioxygenase can efficiently catalyze conversion of various taxane structures in vitro, the framework type and structural diversity of taxane natural products are expanded, a key tool is provided for synthesis of taxane drug lead compounds and new drug development, and the dioxygenase has good application prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of natural product biosynthesis, and particularly relates to a Taxus-derived dioxygenase as well as a preparation method and application thereof. BACKGROUND

[0002] Taxaceae plants are a typical group of gymnosperms, which have important ecological and medicinal values. Taxus plants are widely distributed in Asia, Europe and North America, among which Taxus chinensis var. mairei is a key protected plant in China. It has become an important source of natural medicine research due to its production of paclitaxel, a diterpenoid compound with significant anti-tumor activity. Paclitaxel, as a widely used anticancer drug in clinical practice, represents a classic structural type of taxane natural products. Its tricyclic 6 / 8 / 6 skeleton structure can specifically bind to tubulin protein, regulate microtubule stability, and play an important role in the treatment of various tumors such as breast cancer and ovarian cancer.

[0003] At present, nearly 700 taxane natural products have been isolated and identified from Taxus, covering more than 12 different carbon skeleton structures. In addition to the classic 6 / 8 / 6 skeleton, there are various skeletons (such as 5 / 7 / 6, 6 / 10 / 6, carbon-carbon bond breaking skeleton, cross-ring structure, etc.). The diversity of these skeleton structures determines the rich biological functions and pharmacological activities of taxane compounds. Among them, 5 / 7 / 6 type taxuspine J and other compounds show good anti-tumor drug resistance reversal effect, and have important potential for new drug development. However, the natural content of this new type of skeleton is extremely low, which limits its pharmacological research and development and utilization.

[0004] The paclitaxel biosynthetic pathway has been basically resolved, however, the formation mechanism of 5 / 7 / 6 and other atypical skeletons remains to be elucidated. According to the biosynthetic rules of terpenoids, the diversity of carbon skeleton not only depends on the action of primary cyclase, but also is regulated by post-modification enzymes, especially oxidases such as P450 and Fe(II) / 2-oxoglutarate-dependent dioxygenases (OGD). In recent years, the function of OGD in complex skeleton remodeling has gradually attracted attention. It has been confirmed that OGD can complete various structure remodeling processes such as ring expansion, spirocyclization, hydroxyl transfer and skeleton breaking through free radical-mediated mechanism, and has the potential to participate in the construction of new taxane skeletons.

[0005] Therefore, mining and identifying OGD oxidase with skeleton remodeling function, and revealing its catalytic mechanism in the formation of taxane natural products, not only has important significance for analyzing taxane metabolic network, but also provides important catalytic elements for constructing heterologous synthesis system of taxane drugs in synthetic biology. SUMMARY

[0006] In order to solve the above problems existing in the prior art, the purpose of the present application is to provide a taxus-derived dioxygenase and a preparation method and application thereof.

[0007] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: The present application provides a kind of dioxygenase, the dioxygenase has at least 90% of homology with the amino acid sequence shown as SEQ ID No.3 or SEQ ID No.4.

[0008] Further, the dioxygenase comprises the amino acid sequence shown as SEQ ID No.3 or SEQ ID No.4.

[0009] Further, the amino acid sequence of the dioxygenase is shown as SEQ ID No.3 or SEQ ID No.4.

[0010] Further, the N terminal and / or C terminal of the amino acid sequence are connected to epitope tag.

[0011] The present application also provides a gene fragment encoding the above-mentioned dioxygenase.

[0012] Further, the nucleotide sequence of the gene fragment is shown as SEQ ID No.1 or SEQ ID No.2.

[0013] The present application also provides a recombinant vector comprising the above-mentioned gene fragment.

[0014] The present application also provides a recombinant cell comprising the above-mentioned recombinant vector.

[0015] Further, the cell is Escherichia coli.

[0016] The present application also provides a method for preparing the above-mentioned dioxygenase, comprising the following steps: (1) introducing the above-mentioned recombinant vector into a host cell, screening, and obtaining a recombinant cell; (2) culturing the recombinant cell obtained in step (1), inducing expression, and obtaining it.

[0017] Further, the inducer used in the induction of step (2) is isopropyl-beta-D-thiogalactopyranoside, and the induction condition is 10-20 DEG C, 150-250 rpm, 10-25 hours.

[0018] Further, the induction condition of step (2) is 16 DEG C, 200 rpm, 18 hours.

[0019] The application further provides a method for preparing a taxane compound, which comprises using a taxane natural product as a substrate, and catalyzing a reaction by the above-mentioned dioxygenase to obtain the taxane compound.

[0020] The application further provides applications of the above-mentioned dioxygenase, gene fragment, recombinant carrier and recombinant cell in synthesis of a taxane compound.

[0021] The application has the following beneficial effects: The application identifies dioxygenases TcOGD1 and TcOGD2 from Taxus chinensis var. mairei, and both of them can participate in oxidation modification and skeleton rearrangement reaction of taxane natural products. TcOGD1 shows stronger catalytic activity and wider substrate adaptability, can catalyze various taxane substrates including 6 / 8 / 6 and 6 / 10 / 6 types, synthesize five different types of taxane skeletons, realize in-vitro synthesis of 5 / 7 / 6 type taxuspine J with potential of reversing tumor multi-drug resistance, and obtain a novel 5 / 7 / 6 type taxuspine compound. Although TcOGD2 has weaker catalytic activity, it can still produce selective oxidation products for specific substrates. The dioxygenases of the application can efficiently catalyze conversion of various taxane structures in vitro, expand the skeleton type and structural diversity of taxane natural products, provide a key enzyme tool for synthesis of a taxane drug lead compound and development of a new drug, and have good application prospect.

[0022] Obviously, according to the above content of the application, according to the ordinary technical knowledge and common means in the art, other various forms of modification, replacement or change can be made without departing from the above technical idea of the application.

[0023] The above content of the application is further explained in detail through the following embodiment. However, it should not be understood that the above subject matter of the application is limited to the following examples. Any technology realized based on the above content of the application belongs to the scope of the application. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is a schematic diagram of the oxidation modification and skeleton rearrangement reaction of taxane natural products participated by the dioxygenase.

[0025] Figure 2Structure diagram of pETDuet E. coli expression plasmid containing TcOGD1 gene.

[0026] Figure 3 Structure diagram of pETDuet E. coli expression plasmid containing TcOGD2 gene.

[0027] Figure 4 SDS-PAGE gel map of TcOGD1 and TcOGD2 proteins.

[0028] Figure 5 Enzymatic reaction results of TcOGD1 and TcOGD2 with substrate 1, respectively. A is the UPLC analysis map of the enzymatic reaction; B is the chemical structure of substrate 1 and product 2-7.

[0029] Figure 6 Enzymatic reaction results of TcOGD1 with substrate 8. A is the UPLC analysis map of the enzymatic reaction; B is the chemical structure of substrate 8 and product 9-10.

[0030] Figure 7 Enzymatic reaction results of TcOGD1 with substrate 11. A is the UPLC analysis map of the enzymatic reaction; B is the chemical structure of substrate 11 and product 12. DETAILED DESCRIPTION

[0031] The raw materials and equipment used in the present application are known products, which are obtained by purchasing commercially available products.

[0032] In the following experiments, if the temperature is not specified, it is a room temperature reaction, and the room temperature is 25±5℃.

[0033] The materials and reagents used in the specific embodiments of the present application are obtained from commercial channels or conventional methods, unless otherwise specified. The structural formula of the taxane compound involved is as follows: Example 1: Expression of dioxygenase TcOGD1 and TcOGD2 In the process of the present application in the previous study of Taxus chinensis, through Blast homologous comparison, two dioxygenase genes were found in the genome of Taxus chinensis var. mairei. Taxus chinensis var. mairei Among them: The reading frame nucleotide sequence and amino acid sequence of the dioxygenase gene TcOGD1 are shown in SEQ ID No. 1 and SEQ ID No. 3, respectively.

[0034] SEQ ID No. 1: ATGGCAGAAATTGATCTCGCCGTGATTGACCTTTCACAGTTCCCGAAGGAATTTGACGGAGCTTATTTTCATCATCCCGACGTAGTGAAACTCAGAGAAGCATGCGAAGAATGGGGGTTTTTCCGCGTGGTGAACCACGGATTTCCATCCGATCTTCTGCAAAAAGTTTTGTCTGTGAGCCGAGATTTACTATCTAGGCCGTTAGAGTATAAAGAAAAAGTCCCAACCTATAATACCCTCAAATATAACCCTCTCCACGAGTACATAAGTTTCCTCGCCATGCCCAATCCAGATTTAACTCAGGAAATTTCTCGAAAGATATGGCCCGAAGACGGAAACTCATTCTTTCGTGAAGTAATTGACGCGTACGGTTTATTAATATCAAATCTGGCGATTACAATCACTAAATTTCTTATTGCTGCCCTGAGTTTGGATCCAAGGGCATTCTACCAGTCTAATTTTGGAAAGTGCACAGCGGATTTGGTTATAAGTAGCTTCAGTTTTCTTGAGCATGACAAATGTGTGGGGGATGAGGCTCTGGTTTCTCACGCAGATACAGGTGTTGTTACCATCGTTTACAATGATGACAAGGAAGGGCTTGAGGTTCGCTCCAAACAAGGGCAGTGGGTGAACGTGAAGCCTACACCAGATTCATTCATTGTCAACGTGGGGGATTCCATGAAGGTATGGAGCAACGGCAAGTATCGCAGCGCACATCACCGCGTTCTTTTCAGAGGGTGGCAGACTCGTCTATCTCTTCCCCTTTTTCTAAACTTTCCACTCGACGCGTATATTTACGCCCCTGAAGAGCTTGTCGACAAAGATAATCCCCGTCGCTACAAACCATACACCTTTTCCGAATTACTGATAGAAGTTAACAACAAGAAATCAGGTGGAGAACTCTATGATGCTGTTGAACGTGTGGCGAGCATAATATAG SEQ ID No. 3: MAEIDLAVIDLSQFPKEFDGAYFHHPDVVKLREACEEWGFFRVVNHGFPSDLLQKVLSVSRDLLSRPLEYKEKVPTYNTLKYNPLHEYISFLAMPNPDLTQEISRKIWPEDGNSFFREVIDAYGLLISNLAITITKFLIAALSLDPRAFYQSNFGKCTADLVISSFSFLEHDKCVGDEALVSHADTGVVTIVYNDDKEGLEVRSKQGQWVNVKPTPDSFIVNVGDSMKVWSNGKYRSAHHRVLFRGWQTRLSLPLFLNFPLDAYIYAPEELVDKDNPRRYKPYTFSELLIEVNNKKSGGELYDAVERVASII The reading frame nucleotide sequence and the amino acid sequence of the dioxygenase gene TcOGD2 are shown in SEQ ID No. 2 and SEQ ID No. 4, respectively.

[0035] SEQ ID No. 2: ATGGCAGAAATTGATCTCCCCGTGATTGACATTGCACAGTTCCCAAAGGAATTTGACGGAGGTTATTTTCATCATCCCGACGTGGTGAAACTCAGAGAAGCATGCGAAGAATGGGGGTTTTTCCGCGTGGTGAACCACGGATTTCCTCCCGATCTTCTCCAAAAGGTTTTGTCTGTAAGCCGAGATTTACTATCCAGGCCGTTAGAGTATAAAGAAAAAGTCCCAACCTATAATGCCCTCAAATATAACCCTCTCCACGAGTACATAAGTTTCCTCGCCATGCCCAATCCAGATTTAACTCAGGAAATTTCTCGAAAGATATGGTCCGAAGACGGAAACTCATTCTTTCGCGAAGTGATTGACGCATACGGTGTATTAATATCCGATCTGGCGATTACAATCACTAAATTTCTTATTGCTGCCCTGAATTTGGATCCAAGGGCATTCTACCAGTCTAATTTTGGAAAGTGCACAGCGGATTTGGTTATAAGTAGCTTTAATTTTCTTGAGCATGACAAATGTGCGCGGGATGAGGCTCTGGTTTCTCACGCAGATACATGTGTTGTTACCATCCTTTACAATGATGACAAGGAAGGGCTTCAGGTTCGCTCCAAACAAGGACAGTGGGTGAACGTGAAGCCTACACCAGATTCATTCATTGTAAACGTGGGGGATTCCATGAAGGTATGGAGCAACGGCAGGTATCGCAGCGCACATCACCGCGTTCTTTTAAAAGGGTGGAAGAATCGTTTATCTCTTCCCCTTTTTTTAAACTTTCCACTCGAAGAGCCTATTTACGCCCCCGAGGAGCTTGTGGACAAAGATAATCCCCGATGCTACAAACCTTGTACCTTTTCCGAATTACTCATAGAAATTAAGAACAAGAAATCAGGTGGAGAACTCTATGACGCTGTTGAACGTGTGGCGAGCATAATGTAG SEQ ID No. 4: MAEIDLPVIDIAQFPKEFDGGYFHHPDVVKLREACEEWGFFRVVNHGFPPDLLQKVLSVSRDLLSRPLEYKEKVPTYNALKYNPLHEYISFLAMPNPDLTQEISRKIWSEDGNSFFREVIDAYGVLISDLAITITKFLIAALNLDPRAFYQSNFGKCTADLVISSFNFLEHDKCARDEALVSHADTCVVTILYNDDKEGLQVRSKQGQWVNVKPTPDSFIVNVGDSMKVWSNGRYRSAHHRVLLKGWKNRLSLPLFLNFPLEEPIYAPEELVDKDNPRCYKPCTFSELLIEIKNKKSGGELYDAVERVASIM Total RNA was extracted from Taxus chinensis var. mairei (T. mairei) cultivated in Chengdu University of Traditional Chinese Medicine using an RNA purification kit from Vazyme. Taxus chinensis var. mairei The mRNA was reverse transcribed into cDNA using a HiScript® III first-strand cDNA synthesis kit (Vazyme). The candidate genes (i.e., TcOGD1 and TcOGD2) were amplified using gene-specific primers (Table 1) and 2x Phanta Max Master Mix DNA polymerase (Vazyme). The pETDuet vector was cleaved by SacI and NotI sites, and the restriction enzymes were from Takara. The amplified fragments were then cloned into the pETDuet vector using 2x ClonExpress Mix (Vazyme), i.e., the recombinant plasmids were obtained. The obtained recombinant plasmids were transformed into E. coli DH5a competent cells by heat shock. Positive clones were screened by colony PCR, and the plasmids pETDuet::TcOGD1 and pETDuet::TcOGD2 were extracted and sequenced, respectively. The plasmid maps are shown in Figure 2 and Figure 3

[0036] Table 1 Primer sequences Note: The lower case sequence is the vector sequence, and the upper case sequence is the specific primer sequence.

[0037] The recombinant plasmids pETDuet:: TcOGD1 and pETDuet:: TcOGD2 ​Each of the plasmids was transformed into E. coli BL21 (DE3) competent cells carrying pGro7 plasmid. The positive transformants confirmed by PCR screening were inoculated in Luria-Bertani (LB) broth (composition: 10.0 g / L peptone, 5.0 g / L yeast extract, 10.0 g / L NaCl, pH 7.0) containing 100 mg / L ampicillin and 34 mg / L chloramphenicol and cultured. Then, 1 mL of the bacterial solution was inoculated into 500 mL of fresh LB medium (supplemented with 100 mg / L ampicillin) and cultured at 37°C for 10-12 h until the optical density (OD 600 ) of the culture reached 0.6-0.7. Subsequently, 0.3 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the culture system to induce protein expression, and the induction conditions were 16°C, 200 rpm, and the induction time was 18 h.

[0038] After the culture, the bacterial cells were collected by centrifugation at 8000 rpm and 4°C for 5 min, and resuspended in extraction buffer A (composition: 50 mM Tris, 300 mM NaCl, 15 mM imidazole, and 10% (w / v) glycerol, pH 8.0), and homogenized 3 times to break the cells using a homogenizer. The broken sample was centrifuged at 24000 rpm and 4°C for 60 min in a refrigerated centrifuge. The obtained supernatant was filtered through a 0.45 μm filter membrane, and then loaded onto a HisTraqTM FF (GE Healthcare) affinity chromatography column, and the protein was purified by an AKTA pure system. The target protein was eluted by an equal proportion of buffer A and buffer B (50 mM Tris, 300 mM NaCl, 500 mM imidazole, and 10% (w / v) glycerol, pH 8.0). The purified protein was analyzed by SDS-PAGE, and the gel was stained with Coomassie brilliant blue to show the protein bands Figure 4 ).

[0039] The beneficial effects of the present application are demonstrated by the following experimental examples.

[0040] Experimental Example 1: In vitro enzyme activity test of the dioxygenase of the present application 1. Experimental method: The TcOGD1 or TcOGD2 protein obtained according to the preparation method described in Example 1 was subjected to in vitro enzyme activity test. The total volume of the in vitro enzymatic reaction system (optimized by experiment) was 50 μL, containing 50 mM Tris-HCl buffer (pH 7.0), 0.4 mM substrate (2-Deacetoxytaxinine J, Taxumain W or Taxin B), 4 mM 2-oxoglutaric acid (2OG), 2 mM ascorbic acid, 25 μM FeSO4·7H2O and 50 μg of purified enzyme protein. To exclude the interference of non-enzymatic reactions, a negative control group was set up, and an equal amount of purified enzyme protein was denatured and inactivated by heating in a 99.9°C metal bath for 10 minutes, and then participated in the reaction in parallel. After the reaction system was incubated at 30°C for 12 hours, 100 μL of pre-cooled acetonitrile was added to terminate the reaction, and it was fully vortexed and mixed and centrifuged at 12,000 × g for 10 minutes. 1 μL of supernatant was injected into the ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) system for product analysis.

[0041] The UPLC-MS analysis used a Waters Synapt™ XS high-resolution mass spectrometer equipped with an electrospray ionization (ESI) source and an Agilent Extend-C18 chromatographic column (1.8 μm particle size, 4.6 × 50 mm inner diameter), and gradient elution was performed with 0.1% formic acid aqueous solution (mobile phase A) and 0.1% formic acid acetonitrile solution (mobile phase B). The elution program of mobile phase B was set as follows: 3% 0-1 min, 3%-50% 1-2 min, 50%-100% 2-12 min, 100% 12-16 min, 100%-3% 16-16.5 min and 3% 16.5-20 min. The chromatographic separation conditions were constant flow rate 400 μL / min, column temperature 35°C, injection volume 1 μL. Mass spectrometry detection was performed in positive ion mode, and the mass scan range covered 50-950. The raw data were collected in real time by MassLynx™ (V4.1) software, and qualitative analysis was performed by combining the theoretical molecular weight of the target product and the characteristic fragment ion peak. By comparing the spectrum with the negative control group, background interference was excluded, and the specificity of the enzymatic reaction product was ensured. m / z

[0042] 2、Experimental results (1) TcOGD1 / 2 catalyzes the rearrangement and decarboxylation of 6 / 8 / 6 type taxane skeleton ​2-Deacetoxytaxinine J (compound 1), a 6 / 8 / 6 type taxane isolated from yew, was used as a substrate and reacted in vitro with TcOGD1 / 2, respectively. The results showed that TcOGD1 catalyzed the formation of compound 1 with multiple differential peaks (…). Figure 5 A). Compound 2 is a known 5 / 7 / 6 taxane compound, Taxuspine J. Compounds 3-7 (Nortaxumain EI) all possess a novel 5 / 7 / 6 taxane skeleton with three less carbon atoms (A). Figure 5 (B) Taxanes with this structural feature have not been reported before. Specifically, compounds 3 and 4 have the same planar structure, but the stereoconfiguration of the hydroxyl group at the C-1 position is different. Compared to compound 3, compound 5 introduces an additional α-configured hydroxyl substituent at the C-6 position. Compound 6 exhibits significant structural rearrangement features, with its C-1 hydroxyl group and C-11 double bond possibly forming a new spatial configuration through an allyl radical rearrangement mechanism. Furthermore, compounds 5 and 7 are a pair of positional isomers: in compound 5, the α-hydroxyl and α-cinnamyl groups are located at the C-5 and C-6 positions, respectively, while in compound 7, they are located at the C-6 and C-5 positions, respectively.

[0043] (2) TcOGD1-catalyzed 6 / 8 / 6 type taxane aldehyde alkylation reaction TcOGD1 and TcOGD2 were reacted in vitro with 6 / 8 / 6 type taxumain W(8). The results showed that TcOGD1 could catalyze compound 8 and produce two distinct product peaks ( Figure 6 A). Compared to substrate 1, TcOGD1 exhibits a significantly altered catalytic preference for substrate 8, displaying regioselective oxidation of either the sp2 hybridized carbon atom at C20 or the sp³ hybridized carbon atom at C16, generating aldehyde products 9 and 10, respectively. Figure 6 B).

[0044] (3) TcOGD1 / 2 catalyzes the dehydrogenation reaction of 6 / 10 / 6 type taxane alcohol. TcOGD1 and TcOGD2 were reacted in vitro with the 6 / 10 / 6 taxane substrate TaxinB (11), respectively. TcOGD1 catalyzed the formation of a product peak from the 6 / 10 / 6 taxane substrate 11. Using 6 / 10 / 6 taxane (12) with a carbonyl group at the C-5 position isolated from Taxus chinensis as a standard, the retention time and mass spectrometry data in the UPLS-MS chromatogram were compared to determine that TcOGD1 oxidized the C-5 position of 11 to the carbonyl product 12 (12). Figure 7 A and Figure 7 B). In contrast, TcOGD2 showed only weak catalytic activity towards substrate 11, indicating that its oxidation capacity was relatively weak. Figure 7 A).

[0045] In summary, the present application identifies double oxygenase TcOGD1 and TcOGD2 from Taxus chinensis var. mairei, both of which can participate in the oxidation modification and skeleton rearrangement reaction of taxane natural products. TcOGD1 shows stronger catalytic activity and wider substrate adaptability, can catalyze various taxane substrates including 6 / 8 / 6 and 6 / 10 / 6 types, synthesize five different types of taxane skeletons, realize the in vitro synthesis of 5 / 7 / 6 type taxuspine J with potential to reverse tumor multidrug resistance, and obtain a novel 5 / 7 / 6 type taxane compound. Although TcOGD2 has weaker catalytic activity, it can still produce selective oxidation products for specific substrates. The double oxygenase of the present application can efficiently catalyze the conversion of various taxane structures in vitro, expand the skeleton type and structural diversity of taxane natural products, provide a key enzyme tool for the synthesis of taxane drug lead compounds and the development of new drugs, and have good application prospect.

Claims

1. A dioxygenase, characterized in that, The dioxygenase has at least 90% homology with the amino acid sequence shown as SEQ ID No. 3 or SEQ ID No.

4.

2. The dioxygenase enzyme according to claim 1, characterized in that, The dioxygenase comprises the amino acid sequence shown as SEQ ID No. 3 or SEQ ID No.

4.

3. The dioxygenase enzyme of claim 2, wherein, The amino acid sequence of the dioxygenase is shown as SEQ ID No. 3 or SEQ ID No.

4.

4. The dioxygenase enzyme according to any one of claims 1 to 3, characterized in that, The N-terminus and / or C-terminus of the amino acid sequence is connected with an epitope tag.

5. A gene fragment encoding the dioxygenase according to any one of claims 1 to 4; preferably, the nucleotide sequence of the gene fragment is shown as SEQ ID No. 1 or SEQ ID No.

2.

6. A recombinant vector, characterized in that, The recombinant vector comprises the gene fragment according to claim 5.

7. A recombinant cell, wherein, The recombinant cell comprises the recombinant vector according to claim 6; preferably, the cell is Escherichia coli.

8. A method for preparing the dioxygenase according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: (1) introducing the recombinant vector according to claim 6 into a host cell, screening, and obtaining a recombinant cell; (2) culturing the recombinant cell obtained in step (1), inducing expression, and obtaining the product; Preferably, the inducer for the induction in step (2) is isopropyl-β-D-thiogalactopyranoside, and the induction condition is 10-20°C, 150-250 rpm, and 10-25 hours. More preferably, the induction condition in step (2) is 16°C, 200 rpm, and 18 hours.

9. A process for preparing a taxane compound, characterized by, The method is a reaction using a taxane natural product as a substrate under the catalysis of the dioxygenase according to any one of claims 1 to 4.

10. Use of the dioxygenase according to any one of claims 1 to 4, the gene fragment according to claim 5, the recombinant vector according to claim 6, or the recombinant cell according to claim 7 in the synthesis of a taxane compound.

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