Hydroxylase and application thereof

Abstract

The invention belongs to the field of genetic engineering and synthetic biology, and particularly relates to novel hydroxylase from cephalotaxus plants, a nucleic acid molecule of the hydroxylase, an expression vector containing the nucleic acid molecule, a host cell containing the vector and application of the hydroxylase or the host cell in biosynthesis of cephalotaxus diterpenoid compounds. The hydroxylase provided by the invention can efficiently catalyze C-13 site hydroxylation of cephalotaxene, and a new method is provided for obtaining a cephalotaxane diterpenoid compound by adopting a synthetic biological means.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering and synthetic biology, in particular to a new hydroxylase derived from Gephalotaxus plants, a nucleotide encoding the hydroxylase, a vector containing the nucleotide and a host containing the vector, and the application of the hydroxylase in preparing Cephalotaxus diterpenoids. BACKGROUND

[0002] Cephalotaxus diterpenoids are a class of complex diterpenoids with 7 / 6 / 6 / 5 rigid four-ring skeleton and high oxidation, which are isolated from Gephalotaxus plants. Since the representative compound harringtonolide (also known as hainanolide) was discovered in the 1970s, nearly 80 natural products of this type have been isolated and reported. According to the number of carbon atoms in the diterpene nucleus and the characteristic structure, the reported Cephalotaxus diterpenoids can be divided into five types: C 20 , C 19 , C 19 , C 19 , or C 18 , and dimer type. Among them, the C 19 ring heptatrienone type compounds account for the highest proportion, and the representative compound harringtonolide belongs to this type. Pharmacological studies have found that Cephalotaxus diterpenoids have a wide range of biological activities; harringtonolide as a representative, its pharmacological activities mainly include antioxidant, anti-inflammatory, antibacterial, and antiviral activities, and especially has strong inhibitory activity on various human tumor cells.

[0003] So far, Cephalotaxus diterpenoids have only been isolated from Gephalotaxus plants, which are limited in source and low in content, greatly limiting the acquisition of this class of compounds and restricting their development and application as drugs. Although the chemical total synthesis of this class of compounds has been widely concerned by the academic circle, and in recent years there have been many successful reports, but due to the complex structure of this class of compounds and the presence of multiple chiral centers, chemical synthesis is difficult, the route is long, the yield is low, and often causes environmental pollution and other problems, and there is no large-scale production and commercial application value.

[0004] In recent years, the rapid development of biosynthesis and synthetic biology technology provides an effective means to solve the accessibility and other problems of this class of compounds.

[0005] Recently, the diterpene parent structure of harringtonines has been identified, and the diterpene synthase catalyzing the formation of the diterpene parent structure and the corresponding cyclization mechanism have also been discovered; however, the key enzymes for post-modification based on the diterpene parent structure and the corresponding biosynthetic pathway have not been reported.

[0006] Therefore, it is of great significance to find and functionally identify the key enzyme genes of harringtonine-type diterpene biosynthesis from plants of the genus Cephalotaxus, especially the oxidase genes for post-modification based on the diterpene parent structure, and to analyze and further create the biosynthetic pathway of the compounds. SUMMARY

[0007] To overcome the deficiencies in the prior art, the purpose of the present application is to provide a new hydroxylase, a nucleotide encoding the hydroxylase, a vector containing the nucleotide and a host containing the vector, and to provide the application of the hydroxylase in preparing harringtonine-type diterpene compounds.

[0008] In the first aspect, the present application provides a hydroxylase derived from plants of the genus Cephalotaxus, which has an amino acid sequence as shown in SEQ ID NO. 1 (named as CsCYP1 in the present application), or an amino acid sequence having at least 90% identity with the amino acid sequence as shown in SEQ ID NO: 1 and having catalytic activity. The at least 90% identity in the present application can be substitution, deletion, insertion, insertion-deletion, duplication, etc. of one or more amino acids based on the amino acid sequence as shown in SEQ ID NO. 1.

[0009] The present application also provides a truncated form of the amino acid sequence as shown in SEQ ID NO. 1 or the amino acid sequence having at least 90% identity with the amino acid sequence as shown in SEQ ID NO: 1 and having catalytic activity, in which the signal peptide and / or the transmembrane region are removed. The hydroxylase provided by the present application is a membrane protein having two transmembrane regions, so that the truncated form thereof in which the transmembrane region is removed can be expressed in a soluble form, and is easy to separate and purify.

[0010] The hydroxylase or the truncated form thereof provided by the present application can be chemically modified and / or immobilized to facilitate practical application. The chemical modification can be selected from one or more of acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, and PEG modification. The immobilization modification can be performed by conventional methods such as physical adsorption and chemical crosslinking.

[0011] The hydroxylase or the truncated form thereof provided by the present application can also be connected with a tag for detection and purification, so as to facilitate detection and / or purification. Specifically, the tag can be selected from one or more of the following: fluorescent proteins (such as GFP, CFP, YFP or their enhanced versions), histidine tag His6, maltose binding protein MBP, glutathione thio transferase GST, FLAG, C-Myc, HA, NusA, Strep, Avi, SNAP, Halo, SUMO, IgG, Profinity eXact and a plurality of commonly used protein tags.

[0012] In a second aspect, the present application provides a nucleotide encoding the hydroxylase.

[0013] As a preferred solution, the nucleotide has a nucleotide sequence as shown in SEQ ID NO: 5, which is named as CsCYP1 in the present application.

[0014] In a third aspect, the present application provides a recombinant vector. The vector can be a cloning vector into which the nucleotide of the second aspect is inserted, or an expression vector into which the nucleotide is inserted, or an expression vector capable of expressing the hydroxylase of the present application exogenously.

[0015] As a preferred solution, the vector is an expression vector capable of expressing a target protein in a bacterial, yeast, plant or animal cell. More preferably, the vector is an expression vector capable of expressing a target protein in E. coli, Pichia pastoris, Saccharomyces cerevisiae, Arabidopsis thaliana, Nicotiana benthamiana or insect cells.

[0016] As a specific embodiment, the nucleotide molecule provided by the present application can be connected to an expression vector, such as pEAQ-HT or pEAQ-HT-DEST1 vector capable of expressing a target protein in a plant, by means of homologous recombination.

[0017] In a fourth aspect, the present application provides a host into which the vector of the third aspect is exogenously introduced.

[0018] The host can be a microorganism such as E. coli or yeast, or a plant with a clear genetic background, or an animal cell. Preferably, the host is selected from E. coli, Pichia pastoris, Saccharomyces cerevisiae, Arabidopsis thaliana, Nicotiana benthamiana or insect cells.

[0019] In a fifth aspect, the present application provides a method for preparing the hydroxylase of the present application by using the nucleotide, the vector or the host.

[0020] As a specific embodiment, the method comprises culturing the host, collecting the culture containing the hydroxylase, and extracting the hydroxylase or the tissue containing the hydroxylase (such as microsomes containing membrane proteins). The culture is a conventional method adopted in the art according to the characteristics of the host, such as fermentation culture of bacteria and yeast, tissue culture or planting cultivation of plants, and in vitro cell culture of animal cells. The extraction is a conventional method adopted in the art according to the characteristics of the protein, such as extraction of microsomes containing membrane proteins.

[0021] In a sixth aspect, the present application provides the use of the hydroxylase or the tissue containing the hydroxylase in an enzymatic reaction.

[0022] The enzymatic reaction of the present application can be a biochemical reaction in vitro or a reaction in vivo in a host. Preferably, the host can be bacteria, yeast, plants or animals. More preferably, the host can be selected from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Arabidopsis thaliana, Nicotiana benthamiana or insect cells.

[0023] The enzymatic reaction of the present application can be catalyzed by the hydroxylase or the tissue containing the hydroxylase alone or by the hydroxylase or the tissue containing the hydroxylase together with other hydroxylases.

[0024] The substrate of the enzymatic reaction of the present application is harringtonene, which has the structure shown in I:

[0025]

[0026] In a seventh aspect, the present application provides the use of the hydroxylase or the tissue containing the hydroxylase in the preparation of harringtonane diterpenoids. The preparation is carried out by a biosynthetic method or a combination of biosynthesis and chemical synthesis.

[0027] The harringtonane diterpenoids of the present application can be natural diterpenoids isolated from plants of the genus Cephalotaxus, such as all harringtonane diterpenoids reported in the review article “Jiang C, Xue J, Yuan Y, et al. Progress in structure, synthesis and biological activity of natural cephalotane diterpenoids. Phytochemistry. 2021; 192: 112939. doi: 10.1016 / j.phytochem.2021.112939”, and other natural harringtonane diterpenoids not yet reported which have harringtonene as the diterpene nucleus.

[0028] The triptolide-type diterpene compound of the present application can also refer to an analogue or derivative obtained by structural modification on the basis of natural triptolide-type diterpene. The structural modification is a routine modification such as oxidation, reduction, glycosylation, methylation, acylation, prenylation, etc. by chemical synthesis or biological synthesis known in the art.

[0029] As a preferred solution, the triptolide-type diterpene compound has the structure shown in I as a diterpene nucleus, and is modified by oxidation at least at the C-13 position thereof;

[0030]

[0031] As a preferred solution, the application of the present application comprises the following steps: using the hydroxylase to catalyze the diterpene substrate having the structure shown in I to perform in vitro enzymatic reaction alone, or using the hydroxylase and other hydroxylases to catalyze the diterpene substrate to perform in vitro enzymatic reaction, to prepare a triptolide-type diterpene compound. When the hydroxylase and / or other hydroxylases provided by the present application retain a transmembrane region, the in vitro enzymatic reaction is preferably performed using a membrane structure organization (microsomes) containing the hydroxylase. The cofactors required for enzymatic reaction, such as electron donors, etc. are added during the reaction. The other hydroxylases are preferably one or more cytochrome P450 enzymes derived from plants of the genus Tripterygium.

[0032] As a preferred solution, the application of the present application comprises the following steps: culturing a host into which the expression vector of the present application is exogenously introduced, or culturing a host into which the expression vector of the present application and the expression vector of other hydroxylases are exogenously introduced, and adding the diterpene substrate having the structure shown in I to prepare a triptolide-type diterpene compound in the host.

[0033] Among them, the host can be selected from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Arabidopsis thaliana, Nicotiana benthamiana or insect cells. The expression vector of other biocatalytically active enzymes (mainly hydroxylases) can also be simultaneously introduced into the host, so that the hydroxylase provided by the present application and other hydroxylases jointly exert catalytic function; the other hydroxylases are preferably one or more cytochrome P450 enzymes derived from plants of the genus Tripterygium.

[0034] As a specific solution, the application comprises the following steps: exogenously introducing into Nicotiana benthamiana an expression vector expressing a hydroxylase having the amino acid sequence shown in SEQ ID NO. 1, and adding a diterpene substrate having the structure shown in I to prepare a triptolide-type diterpene compound.

[0035] As a preferred solution, the application comprises the following steps: co-expressing the enzyme capable of catalyzing the generation of a diterpene substrate with the structure shown in I, or co-expressing the enzyme with other hydroxylases, in a host capable of providing diterpene biosynthesis precursors, to prepare a harringtonine-type diterpene compound. The host capable of providing diterpene biosynthesis precursors is preferably Nicotiana benthamiana.

[0036] Specifically, the diterpene substrate is catalyzed by expression of a recombinant vector in the host, and is directly oxidized without isolation. The expression vector of other enzymes with biological catalytic activity (mainly hydroxylases) can also be simultaneously introduced into the host, so that the hydroxylase provided by the application and other hydroxylases jointly exert catalytic functions; the other hydroxylases are preferably one or more cytochrome P450 enzymes derived from plants of the genus Tripterygium.

[0037] As a specific solution, the application comprises the following steps: in Nicotiana benthamiana, exogenously introducing expression vectors expressing a hydroxylase with the amino acid sequence shown in SEQ ID NO. 1 and a harringtonine synthase with the amino acid sequence shown in SEQ ID NO. 2, to prepare a harringtonine-type diterpene compound. The method can further simultaneously introduce an expression vector of 1-deoxyxylulose-5-phosphate synthase (DXS, a rate-limiting enzyme of the MEP pathway of terpene biosynthesis) and / or an expression vector of geranylgeranyl pyrophosphate synthase (GGPPS), to express the corresponding synthases, so as to increase the supply of diterpene biosynthesis precursors in the expression system, thereby increasing the yield of the target product.

[0038] In the above-mentioned solution, other enzymes with biological catalytic activity can be further introduced to modify the structure of the harringtonine-type diterpene compound to obtain a derivative thereof. Specifically, the enzymes can be selected from hydroxylases, reductases, glycosyltransferases, methyltransferases, acyltransferases, and isopentenyltransferases; the hydroxylases can be specifically selected from dehydrogenases, monooxygenases (such as cytochrome P450 enzymes) or dioxygenases (such as α-ketoglutarate-dependent dioxygenases). In the application, the hydroxylases are preferably cytochrome P450 enzymes, and more preferably cytochrome P450 enzymes derived from plants of the genus Tripterygium. The other enzymes with biological catalytic activity can be a single enzyme or a combination of multiple enzymes; the combination of multiple enzymes can be a combination of multiple enzymes with different catalytic principles and types, such as a combination of a hydroxylase and a glycosyltransferase, a combination of a hydroxylase and a methyltransferase, a combination of a hydroxylase and an acyltransferase, etc.; or a combination of multiple enzymes with the same catalytic principle, such as a combination of multiple enzymes of different subfamilies in cytochrome P450 enzymes, or a combination of multiple enzymes belonging to the same subfamily but having different primary structures in cytochrome P450 enzymes.

[0039] The hydroxylase provided by this invention can efficiently catalyze the C-13 hydroxylation of thiabendazole substrates, laying a solid foundation for the efficient production of thiabendazole-type diterpenoids through biosynthesis and synthetic biology. Attached Figure Description

[0040] Figure 1 Gas chromatogram of product I-1;

[0041] Figure 2 EI-MS spectrum of product I-1;

[0042] Figure 3 Product I-1 1 1H NMR spectrum (400MHz, CDCl3);

[0043] Figure 4 Product I-1 13 C NMR spectrum (100MHz, CDCl3);

[0044] Figure 5 1D-NOE spectrum of product I-1 (600MHz, CDCl3). Detailed Implementation

[0045] The following embodiments are provided to further illustrate various aspects of the present invention. These embodiments are non-limiting and should not be construed as limiting any aspect of the invention. The scope of protection of the present invention is limited only by the claims. Various modifications and improvements can be made to various aspects of the present invention without departing from the scope of the claims, and these modifications and improvements also fall within the scope of protection of the present invention. For example, replacing the promoters and expression vectors used in the embodiments with other promoters and expression vectors commonly used in the art is something that those skilled in the art can understand and implement.

[0046] Additionally, it should be noted that, unless otherwise specified, all materials and reagents used in the following embodiments are commonly used in the art and can be obtained through conventional commercial means; all methods used are conventional methods known to those skilled in the art.

[0047] Example 1: Cloning of the target gene

[0048] 1. Extraction of total RNA and synthesis of cDNA first strand

[0049] Fresh leaves of *Cephalotaxus fortunei* were used as material and rapidly transferred to a mortar pre-cooled with liquid nitrogen, where they were thoroughly ground into powder. The powdered sample was then transferred to a centrifuge tube, and 500 μL of Buffer RCL / 100 mg tissue (EZNA) was added. TMPlant RNA Kit), homogenate treatment with vortex and mix with pipette repeatedly; 55℃ incubation for 3 min, 15,000g centrifugation for 5 min, transfer the supernatant to filter column, 14,000g centrifugation for 2 min; add the filtrate to equal volume Buffer RCB, upside down for 5-10 times, transfer the liquid to RNA adsorption column, 10,000g centrifugation for 1 min; discard the filtrate, add 400μL RWC wash Buffer to the adsorption column, 10,000g centrifugation for 1 min; dry the adsorption column at room temperature, then elute the total RNA with 50μL ddH2O. Then use SMARTer TM RACE cDNA amplification kit (Clontech, USA) to synthesize the first strand of cDNA.

[0050] 2. Obtaining of the target gene

[0051] Using the first strand of cDNA as template, the specific primers of CsCYP1 (CsCYP1-F, sequence as shown in SEQ ID NO. 6; CsCYP1-R, sequence as shown in SEQ ID NO. 7) were used for PCR amplification with KOD Plus Neo enzyme; the PCR product was recovered to obtain the target gene fragment, which was ligated to the cloning vector pEASY-Blunt Zero (TransGen Biotech); the ligation product was transformed into E. coli competent cells (DH5α Chemically Competent Cell) and cultured in solid LB medium (kanamycin 50ug / ml); positive clones were selected for sequencing to obtain the pEASY-CsCYP1 plasmid with correct sequence.

[0052] Example 2: Construction of the recombinant expression vector

[0053] The pEASY-CsCYP1 plasmid obtained in Example 1 was used as a template, specific primers with homologous arms (pEAQ-HT-CsCYP1-F, sequence as shown in SEQ ID NO. 8; pEAQ-HT-CsCYP1-R, sequence as shown in SEQ ID NO. 9) were used for PCR amplification using KOD Plus Neo enzyme, and the amplified product was recovered to obtain the target gene fragment; the In-Fusion SnapAssembly Master Mix (Takara) was used to link the target gene fragment with the pEAQ-HT vector fragment treated with restriction enzymes Age I and Xho I; the linking product was transformed into E. coli DH5α competent cells (Qingke Biology), and solid LB medium (kanamycin 50 ug / mL) was used for culture; positive clones were selected for sequencing, and the pEAQ-HT-CsCYP1 expression vector with correct sequence was obtained. A large amount of plasmid was extracted for standby use.

[0054] According to the method provided in the present application, the following recombinant expression vectors were constructed: pEAQ-HT-CsCYP1 (for expressing the oxidase of the amino acid sequence shown in SEQ ID NO. 1), pEAQ-HT-CsCTS (for expressing the triptolide synthase of the amino acid sequence shown in SEQ ID NO. 2), pEAQ-HT-DXS (for expressing the DXS of the amino acid sequence shown in SEQ ID NO. 3), and pEAQ-HT-GGPPS (for expressing the GGPPS of the amino acid sequence shown in SEQ ID NO. 4).

[0055] Example 3: Preparation of triptolide-type diterpenoid compounds using hydroxylase

[0056] 1. Transient expression of enzymes

[0057] The pEAQ-HT-CsCYP1 expression vector provided in Example 2 was transformed into Agrobacterium GV3101 competent cells (Qingke Biology), and solid LB medium (kanamycin 50 ug / mL, rifampicin 20 ug / mL) was used for culture, and positive clones were selected.

[0058] The positive clones were inoculated into liquid LB medium (kanamycin 50 ug / mL, rifampicin 20 ug / mL), and cultured under the condition of 28℃, 220 rpm; the culture was collected, centrifuged at 4℃, 5000 rpm for 5-10 min, the supernatant was discarded, and the bacterial body was collected; the bacterial body was resuspended with MMA buffer (10 mM MES, 10 mM MgCl2, 0.1 mM acetyl-syringone AS) to an OD value of 0.5-0.6. 600

[0059] ​According to the above method, the bacterial liquid of pEAQ-HT-CsCYP1, pEAQ-HT-CsCTS, pEAQ-HT-DXS and pEAQ-HT-GGPPS were obtained respectively, and each bacterial liquid was mixed with equal volume, and then was placed at room temperature and in dark for 1-2 hours. Then, the mixed bacterial liquid with total volume of about 500 mL was injected into the leaves of 5-6 weeks old N. benthamiana by using a syringe with removed needle. After the injection, the tobacco was cultured for 5 days, and then the leaves were collected.

[0060] 2. Preparation of product I-1

[0061] After the collected tobacco leaves were freeze-dried, they were ground into powder, and then were extracted by ultrasonic in ethyl acetate. A small amount of the extract was detected by GC-EI-MS, and obvious product peak of I-1 was observed, while no product was observed in the control group without pEAQ-HT-CsCYP1. The above results are shown in Table 1. Figure 1

[0062] After the above extract was concentrated under reduced pressure, it was subjected to silica gel column chromatography, and was eluted by gradient elution with n-hexane-ethyl acetate system (100:1, 100:3, 100:5, 100:8, 10:1, 4:1, 0:100). Seven fractions (Fr1-Fr7) were obtained. Fraction Fr4 was subjected to reversed-phase semi-preparative HPLC gradient elution (mobile phase A (pure water), mobile phase B (acetonitrile)); elution gradient: 80-85% B, 30 min; 85-100% B, 5 min; 100% B, 5 min; flow rate = 1.0 mL / min; column temperature: 30°C; λ = 210 nm), and compound I-1 was separated. max R = 20.1 min).

[0063] The EI-MS spectrum, H NMR spectrum (400 MHz, CDC13), C NMR spectrum (100 MHz, CDC13) and 1D-NOE spectrum (600 MHz, CDC13) of the prepared product I-1 are shown in Table 2, and the nuclear magnetic data are as follows: 1 H NMR (400 MHz, CDC13) δ 13 Figures 2 to 5 1 H NMR (400 MHz, CDC13) δ H ​​​​1.87 (1H, ddd, J = 12.4, 6.8, 2.4 Hz, H-1a), 1.50 (1H, m, H-1b), 2.67 (1H, m, H-2), 5.33 (1H, d, J = 5.2 Hz, H-4), 2.32 (1H, m, H-5a), 2.04 (1H, m, H-5b), 1.78 (1H, dd, J = 13.6, 2.4 Hz, H-6a), 1.24 (1H, ddd, J = 13.6, 5.6, 2.4 Hz, H-6b), 1.31 (1H, m, H-8a), 0.98 (1H, m, H-8b), 1.59 (1H, m, H-9a), 1.20 (1H, m, H-9b), 1.58 (1H, m, H-11), 1.70 (1H, m, H-12a), 1.38 (1H, m, H-12b), 3.51 (1H, m, H-13), 2.02 (1H, m, H-14), 2.23 (1H, dd, J = 10.4, 9.2 Hz, H-15), 1.75 (1H, m, H-16), 0.94 (3H, s, H-17), 0.80 (3H, d, J = 6.8 Hz, H-18), 1.01 (3H, s, H-19), 1.67 (3H, s, H-20); 13 C NMR (100 MHz, CDC13) δ C 36.4 (C-1), 43.3 (C-2), 137.9 (C-3), 125.5 (C-4), 25.4 (C-5), 40.6 (C-6), 33.9 (C-7), 33.5 (C-8), 31.4 (C-9), 34.1 (C-10), 31.9 (C-11), 37.2 (C-12), 73.4 (C-13), 43.6 (C-14), 47.4 (C-15), 52.9 (C-16), 25.4 (C-17), 16.3 (C-18), 28.9 (C-19), 24.7 (C-20). The structure of compound I-1 was determined as follows, named 13-hydroxy-cephalotene:

[0064]

[0065] Example 4: Preparation of triptolide-type diterpenoid compounds using hydroxylase

[0066] 1. Transient expression of enzymes

[0067] The bacterial solution of pEAQ-HT-CsCYP1 was obtained according to the method provided in Example 3, and after standing at room temperature, away from light, for 1-2 h, the mixed bacterial solution was injected into the leaves of 5-6 week old N. benthamiana using a syringe with the needle removed.

[0068] 2. Injection of substrate

[0069] The tobacco was routinely cultured for 4 days after injection, and the substrate harringtonine aqueous solution (200 μM, a small amount of DMSO was added to assist dissolution) was injected into the leaf injected with the bacterial solution using a syringe with a removed needle. After 24 hours, the leaves were collected.

[0070] 3. Product detection

[0071] The collected tobacco leaves were freeze-dried, ground into powder, and ultrasonically extracted with ethyl acetate. The extract was collected by centrifugation. Analysis was performed using the GC-EI-MS detection conditions in Example 3. A small amount of product I-1 was detected, while no product was detected in the experimental group without the pEAQ-HT-CsCYP1 injection after the substrate injection.

[0072] The product prepared in the present application can be used as an intermediate for further post-modification by enzymatic catalysis, chemical synthesis, or a combination of both, to prepare other harringtonine-type diterpenoid compounds.

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice of the present application. The specific embodiments and preferred methods and materials described herein are illustrative only and not intended to be limiting.

Claims

1. An isolated hydroxylase, characterized in that, It has an amino acid sequence as shown in SEQ ID NO:1, or has an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:1 and has catalytic activity.

2. The hydroxylase according to claim 1, characterized in that, It is an amino acid sequence as shown in SEQ ID NO:1 or a truncated form of an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO:1 and has catalytic activity, with the signal peptide and / or transmembrane region removed.

3. The hydroxylase according to claim 1 or 2, characterized in that, It is chemically modified and / or immobilized, or has a tag attached for detection and / or purification.

4. The isolated nucleotides, characterized in that, It encodes the hydroxylase according to any one of claims 1 to 3, and preferably has the nucleotide sequence shown in SEQ ID NO:

5.

5. A carrier, characterized in that, It is a cloning vector that inserts the nucleotide of claim 4, or an expression vector that inserts the nucleotide of claim 4, or an expression vector that can exogenously express the hydroxylase of any one of claims 1 to 3; Preferably, the expression vector is capable of expressing the target protein in bacterial, yeast, plant, or animal cells; More preferably, the expression vector is capable of expressing the target protein in Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Arabidopsis thaliana, Nicotiana benthamiana, or insect cells.

6. The host of the vector described in claim 5 has been exogenously transferred; Preferably, the host is a bacterium, yeast, plant or animal cell; more preferably, it is an Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Arabidopsis thaliana, Nicotiana benthamiana or insect cell.

7. A method for preparing the hydroxylase of any one of claims 1 to 3 using the nucleotide of claim 4, the vector of claim 5, or the host of claim 6; Preferably, the method includes: The host is cultured, the culture is collected, and the hydroxylase or tissue containing the hydroxylase is extracted.

8. The use of the hydroxylase according to any one of claims 1 to 3, or the hydroxylase prepared by the method of claim 7, or a tissue containing the hydroxylase, in an enzymatic reaction; wherein the enzymatic reaction is an in vitro biochemical reaction or is carried out in vivo; and the substrate of the enzymatic reaction has the structure shown in I:

9. The use of the hydroxylase according to any one of claims 1 to 3, or the hydroxylase prepared by the method of claim 7, or a tissue containing said hydroxylase, in the preparation of thiamethoxam-type diterpenoid compounds, characterized in that, Prepared using biosynthesis or a combination of biosynthesis and chemical synthesis; Preferably, the trifurcanane-type diterpenoid compound has the structure shown in I as the diterpenoid core, and at least the C-13 position therein is modified by oxidation; 10. The application according to claim 9, characterized in that, The hydroxylase was used to catalyze an in vitro enzymatic reaction of a diterpenoid substrate having the structure shown in Figure I to prepare a thiazoline-type diterpenoid compound.

11. The application according to claim 9, characterized in that, A hydroxylase expressing the amino acid sequence shown in SEQ ID NO. 1 was exogenously introduced into *Nicotiana benthamiana*, and a diterpene substrate shown in I was added to prepare a thiamethoxam-type diterpene compound.

12. The application according to claim 9, characterized in that, In *Tobacco Benzoinus*, expression vectors expressing hydroxylase having the amino acid sequence shown in SEQ ID NO. 1 and sphagnum moietin synthase having the amino acid sequence shown in SEQ ID NO. 2 are exogenously introduced to prepare sphagnum moietin-type diterpenoid compounds; preferably, expression vectors of DXS and / or GGPPS are also simultaneously introduced.

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