Abietane type tricyclic diterpenoid C-14 site hydroxylase

A technology of tricyclic diterpenes and compounds, applied in the field of tripterygium wilfordii cytochrome P450 oxidase, can solve problems such as unknown multi-step pathways

Pending Publication Date: 2022-05-13
BEIJING SHIJITAN HOSPITAL CAPITAL MEDICAL UNIVERSTY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the downstream multi-step pathway from

Method used

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  • Abietane type tricyclic diterpenoid C-14 site hydroxylase
  • Abietane type tricyclic diterpenoid C-14 site hydroxylase
  • Abietane type tricyclic diterpenoid C-14 site hydroxylase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, Tripterygium wilfordii CYP82D274 gene cloning ( figure 1 )

[0044] First, use Super Total RNA Extraction Kit (Promega) for operation:

[0045] (1) Take about 10 mg of Tripterygium wilfordii suspended cell sample and place it in a 2 mL centrifuge tube, pulverize it evenly in a liquid nitrogen environment, and quickly pulverize it with a mixing mill (MM400, Retsch) for 2 minutes;

[0046] (2) Quickly add 500 μL RNA Lysis Solution to the EP tube containing the pulverized sample, and invert the centrifuge tube 3-4 times to fully lyse the sample;

[0047] (3) Continue to add 500 μL RNA diluent, mix with a pipette, centrifuge at 14 000 rpm at 4°C for 5 min;

[0048] (4) Take the supernatant into a new sterile 2mL centrifuge tube, add 0.5 times the volume of absolute ethanol, and invert the centrifuge tube to fully mix;

[0049] (5) Transfer the mixed solution to a spin column in stages, centrifuge at 12 000 rpm at 4°C for 1 min, and discard the filtrate; ...

Embodiment 2

[0076] Example 2. In vivo functional characterization of CYP82D274 yeast

[0077] First, construct a eukaryotic expression vector containing P450 and CPR

[0078] (1) Construction of pESC-LEU::TwCPR3

[0079] Since the function of P450 requires cytochrome P450 oxidoreductase (CPR) to provide electrons, tripterygium wilfordii cytochrome P450 oxidoreductase 3 (TwCPR3) was constructed to the multiple cloning site MCS2 (multiple cloning site) terminal. The carrier part is subjected to double digestion reaction with NEB restriction endonucleases BamHI-HF and SalI-HF, and the reaction system is:

[0080]

[0081] After the reaction is over, perform electrophoresis detection together with the undigested carrier and 15K DNA Marker (full gold), and confirm that there is a band of the target size and the expression form is different from that of the undigested sample, and the digestion is considered successful. Use 1.5% agar Glycogel electrophoresis (150V, about 25min) and gel rec...

Embodiment 3

[0118] Example 3, In vitro functional characterization of CYP82D274 yeast ( image 3 )

[0119] Microsomes were extracted from the BY47471 Saccharomyces cerevisiae containing pESC-LEU::(CYP82D274+TwCPR3) and the corresponding empty vector pESC-LEU::TwCPR3, and dehydroabietic acid was used as a substrate to detect the product in vitro.

[0120] The microsome extraction method is as follows:

[0121] (1) Transfer the activated bacterial liquid into 50mL SD-Leu+2%Glc liquid medium, incubate at 200rpm at 30°C for 16-20h, collect the bacterial liquid, and centrifuge at 4000g for 5min at room temperature;

[0122] (2) Resuspend the cells with 100mL inducible SD-Leu+2%Gal liquid medium, induce at 30°C, 200rpm for 12-16h;

[0123] (3) Bacterial solution was centrifuged at 4 000 g for 3 min at 4°C to collect the bacterial cells, resuspended in 10 mL TEK solution (TE (50 mM Tris-HCl, 1 mM EDTA, pH 7.4) + potassium chloride KCl), and allowed to stand at room temperature 5min;

[0124...

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Abstract

The invention relates to an enzyme for catalyzing hydroxylation of an abietane type tricyclic diterpenoid compound C-14, the enzyme is tripterygium wilfordii cytochrome p450 oxidase CYP82D274, and the invention also relates to polynucleotide CYP82D274 for coding the enzyme, and the polynucleotide CYP82D274 is used for catalyzing hydroxylation of the abietane type tricyclic diterpenoid compound C-14. An enzyme catalysis experiment proves that the triptolide gene has the function of catalyzing hydroxylation of triptolide biosynthesis intermediates, namely miltidiene, dehydroabietic acid and triptobenzene terpene D. The triptolide gene has the advantages that triptolide biosynthesis pathway analysis is further promoted, an important gene element is provided for synthesis biological production of the triptolide intermediates, and the triptolide gene has a wide application prospect. The method has important significance on synthesis regulation and control of abietane type tricyclic diterpenoid compounds such as triptolide and the like.

Description

technical field [0001] The invention relates to a tripterygium wilfordii cytochrome P450 oxidase and a polynucleotide encoding the enzyme, the enzyme can be used for the key C-14 biosynthesis of abietane-type tricyclic diterpenoids, and belongs to medicinal plants field of genetic engineering. Background technique [0002] Triptolide is one of the main active ingredients of the medicinal plant Tripterygium wilfordii Hook.f. It is a rosin with 3 epoxy groups and an α, β unsaturated five-membered lactone ring structure Alkyl diterpene compound, also the most active epoxy diterpene lactone compound in Triptolide (Zhou ZL, et al.Triptolide: structural modifications, structure-activity relationships, bioactivities, clinical development and mechanisms[J].Natural Product Reports, 2012, 29(4):457-475). Triptolide has significant anti-inflammatory, immunosuppressive, anti-tumor and other biological activities. Cell or animal model experiments have shown that it can treat autoimmune...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/81C12N1/19C12P15/00C12N15/82C12R1/865
CPCC12N9/0073C12N15/81C12P15/00C12N15/8216
Inventor 高伟张逸风
Owner BEIJING SHIJITAN HOSPITAL CAPITAL MEDICAL UNIVERSTY
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