A cytochrome P450 CYP81AM1 from Tripterygium wilfordii catalyses the C-15 hydroxylation of dehydroabietic acid

Abstract Triptolide is a trace natural product of Tripterygium wilfordii. It has antitumor activities, particularly against pancreatic cancer cells. Identification of genes and elucidation of the biosynthetic pathway leading to triptolide are the prerequisite for heterologous bioproduction. Here, we report a reference-grade genome of T. wilfordii with a contig N50 of 4.36 Mb. We show that copy numbers of triptolide biosynthetic pathway genes are impacted by a recent whole-genome triplication event. We further integrate genomic, transcriptomic, and metabolomic data to map a gene-to-metabolite network. This leads to the identification of a cytochrome P450 (CYP728B70) that can catalyze oxidation of a methyl to the acid moiety of dehydroabietic acid in triptolide biosynthesis. We think the genomic resource and the candidate genes reported here set the foundation to fully reveal triptolide biosynthetic pathway and consequently the heterologous bioproduction.

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Probing the Single Key Amino Acid Responsible for the Novel Catalytic Function of ent-Kaurene Oxidase Supported by NADPH-Cytochrome P450 Reductases in Tripterygium wilfordii

Frontiers in Plant Science ◽

10.3389/fpls.2017.01756 ◽

2017 ◽

Vol 8 ◽

Cited By ~ 12

Author(s):

Ping Su ◽

Hongyu Guan ◽

Yifeng Zhang ◽

Xing Wang ◽

Linhui Gao ◽

...

Keyword(s):

Cytochrome P450 ◽

Amino Acid ◽

The Novel ◽

Tripterygium Wilfordii ◽

Nadph Cytochrome ◽

Catalytic Function

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Author Correction: Genome of Tripterygium wilfordii and identification of cytochrome P450 involved in triptolide biosynthesis

Nature Communications ◽

10.1038/s41467-020-19253-3 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Lichan Tu ◽

Ping Su ◽

Zhongren Zhang ◽

Linhui Gao ◽

Jiadian Wang ◽

...

Keyword(s):

Cytochrome P450 ◽

Tripterygium Wilfordii

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Cytotoxic diterpenes triptolide, tripdiolide, and cytotoxic triterpenes from tissue cultures of Tripterygium wilfordii

Canadian Journal of Chemistry ◽

10.1139/v81-385 ◽

1981 ◽

Vol 59 (17) ◽

pp. 2677-2683 ◽

Cited By ~ 50

Author(s):

James P. Kutney ◽

Gary M. Hewitt ◽

Toshio Kurihara ◽

Phillip J. Salisbury ◽

Robert D. Sindelar ◽

...

Keyword(s):

Acid Methyl Ester ◽

Dehydroabietic Acid ◽

Tissue Cultures ◽

Tripterygium Wilfordii ◽

Quinone Methides ◽

Tripterygium Wilfordii Hook F ◽

Acid Methyl ◽

Plant Tissue Cultures ◽

Tripterygium Wilfordii Hook ◽

Acid Compound

Plant tissue cultures of Tripterygium wilfordii Hook F produced the cytotoxic diterpene triepoxides tripdiolide (1) and triptolide (2) in yields that were 16 and 3 times greater, respectively, than those observed in the plant itself. Other diterpenes, dehydroabietic acid (3) and 15-hydroxy-18-norabieta-3,8,11,13-tetraene-3-oic acid methyl ester (4) were also isolated. Co-occuring in these cultures were the cytotoxic quinone-methides, celastrol (5), compound 6, and compound 7. Other triterpenes produced were oleanolic acid (8) and polpunonic acid (9). β-Sitosterol (17) was also isolated. The proposed structure of 4 was confirmed by synthesis starting from compound 3. Cytotoxic data are reported, and a possible biosynthetic relationship among dehydroabietic acid, compound 4, and tripdiolide (1) is presented.

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A Cytochrome P450 Involved in the Metabolism of Abietane Diterpenoids by Pseudomonas abietaniphila BKME-9

Journal of Bacteriology ◽

10.1128/jb.186.11.3631-3639.2004 ◽

2004 ◽

Vol 186 (11) ◽

pp. 3631-3639 ◽

Cited By ~ 26

Author(s):

Daryl J. Smith ◽

Vincent J. J. Martin ◽

William W. Mohn

Keyword(s):

Cytochrome P450 ◽

Dehydroabietic Acid ◽

Gene Arrangement ◽

Type I ◽

Binding Assays ◽

P450 Monooxygenase ◽

Naturally Occurring ◽

Abietane Diterpenoids ◽

Pharmaceutical Properties ◽

Conserved Gene

ABSTRACT Diterpenoids are naturally occurring plant compounds which have pharmaceutical properties. We have sequenced a 10.4-kbp extension of the dit cluster in Pseudomonas abietaniphila BKME-9, containing genes involved in abietane diterpenoid biodegradation. The ditQ gene was found to encode a cytochrome P450 monooxygenase, P450dit, and to be homologous to the tdtD gene of Pseudomonas diterpeniphila A19-6a. Knocking out ditQ had little effect on growth of BKME-9 on abietic acid but severely impaired growth on dehydroabietic acid (DhA) and palustric acid (PaA), increasing doubling times from 3.8 to 15 h on DhA and from 5.6 to 18.5 h on PaA. A xylE transcriptional fusion showed that transcription of ditQ was induced by a range of diterpenoids. Substrate binding assays of P450dit expressed in Escherichia coli revealed that DhA binds to the enzyme and yields a type I binding spectrum with a Kd of 0.4 μM. These results indicate that P450dit is involved in the metabolism of DhA and PaA and are consistent with its putative role in converting DhA to 7-hydroxy-DhA. Finally, an amino acid sequence identity of greater than 72% and conserved gene arrangement support the hypothesis that the dit gene cluster of P. abietaniphila BKME-9 and the tdt cluster of P. diterpeniphila A19-6a contain functional homologues.

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Molecular evolution of a cytochrome P450 for the synthesis of potential antidepressant (2R,6R)-hydroxynorketamine

Chemical Communications ◽

10.1039/d0cc06729f ◽

2021 ◽

Author(s):

Ansgar Bokel ◽

Michael C. Hutter ◽

Vlada B. Urlacher

Keyword(s):

Cytochrome P450 ◽

Molecular Evolution ◽

Cytochrome P450 Monooxygenase ◽

P450 Monooxygenase ◽

Effective Synthesis

Engineered cytochrome P450 monooxygenase CYP154E1 enables the effective synthesis of the potential antidepressant (2R,6R)-hydroxynorketamine via N-demethylation and regio- and stereoselective hydroxylation of (R)-ketamine.

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