Cytochrome P450-Mediated Cyclization in Eunicellane-Derived Diterpenoid Biosynthesis

09 March 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Terpene cyclization, one of the most complex chemical reactions in nature, is generally catalyzed by two classes of terpene cyclases (TCs). Cytochrome P450s that act as unexpected TC-like enzymes are known but are very rare. Here, we genome-mined a cryptic bacterial terpenoid gene cluster, named ari, from the thermophilic actinomycete strain Amycolatopsis arida. By employing a heterologous production system, we isolated and characterized three highly oxidized eunicellane-derived diterpenoids, aridacins AC (13), that possess a rare 6/7/5-fused tricyclic scaffold. In vivo and in vitro experiments systematically established a non-canonical two-step biosynthetic pathway for diterpene skeleton formation. First, a class I TC (AriE) cyclizes geranylgeranyl diphosphate (GGPP) into a 6/10-fused bicyclic cis-eunicellane skeleton. Next, a cytochrome P450 (AriF) catalyzes cyclization of the eunicellane skeleton into the 6/7/5-fused tricyclic scaffold via C2-C6 bond formation. Quantum chemical computations support a hydrogen abstraction and subsequent oxidation mechanism for AriF catalyzed carbocation cyclization. The biosynthetic logic of skeleton construction in the aridacin diterpenoids is unprecedented, expanding the catalytic capacity and diversity of P450s and setting the stage to investigate the inherent principles of carbocation generation by P450s in the biosynthesis of terpenoids.


Terpene cyclization
cytochrome P450
eunicellane-derived diterpenoids
terpenoid biosynthesis

Supplementary materials

SI for P450-TC
Detailed experimental procedures, primers used and plasmids constructed, gene clusters information, HPLC, EIC and GC-MS profiles, X-ray crystallographic data, and NMR spectra


Comments are not moderated before they are posted, but they can be removed by the site moderators if they are found to be in contravention of our Commenting Policy [opens in a new tab] - please read this policy before you post. Comments should be used for scholarly discussion of the content in question. You can find more information about how to use the commenting feature here [opens in a new tab] .
This site is protected by reCAPTCHA and the Google Privacy Policy [opens in a new tab] and Terms of Service [opens in a new tab] apply.