Four Cytochrome P450 Enzymes Mediate Oxidation Cascades in the Biosynthesis of Cephalotane‐Type Diterpenoids

Abstract Cephalotane‐type diterpenoids, a class of natural products exclusively found in Cephalotaxus plants, are well known for their attractive structures and potent biological activities. However, their low natural abundance and intricate cage‐like structures hinder their accessibility. Recently, the identification of a cephalotene synthase ( Cs CTS) has addressed the first committed step in the biosynthesis. However, the enzymes involved in the complex post‐modification of the cephalotene core into structurally diverse cephalotane‐type diterpenoids remain obscure. In this study, we functionally characterised four novel cytochrome P450 enzymes from C. sinensis . These enzymes demonstrate multiple oxidative functions and cooperatively catalyse a cascade of oxidation reactions, including the formation of signature 13,17‐lactone, 5,19‐lactone, and tropone. We further co‐expressed the characterised CYP450 enzymes in combination with Cs CTS to produce a variety of cephalotane‐type diterpenoids, including hainanolidol ( 2 ), mannolide C ( 3 ), mannolide A ( 4 ), and cephinoid H ( 5 ), in Nicotiana benthamiana . Subsequently, harringtonolide ( 1 ) was chemically converted from hainanolidol (3.9 µmol with 10 equiv of Pb(OAc) 4 ) in 87.5% yield. In this study, the biosynthetic pathways of representative cephalotane‐type diterpenoids were elucidated and reconstructed, thereby establishing a foundation for their sustainable production through biosynthesis and/or chemo‐biosynthesis, highlighting the remarkable efficiency of merely five Cephalotaxus ‐specific enzymes in assembling such structurally complex natural products.