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

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 (CsCTS) 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 CsCTS 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.