Functional Characterization and Cyclization Mechanism of a Diterpene Synthase Responsible for the Biosynthesis of Crinipellins.

Crinipellins are a unique class of natural products distinguished by an unusual 5/5/5/5 tetracyclic skeleton. While the chemical synthesis of several crinipellin diterpenoids has been accomplished, the biochemical and genetic basis underlying their biosynthesis remains elusive. Here, we identify the biosynthetic gene cluster responsible for crinipellin production and establish CriF as the key diterpene synthase mediating the formation of the characteristic skeleton. Computational simulations and site-directed mutagenesis revealed critical amino acid residues governing the carbocation-driven cascade cyclization, enabling the identification of seven additional diterpene products in CriF mutants. Furthermore, isotopic labeling experiments combined with the hybrid quantum mechanical/molecular mechanical (QM/MM) calculations elucidated the cyclization mechanism of CriF. These findings provide the first comprehensive insights into crinipellin biosynthesis, laying the groundwork for future pathway elucidation and engineering of these complex diterpenoids.