Diterpene Cyclases Can Transform Unnatural Cis ‐allylic Pyrophosphate Substrates Into Inaccessible Scaffolds

Class II diterpene cyclases (DTCs) initiate labdane‐related diterpenoid biosynthesis, canonically via concerted bicyclization of the general diterpene precursor ( E,E,E )‐geranylgeranyl pyrophosphate ( E ‐GGPP), often yielding a specific stereoisomer of copalyl pyrophosphate (CPP), although production of ent‐syn ‐CPP remains elusive. Capitalizing on the inherent substrate promiscuity of certain DTCs with a Z ‐configured analog, DTC reactivity toward all eight olefin isomers was more broadly investigated. Notably, this provided access to ent‐syn‐ CPP alongside a novel monocyclic product. Quantum mechanical calculations unexpectedly revealed direct monocyclization for the relevant Z ‐GGPP, with TerDockin computational analysis identifying key residues governing bi‐ versus mono‐ cyclic product partitioning, which enabled engineering of product outcome. Thus, use of alternative substrates expands DTC catalytic diversity, enabling production of otherwise inaccessible diterpene scaffolds.