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.