Engineered geranyl diphosphate methyltransferase produces 2-methyl-dimethylallyl diphosphate as a noncanonical C unit for terpenoid biosynthesis.

Terpenoids constitute the largest class of natural products with complex structures, essential functions, and versatile applications. Creation of new building blocks beyond the conventional five-carbon (C) units, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate, expands significantly the chemical space of terpenoids. Structure-guided engineering of an -adenosylmethionine-dependent geranyl diphosphate (GPP) C2-methyltransferase from yielded variants converting DMAPP to a new C unit, 2-methyl-DMAPP. Mutation of the Gly residue at the position 202 resulted in a smaller substrate-binding pocket to fit DMAPP instead of its native substrate GPP. Replacement of Phe residue at the position 222 with a Tyr residue contributed to DMAPP binding via hydrogen bond. Furthermore, using as the chassis, we demonstrated that 2-methyl-DMAPP was accepted as a start unit to generate noncanonical - and -prenyl diphosphates (C) and terpenoids. This work provides insights into substrate recognition of prenyl diphosphate methyltransferases, and strategies to diversify terpenoids by expanding the building block portfolio.