Repurposing Terpene Synthases for the Conversion of Synthetic Geranylgeranyl Diphosphate Derivatives

Terpenoids are an incredibly diverse class of specialized metabolites with tens of thousands of compounds identified in plants. Despite typically highly complex structures including multiple chiral centers and ring systems, their biosynthesis stems from only a small handful of linear substrates. Nearly all diterpenoids (C20 backbones) are derived from a single substrate, geranylgeranyl diphosphate (GGPP), which is cyclized to hundreds of diterpene backbones through either single-step or multi-step conversion by paired modules of diterpene synthases (diTPSs). Specifically, the second enzyme in these modules was found to display a spectrum of substrate promiscuity, enabling the construction of new-to-nature pathways from GGPP. Here we extend this to demonstrate that diTPSs can convert synthetic GGPP derivatives which are not found in nature into new terpene backbones. Synthesis and screening of twenty synthetic substrates against twenty-four enzymes in 438 unique combinations resulted in fifty-four successful combinations and the identification of fifty-six new compounds. Structural characterization by NMR for six select compounds was carried out, indicating that many of these products are a direct translation of the synthetic substrate into a respective derivative of the enzymes native products. This screening of substrate tolerance revealed trends to inform future design principles for TPS biocatalysis. A high degree of promiscuity in several enzymes with native functions involved in the biosynthesis of medicinal natural products indicates a remarkable potential of diTPSs as biocatalysts in the semi-biosynthesis of non-natural compounds with predictable structures. The integration of synthetic and biosynthetic approaches can afford access to derivatives of natural terpene products with bioactivities and which would be difficult to achieve strictly through chemical synthesis.