FoTO1 orchestrates Taxol biosynthesis through catalytic and non-catalytic mechanisms

Taxol is a blockbuster chemotherapeutic derived from the Pacific Yew tree. Recent work in our group has identified a complete pathway to baccatin III, a key intermediate, that hinges on a novel accessory protein, Facilitator of Taxane Oxidation (FoTO1). This protein dramatically improves yield and alters enzyme product profiles when reconstituting the Taxol pathway in N. benthamiana. FoTO1 has been shown to act early in the biosynthetic pathway improving the yields of product generated by the combination of a plastidial diterpene synthase (taxadiene synthase) and an endoplasmic reticulum (ER) localized cytochrome P450 (T5-alpha-hydroxylase). Here, we show that FoTO1 is an enzyme capable of converting taxadiene-(4),5-epoxide, the likely product of T5H oxidation, into taxadien-5-ol. FoTO1 is also functional in yeast, resolving a key bottleneck for development of a bioproduction route to Taxol in this host. Targeted mutagenesis of key catalytic residues in FoTO1 abrogates function in vitro but not in planta, suggesting non-catalytic contributions of FoTO1 to the taxane pathway. A combination of proximity labelling, bimolecular fluorescence complementation assays, and co-immunoprecipitation studies revealed that FoTO1 interacts with and organizes various P450s in the Taxol pathway. These approaches highlight the importance of both FoTO1s catalytic and non-catalytic functions in improving yields in the early Taxol pathway. Beyond Taxol biosynthesis, FoTO1 boosts yields for diverse diterpene pathways from across phylogeny, suggesting a general role of this protein class in mediating metabolism across the plastid and ER in plants.