Salvia divinorum leaf trichome transcripts

Salvia divinorum or diviner's sage, is an ethnomedical plant of the mint family rich in neoclerodane diterpenoid natural products that accumulate primarily in leaf glandular trichomes. The main active ingredient, salvinorin A, is the first non-nitrogenous natural compound known to act as an opioid-receptor agonist, and is undergoing clinical trials as a potential therapy of neuropsychiatric diseases and addictions. We report the discovery and functional characterization of two S. divinorum diterpene synthases (diTPSs), the ent-copalyl diphosphate (ent-CPP) synthase SdCPS1, and the kolavenyl diphosphate synthase SdCPS2 catalyzing the committed reaction in salvinorin A biosynthesis. Mining of tissue-specific transcriptome inventories revealed five diTPSs, comprising three class I and two class II diTPSs. Combinatorial transient expression in Nicotiana benthamiana identified SdCPS1 as an ent-CPP synthase with a possible dual role in general and specialized diterpenoid metabolism given its prevalent expression in roots. In vivo co-expression and in vitro assays combined with NMR analysis identified SdCPS2 as a kolavenyl diphosphate synthase, which, together with the trichome-specific expression of SdCPS2 demonstrates its function in salvinorin A formation. Structure-guided point mutations of three catalytic residues enabled the re-programming of SdCPS2 activity to yield four distinct products, providing deeper insight into the evolutionary divergence of specialized diterpenoid metabolism and offering new opportunities for developing synthetic biology platforms toward a broader array of diterpenoid bioproducts.