Three cytochrome P450 enzymes consecutively catalyze the biosynthesis of furanoclerodane precursors in Salvia species

Salvia species native to the Americas are rich in valuable bioactive furanoclerodanes, like the psychoactive salvinorin A found in Salvia divinorum, which is used in treatment of opioid addiction. However, there is relatively little known about their biosynthesis. To address this, we investigated the biosynthesis of salviarin, the most abundant furanoclerodane structure in the ornamental sage Salvia splendens. Using a self-organizing map and mutual rank analysis of RNA-seq co-expression data, we identified three cytochrome P450 enzymes responsible for converting kolavenol into salviarin precursors, consecutively: annonene, hardwickiic acid and hautriwaic acid. As annonene and hardwickiic acid have been proposed as intermediates in the biosynthesis of salvinorin A, and to test for a common evolutionary origin of the furanoclerodane pathway in these Salvia species, we searched for homologous genes in available data for S. divinorum. The enzymes encoded by orthologous genes from S. divinorum displayed kolavenol synthase (SdKLS), annonene synthase (SdANS), and hardwickiic acid synthase (SdHDAS) activity respectively, supporting the view that these are intermediate steps in the biosynthesis of salvinorin A. We investigated the origin of annonene synthase and the role of gene duplication in the evolution of this specific activity. Our work shows how S. splendens can serve as a model species for studying furanoclerodanes biosynthesis in Salvia species, contributes to understanding the evolution of specialized metabolism in plants, and provides new tools to produce salvinorin A in biotechnological chassis.