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 the treatment of opioid addiction. However, relatively little is 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 the consecutive conversion of kolavenol into the salviarin precursors: annonene, hardwickiic acid, and hautriwaic acid. Annonene and hardwickiic acid have been proposed as intermediates in the biosynthesis of salvinorin A, and we therefore tested for a common evolutionary origin of the furanoclerodane pathway in these Salvia species by searching for homologous genes in available data for S. divinorum. The enzymes encoded by orthologous genes from S. divinorum displayed kolavenol synthase, annonene synthase, and hardwickiic acid synthase activity, respectively, supporting the view that these are intermediate steps in the biosynthesis of salvinorin A. We further 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 the study of furanoclerodane biosynthesis in Salvia species, contributes to understanding the evolution of specialized metabolism in plants, and provides new tools for the production of salvinorin A in biotechnological chassis.