title: Biosynthesis of the psychotropic plant diterpene salvinorin A: Discovery and characterization of the Salvia divinorum clerodienyl diphosphate synthase.
abstract: Salvia divinorum commonly known as diviner's sage, is an ethnomedicinal plant of the mint family (Lamiaceae). Salvia divinorum is rich in clerodane-type diterpenoids, which accumulate predominantly in leaf glandular trichomes. The main bioactive metabolite, salvinorin A, is the first non-nitrogenous natural compound known to function as an opioid-receptor agonist, and is undergoing clinical trials for potential use in treating neuropsychiatric diseases and drug addictions. We report here the discovery and functional characterization of two S. divinorum diterpene synthases (diTPSs), the ent-copalyl diphosphate (ent-CPP) synthase SdCPS1, and the clerodienyl diphosphate (CLPP) synthase SdCPS2. Mining of leaf- and trichome-specific transcriptomes revealed five diTPSs, two of which are class II diTPSs (SdCPS1-2) and three are class I enzymes (SdKSL1-3). Of the class II diTPSs, transient expression in Nicotiana benthamiana identified SdCPS1 as an ent-CPP synthase, which is prevalent in roots and, together with SdKSL1, exhibits a possible dual role in general and specialized metabolism. In vivo co-expression and in vitro assays combined with nuclear magnetic resonance (NMR) analysis identified SdCPS2 as a CLPP synthase. A role of SdCPS2 in catalyzing the committed step in salvinorin A biosynthesis is supported by its biochemical function, trichome-specific expression and absence of additional class II diTPSs in S. divinorum. Structure-guided mutagenesis revealed four catalytic residues that enabled the re-programming of SdCPS2 activity to afford four distinct products, thus advancing our understanding of how neo-functionalization events have shaped the array of different class II diTPS functions in plants, and may promote synthetic biology platforms for a broader spectrum of diterpenoid bioproducts.
authors.affiliation: Department of Plant Biology, University of California-Davis, 1 Shields Avenue, Davis, CA, 95616, USA.
authors.forename: Kyle A
authors.initials: KA
authors.lastname: Pelot
authors.affiliation: Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, AB, T2N1N4, Canada.
authors.forename: Rod
authors.initials: R
authors.lastname: Mitchell
authors.affiliation: Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, AB, T2N1N4, Canada.
authors.forename: Moonhyuk
authors.initials: M
authors.lastname: Kwon
authors.affiliation: Department of Plant Biology, University of California-Davis, 1 Shields Avenue, Davis, CA, 95616, USA.
authors.forename: Lynne M
authors.initials: LM
authors.lastname: Hagelthorn
authors.affiliation: Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, AB, T2N1N4, Canada.
authors.forename: Jacob F
authors.initials: JF
authors.lastname: Wardman
authors.affiliation: Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC, V6T 1Z4, Canada.
authors.forename: Angela
authors.initials: A
authors.lastname: Chiang
authors.affiliation: Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC, V6T 1Z4, Canada.
authors.forename: Jörg
authors.initials: J
authors.lastname: Bohlmann
authors.affiliation: Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary, AB, T2N1N4, Canada.
authors.forename: Dae-Kyun
authors.initials: DK
authors.lastname: Ro
authors.affiliation: Department of Plant Biology, University of California-Davis, 1 Shields Avenue, Davis, CA, 95616, USA.
authors.forename: Philipp
authors.initials: P
authors.lastname: Zerbe
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism*; D001688:Biological Products / Q000378:metabolism; D004224:Diterpenes / Q000378:metabolism*; D045785:Diterpenes, Clerodane / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D027544:Salvia / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001688:Biological Products; D004224:Diterpenes; D045785:Diterpenes, Clerodane; D010940:Plant Proteins; D019883:Alkyl and Aryl Transferases; C090499:salvinorin A
keywords: 
  Salvia divinorum
  ; botanicals; diterpene synthases; diterpenoid metabolism; pathway discovery; plant natural product; salvinorin A