Premium
Biosynthesis of the psychotropic plant diterpene salvinorin A: Discovery and characterization of the Salvia divinorum clerodienyl diphosphate synthase
Author(s) -
Pelot Kyle A.,
Mitchell Rod,
Kwon Moonhyuk,
Hagelthorn Lynne M.,
Wardman Jacob F.,
Chiang Angela,
Bohlmann Jörg,
Ro DaeKyun,
Zerbe Philipp
Publication year - 2017
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.13427
Subject(s) - diterpene , atp synthase , salvia , biology , biochemistry , nicotiana benthamiana , chemistry , stereochemistry , enzyme , botany , gene
Summary 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 (di TPS s), the ent ‐copalyl diphosphate ( ent ‐ CPP ) synthase Sd CPS 1, and the clerodienyl diphosphate ( CLPP ) synthase Sd CPS 2. Mining of leaf‐ and trichome‐specific transcriptomes revealed five di TPS s, two of which are class II di TPS s (Sd CPS 1‐2) and three are class I enzymes (Sd KSL 1‐3). Of the class II di TPS s, transient expression in Nicotiana benthamiana identified Sd CPS 1 as an ent ‐ CPP synthase, which is prevalent in roots and, together with Sd KSL 1, 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 Sd CPS 2 as a CLPP synthase. A role of Sd CPS 2 in catalyzing the committed step in salvinorin A biosynthesis is supported by its biochemical function, trichome‐specific expression and absence of additional class II di TPS s in S. divinorum . Structure‐guided mutagenesis revealed four catalytic residues that enabled the re‐programming of Sd CPS 2 activity to afford four distinct products, thus advancing our understanding of how neo‐functionalization events have shaped the array of different class II di TPS functions in plants, and may promote synthetic biology platforms for a broader spectrum of diterpenoid bioproducts.