title: Exploring diterpene metabolism in non-model species: transcriptome-enabled discovery and functional characterization of labda-7,13E-dienyl diphosphate synthase from Grindelia robusta.
abstract: Grindelia robusta or gumweed, is a medicinal herb of the sunflower family that forms a diverse suite of diterpenoid natural products. Its major constituents, grindelic acid and related grindelane diterpenoids accumulate in a resinous exudate covering the plants' surfaces, most prominently the unopened composite flower. Recent studies demonstrated potential pharmaceutical applications for grindelic acid and its synthetic derivatives. Mining of the previously published transcriptome of G. robusta flower tissue identified two additional diterpene synthases (diTPSs). We report the in vitro and in vivo functional characterization of an ent-kaurene synthase of general metabolism (GrTPS4) and a class II diTPS (GrTPS2) of specialized metabolism that converts geranylgeranyl diphosphate (GGPP) into labda-7,13E-dienyl diphosphate as verified by nuclear magnetic resonance (NMR) analysis. Tissue-specific transcript abundance of GrTPS2 in leaves and flowers accompanied by the presence of an endocyclic 7,13 double bond in labda-7,13E-dienyl diphosphate suggest that GrTPS2 catalyzes the first committed reaction in the biosynthesis of grindelic acid and related grindelane metabolites. With the formation of labda-7,13E-dienyl diphosphate, GrTPS2 adds an additional function to the portfolio of monofunctional class II diTPSs, which catalytically most closely resembles the bifunctional labda-7,13E-dien-15-ol synthase of the lycopod Selaginella moellendorffii. Together with a recently identified functional diTPS pair of G. robusta producing manoyl oxide, GrTPS2 lays the biosynthetic foundation of the diverse array of labdane-related diterpenoids in the genus Grindelia. Knowledge of these natural diterpenoid metabolic pathways paves the way for developing biotechnology approaches toward producing grindelic acid and related bioproducts.
authors.affiliation: Department of Plant Biology, University of California-Davis, 1 Shields Avenue, Davis, CA, 95616, USA.|Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC, V6T 1Z4, Canada.
authors.forename: Philipp
authors.initials: P
authors.lastname: Zerbe
authors.affiliation: Department of Plant Biology, University of California-Davis, 1 Shields Avenue, Davis, CA, 95616, USA.
authors.forename: Selina M
authors.initials: SM
authors.lastname: Rodriguez
authors.affiliation: Department of Plant Biology, University of California-Davis, 1 Shields Avenue, Davis, CA, 95616, USA.
authors.forename: Sibongile
authors.initials: S
authors.lastname: Mafu
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: Harpreet K
authors.initials: HK
authors.lastname: Sandhu
authors.affiliation: Sequoia Sciences, 1912 Innerbelt Business Center Drive, Saint Louis, MO, 63114, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: O'Neil-Johnson
authors.affiliation: Sequoia Sciences, 1912 Innerbelt Business Center Drive, Saint Louis, MO, 63114, USA.
authors.forename: Courtney M
authors.initials: CM
authors.lastname: Starks
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
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism*; D004224:Diterpenes / Q000378:metabolism*; D045786:Diterpenes, Kaurane / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D036623:Grindelia / Q000235:genetics* / Q000378:metabolism*; D019753:Intramolecular Lyases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D014026:Nicotiana / Q000235:genetics
chemical_list: D004224:Diterpenes; D045786:Diterpenes, Kaurane; D010940:Plant Proteins; D019883:Alkyl and Aryl Transferases; C021459:kaurene synthetase; D019753:Intramolecular Lyases; C000608069:labda-7,13-dienyl diphosphate synthase, Grindelia robusta
keywords: Grindelia robusta; diterpene synthase; diterpenoid biosynthesis; grindelic acid; medicinal plants; plant natural products; plant-specialized metabolism