title: Diterpene synthases of the biosynthetic system of medicinally active diterpenoids in Marrubium vulgare.
abstract: Marrubium vulgare (Lamiaceae) is a medicinal plant whose major bioactive compounds, marrubiin and other labdane-related furanoid diterpenoids, have potential applications as anti-diabetics, analgesics or vasorelaxants. Metabolite and transcriptome profiling of M. vulgare leaves identified five different candidate diterpene synthases (diTPSs) of the TPS-c and TPS-e/f clades. We describe the in vitro and in vivo functional characterization of the M. vulgare diTPS family. In addition to MvEKS ent-kaurene synthase of general metabolism, we identified three diTPSs of specialized metabolism: MvCPS3 (+)-copalyl diphosphate synthase, and the functional diTPS pair MvCPS1 and MvELS. In a sequential reaction, MvCPS1 and MvELS produce a unique oxygenated diterpene scaffold 9,13-epoxy-labd-14-ene en route to marrubiin and an array of related compounds. In contrast with previously known diTPSs that introduce a hydroxyl group at carbon C-8 of the labdane backbone, the MvCPS1-catalyzed reaction proceeds via oxygenation of an intermediate carbocation at C-9, yielding the bicyclic peregrinol diphosphate. MvELS belongs to a subgroup of the diTPS TPS-e/f clade with unusual βα-domain architecture. MvELS is active in vitro and in vivo with three different prenyl diphosphate substrates forming the marrubiin precursor 9,13-epoxy-labd-14-ene, as identified by nuclear magnetic resonance (NMR) analysis, manoyl oxide and miltiradiene. MvELS fills a central position in the biosynthetic system that forms the foundation for the diverse repertoire of Marrubium diterpenoids. Co-expression of MvCPS1 and MvELS in engineered E. coli and Nicotiana benthamiana offers opportunities for producing precursors for an array of biologically active diterpenoids.
authors.affiliation: Michael Smith Laboratories, University of British Columbia, 301-2185 East Mall, Vancouver, BC, V6T 1Z4, Canada.
authors.forename: Philipp
authors.initials: P
authors.lastname: Zerbe
authors.forename: Angela
authors.initials: A
authors.lastname: Chiang
authors.forename: Harpreet
authors.initials: H
authors.lastname: Dullat
authors.forename: Mark
authors.initials: M
authors.lastname: O'Neil-Johnson
authors.forename: Courtney
authors.initials: C
authors.lastname: Starks
authors.forename: Björn
authors.initials: B
authors.lastname: Hamberger
authors.forename: Jörg
authors.initials: J
authors.lastname: Bohlmann
mesh_terms: D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D004224:Diterpenes / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D027522:Marrubium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010755:Organophosphates; D010802:Phylogeny; D010940:Plant Proteins; D010946:Plants, Medicinal
chemical_list: D004224:Diterpenes; D010755:Organophosphates; D010940:Plant Proteins; C408747:copalyl diphosphate; C433017:labdane; C475535:manoyl oxide; C573600:miltiradiene; C105731:marrubiin; D019883:Alkyl and Aryl Transferases; C090963:ent-kaurene synthetase A; C105712:ent-kaurene synthetase B
keywords: Marrubium vulgare; diterpene synthase; diterpenoid biosynthesis; marrubiin; medicinal plants; natural product biosynthesis; plant specialized metabolism