Diterpene synthases of the biosynthetic system of medicinally active diterpenoids in Marrubium vulgare

Summary 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 (di TPS s) of the TPS –c and TPS –e/f clades. We describe the in vitro and in vivo functional characterization of the M. vulgare di TPS family. In addition to Mv EKS ent –kaurene synthase of general metabolism, we identified three di TPS s of specialized metabolism: Mv CPS 3 (+)‐copalyl diphosphate synthase, and the functional di TPS pair Mv CPS 1 and Mv ELS . In a sequential reaction, Mv CPS 1 and Mv ELS 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 di TPS s that introduce a hydroxyl group at carbon C–8 of the labdane backbone, the Mv CPS 1‐catalyzed reaction proceeds via oxygenation of an intermediate carbocation at C–9, yielding the bicyclic peregrinol diphosphate. Mv ELS belongs to a subgroup of the di TPS TPS –e/f clade with unusual βα–domain architecture. Mv ELS 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. Mv ELS fills a central position in the biosynthetic system that forms the foundation for the diverse repertoire of Marrubium diterpenoids. Co‐expression of Mv CPS 1 and Mv ELS in engineered E. coli and Nicotiana benthamiana offers opportunities for producing precursors for an array of biologically active diterpenoids.