Background: Sclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea) and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species. Results: Based on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS) enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS) produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP) with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S)-copalyl diphosphate. A class I diTPS (SsSS) then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae). Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants. Conclusions: SsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid specialized metabolite sclareol in a two-step process. They represent two of the first characterized hydroxylating diTPSs in angiosperms and generate the dihydroxylated labdane sclareol without requirement for additional enzymatic oxidation by activities such as cytochrome P450 monoxygenases. Yeast-based production of sclareol by co-expresssion of SsLPPS and SsSS was efficient enough to warrant the development and use of such technology for the biotechnological production of scareol and other oxygenated diterpenes.

Sclareol is a high-value natural product obtained by solid/liquid extraction of clary sage (Salvia sclarea L.) inflorescences. Because processes of excretion and accumulation of this labdane diterpene are unknown, the aim of this work was to gain knowledge on its sites of accumulation in planta. Samples were collected in natura or during different steps of the industrial process of extraction (steam distillation and solid/liquid extraction). Samples were then analysed with a combination of complementary analytical techniques (gas chromatography coupled to a mass spectrometer, polarized light microscopy, environmental scanning electron microscopy, two-photon fluorescence microscopy, second harmonic generation microscopy). According to the literature, it is hypothesized that sclareol is localized in oil pockets of secretory trichomes. This study demonstrates that this is not the case and that sclareol accumulates in a crystalline epicuticular form, mostly on calyces.