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Accueil > Productions scientifiques > Autres ... > Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture

Farfour, E. ; Leto, J. ; Barritault, M. ; Barberis, C. ; Meyer, J. ; Dauphin, B. ; Guern, A.-S. Le ; Leflèche, A. ; Badell, E. ; Guiso, N. ; Leclercq, A. ; Monnier, A. Le ; Lecuit, M. ; Rodriguez-Nava, V. ; Bergeron, E. ; Raymond, J. ; Vimont, S. ; Bille, E. ; Carbonnelle, E. ; Guet-Revillet, H. ; Lécuyer, H. ; Beretti, J.-L. ; Vay, C. ; Berche, P. ; Ferroni, A. ; Nassif, X. ; Join-Lambert, O.

Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture

Journal of Clinical Microbiology

Sclareol is a diterpene natural product of high value for the fragrance industry. Its labdanecarbon skeleton and its two hydroxyl groups also make it a valued starting material forsemisynthesis of numerous commercial substances, including production of Ambrox® andrelated ambergris substitutes used in the formulation of high end perfumes. Most of thecommercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea) andextraction of the plant material. In clary sage, sclareol mainly accumulates in essential oilproducingtrichomes that densely cover flower calices. Manool also is a minor diterpene ofthis species and the main diterpene of related Salvia species. RESULTS : Based on previous general knowledge of diterpene biosynthesis in angiosperms, and based onmining of our recently published transcriptome database obtained by deep 454-sequencing ofcDNA from clary sage calices, we cloned and functionally characterized two new diterpenesynthase (diTPS) enzymes for the complete biosynthesis of sclareol in clary sage. A class IIdiTPS (SsLPPS) produced labda-13-en-8-ol diphosphate as major product fromgeranylgeranyl diphosphate (GGPP) with some minor quantities of its non-hydroxylatedanalogue, (9 S, 10 S)-copalyl diphosphate. A class I diTPS (SsSS) then transformed theseintermediates into sclareol and manool, respectively. The production of sclareol wasreconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPPstarting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomycescerevisiae). Tobacco-based transient expression assays of green fluorescent protein-fusionconstructs revealed that both enzymes possess an N-terminal signal sequence that activelytargets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production inplants. CONCLUSIONS : SsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoidspecialized metabolite sclareol in a two-step process. They represent two of the firstcharacterized hydroxylating diTPSs in angiosperms and generate the dihydroxylated labdanesclareol without requirement for additional enzymatic oxidation by activities such ascytochrome P450 monoxygenases. Yeast-based production of sclareol by co-expresssion ofSsLPPS and SsSS was efficient enough to warrant the development and use of suchtechnology for the biotechnological production of scareol and other oxygenated diterpenes.

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