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

Abstract

BackgroundSclareol 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.ResultsBased 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.ConclusionsSsLPPS 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.

Manoyl Oxide (13R), the Biosynthetic Precursor of Forskolin, Is Synthesized in Specialized Root Cork Cells in Coleus forskohlii

“…A separate clade contains members involved in specialized metabolism, including tobacco NtCAS, converting copal-8-ol diphosphate to the specialized metabolite cis-abienol (Sallaud et al, 2012). The enzymes from S. sclarea and S. miltiorrhiza, producing the labdanespecialized metabolites sclareol (SsSCS) and the abietane miltiradiene, respectively (Gao et al, 2009;Caniard et al, 2012), are members of a subgroup consisting of Lamiaceae enzymes. A second pair of C. forskohlii diTPS, CfTPS3 and CfTPS4, is clustered with this clade.…”

“…Also in this clade are paralogous gene pairs from pumpkin (Cucurbita maxima; CmCPS1 and CmCPS2) and Isodon eriocalyx (IeCPS1 and IeCPS2), which resulted from gene duplications potentially allowing for neofunctionalization of diTPS in the biosynthesis of specialized metabolites (Li et al, 2012a). In the second and apparently more divergent clade, enzymes of rock rose (Cistus cresticus) copal-8-ol diphosphate synthase [CcCLS], tobacco (Nicotiana tabacum; labda-13-en-8-ol diphosphate synthase [LPPS]; NtLPPS), and two closely related Lamiaceae (clary sage [Salvia sclarea; SsLPPS] and Salvia miltiorrhiza, SmCPS1) are involved in specialized metabolism and have been shown to produce copal-8-ol diphosphate and (+)-copalyl diphosphate, respectively (Gao et al, 2009;Caniard et al, 2012). In C. forskohlii, we identified CfTPS15, a single-copy class II diTPSs grouping with the bona fide CPS (general metabolism), and CfTPS1 and CfTPS2, a pair of diTPSs grouping with the class II enzymes involved in specialized metabolism.…”

“…As with the class II diTPSs, deprotonation or water capture terminate the class I diTPS reaction. Water capture as a mechanism of diTPSs that leads to oxygen functionalities in the diterpene products has been described for the class II active site of bifunctional class I/II gymnosperm diTPSs (Keeling et al, 2011;Zerbe et al, 2012Zerbe et al, , 2013, class II angiosperm diTPSs (Falara et al, 2010;Caniard et al, 2012;Zerbe et al, 2013), and class I angiosperm diTPS (Caniard et al, 2012). Further oxidative functionalization of diterpenoids is typically catalyzed by cytochrome P450-dependent monooxygenase enzymes (P450s; Ro et al, 2005;Swaminathan et al, 2009;Hamberger et al, 2011;Wang et al, 2011;Guo et al, 2013;Hamberger and Bak, 2013), which then provide molecular handles for addition of auxiliary functional groups, leading ultimately to highly complex and decorated products such as forskolin and its derivatives.…”

Expanding the Landscape of Diterpene Structural Diversity through Stereochemically Controlled Combinatorial Biosynthesis

“…The formation of structurally distinct bicyclic diterpenes, such as sclareol, which is used in high‐end fragrance manufacture, is catalyzed in S. sclarea by SsLPPS and SsSCS 9. Antibiotic activities have been reported for a compound of this class, (+)‐manool,10 which is also recognized as a starting material for the semisynthesis of a wide range of diterpenoids with pharmaceutical properties 11.…”

“…Antibiotic activities have been reported for a compound of this class, (+)‐manool,10 which is also recognized as a starting material for the semisynthesis of a wide range of diterpenoids with pharmaceutical properties 11. SsSCS was used as a representative of class I diTPSs that, following rearrangement, facilitate neutralization of the carbocation through water quenching at C13 to give regiospecific introduction of a hydroxy group 9. When offered the three known stereoisomers of copalyl diphosphate (CPP), SsSCS formed products with the stereochemistry preserved at the chiral centers C9 and C10, as defined by the class II diTPSs.…”