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. 2012 Jul 26:12:119.
doi: 10.1186/1471-2229-12-119.

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

Anne Caniard  1 , Philipp Zerbe, Sylvain Legrand, Allison Cohade, Nadine Valot, Jean-Louis Magnard, Jörg Bohlmann, Laurent Legendre
Affiliations

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

Anne Caniard et al. BMC Plant Biol. .

Abstract

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.

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Figures

Figure 1
Figure 1
Proposed biosynthetic pathway of sclareol and related diterpenes in Salvia sclarea. The suggested biosynthetic pathway of sclareol 4 as the predominant diterpene in S. sclarea and other minor constituents detected in planta, such as manool 6 as well as manoyl oxide 7 and 13-epi-manoyl oxide 8, requires the activity of at least two monofunctional diTPSs. A class II enzyme catalyzes the protonation-initiated cyclization of (E,E,E)-geranylgeranyl diphosphate (GGPP) 1 to form labda-13-en-8- ol diphosphate (LPP)3 or (9 S,10 S)-copalyl diphosphate (CPP) 5 (i.e., CPP of normal or (+)-stereochemistry) via a labda-13-en-8-yl diphosphate 2 carbocation. Catalyzed by class I diTPS activity, ionization of the diphosphate ester of LPP 3 or CPP 5 results in the formation of sclareol 4 and manool 6, respectively. In addition, manoyl oxide 7 and 13-epi-manoyl oxide 8 may occur as a product of this biosynthetic pathway.
Figure 2
Figure 2
Figure 2 – Phylogenetic analysis of Salvia sclarea diterpene synthases. The phylogenetic tree was generated based on multiple amino acid sequence alignments (DIALIGN-TX), phylogenetic analysis (PhyML, four rate substitution categories, LG substitution model, BIONJ starting tree, 100 bootstrap repetitions, rooted with the outgroup copalyl diphosphate synthase/kaurene synthase from the moss Physcomitrella patens PpCPS/KS [BAF61135]), and visualization in treeview. Asterisks indicate nodes supported by > 90% bootstrap values. Protein abbreviations [NCBI GenBank accession no. ]: JsCPS/KS, Jungermannia subulata ent-copalyl diphosphate/ent-kaurene synthase [BAJ39816]; SmCPS/KS1, Selaginella moellendorfii labda-7,13E-dien-15-olsynthase [AEK75338]; TbTS, Taxus brevifolia taxadiene synthase [AAC49310]; GbLS, Ginkgo biloba levopimaradiene synthase [AAL09965]; AbCAS, Abies balsamea cis-abienol synthase [JN254808]; AgAS, Abies grandis abietadiene synthase [AAK83563]; PaLAS, Picea abies levopimaradiene/abietadiene synthase [AAS47691]; PaIso, Picea abies isopimaradiene synthase [AAS47690]; CmKS, Cucurbita maxima kaurene synthase [AAB39482]; AtKS, Arabidopsis thaliana ent-kaurene synthase [AF034774]; PtKS, Populus trichocarpa kaurene synthase [XM_002311250]; RcKS, Ricinus communis kaurene synthase-like [XP_002533694]; HaKS, Helianthus annuus kaurene synthase [CBL42917]; SdKS, Scoparia dulcis kaurene synthase-like [AEF33360]; OsKS1, Oryza sativa ent-kaurene synthase [AY347876]; OsKSL4, O. sativa syn-pimaradiene synthase [AY616862]; OsKSL8, O. sativa syn-stemarene synthase [AB118056]; OsKSL10, O. sativa ent-pimaradiene synthase [DQ823355]; RcKSL1, R. communis kaurene synthase-like [XM_002525795]; RcKSL2, R. communis kaurene synthase-like [XM_002525790]; RcKSL3, R. communis kaurene synthase-like [XM_002525796]; SmKSL, Salvia miltiorhizza kaurene synthase-like [EF635966]; NtKSL, N. tabacum kaurene synthase-like [CCD33019]; SlPHE, Solanum lycopersicum phellandrene synthase [FJ797957]; ShSBS, Solanum habrochaites bergamotene/santalene synthase [FJ194970]; CcCLS, Cistus creticus copal-8-ol synthase [DJ93862]; NtCPSL, Nicotiana tabacum 8-hydroxy copalyl diphosphate synthase [CCD33018]; SmCPSL, S. miltiorhizza copalyl diphosphate synthase-like [EU003997]; HaCPS, H. annuus copalyl diphosphate synthase [CBL42915]; AtCPSL, A. thaliana ent-copalyl diphosphate synthase [AAA53632]; CmCPS, C. maxima copalyl diphosphate synthase [AF049905]; SdCPSL, S. dulcis copalyl diphosphate synthase-like [AB046689]; SlCPSL, S.lycopersicum copalyl diphosphate synthase-like [AB015075]; ZmCPSL, Zea mays ent-copalyl diphosphate synthase [AY562491]; OsCPS1, O. sativa ent-copalyl diphosphate synthase [Q6ET36]; OsCPS2, O. sativa ent-copalyl diphosphate synthase [Q6Z5I0]; OsCPS4, O. sativa syn-copalyl diphosphate synthase [Q6E7D7].
Figure 3
Figure 3
GC-MS analysis of SsLPPS reaction products. Shown are total ion chromatograms (TIC) of [A] reaction products obtained from in vitro assays with Ni2+-affinity-purified SsLPPS using GGPP 1 as a substrate and subsequent dephosphorylation, [B] in vitro assay products without dephosphorylation, [C] dephosphorylated GGPP, and [D] authentic sclareol standard (Sigma). GC-MS analysis was performed on an Agilent HP5ms column with electronic impact ionization at 70 eV. Results were confirmed with three independent experiments. Identification of reaction products was achieved by comparison to authentic standards or reference mass spectra from the National Institute of Standards and Technology MS library searches (Wiley W9N08L): peak a, epi-manoyl oxide 8; peak b, manoyl oxide 7; peak c, putative 13(16),-14-labdien-8-ol; peak d, putative copalol; peak e, sclareol 4; peak f, unknown compound; peak g, labda-13-en-8,15-diol; peak h, geranylgeraniol.
Figure 4
Figure 4
LC-MS analysis of SsLPPS reaction products. Normal phase LC-APCI-MS analysis in negative mode was used to directly detect the SsLPPS reaction product from GGPP as substrate without dephosphorylation. Results are depicted as extracted ion chromatogram (EIC) of the parent mass m/z 467, which is consistent with labda-13-en-8-ol diphosphate (LPP) 3.
Figure 5
Figure 5
GC-MS analysis of SsSS and SsdiTPS3 reaction products. Shown are total ion chromatograms (TIC) of reaction products from coupled enzyme assays with equal amounts of SsLPPS and either SsSS or SsdiTPS3 using GGPP 1 as a substrate. [A] Direct reaction products from coupled assays with SsLPPS and SsSS, [B] direct reaction products from coupled assays with SsLPPS and SsdiTPS3, [C] dephosphorylated reaction products of SsLPPS alone, [D] authentic manool standard (GlycoSyn, Gracefield, NZ), and [E] authentic sclareol standard (Sigma). GC-MS analysis was performed on an Agilent HP5ms column with electronic impact ionization at 70 eV. Results were confirmed with three independent experiments. Identification of reaction products was achieved by comparison to authentic standards or reference mass spectra from the National Institute of Standards and Technology MS library searches (Wiley W9N08L): peak a, epi-manoyl oxide 8; peak b, manoyl oxide 7; peak c, putative 13(16),-14-labdien-8-ol; peak d, putative copalol; peak e, sclareol 4; peak f, unknown compound; peak g, labda-13-en-8,15-diol; peak i, manool 6.
Figure 6
Figure 6
Production of sclareol in engineered yeast cells. Shown are GC-MS total ion chromatograms (TIC) of extracts from engineered yeast strains expressing [A] yeast GGPP synthase (ScGGPPS), [B] ScGGPPS and SsLPPS, [C-D] ScGGPPS, SsLPPS and SsSS, and [E-F] ScGGPPS, SsLPPS and SsSdiTPS3.Sclareol was detected at similar levels in both the media [D &F] and harvested yeast cells [C &E] of each yeast culture, with a lower amount of impurities being observed in the media. GC-MS analysis was performed on an Agilent HP5ms column with electronic impact ionization at 70 eV. Identification of reaction products was achieved by comparison to authentic standards or reference mass spectra from the National Institute of Standards and Technology MS library searches (Wiley W9N08L): peak a, epi-manoyl oxide 8; peak b, manoyl oxide 7; peak c, putative 13(16),-14-labdien-8-ol; peak d, putative copalol; peak e, sclareol 4; peak f, unknown compound; peak g, labda-13-en-8,15-diol; peak i, manool 6.
Figure 7
Figure 7
Subcellular localization of SsLPPS and SsSS. The first 102 bp of SsLPPS (SsLPPS(SP)-GFP) and the first 99 bp of SsSS (SsSS(SP)-GFP) were fused to a downstream GFP and transiently expressed in tobacco leaves. GFP fluorescence (green) was detected at an emission wavelength of 485–520 nm as compared to chlorophyll autofluorescence (red) at an emission wavelength of 600–700 nm. The column labeled “Merged signals” provides a view of all fluorescent signals obtained for this sample.
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