Isolated from the plant Salvia sclarea (clary sage). Originally thought to be synthesized in one step from geranylgeranyl diphosphate it is now known to require two enzymes, EC 4.2.1.133, copal-8-ol diphosphate synthase and EC 4.2.3.141, sclareol synthase. Sclareol is used in perfumery.
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The expected taxonomic range for this enzyme is: Salvia sclarea
Isolated from the plant Salvia sclarea (clary sage). Originally thought to be synthesized in one step from geranylgeranyl diphosphate it is now known to require two enzymes, EC 4.2.1.133, copal-8-ol diphosphate synthase and EC 4.2.3.141, sclareol synthase. Sclareol is used in perfumery.
in coupled enzyme assays with labda-13-en-8-ol diphosphate synthase, sclareol is the major product with minor amounts of manool, manoyl oxides and epi-manoyl oxide as secondary products
in coupled enzyme assays with labda-13-en-8-ol diphosphate synthase, sclareol is the major product with minor amounts of manool, manoyl oxides and epi-manoyl oxide as secondary products
in angiosperms, class II diTPSs that enable water quenching of the labda-13-en-8-yl carbocation are found in six different plant families, phylogenetic tree, overview
mutants N431D and N431Q selectively produce (13S)-sclareol, with SsSS:N431Q exhibiting almost complete reversal of the stereoselective addition of water, i.e., while wild-type SsSS produces (13R)-sclareol in 80% enantiomeric excess (ee), SsSS:N431Q produces 70% ee of (13S)-sclareol. The single residue change N431Q essentially flips the stereochemical outcome, changing the addition of water to the si face of the double bond in (13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate providing biosynthetic access to (13S)-sclareol
the enzyme substrate is derived from bicyclization of the general diterpene precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) by class II diterpene cyclase copal-8-ol diphosphate hydratase, EC 4.2.1.133
enzymes labda-13-en-8-ol diphosphate synthase and sclareol synthase are two monofunctional diterpene synthases which, together, produce the diterpenoid specialized metabolite sclareol in a two-step process. Labda-13-en-8-ol diphosphate synthase produces labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S)-copalyl diphosphate. Sclareol synthase then transformes these intermediates into sclareol and manool, respectively
in nature, diterpene synthase (diTPS) enzymes are essential for generating diverse diterpene hydrocarbon scaffolds. Major commercial diterpenoids include sclareol from clary sage (Salvia sclarea) used as a precursor for ambroxide fragrance and fixatives in perfume manufacture. In angiosperms, class II diTPSs that enable water quenching of the labda-13-en-8-yl carbocation are found in six different plant families, these class II diTPSs can pair with class I enzymes, converting oxygenated prenyl diphosphates through ionization with or without secondary cyclization or additional hydroxylation to afford diterpene alcohols, including manoyl oxide, sclareol and cis-abienol, that are of interest in the fragrance industry
site-directed mutagenesis, the mutant exclusively produces isoabienol, resullting from direct deprotonation of the initially generated 13-yl+ intermediate at the neighboring methyl group. There is a below 10% decrease in yield for this mutant relative to wild-type SsSS in the engineered bacterial culture
site-directed mutagenesis, the mutant selectively produces epimeric (13S)-sclareol, with SsSS:N431Q exhibiting almost complete reversal of the stereoselective addition of water, i.e., while wild-type SsSS enzyme produces (13R)-sclareol in 80% enantiomeric excess (ee), SsSS:N431Q produced 70% ee of (13S)-sclareol
coexpression of gene SsLPPS encoding labd-13-en-8-ol diphosphate synthase (EC 4.2.1.133) and gene SsSS encoding sclareol synthase, recombinant enzyme expression in Physcomitrella patens from vectors pUNI33-SsLPPS-2A-SsSS and pBK3-SsLPPS-2A-SsSS. The transcript levels of SsSS and SsLPPS cannot be directly correlated to the final yields for sclareol of the different transgenic lines, but it is observed that lines with high transcript levels of the genes also have high sclareol levels. The highest yield is 2.84 lg/mg dry weight (2.28 lg/ml) in a liquid culture of wild-type line 2 (WTF2), including sclareol found in the media and in the moss cells. Enzyme expression analysis, overview. Transcriptional changes in the 14-day-old WTF2 shows a similar down-regulation for most genes, but not for HDR, IDI, and HMGS. The HDR, IDI, and HMGS show a higher expression level than in those in the wild-type. Besides sclareol, the three endogenous ent-kaurenoids (ent-kaur-15-ene, ent-kaur-16-ene and ent-16-hydroxy- kaurene) are also observed in the WTF2, but the peaks are significantly lower than in the wild-type line. GC-MS ligand analysis. Of all transgenic lines, the WTF2 line has the most growth impairment and stops growing after 15 days. general, general growth deficiency for transformed lines
usage of fed-batch fermentation along with an organic solvent overlay for in situ product removal to develop an industrial-scale platform for sclareol production
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant N-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli strain C41 OverExpress by nickel affinity chromatography and dialysis
as biotechnological production systems for sesquiterpenoids, a number of diterpenoid engineering platforms are described that use plant diTPSs (and in some cases P450s) to produce, for example, the fragrance precursors sclareol and cis-abienol in the prefume industry. Sclareol from clary sage (Salvia sclarea) is used as a precursor for ambroxide fragrance and fixatives in perfume manufacture
Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture