Abstract
Sandalwood oil is one of the worlds most highly prized fragrances. To identify the genes and encoded enzymes responsible for santalene biosynthesis, we cloned and characterized three orthologous terpene synthase (TPS) genes SaSSy, SauSSy, and SspiSSy from three divergent sandalwood species; Santalum album, S. austrocaledonicum, and S. spicatum, respectively. The encoded enzymes catalyze the formation of α-, β-, epi-β-santalene, and α-exo-bergamotene from (E,E)-farnesyl diphosphate (E,E-FPP). Recombinant SaSSy was additionally tested with (Z,Z)-farnesyl diphosphate (Z,Z-FPP) and remarkably, found to produce a mixture of α-endo-bergamotene, α-santalene, (Z)-β-farnesene, epi-β-santalene, and β-santalene. Additional cDNAs that encode bisabolene/bisabolol synthases were also cloned and functionally characterized from these three species. Both the santalene synthases and the bisabolene/bisabolol synthases reside in the TPS-b phylogenetic clade, which is more commonly associated with angiosperm monoterpene synthases. An orthologous set of TPS-a synthases responsible for formation of macrocyclic and bicyclic sesquiterpenes were characterized. Strict functionality and limited sequence divergence in the santalene and bisabolene synthases are in contrast to the TPS-a synthases, suggesting these compounds have played a significant role in the evolution of the Santalum genus.
Highlights
Forest Products Commission of Western Australia (FPC) as part of linkage project LP0882690
Primers based on the open reading frame (ORF) of previously identified S. album terpene synthase (TPS) genes were used for amplification [7]
Low Km values for each santalene synthase indicated a high affinity for (E,E)-FPP, while no active site saturation was apparent with geranyl diphosphate (GPP), indicating they are genuine sesquiterpene synthases
Summary
Chemicals and Reagents—All reagents, solvents, antibiotics, cloning kits, modifying enzymes, and precursor chemicals were purchased from commercial sources. (E,E)-FPP and geranyl diphosphate (GPP) were from Sigma. (Z,Z)-FPP was synthesized using standard methods [9, 10] and products verified by GC-MS, NMR, and IR spectroscopy. Primers based on the open reading frame (ORF) of previously identified S. album TPS genes were used for amplification [7] (supplemental Fig. S1). The same ORF primers used for successful cDNA amplifications were used on genomic DNA extracted from the same individuals from which RNA extractions were performed These larger gDNA fragments (3– 4 kb) were cloned into pJET1.2 vectors and sent for sequencing (Macrogen, Korea). Genomic DNA sequences of TPS genes from the three oil-bearing species were spliced insilico with introns following the usual 3Ј n/GT and 5Ј AG/n pattern [12]. Product losses due to extraction inefficiency was accounted for by first adding the standard to assay buffer as an EtOH stock and extracting into the hexane layer as per sample assays
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