Sesquiterpene Variation in West Australian Sandalwood (Santalum spicatum).

West Australian sandalwood (Santalum spicatum) has long been exploited for its fragrant, sesquiterpene-rich heartwood; however sandalwood fragrance qualities vary substantially, which is of interest to the sandalwood industry. We investigated metabolite profiles of trees from the arid northern and southeastern and semi-arid southwestern regions of West Australia for patterns in composition and co-occurrence of sesquiterpenes. Total sesquiterpene content was similar across the entire sample collection; however sesquiterpene composition was highly variable. Northern populations contained the highest levels of desirable fragrance compounds, α- and ß-santalol, as did individuals from the southwest. Southeastern populations were higher in E,E-farnesol, an undesired allergenic constituent, and low in santalols. These trees generally also contained higher levels of α-bisabolol. E,E-farnesol co-occurred with dendrolasin. Contrasting α-santalol and E,E-farnesol chemotypes revealed potential for future genetic tree improvement. Although chemical variation was evident both within and among regions, variation was generally lower within regions. Our results showed distinct patterns in chemical diversity of S. spicatum across its natural distribution, consistent with earlier investigations into sandalwood population genetics. These results are relevant for plantation tree improvement and conservation efforts.

The transcriptome of sesquiterpenoid biosynthesis in heartwood xylem of Western Australian sandalwood (Santalum spicatum).

The fragrant heartwood oil of West Australian sandalwood (Santalum spicatum) contains a mixture of sesquiterpene olefins and alcohols, including variable levels of the valuable sesquiterpene alcohols, α- and ß-santalol, and often high levels of E,E-farnesol. Transcriptome analysis revealed sequences for a nearly complete set of genes of the sesquiterpenoid biosynthetic pathway in this commercially valuable sandalwood species. Transcriptome sequences were produced from heartwood xylem tissue of a farnesol-rich individual tree. From the assembly of 12,537 contigs, seven different terpene synthases (TPSs), several cytochromes P450, and allylic phosphatases were identified, as well as transcripts of the mevalonic acid and methylerythritol phosphate pathways. Five of the S. spicatum TPS sequences were previously unknown. The full-length cDNA of SspiTPS4 was cloned and the enzyme functionally characterized as a multi-product sesquisabinene B synthase, which complements previous characterization of santalene and bisabolol synthases in S. spicatum. While SspiTPS4 and previously cloned sandalwood TPSs do not explain the prevalence of E,E-farnesol in S. spicatum, the genes identified in this and previous work can form a basis for future studies on natural variation of sandalwood terpenoid oil profiles.

Biosynthesis of Sandalwood Oil: Santalum album CYP76F cytochromes P450 produce santalols and bergamotol.

Sandalwood oil is one of the world's most highly prized essential oils, appearing in many high-end perfumes and fragrances. Extracted from the mature heartwood of several Santalum species, sandalwood oil is comprised mainly of sesquiterpene olefins and alcohols. Four sesquiterpenols, α-, ß-, and epi-ß-santalol and α-exo-bergamotol, make up approximately 90% of the oil of Santalum album. These compounds are the hydroxylated analogues of α-, ß-, and epi-ß-santalene and α-exo-bergamotene. By mining a transcriptome database of S. album for candidate cytochrome P450 genes, we cloned and characterized cDNAs encoding a small family of ten cytochrome P450-dependent monooxygenases annotated as SaCYP76F37v1, SaCYP76F37v2, SaCYP76F38v1, SaCYP76F38v2, SaCYP76F39v1, SaCYP76F39v2, SaCYP76F40, SaCYP76F41, SaCYP76F42, and SaCYP76F43. Nine of these genes were functionally characterized using in vitro assays and yeast in vivo assays to encode santalene/bergamotene oxidases and bergamotene oxidases. These results provide a foundation for production of sandalwood oil for the fragrance industry by means of metabolic engineering, as demonstrated with proof-of-concept formation of santalols and bergamotol in engineered yeast cells, simultaneously addressing conservation challenges by reducing pressure on supply of sandalwood from native forests.

Sandalwood fragrance biosynthesis involves sesquiterpene synthases of both the terpene synthase (TPS)-a and TPS-b subfamilies, including santalene synthases.

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-, ß-, epi-ß-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)-ß-farnesene, epi-ß-santalene, and ß-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.