Elizabeth L. Barbour

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-, 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.

Isolation of cDNAs and functional characterisation of two multi-product terpene synthase enzymes from sandalwood, Santalum album L

Sandalwood, Santalum album (Santalaceae) is a small hemi-parasitic tropical tree of great economic value. Sandalwood timber contains resins and essential oils, particularly the santalols, santalenes and dozens of other minor sesquiterpenoids. These sesquiterpenoids provide the unique sandalwood fragrance. The research described in this paper set out to identify genes involved in essential oil biosynthesis, particularly terpene synthases (TPS) in S. album, with the long-term aim of better understanding heartwood oil production. Degenerate TPS primers amplified two genomic TPS fragments from S. album, one of which enabled the isolation of two TPS cDNAs, SamonoTPS1 (1731bp) and SasesquiTPS1 (1680bp). Both translated protein sequences shared highest similarity with known TPS from grapevine (Vitis vinifera). Heterologous expression in Escherichia coli produced catalytically active proteins. SamonoTPS1 was identified as a monoterpene synthase which produced a mixture of (+)-alpha-terpineol and (-)-limonene, along with small quantities of linalool, myrcene, (-)-alpha-pinene, (+)-sabinene and geraniol when assayed with geranyl diphosphate. Sesquiterpene synthase SasesquiTPS1 produced the monocyclic sesquiterpene alcohol germacrene D-4-ol and helminthogermacrene, when incubated with farnesyl diphosphate. Also present were alpha-bulnesene, gamma-muurolene, alpha- and beta-selinenes, as well as several other minor bicyclic compounds. Although these sesquiterpenes are present in only minute quantities in the distilled sandalwood oil, the genes and their encoded enzymes described here represent the first TPS isolated and characterised from a member of the Santalaceae plant family and they may enable the future discovery of additional TPS genes in sandalwood.

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

Abstract 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-, β-, and epi-β-santalol and α-exo-bergamotol, make up approximately 90% of the oil of Santalum album. These compounds are the hydroxylated analogues of α-, β-, and epi-β-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.

Heartwood-specific transcriptome and metabolite signatures of tropical sandalwood (Santalum album) reveal the final step of (Z)-santalol fragrance biosynthesis

Tropical sandalwood (Santalum album) produces one of the worlds most highly prized fragrances, which is extracted from mature heartwood. However, in some places such as southern India, natural populations of this slow-growing tree are threatened by over-exploitation. Sandalwood oil contains four major and fragrance-defining sesquiterpenols: (Z)-α-santalol, (Z)-β-santalol, (Z)-epi-β-santalol and (Z)-α-exo-bergamotol. The first committed step in their biosynthesis is catalyzed by a multi-product santalene/bergamotene synthase. Sandalwood cytochromes P450 of the CYP76F sub-family were recently shown to hydroxylate santalenes and bergamotene; however, these enzymes produced mostly (E)-santalols and (E)-α-exo-bergamotol. We hypothesized that different santalene/bergamotene hydroxylases evolved in S. album to stereo-selectively produce (E)- or (Z)-sesquiterpenols, and that genes encoding (Z)-specific P450s contribute to sandalwood oil formation if co-expressed in the heartwood with upstream genes of sesquiterpene biosynthesis. This hypothesis was validated by the discovery of a heartwood-specific transcriptome signature for sesquiterpenoid biosynthesis, including highly expressed SaCYP736A167 transcripts. We characterized SaCYP736A167 as a multi-substrate P450, which stereo-selectively produces (Z)-α-santalol, (Z)-β-santalol, (Z)-epi-β-santalol and (Z)-α-exo-bergamotol, matching authentic sandalwood oil. This work completes the discovery of the biosynthetic enzymes of key components of sandalwood fragrance, and highlights the evolutionary diversification of stereo-selective P450s in sesquiterpenoid biosynthesis. Bioengineering of microbial systems using SaCYP736A167, combined with santalene/bergamotene synthase, has potential for development of alternative industrial production systems for sandalwood oil fragrances.

Non-destructive sampling of Indian sandalwood (Santalum album L.) for oil content and composition

Abstract East Indian sandalwood, Santalum album, is renowned for its fragrant heartwood. Current methods of heartwood oil assessment of standing trees remain largely untested and unreliable. Core sampling of 22 S. album trees at fixed heights followed by solvent extraction of core samples and subsequent analysis was correlated to distilled yields of wood from the same trees. Oil contents of 10-year-old sandalwood trees generally decreased exponentially up the height of the tree, with considerable variation amongst the population. From these trends, core sampling was used to estimate oil yields but large discrepancies existed, and only relative differences could be described. Gas chromatography yielded the most informative results for each core, particularly that the oil composition varied little at 30 cm or at 100 cm, however, there was a slightly higher proportion of sesquiterpene hydrocarbons in samples taken from 100 cm than those from 30 cm. The proportion of sesquiterpene alcohols was generally higher in 30 cm wood cores than in 100 cm samples.

Genetic Diversity of an Australian Santalum album Collection - Implications for Tree Improvement Potential

Abstract The Forest Products Commission of Western Australia manages a sandalwood (Santalum spp.) core germplasm collection at Kununurra in the states far north. This collection serves as a significant seed source for sandalwood plantations in the area and remains an important resource for ongoing research. The collection contains S. album trees sourced from Indian arboreta, along with a few trees from West Timor, Indonesia. Also present are representatives of S. macgregorii from Papua New Guinea and S. austrocaledonicum from Vanuatu and/or New Caledonia. Despite the apparently diverse seed origins, the genetic background of many of the accessions remains vague. In this study, diversity and relatedness was assessed by nuclear and chloroplast RFLPs and a phylogeny was inferred. Nuclear RFLPs revealed very low levels of genetic diversity for a tree species, with an observed and expected heterozygosity (Ho and He) of 0.047. Nineteen genotypes were identified within the 233 S. album individuals sampled, with only one tree known to have originated from Timor being differentiated from Indian material. Other trees thought to have come from Timor grouped with those believed to be from India, indicating they were either incorrectly labelled or sourced from heavily modified populations. Despite the poor sample size, chloroplast RFLP analysis revealed no genetic distinction between the Timorese and Indian S. album, which supports the theory of human mediated seed dispersal from Timor to India. The structure of the phylogeny and associated relatedness has assisted in the establishment of seed orchards, designed to ensure maximum diversity is maintained through limiting the proximity of highly related trees. Finally, in light of these and other findings, a hypothesis concerning the evolution of S. album is proposed.

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 β-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.

Effect of genetic relatedness among parents on gain in salt tolerance in progeny of crosses of Eucalyptus occidentalis

Abstract Genetic diversity of a Eucalyptus occidentalis breeding population screened for salt and waterlogging tolerance was examined using eight microsatellite loci. Mating using an immature style ‘one stop pollination’ method between parents was carried out to produce progeny for testing under 500 mM salt waterlogging. The effect of parental genetic distance on seed production and early seedling survival was examined and inheritance of salt/or waterlogging tolerance was assessed by testing performance of progeny in comparison to parents. Diversity was moderate among the nine provenances and the families, and most of the diversity was distributed within rather than between families. Genetic relationships showed no structure in relation to provenance indicating any adaptation to saline environments has not affected genetic similarity. Breeding for salt and water tolerance may be achieved without decline in genetic diversity. There was a significant correlation between capsule production and parental genetic distance and a positive trend between increasing parental genetic distance and increasing number of germinated seeds/capsule, and seedling survival at 2 weeks and 9 months. These trends indicate expression of inbreeding depression in crosses between genetically similar parents. Heritability values, under 500 mM salt-waterlogging treatment, indicated that height had moderate heritability (h2 = 0.5). Crosses with the widest parental genetic distance produced progeny with considerable height increase above parents and this trend was evident even with moderate genetic distance indicating crossing at this level of differentiation may achieve optimum breeding gain.