Christopher G. Jones

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.

Hydrolysis of plant polysaccharides and GLC analysis of their constituent neutral sugars

Abstract The release and degradation of sugars from onion cell walls and potato cell wall polysaccharides were followed during hydrolysis with trifluoroacetic acid so that the optimum hydrolysis conditions could be determined. After 6 hr hydrolysis in 2 M acid at 100°, calculated recovery factors of different monosaccharides from potato pectic fractions ranged from 61 to 96%. Lower yields of monosaccharides were obtained from intact onion cell walls, while the yield from cellulose was less than 0.2%. A new GLC column for the separation of alditol acetates derived from cell wall sugars is described.

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.

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.

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 β-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.

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.

Analytical Approaches to Improve Accuracy in Solving the Protein Topology Problem

To take advantage of recent advances in genomics and proteomics it is critical that the three-dimensional physical structure of biological macromolecules be determined. Cryo-Electron Microscopy (cryo-EM) is a promising and improving method for obtaining this data, however resolution is often not sufficient to directly determine the atomic scale structure. Despite this, information for secondary structure locations is detectable. De novo modeling is a computational approach to modeling these macromolecular structures based on cryo-EM derived data. During de novo modeling a mapping between detected secondary structures and the underlying amino acid sequence must be identified. DP-TOSS (Dynamic Programming for determining the Topology Of Secondary Structures) is one tool that attempts to automate the creation of this mapping. By treating the correspondence between the detected structures and the structures predicted from sequence data as a constraint graph problem DP-TOSS achieved good accuracy in its original iteration. In this paper, we propose modifications to the scoring methodology of DP-TOSS to improve its accuracy. Three scoring schemes were applied to DP-TOSS and tested: (i) a skeleton-based scoring function; (ii) a geometry-based analytical function; and (iii) a multi-well potential energy-based function. A test of 25 proteins shows that a combination of these schemes can improve the performance of DP-TOSS to solve the topology determination problem for macromolecule proteins.

Genetic and environmental parameters show associations with essential oil composition in West Australian sandalwood (Santalum spicatum)

Santalum spicatum (R.Br.) A.DC is a West Australian sandalwood species highly valued for the sesquiterpene-rich oil in mature heartwood. The oil composition, particularly levels of the valuable sesquiterpenoids α- and β-santalol and the allergenic E,E-farnesol, are known to vary across its natural distribution. Our study investigated associations of oil characteristics in 186 S. spicatum trees in semiarid and arid regions of Western Australia with genetic structure, environmental parameters and morphological features. We found associations between oil composition and genetic structure, as well as between oil composition and environmental factors. Analysis of individuals using STRUCTURE revealed two major genetic clusters (K = 2), comprising trees from the arid north clustered together, and the semiarid south-west clustered separately. Mantel tests revealed a significant association between oil characteristics and genetic distance (r = 0.129, P = 0.02). There was considerable variation in the growing environment of S. spicatum. An Adonis test showed a significant association between oil composition and provenance (P = 0.001) and between oil composition and soil type (P = 0.002) but not oil composition and other environmental characters. Soil type was significantly related to santalol and E,E-farnesol content. No significant associations between oil composition and morphological features were identified.