Introduction of Sandalwood
Sandalwood is an evergreen tree native to India and Indonesia and grows to 8 to 12 m in height and 2.5 m in girth. The bark is smooth and gray-brown in color, and the small flowers have numerous short stalks. Read more Sandalwood Oil
1. Sandalwood oil is it used for
Sandalwood oil has a warm, woody odor and is commonly used as a fragrance in incense, cosmetics, perfumes, and soaps. It also is used as a flavor for foods and beverages. The wood has been valued in carving because of its dense character.
In traditional medicine, sandalwood oil has been used as an antiseptic and astringent, and for the treatment of headache, stomachache, and urinary and genital disorders.
In India, the essential oil, emulsion, or paste of sandalwood is used in the treatment of inflammatory and eruptive skin diseases. The oil has been used in the traditional Ayurvedic medicinal system as a diuretic and mild stimulant, and for smoothing the skin.
The leaves and bark were used by early Hawaiians to treat dandruff, lice, skin inflammation, and sexually transmitted diseases. Sandalwood oil has also demonstrated repellency against the crop pest Tetranychus urticae (two-spotted spider mite).
2. Biosynthesis of Sandalwood Oil: Santalum album CYP76F Cytochromes P450 Produce Santalols and Bergamotol
Maria L Diaz-Chavez 1, Jessie Moniodis 2,3, Lufiani L Madilao 1, Sharon Jancsik 1, Christopher I Keeling 1, Elizabeth L Barbour 2, Emilio L Ghisalberti 3, Julie A Plummer 2, Christopher G Jones 2, Jörg Bohlmann 1,*
1. 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.
2. Introduction
Sandalwood is the general name for woody perennials of the Santalum genus (Santalaceae), which are exploited for their fragrant heartwood. Sandalwoods are slow growing hemiparasitic trees distributed throughout the tropical and temperate regions of India, Indonesia, Australia and the Pacific Islands [1],[2].
The oil extracted from the stems and roots are highly sought after by the fragrance and perfume industry. Santalum album, also known as tropical or Indian sandalwood, is the most valuable of the commercially used species due to the high heartwood oil content (6–10% by dry weight) and desirable odor characteristics. Approximately 90% of S. album essential oil is composed of the sesquiterpene alcohols α-, β-, and epi-β-santalol and α-exo-bergamotol (Figure 1).
The α- and β-santalols are the most important contributors to sandalwood oil fragrance [3]–[5]. Lanceol and α-bisabolol are also found in modest concentrations [6].
While the demand for sandalwood oil is increasing, disease, grazing animals and unsustainable exploitation of sandalwood trees has led to the demise of many natural populations. Plantations provide a more sustainable alternative to wild harvesting; however, slow growth rates, high potential for disease and substantial variation in oil yield hamper productivity.
Alternatively, chemical approaches to synthesize the santalols have been attempted [7]–[9], but multiple low-recovery steps make chemical synthesis uneconomical at an industrial scale.
3. Results
1. Gene Discovery and Full-Length (FL)cDNA Cloning
A S. album trancriptome assembly of 31,461 isotigs was blastx searched for candidate CPRs and P450s potentially involved in the hydroxylation of santalenes and bergamotene. Two SaCPRs were identified using Arabidopsis thaliana CPRs (CAB58575.1, CAB58576.1) as search sequences. FLcDNAs SaCPR1 and SaCPR2 were 70% identical and 82% similar at the amino acid level. Searches for P450s were performed with a set of known plant P450s of the CYP71, CYP72 and CYP76 families, which include P450s with known functions in terpenoid biosynthesis [11]–[13].
2. Effect of CPR1 and CPR2
To test if substituting SaCPR1 and SaCPR2, which are 70% identical at the protein level, could affect changes in product profiles, we tested both CPRs in yeast in vivo experiments with representative class I and class II SaCYP76F, CYP76F39v1 and CYP76F38v1. No differences were observed in the products and their relative abundances.
3. Discussion
Using transcriptome analysis, cloning and functional characterization of recombinant P450s, we identified a new CYP76F subfamily in S. album involved in the biosynthesis of α-, β- and epi-β-santalols and bergamotols. The different SaCYP76Fs catalyze hydroxylations of santalenes and/or bergamotene products of SaSSy at the terminal allylic methyl groups. Clade I SaCYP76F enzymes produced both (Z) and (E) stereoisomers of α-, β- and epi-β-santalols and bergamotols.
The P450 product ratios of (Z) and (E) stereoisomers of α- and β-santalol were approximately 1∶5 and 1∶4, respectively, while the oil harvested from the mature heartwood of S. album trees contained mainly the (Z) alcohols [17],[18]. There are several possible explanations for the difference in the ratio of stereoisomers found in the enzyme product profile and in the oil extracted from trees. Importantly, we excluded the possibility that the activity of SaCYP76Fs was non-specific towards a range of different substrates, since only products of SaSSy were preferred substrates when compared with other similar sesquiterpenes.
However, it is important to note that conditions of yeast cells and in vitro assays are different compared to the physiological conditions in planta, which might explain the differences of product stereoisomers observed. It is possible that subtle changes in the shape and size of the active site under different conditions might result in the olefin precursors being oxidized in different configurations. It is also important to note that the products detected in in vitro microsome assays and in yeast in vivo assays were formed and accumulated over a period of minutes to hours.
In contrast, the oil extracted from mature heartwood is the product of biosynthesis and accumulation that occurs over a much longer time period of many years. Isomerization, perhaps catalyzed by an isomerase, may be possible in the trees, however may not have been mimicked with the conditions of the in vitro or yeast in vivo enzyme assays used here.
Although the ten P450s isolated in this work are the most abundant P450s in the sandalwood transcriptome sequences, it is also possible that additional sandalwood P450s exist that are similarly active on the santalenes and bergamotene substrates, but generating predominantly the (Z) stereoisomer. We will be exploring this possibility with further screening of the S. album P450 family.
The cloned terpene synthases [10],[24] and P450s (this study) of sandalwood oil biosynthesis can also be explored as biomarkers to monitor the onset of oil formation in sandalwood plantations or for the development of genetic markers for tree improvement. In this context, it is important to note that very little is known about the cell types and the molecular events that control spatial and temporal patterns of the onset of biosynthesis of sandalwood oil.
In fact, the spatial and temporal patterns of the onset of sandalwood oil biosynthesis are not well known, beyond the association of oil accumulation in the aging heartwood of sandalwood stems and roots. The aging heartwood of sandalwood trees provides an extremely difficult system to study with biochemical tools. Thus, the genes described here and in previous work [10] and their possible applications for metabolic engineering of sandalwood oil biosynthesis and the development of molecular markers are likely to become more important as worldwide demand for sandalwood products increase and as natural resources of S. album continue to decline.
4. Materials and Methods
The Saccharomyces cerevisiae yeast strain used in this study was BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0). Escherichia coli α-Select Chemically Competent Cells (Bioline) were used for routine cloning and plasmid propagation. The sesquiterpene olefins α-, β- and epi-β-santalene, and α-exo-bergamotene are not commercially available, but can be produced by expression of SaSSy in yeast [10].
A sesquiterpene oil containing α-, β- and epi-β-santalene, and α-exo-bergamotene was produced in an industrial scale fermentation system by Allylix, Inc. (Kentucky, USA). The mixture was separated using silver nitrate impregnated TLC plates according to Daramwar et al. [25]; fractions were scraped from TLC plates and sesquiterpenes eluted with pentane followed by GCMS analysis for purity.
Other sesquiterpenes, specifically bisabolol, trans-β-farnesene and trans-nerolidol were purchased from SIGMA. Zingiberine, α-curcumene, β-bisabolene and β-sesquiphellandrene were from our in house collection of sesquiterpene standards isolated from natural sources.
1. Transcriptome Sequences
A cDNA library made from Santalum album xylem was sequenced with Sanger technologies generating 11,520 paired end sequences [10]. 454 Titanium sequencing of the cDNA library generated an additional 902,111 sequence reads. The transcriptome assembly was done using both the 454 and Sanger sequences with Roche Newbler assembler version 2.6 under default parameters, which generated a total of 31,461 isotigs.
2. Cloning of P450 and CPR FLcDNAs and Yeast Transformation
FLcDNAs were amplified by PCR using Phusion Hot Start II DNA Polymerase (Thermo Scientific) with gene specific primers (Table S2) and cDNA prepared from S. album wood cores and leaves as template. PCR conditions included initial denaturing at 98°C for 3 min, two cycles at 98°C for 10 sec, Tm-2°C for 20 sec, and 72°C for 30 sec, followed by 30 cycles at 98°C for 10 sec, Tm for 20 sec and 72°C for 30 sec, and termination for 7 min at 72°C. PCR products were gel purified and cloned into the pJET1.2 vector (Fermentas).
Constructs designated pJET1.2-SaCYP76F37 through pJET1.2-SaCYP76F43, pJET1.2-SaCPR1 and pJET1.2-SaCPR2 were sequence verified. SaCYP76F FLcDNAs were subcloned into yeast expression vector pYEDP60 following the User Cloning method [26]. SaSSY (HQ343276) and SaFPPS (HQ343283) cDNAs [10] were cloned, respectively, into the NotI-Bgl II and BamHI-XhoI sites of the dual expression vector pESC-LEU2d by In-Fusion Cloning (Clontech). SaCPR1 and SaCPR2 were cloned individually into the EcoRI-NotI sites of the dual expression vector pESC-HIS (Stratagene).
Plasmid transformation of yeast strain BY4741 was done using the LiCl method Gietz et al. [27]. Transformed yeast strains were selected on plates with appropriate synthetic complete drop-out selection medium and grown at 30°C for 48 h.
3. Microsome Preparation
For microsome isolation, BY4741 cells were transformed with plasmids harboring P450 or CPR. Microsome membranes were prepared from 250 ml cultures according to Pompom et al. [28]. In brief, a 5 ml overnight culture was used to inoculate 50 ml of SD-selective media starting at an OD600 of 0.2 and grown at 30°C, 170 rpm for 24 h.
A volume of 200 ml YPDE medium (1% yeast extract, 2% bacto-peptone, 5% ethanol, 2% dextrose) was inoculated with the 50 ml culture and incubated for another 24 h at 30°C, 170 rpm. Cells were collected by centrifugation for 10 min at 1,000×g and induced with 2% galactose in 250 ml YP medium at 30°C, 170 rpm for 12–16 h.
Yeast cells were pelleted by centrifugation at 2,000×g for 10 min, washed once with 5 ml TEK (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 100 mM KCl) and suspended in TES2 buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 600 mM sorbitol, 5 mM DTT and 0.25 mM PMSF). All subsequent steps were performed at 4°C. Yeast cell were disrupted mechanically using acid-washed glass beads (425–600 µm, Sigma) and vigorous manual shaking for 3×30 sec.
4. CPR Activity and P450 CO Spectra
Activity of recombinant SaCPRs was assayed using the Cytochrome C Reductase (NADPH) assay kit (Sigma). CO difference spectra of recombinant P450s were measured according to Guengerich et al. [29].
5. In Vitro P450 Assays
Micro some preparations containing candidate P450 and CPR were assayed for their capacity to oxidize sesquiterpenes. The reaction mixtures contained 50 mM potassium phosphate pH 7.5, 0.8 mM NADPH and 40 µM of substrate in a total volume of 400 µl.
Enzyme reactions were initiated by adding 50 µl of the microsome preparation, incubated at 30°C for 2 h with shaking and stopped by adding 500 µl of hexane. The organic layer was transferred to a new GC vial and concentrated under N2 gas to about 100 µl followed by GCMS analysis. For kinetic analysis, enzyme assays were performed as above with the following modifications:
6. Yeast Metabolic Engineering
To assess the production of santalols/bergamotol in a yeast system, the yeast strain BY4741 was co-transformed with plasmids containing cDNAs for SaFPPS, SaSSY, SaCPR, and a candidate CYP76F. Recombinant yeast was initially grown overnight at 30°C in 5 ml of 2% dextrose in minimal selective media.
The next day, a 50 ml culture was initiated at a starting OD600 of 0.2 and grown at 30°C with shaking at 170 rpm until the culture reached an OD600 of 0.6–0.8. Expression was initiated by transfer into minimal selective media with 2% galactose and grown for 14–16 h. Yeast cells were harvested by centrifugation at 1,000×g for 10 min and washed once with 5 ml sterile ddH2O. Cells were extracted twice by vortexing for 1 min with 2 ml hexane and 250 µl acid-washed glass beads (425–600 µm, Sigma).
7. GCMS Analysis
https://www.google.co.uk/GCMS analysis was carried out on an Agilent 7890A/5975C GCMS system operating in electron ionization selected ion monitoring (SIM)-scan mode. Samples were analyzed on both an HP5 (non-polar; 30 m×0.25 mm ID×0.25 µm thickness) and a DB-Wax fused silica column (polar; 30 m×0.25 mm ID×0.25 µm thickness). In both cases, the injector was operated in pulsed splitless mode with the injector temperature maintained at 250°C. Helium was used as the carrier gas with a flow rate of 0.8 ml min−1 and pulsed pressure set at 25 psi for 0.5 min. Scan range: m/z 40–500; SIM: m/z 93, 94, 105, 107, 119, 122 and 202 [dwell time 50 msec].
The oven program for the HP5 column was: 40°C for 3 min; ramp of 10°C min−1 to 130°C, 2°C min−1 to 180°C, 50°C min−1 to 300°C; 300°C for 10 min. The oven program for the DB-wax column was: 40°C for 3 min; ramp of 10°C min−1 to 130°C, 2°C min−1 to 200°C, 50°C min−1 to 250°C; 250°C for 15 min.
8. Phylogenetic Analysis
Phylogenetic analysis was performed using the software MEGA version 4 [30] employing the neighbor-joining (NJ) algorithm with default parameters. Bootstrap (500 replications) confidence values over 50% are displayed at branch points.
4. Shaking up ancient scents: Insights into santalol synthesis in engineered Escherichia coli
Author links open overlay panelChonglong Wang, Seon-Won Kim
Abstract
Sandalwood oil is an essential oil that is derived from sandalwood and has important uses in cosmetics and medicine. Sandalwood oil is mainly composed of α-, β- and epi-β-santalols, which are responsible for the pleasant woody aroma. The syntheses of santalols begin with the condensation of the universal C5 precursors dimethylallyl diphosphate and isopentenyl diphosphate via farnesyl diphosphate (FPP) synthase. The resulting FPP undergoes multiple rearrangements and cyclization via santalene synthase to generate α-, β- and/or epi-β-santalenes,
which are finally hydroxylated at the C12 position by cytochrome P450 monooxygenase in cooperation with an NADPH-dependent cytochrome P450 reductase (CPR) to form santalols. Advances in metabolic engineering and protein engineering can increase the use of enzymes to synthesize valuable chemicals. In this review, we summarize the metabolic pathway for santalol synthesis and associated enzymes.
We also review the advances in metabolic engineering and biotechnology that can be utilized to manipulate Escherichia coli for santalol synthesis. We expect that the insights brought out by this review will shed light on metabolic engineering processes for santalol production in E. coli.
Introduction
Sandalwood oil is a precious essential oil with characteristic soft, warm, woody and milky-nutty scents [1]. It is obtained from the heartwood and roots of mature (>25 years), oil-producing Santalaceae (Santalum genus) via steam distillation (Fig. 1A). Four Santalum species contribute to the sandalwood oil market, including Santalum album,
which is native to South Asia; S. spicatum, which is grown in Western Australia; S. austrocaledonicum, which is grown in Vanuatu and New Caledonia; and S. yasi, which is grown in Fiji and Tonga [2]. Sandalwood oil is a mixture of sesquiterpenoid olefins and alcohols (>90%) and mainly consists of α- and β-santalols (50–70%) [3], which are mainly responsible for the pleasant woody aroma. The oil compositions are variable, depending on extraction methods and biological sources [2], [4]. The well-known East Indian sandalwood oil contains approximately 80% santalols (α-, β- and epi-β-) with minor amounts of α-bergamotol, α- and β-santalenes, α-bergamotene, etc. (Fig. 1B and C) [5], which is standardized in ISO (3518:2002) for quality control [6].
Sandalwood and its oil have been used in religious rituals and Ayurvedic medicine for millennia [1]. The current interests in sandalwood oil are growing in the aromatherapy, cosmetics and food industries due to its sedative action and fragrance [2].
Section snippets
1. Pathway engineering for santalol synthesis
The pathways and enzymes associated with santalol biosynthesis have been elucidated. Metabolic engineering of E. coli can be adopted to produce santalols, the main components of sandalwood oil. The recently developed strategies for the production of terpenoids could offer insight into the engineering process to improve santalol yields…
2. Protein engineering for santalol synthesis
The biosynthesis of santalols might also be challenged by deficiencies associated with the constituent enzymes of the santalol synthetic pathway. However, Advances in protein engineering offer possibilities for improving the turnover rate and reaction specificity of santalene synthases and expressing of CYP76F/CPRs in a functional form in a prokaryotic host.
Conclusions
Santalols are the major components of sandalwood oil, a naturally occurring essential oil with wide industry uses. As an alternative to extraction from sandalwood, heterologous production of santalols could be anticipated by engineering santalol synthetic pathway in E. coli. The enabling technologies (Fig. 2B) allow assembling and modulating the pathway enzymes in a desired manner. It is also possible to tailor enzymes such as santalene synthase and CYP76Fs with more efficiency and specificity…
Questions
1. What are the chemicals in sandalwood oil?
Sandalwood oil contains more than 90% sesquiterpenic alcohols of which 50–60% is the tricyclic α-santalol. β-Santalol comprises 20–25%. The composition of the oil will depend on the species, region grown, age of tree, and possibly the season of harvest and details of the extraction process used.
2. What is the use of Sandenol in perfume?
Sandenol (also known as Isobornyl cyclohexanol), an organic molecule known for its olfactory qualities, is an important component in the fragrance business, where it is primarily used to imitate the rich, woody, and sumptuous perfume of real sandalwood oil.
3. What is the origin of sandalwood oil?
Sandalwood essential oil is in many perfumes and air fresheners. It comes from the wood and roots of Santalum album, or the East Indian sandalwood tree. Manufacturers have produced West Indian and African sandalwood oils in the past, but they’re no longer widely available.18-Sept-2024
4. What are the properties of sandalwood?
Renowned for its fragrance and therapeutic properties, sandalwood has been treasured in ancient civilizations such as India, Egypt, China, and Greece. From religious ceremonies to medicinal applications, sandalwood has been revered for its ability to calm the mind, soothe the spirit, and heal the body.07-Jun-2024
5. Which type of sandalwood is best?
Indian sandalwood (Santalum album) and Australian sandalwood (Santalum spicatum or Fusanus spicatus) are considered two of the most coveted varieties. The heartwood contains sandalwood’s healing and aromatic properties which are due to two key compounds: alpha and beta santalol.24-Jan-2022.