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

Abstract

SUMMARYTropical sandalwood (Santalum album) produces one of the world's 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: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)-a-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)-a-santalol, (Z)-b-santalol, (Z)-epi-b-santalol and (Z)-a-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.

Multi-Omics Driven Assembly and Annotation of the Sandalwood (Santalum album) Genome

“…Genes identified in this study validate the findings reported earlier and will provide a foundation for the production of sandalwood oil for the essential oil industry by means of metabolic engineering (synthetic biology) under in vitro conditions. Such proof of concepts of essential oil biosynthesis have been demonstrated previously (Jones et al, 2011;Diaz-Chavez et al, 2013;Srivastava et al, 2015;Celedon et al, 2016). As discussed by Diaz Chavez et al (2013), genes identified in their study and genes described here may be used as biomarkers to monitor the onset of oil formation in sandalwood plantations and in the development of genetic markers for tree improvement breeding strategies.…”

“…Around 184 sandalwood cytochrome P450 genes were identified in the genome. Out of these, four genes (SaT19792-R1, SaT24185-R1, SaT24441-R1, and SaT11750-R1) were found to be homologous with already cloned genes reported previously (Diaz-Chavez et al, 2013;Celedon et al, 2016).…”

“…We performed a phylogenetic analysis of these 184 genes along with the previously characterized genes (Diaz-Chavez et al, 2013;Celedon et al, 2016). Phylogenetic analysis of the CYP450 genes identified in our study and cloned genes showed that they were clustered in two major clades (Supplemental Fig.…”

“…7). Mainly, four sesquiterpenols, a-, b-, and epi-b-santalol and a-exo-bergamotol, together constitute approximately 80% to 90% of heartwood oil obtained from mature trees (Celedon et al, 2016). Around 184 sandalwood cytochrome P450 genes were identified in the genome.…”

“…Currently, large-scale transcriptomic data sets are available for sandalwood (Diaz-Chavez et al, 2013;Srivastava et al, 2015;Zhang et al, 2015Zhang et al, , 2017Celedon et al, 2016) and Santalum spicatum (Moniodis et al, 2015). Most of these genomic resources have been utilized to identify genes involved in sandalwood oil biosynthesis, cold stress response, and the hemiparasitic nature of its roots.…”