Functional Characterization of Novel Sesquiterpene Synthases from Indian Sandalwood, Santalum album

Abstract

Indian Sandalwood, Santalum album L. is highly valued for its fragrant heartwood oil and is dominated by a blend of sesquiterpenes. Sesquiterpenes are formed through cyclization of farnesyl diphosphate (FPP), catalyzed by metal dependent terpene cyclases. This report describes the cloning and functional characterization of five genes, which encode two sesquisabinene synthases (SaSQS1, SaSQS2), bisabolene synthase (SaBS), santalene synthase (SaSS) and farnesyl diphosphate synthase (SaFDS) using the transcriptome sequencing of S. album. Using Illumina next generation sequencing, 33.32 million high quality raw reads were generated, which were assembled into 84,094 unigenes with an average length of 494.17 bp. Based on the transcriptome sequencing, five sesquiterpene synthases SaFDS, SaSQS1, SaSQS2, SaBS and SaSS involved in the biosynthesis of FPP, sesquisabinene, β-bisabolene and santalenes, respectively, were cloned and functionally characterized. Novel sesquiterpene synthases (SaSQS1 and SaSQS2) were characterized as isoforms of sesquisabinene synthase with varying kinetic parameters and expression levels. Furthermore, the feasibility of microbial production of sesquisabinene from both the unigenes, SaSQS1 and SaSQS2 in non-optimized bacterial cell for the preparative scale production of sesquisabinene has been demonstrated. These results may pave the way for in vivo production of sandalwood sesquiterpenes in genetically tractable heterologous systems.

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

“…Six genes were predicted to encode SS (SaT03849-R1, SaT03530-R1, SaT22220-R1, SaT22217-R1, SaT23033-R1, and SaT22976-R1). We performed a BLASTP search of the predicted proteins corresponding to genes identified in our study with previously characterized genes (Jones et al, 2011;Srivastava et al, 2015). We found that SaT22220-R1, SaT22217-R1, SaT02056-R1, SaT23033-R1, and SaT22976-R1 genes were highly similar (greater than 85%) to SaSQS1 (KJ665776), SaSQS2 (KJ665777), SaFDS (KF011939), SaBS (KJ665778), SaSS (KF011938), and SaSSy (HQ343276) (Supplemental Fig.…”

“…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.…”

Substitution of Aromatic Residues with Polar Residues in the Active Site Pocket of epi-Isozizaene Synthase Leads to the Generation of New Cyclic Sesquiterpenes

“…45 This essential oil comprises a blend of sesquiterpene hydrocarbons and alcohols and exhibits useful antimicrobial, antiinflammatory, and antitumor properties. 46–48 SASQS1 and SASQS2 each contain 566 residues and are related by 80% amino acid sequence identity; based on amino acid sequence analysis, each enzyme adopts the characteristic αβ domain architecture 49,50 of a plant sesquiterpene cyclase.…”

Enhancing epi‐cedrol production in Escherichia coli by fusion expression of farnesyl pyrophosphate synthase and epi‐cedrol synthase

“…The plasmids were transferred into E. coli Rosetta DE3α cells. The cells carrying the plasmids FPPS‐pRSETB , ECS‐pET28a , and FPPS‐ECS‐pET32a were grown in 1 L Terrific broth (Hi‐media, India), with ampicillin (100 µg/mL) and chloramphenicol (34 µg/mL), at 37°C. At OD 600 of ∼0.8, IPTG was added to the final concentration of 1 mM.…”

“…The plasmids were transferred into E. coli Rosetta DE3 cells. The cells carrying the plasmids FPPS-pRSETB [29], ECS-pET28a [28], and FPPS-ECS-pET32a were grown in 1 L Terrific broth (Hi-media, India), with ampicillin (100 μg/mL) and chloramphenicol (34 μg/mL), at 37 • C. At OD 600 of ∼0.8, IPTG was added to the final concentration of 1 mM. The cells were harvested after overnight incubation at 16 • C by centrifugation at 6500 × g for 10 min at 16 • C, and pellets were resuspended in 15 mL lysis buffer (50 mM Tris/HCl pH 7.8, containing 300 mM NaCl and 10% glycerol, 5 mM imidazole, 1 mM dithiothreitol), lysozyme (1 mg/mL), CHAPS (0.01%), and Triton X 100 (0.1%) were added.…”