Genomic characterization, molecular cloning and expression analysis of two terpene synthases from Thymus caespititius (Lamiaceae)

Abstract

The identification, isolation and functional characterization of two genes encoding two monoterpene synthases-γ-terpinene synthase (Tctps2) and α-terpineol synthase (Tctps5)-from three chemically distinct Thymus caespititius (Lamiaceae) genotypes were performed. Genomic exon-intron structure was also determined for both terpene synthase genes, revealing an organization with seven exons and six introns. The cDNA of Tctps2 was 2,308 bp long and had an open reading frame of 1,794 bp encoding for a protein with 598 amino acids. Tctps5 was longer, mainly due to intron sequences, and presented high intraspecific variability on the plants analyzed. It encoded for a protein of 602 amino acids from an open reading frame of 1,806 bp comprising a total of 2,507 bp genomic sequence. The amino acid sequence of these two active Tctps genes shared 74 % pairwise identity, ranging between 42 and 94 % similarity with about 50 known terpene synthases of other Lamiaceae species. Gene expression revealed a multi-product Tctps2 and Tctps5 enzymes, producing γ-terpinene and α-terpineol as major components, respectively. These enzymatic results were consistent with the monoterpene profile present in T. caespititius field plants, suggesting a transcriptional regulation in leaves. Herewith reported for the first time for this species, these two newly characterized Tctps genes improve the understanding of the molecular mechanisms of reaction responsible for terpene biosynthesis and chemical diversity found in T. caespititius.

Functional characterization of a new terpene synthase from Plectranthus amboinicus

“…Plants in the Lamiaceae family are known to produce a large variety of terpenoids, and this diversity could be due to expression of multiple terpene synthases and formation of multiple products by individual terpene synthases as previously reported [12][13][14][15]. Terpene synthase genes have been previously isolated and characterized from several important Lamiaceae members including Thymus caespititius [13,14], T. vulgaris [16], Coriandrum sativum L. [15], Origanum vulgarae [12] and Salvia officinalis [17]; however the terpene synthases responsible for terpenoids production in P. amboinicus have not been functionally characterized. Thus, the main objectives of this present study were to isolate and clone the full-length transcript of P. amboinicus monoterpene synthase, and to functionally express and characterize the recombinant terpene synthase in both in vivo and in vitro systems.…”

“…High yield expression of soluble monoterpene synthases can be accomplished by truncation of the transit peptide from the coding region to remove the target sequence [11]. Previous studies on bacterial expression of terpene synthases isolated from other members of Lamiaceae such as M. spicata limonene synthase and T. caespititius γ-terpinene synthase demonstrated the expression of functional enzymes upon removal of the transit peptide upstream of the double arginine motif [13,29]. Therefore, the signal peptide was removed from PamTps1 sequence during cloning in order to achieve expression of a soluble and functional putative monoterpene synthase in an E. coli system.…”

Biological Efficacy of Essential Oils and Plant Extracts of Cultivated and Wild Ecotypes of Origanum vulgare L.

“…Origanum vulgare L. is a highly variable species having different chemotypes and exploitation of genetic variability that can be utilized for development of new cultivars rich in monoterpene and sesquiterpene [32,33]. is disparity in essential oil components in genotypes within different habitats can be correlated with different gene expressions of key enzymes implicated in terpene biosynthesis [39,40]. e disparity in essential composition can be due to diverse geographical location [1], varied climatic zones [41], and different ecological variables [42].…”

Expression, crystallization and structure elucidation of γ-terpinene synthase fromThymus vulgaris

“…0.5 mM isopropyl -d-1-thiogalactopyranoside (IPTG) was then added to induce the expression of TvTPS. Cells were subsequently cultivated at 20 C for up to 18 h for optimal expression of recombinant TvTPS (Lima et al, 2013) and were then harvested by centrifugation for 20 min at 4000g and 4 C. The pellet was frozen and kept at À20 C. After thawing on ice, the cells were resuspended in 10 ml lysis buffer (50 mM Proposed reaction scheme of -terpinene synthesis, as catalyzed by TvTPS1, providing a precursor for thymol and carvacrol biosynthesis (based on Lima et al, 2013).…”

“…To date, TPSs have been described as multifunctional enzymes, with single amino-acid substitutions being sufficient to result in an altered metabolic profile (Kö llner et al, 2004), and a number of TPSs and other terpene-modifying enzymes have been described (Keszei et al, 2008). Recently, TPS genes have been identified and characterized from several plant species from the Lamiaceae family (summarized in Lima et al, 2013): TPS from Lavandula (Muñ oz- Bertomeu et al, 2008;Lane et al, 2010), Mentha (Turner & Croteau, 2004), Origanum (Crocoll et al, 2010;Lukas et al, 2010), Perilla (Ito & Honda, 2007), Coriandrum (Galata et al, 2014) and Salvia (Kampranis et al, 2007;Schmiderer et al, 2010). Within the genus Thymus, only TPS from T. caespititius (including several isoforms) has been described previously (Lima et al, 2010(Lima et al, , 2011(Lima et al, , 2013Mendes et al, 2014).…”