Cloning and functional characterization of β-phellandrene synthase from Lavandula angustifolia

Abstract

En route to building genomics resources for Lavandula, we have obtained over 14,000 ESTs for leaves and flowers of L. angustifolia, a major essential oil crop, and identified a number of previously uncharacterized terpene synthase (TPS) genes. Here we report the cloning, expression in E. coli, and functional characterization of β-phellandrene synthase, LaβPHLS. The ORF--excluding the transit peptide--for this gene encoded a 62.3 kDa protein that contained all conserved motifs present in plant TPSs. Expression in bacteria resulted in the production of a soluble protein that was purified by Ni-NTA agarose affinity chromatography. While the recombinant LaβPHLS did not utilize FPP as a substrate, it converted GPP (the preferred substrate) and NPP into β-phellandrene as the major product, with K (m) and k (cat) of 6.55 μM and 1.75 × 10(-2) s(-1), respectively, for GPP. The LaβPHLS transcripts were highly abundant in young leaves where β-phellandrene is produced, but were barely detectable in flowers and older leaves, where β-phellandrene is not synthesized in significant quantities. This data indicate that β-phellandrene biosynthesis is transcriptionally and developmentally regulated. We also cloned and expressed in E. coli a second TPS-like protein, LaTPS-I, that lacks an internal stretch of 73 amino acids, including the signature DDxxD divalent metal binding motif, compared to other plant TPSs. The recombinant LaTPS-I did not produce detectable products in vitro when assayed with GPP, NPP or FPP as substrates. The lack of activity is most likely due to the absence of catalytically important amino acid residues within the missing region.

Biosynthesis of volatile terpenes that accumulate in the secretory cavities of young leaves of Japanese pepper (<i>Zanthoxylum piperitum</i>): Isolation and functional characterization of monoterpene and sesquiterpene synthase genes

“…The ORF for ZpTPS3 encoded a protein 610 amino acids in length with a molecular mass of 70.7 kDa. ZpTPS3 contained a predicted N-terminal chloroplast transit peptide (ChloroP 1.1; www.cbs.dtu.dk/services/ ChloroP) composed of 42 amino acids, which resembled other plant monoterpene synthase chloroplast transit peptides (Demissie et al 2011;Lücker et al 2002). In contrast, no chloroplast transit peptides were predicted in ZpTPS1 and ZpTPS2.…”

The Biosynthetic Origin of Irregular Monoterpenes in Lavandula

“…For example ␤-phellandrene synthase of L. angustifolia shares 48 and 47% identity with linalool and limonene synthases of the same species, respectively. However, the gene only shares 31 and 25% similarity with ␤-phellandrene synthases of grand fir and tomato, respectively (30). In this study, we observed that L. x intermedia trans-PDPS homologs (GPPS, FPPS, and GGPPS) share a higher sequence similarity among each other and with other trans-PDPSs cloned from genetically unrelated species than they do with LiLPPS.…”

“…The PCR program used was 95°C for 5 min, followed by 37 cycles of 95°C for 1 min, 58°C for 30 s, and 72°C for 1 min, and a 5-min final extension at 72°C. The amplified fragments were cloned into the NdeI/ EcoRI sites of the pET41b(ϩ) expression vector and expressed in Escherichia coli BL21(DE3) strain (EMD Chemicals, Darmstadt, Germany) following previously described procedures (30,31). Except for the bind buffer that was slightly modified (0.5 M NaCl, 50 mM KH 2 PO 4 , pH 8.0) the procedure described in the aforementioned papers was followed to enrich the expressed protein.…”

“…Consequently, these oils are industrially utilized as ingredients of various cosmetic and antiseptic products (29). The biosynthetic pathway leading to these monoterpenes from IPP and DMAPP has been defined both experimentally (30 -32) and through in silico analysis of Lavandula EST databases (30,31,33). In this regard, four cDNAs encoding for mTPSs that transform GPP and NPP into linalool, 1,8-cineole, limonene, and ␤-phellandrene in vitro have been cloned from L. angustifolia and L. x intermedia species (30 -32).…”

Essential Oils as Antimicrobials

“…The 'omic' techniques in terms of genomics, transcriptomics and proteomics have been developed to follow the antimicrobial activities of EOs and their components against single pathogen in particular (Demissie et al 2011;Sertel et al 2011;Efferth and Koch 2011). These novel techniques coupled with meta-genomics, transcriptomics and proteomics help to understand how microbial communities respond to changes in their environment so that they could become a prerequisite for better understanding of the EOs' action mechanism by identification of their targets and the disrupted molecular functions and pathways (Gillbert and Hughes 2011; Mitra et al 2011).…”