Functional characterization of a new terpene synthase from Plectranthus amboinicus

Abstract

Plectranthus amboinicus (Lour.) Spreng is an aromatic medicinal herb known for its therapeutic and nutritional properties attributed by the presence of monoterpene and sesquiterpene compounds. Up until now, research on terpenoid biosynthesis has focused on a few mint species with economic importance such as thyme and oregano, yet the terpene synthases responsible for monoterpene production in P. amboinicus have not been described. Here we report the isolation, heterologous expression and functional characterization of a terpene synthase involved in P. amboinicus terpenoid biosynthesis. A putative monoterpene synthase gene (PamTps1) from P. amboinicus was isolated with an open reading frame of 1797 bp encoding a predicted protein of 598 amino acids with molecular weight of 69.6 kDa. PamTps1 shares 60-70% amino acid sequence similarity with other known terpene synthases of Lamiaceae. The in vitro enzymatic activity of PamTps1 demonstrated the conversion of geranyl pyrophosphate and farnesyl pyrophosphate exclusively into linalool and nerolidol, respectively, and thus PamTps1 was classified as a linalool/nerolidol synthase. In vivo activity of PamTps1 in a recombinant Escherichia coli strain revealed production of linalool and nerolidol which correlated with its in vitro activity. This outcome validated the multi-substrate usage of this enzyme in producing linalool and nerolidol both in in vivo and in vitro systems. The transcript level of PamTps1 was prominent in the leaf during daytime as compared to the stem. Gas chromatography-mass spectrometry (GC-MS) and quantitative real-time PCR analyses showed that maximal linalool level was released during the daytime and lower at night following a diurnal circadian pattern which correlated with the PamTps1 expression pattern. The PamTps1 cloned herein provides a molecular basis for the terpenoid biosynthesis in this local herb that could be exploited for valuable production using metabolic engineering in both microbial and plant systems.

Fig 1. Total ion chromatogram of Plectranthus amboinicus leaf volatiles harvested at 8.00 AM extracted using headspace solid phase microextraction (HS-SPME).

The numbers corresponded to the compounds detected as described in Table 1.

Fig 2. Alignment of PamTps1 amino acid sequence without transit peptide with other plant monoterpene synthases using Clustal Omega and BoxShade server.

Conserved domains of RRx8W, LQLYEASFL, DDxxD, GTLxEL and DTE were labelled. AGK88250.1: T. caespititius α-terpineol synthase; ARA91313.1: L.x intermedia 3-carene synthase; ABD77417.1: L. latifolia linalool synthase; AID51195.1: T. caespititius γ-terpinene synthase; ADK73617.1: O. vulgare terpene synthase 5; AGT29345.1: T. serpyllum γ-terpinene synthase; AEO27879.1: O. syriacum γ-terpinene synthase; ABP01684.1: S. rosmarinus pinene synthase; ACN42010.1: P. setoyensis geraniol synthase; ACN42013.2: P. frutescens var. hirtella linalool synthase and AAX16075.1: P. citriodora linalool synthase.

Fig 3. GC-MS chromatograms (selected ion, m/z = 93) of PamTps1 products generated both in in vitro and in vivo systems.

PamTps1 enzymatic reaction incubated with (A) GPP or (B) FPP; products generated in E. coli harboring (C) empty vector and (D) PamTps1; (E) (-)-linalool standard and (F) cis- and trans-nerolidol standards. In vitro PamTps1 reactions produced exclusively linalool and nerolidol when incubated with GPP and FPP, respectively. In recombinant E. coli, PamTps1 produced both linalool and nerolidol. Only sample peaks higher than the negative control were marked with numbers. Corresponding compounds are: 1 = linalool (retention time = 6.7 min); 2 = trans-nerolidol (retention time = 13.0 min) and 3 = cis–nerolidol (retention time = 12.6 min).

Fig 4. Correlation between linalool and nerolidol emissions and PamTps1 expression in leaves and stems of P. amboinicus within a 24 h day/night cycle.

Relative expression analysis was performed by qRT-PCR using EFG, TUB and APRT as reference genes. The relative transcription level in tissue with the highest expression quantity was set to 1 (100%). Each bar represents the mean value ±SE of three biological and three technical replicates. The linalool and nerolidol emission data presented are means ± SE of duplicate experiments.

Fig 5. Phylogenetic relationship of PamTps1 with selected plant terpene synthases from different subfamilies.

Target sequences upstream of the RRx8W motif of the alignment were removed. Selection of terpene synthases subfamilies were based on previous literatures [11,100,101]. The Tpsc and Tpse subfamilies were chosen as outgroups. Evolutionary relationship was inferred using Neighbor-Joining method with 1000 replicates for bootstrapping. The numbers indicated were the actual bootstrap values of the branches.