Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus.

Linalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis-Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10-3 µM-1 s-1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.

Functional characterization of a new terpene synthase from Plectranthus amboinicus.

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.

Cloning and expression analysis of chalcone synthase gene from Coleus forskohlii.

Flavonoids are an important class of secondary metabolites that play various roles in plants such as mediating defense, floral pigmentation and plant-microbe interaction. Flavonoids are also known to possess antioxidant and antimicrobial activities. Coleus forskohlii (Willd.) Briq. (Lamiaceae) is an important medicinal herb with a diverse metabolic profile, including production of a flavonoid, genkwanin. However, components of the flavonoid pathway have not yet been studied in this plant. Chalcone synthase (CHS) catalyses the first committed step of flavonoid biosynthetic pathway. Full-length cDNA, showing homology with plant CHS gene was isolated from leaves of C. forskohlii and named CfCHS (GenBank accession no. KF643243). Theoretical translation of CfCHS nucleotide sequence shows that it encodes a protein of 391 amino acids with a molecular weight of 42.75 kDa and pI 6.57. Expression analysis of CfCHS in different tissues and elicitor treatments showed that methyl jasmonate (MeJA) strongly induced its expression. Total flavonoids content and antioxidant activity of C. forskohlii also got enhanced in response to MeJA, which correlated with increased CfCHS expression. Induction of CfCHS by MeJA suggest its involvement in production of flavonoids, providing protection from microbes during herbivory or mechanical wounding. Further, our in silico predictions and experimental data suggested that CfCHS may be posttranscriptionally regulated by miR34.

Defence strategies adopted by the medicinal plant Coleus forskohlii against supplemental ultraviolet-B radiation: Augmentation of secondary metabolites and antioxidants.

Supplementary ultraviolet-B (ambient+3.6  kJ m(-2) day(-1)) induced changes on morphological, physiological, and biochemical characteristics (specifically the defence strategies: UV-B protective compounds and antioxidants) of Coleus forskohlii were investigated under field conditions at 30, 60, and 90 days after transplantation. Levels of secondary metabolites increased under s-UV-B stress; flavonoids and phenolics (primary UV-B screening agents) were recorded to be higher in leaves which are directly exposed to s-UV-B. This was also verified by enhanced activities of phenylpropanoid pathway enzymes: phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), 4-coumarate-CoA ligase (4CL), chalcone-flavanone isomerase (CHI), and dihydroflavonol reductase (DFR). Antioxidants, both enzymatic (ascorbate peroxidase, catalase, glutathione reductase, peroxidase, polyphenol oxidase, and superoxide dismutase) and non-enzymatic (ascorbic acid and α-tocopherol) also increased in the treated organs of the test plant, higher contents being recorded in roots except for ascorbic acid. On the contrary, protein and chlorophyll content (directly implicated in regulating plant growth and development) declined under s-UV-B. These alterations in plant biochemistry led the plant to compromise on its photosynthate allocation towards growth and biomass production as evidenced by a reduction in its height and biomass. The study concludes that s-UV-B is a potent stimulating factor in increasing the concentrations of defense compounds and antioxidants in C. forskohlii to optimize its performance under stress.

Metabolic Engineering of Synechocystis sp. PCC 6803 for Production of the Plant Diterpenoid Manoyl Oxide.

Forskolin is a high value diterpenoid with a broad range of pharmaceutical applications, naturally found in root bark of the plant Coleus forskohlii. Because of its complex molecular structure, chemical synthesis of forskolin is not commercially attractive. Hence, the labor and resource intensive extraction and purification from C. forskohlii plants remains the current source of the compound. We have engineered the unicellular cyanobacterium Synechocystis sp. PCC 6803 to produce the forskolin precursor 13R-manoyl oxide (13R-MO), paving the way for light driven biotechnological production of this high value compound. In the course of this work, a new series of integrative vectors for use in Synechocystis was developed and used to create stable lines expressing chromosomally integrated CfTPS2 and CfTPS3, the enzymes responsible for the formation of 13R-MO in C. forskohlii. The engineered strains yielded production titers of up to 0.24 mg g(-1) DCW 13R-MO. To increase the yield, 13R-MO producing strains were further engineered by introduction of selected enzymes from C. forskohlii, improving the titer to 0.45 mg g(-1) DCW. This work forms a basis for further development of production of complex plant diterpenoids in cyanobacteria.