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Systematic analysis of a Monoterpene synthase from bangun-bangun (Plectranthus amboinicus lour. spreng)


Ashaari, Nur Suhanawati (2021) Systematic analysis of a Monoterpene synthase from bangun-bangun (Plectranthus amboinicus lour. spreng). Doctoral thesis, Universiti Putra Malaysia.


Plectranthus amboinicus (Lour.) Spreng, is an aromatic medicinal herb noted for its therapeutic and nutritional properties attributed by the presence of terpenoids. To date, molecular research on terpenoids biosynthesis from this herb is still limited and the terpene synthases responsible for terpenoids production in P. amboinicus have yet to be described. Terpenoids have high demands in their applications as flavours and fragrances in food and cosmetics, and also as therapeutic agents in pharmaceutical products. Unfortunately, nature rarely produces these compounds in abundance. Hence, isolating the desired gene from the P. amboinicus for production of the terpenoid compounds in a suitable heterologous system poses an alternative way to overcome such limitations in the natural sources. The aim of this study was to isolate and characterise the P. amboinicus terpene synthase. Among the approaches involved are profiling of volatiles that contributed to the distinctive aroma of P. amboinicus conducted using GC-MS, accompanied by characterisation of the recombinant terpene synthase and terpenoids production in an Escherichia coli system, gene expression analysis in planta using quantitative real-time PCR and in silico structure prediction using homology modelling and protein docking. Volatiles profiling of P. amboinicus leaves revealed the presence of 46 % monoterpenes and 53 % sesquiterpenes comprising of α-bergamotene, carvacrol, caryophyllene, ρ-cymene, γ-terpinene and terpene alcohols. A putative monoterpene synthase coding sequences (designated as PamTps1) with an open reading frame of 1797 bp encoding a predicted protein of 598 amino acids with more than 60 % identity to known terpene synthases of Lamiaceae was isolated. A soluble recombinant PamTps1 with estimated size of ~79 kDa was successfully expressed via pET-32b(+) expression vector in Escherichia coli Rosetta™ 2(DE3) system at 28 °C with 0.5 mM IPTG induction. Enzyme characterisation revealed that the PamTps1 catalysed the conversion of geranyl pyrophosphate (GPP, C10) and farnesyl pyrophosphate (FPP, C15) into linalool and nerolidol, respectively. Biochemical properties of PamTps1 exhibited the highest activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, in the presence of 20 mM magnesium as a cofactor. The Michaelis-Menten constant (Km) and catalytic efficiency (kcat /Km) were 16.72 ± 1.32 μM and 9.57 x 10-3 μM-1 s-1, respectively showed that the PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The recombinant E. coli harbouring PamTps1 produced 13.6 ± 0.2 μg/ml and 10.6 ± 0.1 μg/ml linalool and nerolidol after 72 h and 24 h incubation, respectively. These outcomes validated the multi-substrate use of this enzyme in producing linalool and nerolidol, both in the in vivo and in vitro systems. Transcript expression analysis revealed that PamTps1 was up-regulated in leaves (42-fold of expression level) instead of stems which were associated with linalool emission following a diurnal rhythm regulated by circadian clock of the plant. Homology modelling of PamTps1 was predicted using BPPS (1N24) with 67 % sequence identity as a template. PamTps1 active site pocket analysis revealed nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517, and Y523) that defined the hydrophobic walls of the active site cavity and shaped the active site for proper substrate binding and folding. While residues from the RRx8W motif, RxR motif, H-α1, and J-K loops formed the active site lid, protecting the highly reactive carbocationic intermediates from solvents. The dual-substrates used by PamTps1 were hypothesised due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by protein modelling and docking analysis. The PamTps1 herein was identified as a linalool/nerolidol synthase, and this is the first study describing isolation and characterisation of such substrate promiscuity terpene synthase from the P. amboinicus. This study provides an initial insight into terpenoids biosynthesis in this herb that can be exploited for production of these natural products using metabolic engineering in both microbial and plant systems.

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Additional Metadata

Item Type: Thesis (Doctoral)
Subject: Medicinal plants
Subject: Lamiaceae
Subject: Herbs - Therapeutic use
Call Number: FBSB 2021 14
Chairman Supervisor: Janna Ong Abdullah, PhD
Divisions: Faculty of Biotechnology and Biomolecular Sciences
Depositing User: Ms. Nur Faseha Mohd Kadim
Date Deposited: 20 Apr 2022 01:30
Last Modified: 20 Apr 2022 01:30
URI: http://psasir.upm.edu.my/id/eprint/92670
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