Functional Characterisation of New Sesquiterpene Synthase from the Malaysian Herbal Plant, Polygonum Minus
Polygonum minus (syn. Persicaria minor) is a herbal plant that is well known for producing sesquiterpenes, which contribute to its flavour and fragrance. This study describes the cloning and functional characterisation of PmSTPS1 and PmSTPS2, two sesquiterpene synthase genes that were identified from P. minus transcriptome data mining. The full-length sequences of the PmSTPS1 and PmSTPS2 genes were expressed in the E. coli pQE-2 expression vector. The sizes of PmSTPS1 and PmSTPS2 were 1098 bp
... TPS2 were 1098 bp and 1967 bp, respectively, with open reading frames (ORF) of 1047 and 1695 bp and encoding polypeptides of 348 and 564 amino acids, respectively. The proteins consist of three conserved motifs, namely, Asp-rich substrate binding (DDxxD), metal binding residues (NSE/DTE), and cytoplasmic ER retention (RxR), as well as the terpene synthase family N-terminal domain and C-terminal metal-binding domain. From the in vitro enzyme assays, using the farnesyl pyrophosphate (FPP) substrate, the PmSTPS1 enzyme produced multiple acyclic sesquiterpenes of β-farnesene, α-farnesene, and farnesol, while the PmSTPS2 enzyme produced an additional nerolidol as a final product. The results confirmed the roles of PmSTPS1 and PmSTPS2 in the biosynthesis pathway of P. minus, to produce aromatic sesquiterpenes. Molecules 2018, 23, 1370 2 of 15 (FPP), which is catalysed by the enzyme FPP synthase. The resulting linear FPP undergoes electrophilic cyclisation and rearrangement to form acyclic and non-acyclic sesquiterpenes, based on the sesquiterpene synthase enzyme reactions [12, 13] . In particular, monoterpenes and sesquiterpenes are commonly present in plant essential oils, which are widely utilised for commercial purposes, such as fragrances, cosmetics, pharmaceuticals, medicine, biofuel precursors, and industrial materials [14, 15] . To date, over 7000 sesquiterpenes (C15) with different hydrocarbon skeletons and stereo-chemically structures have been reported  . Studies on naturally derived 15-carbon terpenoids in aromatic herbal plants as anti-malarial, anti-microbial, and insecticidal agents have also increased over the last several years  . Hence, many sesquiterpenes are founds as major components of fruits and vegetables, floral scents, and essential oils of herbs. In previous studies, a number of genes encoding the sesquiterpene enzymes, which control the key steps of secondary metabolic pathways, have been extensively cloned and characterized from a number of herbal plant species. For example, squalene synthase from Tripterygium wilfordii , germacrene A synthase from Achillea millefolium , drimenol synthase from Valeriana officinalis , E-E-farnesol synthase and α-bisabolene synthase from Gingko biloba , α-humulene synthase from Zingiber zerumbet Smith , (+)-epi-α-bisabolol synthase from Lippia dulcis , β-caryophyllene synthase from Ocimum basilicum L. , α-humulene synthase from Zingiber zerumbate Smith , and Germacrene D synthase from Zingiber officinale . Polygonum minus (syn. Persicaria minor) is a herbal plant that originated from Southeast Asia and belongs to the Polygonaceae family. In Malaysia, P. minus is locally known as a 'kesum', and is commonly used as a food additive and flavouring agent. Many studies have been carried out because of the popularity of the P. minus as a potential medicinal plant with high antioxidant and antimicrobial activities and strong anti-inflammatory properties     . P. minus is an economically important herbal plant because of its essential oils, which cause it to emit a strong scent from a simple mixture of terpenoid hydrocarbons (monoterpenoids and sesquiterpenoids), including β-farnesene, α-farnesene, nerolidol, farnesol, caryophyllene, α-bergamotene, and drimenol [31, 32] . Other characteristic components that have been identified from P. minus include aldehydes (decanal and dodecanal), esters, and organic acids  . Moreover, recent studies have revealed that the sesquiterpenes compounds are the main contributors to the characteristic fragrance of this plant  . These studies have shown the potential for developing P. minus as a resource to produce natural products. While many structurally diverse secondary metabolites, especially terpenoid compounds, have been identified in P. minus, its biosynthesis of major constituents remains unclear because of the limited genomic information that is available for this plant. Therefore, this plant is investigated for the isolation and characterisation of novel sesquiterpene synthase genes. Additionally, there is not much work on sesquiterpene synthase from P. minus, especially at the genetic level, as it is still very scarce. In previous studies, several works on sesquiterpene synthases were identified. The first P. minus putative sesquiterpene synthase gene was cloned and expressed in E. coli systems  . Song et al.  successfully overexpressed a sesquiterpene synthase, PmSTS, in metabolically engineered gram-positive bacteria, Lactococcus lactis, with the MVA pathway. Then, a structural study was performed that demonstrated an active catalytic site with the same gene sequence  . The encoded enzyme was named β-sesquiphellandrene synthase, based on the principal product that was formed. In a subsequent study, the influence of jasmonic acid treatment on the expression level of the Persicaria minor sesquiterpene synthase (PmSS) gene was reported  . Until very recently, the purification and overexpression of P. minor sesquiterpene synthase encoded as PmSTS recombinant protein in pET28b vector, using the E. coli BL21 (DE3) strain, were reported  . All of the studies on P. minus that have been mentioned above were based on the same sesquiterpene synthase. In order to understand the terpenoid metabolism of P. minus, the potential gene for terpene synthases must be isolated and studied. Therefore, in this study, we described the cDNAs isolation and characterisation of two new sesquiterpene synthases (STPS) that were responsible for the formation of two key aromatic compounds, which made substantial contributions to the flavour and fragrance of P. minus essential oil. The results could have provided a foundation for the further exploration of gene function in P. minus, and helped to reveal the regulation of terpenoid biosynthesis.