Molecular cloning and characterization of farnesyl pyrophosphate synthase gene from Panax sokpayensis, a new Panax species from Sikkim Himalaya

• Published in: Journal of applied research on medicinal and aromatic plants 2019-09, Vol.14, p.100215, Article 100215
• Main Authors: Gurung, Bhusan, Bains, Savita, Saha, Dipanwita, Singh, Kashmir, Bhardwaj, Pardeep K., Sahoo, Dinabandhu
• Format: Article
• Language: English
• Subjects: Farnesyl pyrophosphate synthase; Ginsenosides; Panax sokpayensis; Polyadenylation signal; RACE
• ISSN: 2214-7861; 2214-7861
• DOI: 10.1016/j.jarmap.2019.100215

•Full-length cDNA of farnesyl pyrophosphate synthase from Panax sokpayensis (PsFPS) was cloned through RACE.•In silico analyses revealed the motifs of FARM and SARM with highly conserved catalytic site “DDXXD” present in the sequence.•Three polyadenylation signals (PASs) present in 3`UTR region suggested the existence of alternative polyadenylation signal guided PsFPS mRNA isoforms.•Heterologous expression and production of functional PsFPS in E. coli. This study describes the molecular characterization and heterologous expression of farnesyl pyrophosphate synthase (PsFPS) from Panax sokpayensis Shiva K. Sharma & Pandit. FPS is a key regulatory enzyme of ginsenoside biosynthetic pathway that directs the metabolic flux from mevalonate (MVA) and methylerythritol phosphate (MEP) pathway towards the synthesis of ginsenosides and phytosterols. Using Rapid Amplification of cDNA Ends (RACE), full-length PsFPS of 1437 bp with an open reading frame (ORF) of 1026 bp coding for 342 amino acids was cloned. Deduced amino acid sequence of PsFPS contained two highly conserved aspartate rich motifs with consensus DDXXD sequence that constitute the main catalytic site of FPS. Full length cDNA of PsFPS has 5′ untranslated region (UTR) with 142 bp and 3′ UTR with 234 bp followed by a poly (A) tail. In silico analysis detected three identical polyadenylation signals (PAS) with canonical sequence, AATAAA in the 3′ UTR. Alignment of 3′ UTR of PsFPS with the FPS from other Panax species indicated the presence of alternative PAS which could have possible role in the formation of different isoforms of FPS in Panax species. Open reading frame of PsFPS was expressed in Escherichia coli (E. coli) and the recombinant protein was produced in vitro. High expression of recombinant protein was observed after 1 h of induction with 1 mM IPTG. HPLC mediated enzyme assay confirmed the production of functional PsFPS in E. coli. This is the first report on molecular characterization, heterologous expression and functional validation of PsFPS from P. sokpayensis.