Cloning and Functional Characterization of Three Branch Point Oxidosqualene Cyclases From Withania somnifera (L.) Dunal [Genomics and Proteomics]

April 25th, 2014 by Dhar, N., Rana, S., Razdan, S., Bhat, W. W., Hussain, A., Dhar, R. S., Vaishnavi, S., Hamid, A., Vishwakarma, R., Lattoo, S. K.

Oxidosqualene cyclases (OSCs) positioned at a key metabolic sub-dividing junction execute indispensable enzymatic cyclization of 2, 3-oxidosqualene for varied triterpenoid biosynthesis. Such branch-points present favourable gene targets for redirecting metabolic flux towards specific secondary metabolites. However, detailed information regarding the candidate OSCs covering different branches and their regulation is necessary for desired genetic manipulation. The aim of the present study, therefore, was to characterize members of OSC superfamily from Withania somnifera (Ws), a medicinal plant of immense repute known to synthesize a large array of biologically active steroidal lactone triterpenoids called withanolides. Three full length OSC cDNAs, β-amyrin synthase (WsOSC/BS), lupeol synthase (WsOSC/LS) and cycloartenol synthase (WsOSC/CS) having open reading frames of 2289 bp, 2268 bp and 2277 bp were isolated. Heterologous expression in Schizosaccharomyces pombe, LC-MS analyses and kinetic studies confirmed their mono-functionality. The three WsOSCs were found to be spatially regulated at transcriptional level with WsOSC/CS being maximally expressed in leaf tissue. Promoter analysis of three WsOSCs genes resulted in identification of distinct cis-regulatory elements. Further, transcript-profiling under methyl jasmonate (MeJA), gibberellic acid (GA3) and yeast extract (YE) elicitations displayed differential transcriptional regulation of each of the OSCs. Changes were also observed in mRNA levels under elicitations and further substantiated with protein expression levels by western blotting. Negative regulation by YE resulted in significant increase in withanolide content. Empirical evidence suggests that repression of competitive branch OSCs like WsOSC/BS and WsOSC/LS possibly leads to diversion of substrate pool towards WsOSC/CS for increased withanolide production.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Cloning and Functional Characterization of Three Branch Point Oxidosqualene Cyclases From Withania somnifera (L.) Dunal [Genomics and Proteomics]