Abstract
Oxidosqualene cyclases (OSCs) positioned at a key metabolic subdividing junction execute indispensable enzymatic cyclization of 2,3-oxidosqualene for varied triterpenoid biosynthesis. Such branch points present favorable gene targets for redirecting metabolic flux toward specific secondary metabolites. However, detailed information regarding the candidate OSCs covering different branches and their regulation is necessary for the 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, 2268, and 2277 bp, were isolated. Heterologous expression in Schizosaccharomyces pombe, LC-MS analyses, and kinetic studies confirmed their monofunctionality. 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, gibberellic acid, and yeast extract 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 yeast extract 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 toward WsOSC/CS for increased withanolide production.
Highlights
Pharmacological investigations position withanolides as important bioactive molecules demanding their copious production
In Withania somnifera (Ws), this branching point leads to the division of 2,3-oxidosqualene between cycloartenol synthase ((S)-2,3-epoxysqualene mutase, EC 5.4.99.8), which leads to the formation of cycloartenol, a pentacyclic triterpene that contains nine chiral centers and acts as the precursor to phytosterols and apparently to withanolides and other array of Oxidosqualene cyclases (OSCs) that shape a range of diverse triterpenoids
Full-length gene specific primers (GSP) were designed and used to obtain 2.289, 2.268, and 2.277 kb ORFs, which were designated as WsOSC/BS, WsOSC/LS, and WsOSC/CS
Summary
Pharmacological investigations position withanolides as important bioactive molecules demanding their copious production. In Ws, this branching point leads to the division of 2,3-oxidosqualene between cycloartenol synthase ((S)-2,3-epoxysqualene mutase (cyclizing, cycloartenol forming), EC 5.4.99.8), which leads to the formation of cycloartenol, a pentacyclic triterpene that contains nine chiral centers and acts as the precursor to phytosterols and apparently to withanolides and other array of OSCs that shape a range of diverse triterpenoids Such branch point genes become prospective candidates for perturbation, which may impact respective branch flux by redirecting the precursor pool toward desired secondary metabolite(s) and concurrently decrease the flux through competitive pathways [12]. On the basis of cis-regulatory elements identified in the isolated promoters, plant-derived and microbe-derived elicitors altered WsOSCs expression pattern both at the mRNA and protein levels, which corresponded with the change in the accumulation of three key withanolides namely withanolide A (WS-1), withanone (WS-2), and WS-3 This suggests that repression of OSCs covering different branches like WsOSC/BS and WsOSC/LS lead to diversion of common precursor pools toward WsOSC/CS, leading to an increase in the withanolide production. This approach has the potential to become a useful predictive guide for future metabolic engineering efforts aimed at enhanced withanolide biosynthesis
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