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| Content Provider | Journal of Biological Chemistry (JBC) |
|---|---|
| Author | Dhar, Niha Rana, Satiander Razdan, Sumeer Bhat, Wajid Waheed Hussain, Aashiq Dhar, Rekha S. Vaishnavi, Samantha Hamid, Abid Vishwakarma, Ram Lattoo, Surrinder K. |
| 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. |
| Related Links | http://www.jbc.org/content/289/24/17249.abstract |
| Ending Page | 17267 |
| Starting Page | 17249 |
| Page Count | 19 |
| File Format | HTM / HTML PDF |
| ISSN | 00219258 |
| Journal | Journal of Biological Chemistry (JBC) |
| Issue Number | 24 |
| Volume Number | 289 |
| DOI | 10.1074/jbc.M114.571919 |
| e-ISSN | 1083351X |
| Language | English |
| Publisher | American Society for Biochemistry and Molecular Biology |
| Publisher Date | 2014-06-13 |
| Access Restriction | Open |
| Subject Keyword | Gene Regulation Isoprenoid Mass Spectrometry (MS) Metabolic Engineering Western Blot Genomics and Proteomics |
| Content Type | Text |
| Resource Type | Article |
| Subject | Cell Biology Biochemistry Molecular Biology |
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