Cloning and functional characterization of three branch point oxidosqualene cyclases from Withania somnifera (L.) dunal.
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.
From the Divisions of Plant Biotechnology.
Niha
N
Dhar
From the Divisions of Plant Biotechnology.
Satiander
S
Rana
From the Divisions of Plant Biotechnology.
Sumeer
S
Razdan
From the Divisions of Plant Biotechnology.
Wajid Waheed
WW
Bhat
Cancer Pharmacology, and.
Aashiq
A
Hussain
From the Divisions of Plant Biotechnology.
Rekha S
RS
Dhar
the School of Biotechnology, Shri Mata Vaishno Devi University, Katra-182320, India.
Samantha
S
Vaishnavi
Cancer Pharmacology, and.
Abid
A
Hamid
Medicinal Chemistry, CSIR-Indian Institute of Integrative Medicine, Jammu Tawi-180001, India and.
Ram
R
Vishwakarma
From the Divisions of Plant Biotechnology, sklattoo@iiim.ac.in.
Surrinder K
SK
Lattoo
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018506:Gene Expression Regulation, Plant; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D014158:Transcription, Genetic; D032341:Withania / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D054358:Withanolides / Q000378:metabolism
D010940:Plant Proteins; D054358:Withanolides; D019751:Intramolecular Transferases; C039924:2,3-oxidosqualene-beta-amyrin-cyclase; C117114:lupeol synthase; C021923:cycloartenol synthase
Gene Regulation; Isoprenoid; Mass Spectrometry (MS); Metabolic Engineering; Western Blot