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somnifera leads to enhanced withanolide biosynthesis

Genetic engineering of secondary metabolic pathways is an emerging area of research for production and improvement of natural products in plant biotechnology. Here, we describe a systematic approach to manipulate a key regulatory step of isoprenoid biosynthetic pathway in Withania somnifera to study its effect on withanolide production. We generated T0 W. somnifera plants overexpressing squalene synthase (WsSQS) by Agrobacterium tumefaciens mediated transformation, which were analyzed by Gus biochemical assay and PCR of hygromycin phosphotransferase (hptII) and WsSQS. qRT-PCR analyses of various transformed tissues indicated 2–5 fold increase in WsSQS transcripts in both T0 and T1 generations. The tissue specific protein expression studies revealed 2–3 fold increase in WsSQS, which was further confirmed by enzyme activity. These observations were corroborated with the 1.5–2 fold increase in total withanolide content of the transformed tissues. However, in leaf tissue, the levels of Withaferin A and Withanolide A increased significantly up to 4–4.5 fold. These findings demonstrate genetic engineering of isoprenoid pathway in W. somnifera resulting in enhanced production of withanolides, and also provide insights into such metabolic pathways for their manipulation to improve the pharmacological content of different medicinally important plants.


Key message The study elucidates significant contribution of DOXP pathway to withanolide biosynthesis. A new connotation of biosynthetic load-based role of DOXP/MVA recruitment in isoprenoid biosynthesis has been proposed.

Enhanced Biosynthesis of Withanolides by Elicitation …

Cycloartenol synthase (CAS) is an important enzyme in the withanolide biosynthetic pathway, catalyzing cyclization of 2, 3 oxidosqualene into cycloartenol.

somnifera at whole plant level as per our knowledge and it will be further useful to understand the regulatory role of different genes involved in the biosynthesis of withanolides.
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Molecular studies on withanolide biosynthetic pathway …

Withanolides are pharmaceutically important C28-phytochemicals produced in most prodigal amounts and diversified forms by Withania somnifera. Metabolic origin of withanolides from triterpenoid pathway intermediates implies that isoprenogenesis could significantly govern withanolide production. In plants, isoprenogenesis occurs via two routes: mevalonate (MVA) pathway in cytosol and non-mevalonate or DOXP/MEP pathway in plastids. We have investigated relative carbon contribution of MVA and DOXP pathways to withanolide biosynthesis in W. somnifera. The quantitative NMR-based biosynthetic study involved tracing of 13C label from 13C1--glucose to withaferin A in withanolide producing in vitro microshoot cultures of the plant. Enrichment of 13C abundance at each carbon of withaferin A from 13C1-glucose-fed cultures was monitored by normalization and integration of NMR signal intensities. The pattern of carbon position-specific 13C enrichment of withaferin A was analyzed by a retro-biosynthetic approach using a squalene-intermediated metabolic model of withanolide (withaferin A) biosynthesis. The pattern suggested that both DOXP and MVA pathways of isoprenogenesis were significantly involved in withanolide biosynthesis with their relative contribution on the ratio of 25:75, respectively. The results have been discussed in a new conceptual line of biosynthetic load-driven model of relative recruitment of DOXP and MVA pathways for biosynthesis of isoprenoids.

Present study describes isolation and molecular characterization of DWF1 gene from Withania somnifera and also elucidates its role in withanolide biosynthesis. Using the degenerate-reverse transcriptase and RACE strategy full length cDNA of DWF1 was isolated from W. somnifera and subsequently cloned in pGEX-4T-2 vector and expressed in E. coli. Effect of various abiotic and biotic chemicals such as methyl jasmonate (MeJA), salicylic acid (SA), 2,4-dichlorophenoxyacetic acid (2,4-D) and microbe-derived exogenous elicitor yeast extract (YE) was analyzed to establish any correlation between expression levels of transcript and withanolide accumulation. Effects on the transcript levels of WsDWF1 with each progressing ontogenic stages of W. somnifera was also carried out. Tissue-specific expression studies analyzing expression of WsDWF1 in various tissues such as leaf, root and stalk was also carried out. Full length cDNA of WsDWF1 contained an ORF of 1707 bp and encoded 65.8 kDa protein. Based upon elicitation studies using various abiotic and biotic chemicals such MeJA, SA, 2,4-D and YE, a positive correlation between withanolide accumulation and transcript profile of WsDWF1 was observed. Tissue-specific expression studies suggested higher expression of WsDWF1 in leaves followed by stalk and root tissues. A uniform increase in the transcript levels of WsDWF1 with each progressing developemental stage indicated increased substrate pool for phytosterol biosynthesis. Use of YE a known inhibitor of oxidosqualene cyclases (OSCs), showed correspondence with the increased transcript levels of WsDWF1 and an enhanced withanolide accumulation, possibly by re-routing of metabolite flux towards the downstream phytosterol biosynthesis. Present work describes WsDWF1 as a new player in withanogenesis, which can open up a fresh prospect for pathway engineering of W. somnifera.

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Enhancement of withanolide biosynthesis in Withania somnifera

Withania somnifera (L.) Dunal (Family, Solanaceae), is among the most valuable medicinal plants used in Ayurveda owing to its rich reservoir of pharmaceutically active secondary metabolites known as withanolides. Withanolides are C28-steroidal lactones having a triterpenoidal metabolic origin synthesised via mevalonate (MVA) pathway and methyl-D-erythritol-4-phosphate (MEP) pathway involving metabolic intermediacy of 24-methylene (C30-terpenoid) cholesterol. Phytochemical studies suggest differences in the content and/or nature of withanolides in different tissues of different chemotypes. Though development of genomic resources has provided information about putative genes encoding enzymes for biosynthesis of intermediate steps of terpenoid backbone, not much is known about their regulation and response to elicitation. In this study, we generated detailed molecular information about genes catalysing key regulatory steps of withanolide biosynthetic pathway. The full-length sequences of genes encoding enzymes for intermediate steps of terpenoid backbone biosynthesis and their paralogs have been characterized for their functional and structural properties as well as phylogeny using bioinformatics approach. The expression analysis suggests that these genes are differentially expressed in different tissues (with maximal expression in young leaf), chemotypes and in response to salicylic acid (SA) and methyl jasmonate (MJ) treatments. Sub-cellular localization studies suggest that both paralogs of sterol ∆-7 reductase (WsDWF5-1 and WsDWF5-2) are localized in the endoplasmic reticulum (ER) thus supporting their indispensible role in withanolide biosynthesis. Comprehensive information developed, in this study, will lead to elucidation of chemotype- as well as tissue-specific withanolide biosynthesis and development of new tools for functional genomics in this important medicinal plant.

and proteomic elements involved in withanolide biosynthesis.

(2013)Molecular studies on withanolide biosynthetic pathway gene(s) (Squalene synthase) from Withania somnifera. PhD thesis, CSIR-National Chemical Laboratory, Pune, India.

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