Abstract
Withania somnifera (Ashwagandha) synthesizes a wide spectrum of triterpenoids that are produced via an intricate isoprenoid pathway whose biosynthetic and regulatory mechanism remains elusive. Their pharmacological examination position them as potent bioactive molecules, hence demanding their copious production. Previous investigations have revealed that P450 monooxygenases are pivotal enzymes involved in the biosynthetic machinery of various metabolites and assist in decorating their core skeletal structures. The present study entails the isolation and functional characterization of castasterone synthase (CYP85A69) from W. somnifera. The full length WsCYP85A69, having an open reading frame of 1413 bp, encodes 470 amino acid residues. Further, in vitro conversion of 6-deoxocastasterone into castasterone validated its oxidative functionality. Product formation was confirmed using LC-PDA-MS with a m/z value of 506 [M+ACN]+. In planta transient over-expression of WsCYP85A69 significantly enhanced castasterone, stigmasterol and withanolides (WS-I, WS-II, WS-III). Artificial micro-RNA mediated silencing of WsCYP85A69 resulted in the reduced accumulation of castasterone, stigmasterol and withanolides (WS-I, WS-II, WS-III). Altogether, these non-complementary approaches plausibly suggest a key role of WsCYP85A69 in the biosynthesis of castasterone and the accumulation of withanolides and stigmasterol. Furthermore, a promoter analysis of WsCYP85A69 resulted in the identification of several potential cis-regulatory elements. Elicitations, given on the basis of identified cis-regulatory elements, demonstrated methyl jasmonate as an effective inducer of WsCYP85A69. Overall, these empirical findings suggest that functional characterization of WsCYP85A69 may conceivably be helpful to unravel the mechanism of brassinosteroids biosynthesis and could also pave the way for targeted metabolic engineering.
Highlights
Triterpenoids are 30-carbon compounds that have fascinating structural frameworks with indispensable pharmacological properties (Sawai and Saito, 2011; Knoch et al, 2018)
A full-length Open reading frame (ORF) of the WsCYP85A69 (MK410296) gene included 1,413 bp nucleotides that codes for a protein of 470 amino acids. This sequence was submitted to BLASTx for a similarity search and revealed similarities with homologs of the CYP85A1 gene of Capsicum annum (GenBank accession number PHT91631.1) Solanum lycopersicum (GenBank accession number NP_001234263.1) and Nicotiana tabacum (GenBank accession number NP_001312136.1)
Active residues in ligand binding sites were predicted using the GALAXY web server and displayed as I112, H120, M244, T269, L270, S273, T277, E340, V344, R346, L403, F404, R409, C411, P412, G413, L416, G417 (Supplementary Figure S2). These entire features substantiate that WsCYP85A69 belongs to the cytochrome P450 monooxygenases (P450s) superfamily which mediates the biosynthesis of various secondary metabolites
Summary
Triterpenoids are 30-carbon compounds that have fascinating structural frameworks with indispensable pharmacological properties (Sawai and Saito, 2011; Knoch et al, 2018) These are pervasive in the plant kingdom and have become significant targets for metabolic engineering because of their diverse pharmacological properties (Sandjo and Kuete, 2013; Seki et al, 2015). Withania somnifera (L.) Dunal (Solanaceae) is a small shrub which grows copiously in various climatic conditions in India including tropical, sub-tropical and semi-temperate climates (Misra et al, 2010) It is a reputed multipurpose medicinal plant and is unique in synthesizing various types of secondary metabolites including withanolides, alkaloids and steroids, and phytosterols etc., (Bhat et al, 2012; Gupta et al, 2015). Owing to the endemic and therapeutic potential of W. somnifera, a metabolic engineering program is being executed for a higher and purposeful production of its characteristic phytoconstituents
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