## 2 Biosynthesis of Withaferin A (WA)  
WA is a steroidal lactone belonging to the withanolide group, which was first isolated from the familiar medicinal plant _W. somifera_ in 1965 by [PERSON]. It is formed from naturally occurring withanolides, which are C-28 steroids with an ergostane-based skeleton functionalized o at C-26 and C-22 to form a \(\delta\) lactone. Furthermore, the ketone and hydroxyl groups functionally oxidize the C-1 group to form a 1-one [31; 32]. WA was also biosynthetically isolated from _Acnistus brevitforus_[33]. Chemoinformatic differentiation sterols, triterpenes, and withanolides indicated that withanolides may have originated from 24-methylene lophenol, lanosterol, campesterol, 24-methylene cholesterol metabolite analogs, or from the triterpene pathway's upstream intermediates [34]. Although conversion of the 24-methylene cholesterol into withanolides involves a complicated metabolic pathway, which includes glycosylation, hydroxylation, chain elongation, desaturation, cyclization, and epoxidation, that is not fully understood, several researchers have specified the biosynthesis of the responsible gene. However, the triterpeneoid pathway occurs by the isoprenogenesis process, which is a biogeochemical metabolic course for the determination of isoprene units such as dimethylallyl pyrophosphate and isopentenyl diphosphate [35; 36; 37; 38]. Similarly, isoprenogenesis arises in two separate ways in plants. The first is the classical mevalonate (MVA) pathway, which occurs in the cytosol, and the other is the non-mevalonate pathway, also known as the deoxyxylulose pathway (DOXP) or the newly discovered methyl erythreitol pathway (MEP) in plastids [39; 40]. In the MVA route, endophyte inoculation up-regulates the expression of HMGR with the concerned isopentenyl pyrophosphate (IPP), which is the predominant pathway [41]. In this case, the enzymes responsible for gene encoding in the transcription process have no effect on 1-deoxy-D-xylulose-5-phosphate reductase (DXR) and 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and may be the primary reason for the maximum productivity of withanolide components [41]. Biosynthesis, isopentenyl diphosphate (IPP) is a five-carbon precursor, a common compound in both MVA and MEP processes [42]. In the MVA pathway, cholesterol is the pertinent starting point for the biosynthesis of withanolides. Acetyl co-enzyme A obtained from activation of acetate is the first step of biotransformation of cholesterol. Two units of acetyl-CoA then convert to mevalonate through metabolization, and by losing one carbon atom, are metamorphosed into isopentenyl pyrophosphate (IPP). It is worth mentioning that the S-form of mevalonate is inert metabolically, while the R-form can produce terpen in the living system [42]. In contrast, pyruvate integrates with glyceraldehyde-3-phosphate in the MEP pathway, and the presence of DXS enzyme produces 1-deoxy-D-xylulose-5-phosphate (DOXP). Furthermore, DOXP is converted into 2-methylerythitol-4-phosphate (MEP) in the presence of DXR catalyst and finally turns into IPP [42; 43; 44]. Head-to-tail condensation of IPP with 3,3-dimethylallyl pyrophosphate (DMAPP) produces geranyl pyrophosphate (GPP). Further, farnesyl pyrophosphate (GPP) is obtained by the condensation of trans-geranyl pyrophosphate with another IPP molecule. Similarly, in a head-to-head manner, condensation of two molecules of farnesyl pyrophosphate catalyzed by the enzyme squalene synthase in the presence of NADPH yields squalene [45; 46]. In turn, squalene 2,3-epoxide is formed by atmospheric oxygen through an oxidation process that is catalyzed by NADPH-linked oxide. Then, ring closure leads to the formation of lanosterol, which can be converted into a few skeletons of obtusifoliol to steroidal triterpenoid (Delta 8, 14 sterol). Furthermore, the 24-methylenedolesterol biotransformation from lanosterol is not yet fully understood. However, 24-methylenedolesterol may be a biosynthetic precursor to steroidal lactones.