Fig. 7: Lactone formation by CYP749B2 (W26O) and SDR (W26DH) generating withanoside V aglycone (16).

a Proposed lactone formation from (22R)-ergosta-5,24-diene-1α,3β,22-triol (14) to withanoside V aglycone (16) by CYP749B2 (W26O) and SDR (W26DH) and semisynthetic conversion of 16 to (22R)-ergosta-5,24-diene-1α,3β,22,26-tetrol (17). b Co-expression of CYPs from P. pruinosa (Pp) in N. benthamiana leading to formation of withanoside V aglycone (16) in comparison to an authentic reference compound. Extracted ion LC-MS chromatograms of m/z 443 (low resolution) are shown with the respective ion levels. c Co-expression of CYPs from P. pruinosa (Pp) in yeast leading to formation of (22R)-ergosta-5,24-diene-1α,3β,22,26-tetrol (17). Extracted ion LC-MS chromatograms of m/z 469 are shown with the respective ion levels. Withanoside V aglycone (16) was not detected in these strains and was only produced when SDR was additionally included (Supplementary Fig. 24). d Increased formation of withanoside V aglycone (16) in N. benthamiana upon co-expression of SDR homologues with 24ISO and CYP87G1/CYP88C7/CYP749B2 genes. Comparable results were obtained when genes only from W. somnifera (left) or only from P. pruinosa (right) were used, with the exception of PpSDR-b. The bar plots show means ± SEM and data points of six biological replicates. e In vitro assays with PpCYP749B2 (obtained as yeast microsomes containing a cytochrome P450 reductase (CPR)) and PpSDR-a/b (purified soluble protein produced in E. coli). Semisynthetic intermediate 17 was used as a substrate. Extracted ion LC-MS chromatograms of m/z 443 to monitor formation of withanoside V aglycone (16) are shown with the respective ion levels. W26O: Withanolide biosynthesis 26-oxidase; W26DH: withanolide biosynthesis 26-dehydrogenase. Budding_yeast icon by umasstr (https://github.com/umasstr) is licensed under CC0 (https://creativecommons.org/publicdomain/zero/1.0/) and was used without modifications. Nicotiana_benthamiana icon by Connor-Tansley (https://github.com/Ctansley) is licensed under CC-BY 4.0 Unported (https://creativecommons.org/licenses/by/4.0/) and was used without modifications. Source data are provided as a Source Data file.