Data Sheet 1_Subcellular localization of enzymes involved in the biosynthesis of digoxin in Digitalis lanata.pdf

Digoxin is a cardiac glycoside derived from the foxglove plant Digitalis lanata and is widely prescribed for treating heart failure and atrial fibrillation. Despite its medical importance, the cardiac glycoside biosynthetic pathway is only partially understood. Furthermore, the subcellular localizations of the three known enzymes for digoxin biosynthesis have escaped investigation. In this study, we identified the subcellular localization of the three known enzymes in the digoxin biosynthetic pathway, including the cytochrome P450 sterol side-chain cleaving enzyme (P450scc), the 3β-hydroxysteroid dehydrogenase (3βHSD), and the progesterone-5β-reductase 2 (P5βR2). Expressing these enzymes with a fluorescent tag in tobacco leaves revealed that the P450scc localized to the endoplasmic reticulum (ER), and the 3βHSD and P5βR2 localized to the cytosol. The ER-localization of the foxglove P450scc is of particular interest because the mammalian P450scc, or CYP11A1, is localized to the mitochondria. These findings provide key insights into the spatial organization of digoxin biosynthesis and guide future synthetic biology and metabolic engineering of digoxin biosynthesis in plants and microbes.