Subcellular localization of enzymes involved in the biosynthesis of digoxin in Digitalis lanata

Digoxin is a cardiac glycoside derived from the foxglove plant <i>Digitalis lanata</i> 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 (P450_scc), the 3<i>β</i>-hydroxysteroid dehydrogenase (3<i>β</i>HSD), and the progesterone-5<i>β</i>-reductase 2 (P5<i>β</i>R2). Expressing these enzymes with a fluorescent tag in tobacco leaves revealed that the P450_scc localized to the endoplasmic reticulum (ER), and the 3<i>β</i>HSD and P5<i>β</i>R2 localized to the cytosol. The ER-localization of the foxglove P450_scc is of particular interest because the mammalian P450_scc, 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.