Elucidation of the plant progesterone biosynthetic pathway and its application in a yeast cell factory

Progesterone and its steroidal derivatives, including androgens, estrogens, glucocorticoids and mineralocorticoids are extensively utilized in pharmacotherapy, serving as predominant agents in anti-inflammatory, contraceptive, and anticancer treatments. From the 1990s to the present, scientists attempted to biosynthesize steroids such as progesterone and hydrocortisone from sugars in engineered microbial strains expressing pathway enzymes derived from animal sources. However, the low activity of the cytochrome P450 sterol side-chain cleavage (P450scc) system and their mitochondrial compartmentalization have limited the development of this route. Therefore, discovering an efficient P450scc system and developing innovative strategies will be necessary to overcome this bottleneck. Here, we elucidated the complete biosynthetic pathway of progesterone in Marsdenia tenacissima, a medicinal plant rich in steroids. The pathway comprises four enzymes, the two P450scc enzymes MtCYP108 and MtCYP150, as well as the two 3β-hydroxysteroid dehydrogenase/Δ⁵-Δ⁴ isomerases (HSDs) MtHSD5 and MtHSD6. Unlike their animal counterparts, the plant-derived P450scc enzymes were found to be localized to the endoplasmic reticulum in yeast and plants, and using the plant-type cytochrome P450 reductase (CPR) as electron transfer chain. The plant-derived HSDs are cytoplasmic in yeast and plants, whereas animal-derived HSDs localize to the endoplasmic reticulum. Based on this discovery, we engineered a yeast-based cell factory capable of synthesizing 1.06 g/L of progesterone from a simple carbon source. This discovery lays the groundwork for the sustainable synthesis of steroid hormone drugs through the use of plant-based systems or microbial host cells.