Rewiring CYP11A1-coupled electron transfer boosts de novo progesterone production in yeast.

CYP11A1-mediated catalysis is the rate-limiting step in the de novo biosynthesis of progesterone in yeast, and its relatively low catalytic efficiency largely results from insufficient electron transfer efficiency. In this study, we addressed this bottleneck by systematically reconstructing the electron transfer system (ETS) of CYP11A1. We found that, beyond its native Class I-type ETS (Adx/AdR), CYP11A1 can also function with diverse Class II-type ETSs (CPRs) across species. Screening identified the combination of CYP11A1 from Sus scrofa (SsCYP11A1) and CPR from Absidia orchidis (AoCPR) as the most effective pair. Furthermore, fusion of the two proteins via a flexible GGGS linker significantly enhanced catalytic efficiency. Subsequently, single-site mutational scanning of SsCYP11A1 using the protein language model ESM-2 identified the F75S variant, which increased progesterone titer by 2.43-fold compared to the wild type. Further analysis integrating residue flexibility prediction and site-directed mutagenesis revealed that the F75S mutation improves overall catalytic efficiency by strengthening interfacial interactions between SsCYP11A1 and AoCPR and stabilizing a catalytically favorable conformation. In addition, deletion of the vacuolar iron transporter CCC1 increased the availability of [2Fe-2S] clusters in the yeast cytosol, thereby enhancing Adx functionality and enabling the coordinated operation of Class I-type and Class II-type ETSs. Ultimately, in a 5 L bioreactor fermentation system, the engineered yeast chassis achieved a progesterone titer of 1594.15 ± 71.36 mg/L, representing the highest reported level of de novo progesterone production in yeast to date.