Electricity-driven 7α-Hydroxylation of Steroid Catalyzed by Cytochrome P450 Monooxygenase in Engineered Yeast
NAD(P)H are the cofactors of the cytochrome P450 monooxygenase for the catalytic hydroxylation of steroids, which is notoriously challengeable to traditional synthetic chemistry due to the requirement of specificity and chirality. However, undersupply of NAD(P)H is a crucial bottleneck in restricting the conversion rate of P450-catalyzed steroidal hydroxylation. To address the problem of cofactors undersupply and enhance the biotransformation from dehydroepiandrosterone (DHEA) to 7α-OH-DHEA, an electricity-driven NAD(P)H regeneration shortcut was incorporated into a recombinant Saccharomyces cerevisiae harbouring 7α-hydroxylase (CYP7B1) to construct the P450 bioelectrocatalysis system (BES). The BES could collaboratively utilize electrons derived from both electrodes and the oxidation of carbon sources, enabling intracellular NAD(P)H regeneration via the concomitant electron shuttle (i.e., neutral red (NR))-mediated extracellular electron transfer (EET) pathway and the oxidation of glucose to facilitate the P450 enzyme-catalyzed hydroxylation of steroids. Thus, the 7α-OH-DHEA yield reached 288.6 ± 7.8 mg/L in the BES, ~2.4-folds of the counterpart in the absence of the EET (122.1 ± 3.7 mg/L). For the first time, the NR-mediated BES was exhibited to be an efficient strategy to enhance the steroids hydroxylation in engineered yeast, being promising for steroid conversion in pharmaceutical engineering.