Simplification of Corticosteroids Biosynthetic Pathway by Engineering P450BM3

Synthesis of corticosteroids, particularly hydrocortisone, is challenging owing to the complex network requiring pairing of cytochrome P450s with cytochrome P450 reductase (CPR) for achieving regionally selective hydroxylation modifications at multiple sites. Herein, we engineered a self-sufficient P450BM3 (CYP102A1 from Bacillus megaterium) for effectively reducing the traditionally complex, multienzyme cascade process (three steps and six enzymes) of hydrocortisone synthesis from progesterone (PG) to a simplified two-step process involving at least two enzymes. Driven by computational simulation-guided substrate access channel and heme center pocket engineering, a series of P450BM3 variants were gradually designed with the ability to catalyze C16 beta, C17 alpha, C21, and C17 alpha/21 oxidation of PG and C11 alpha oxidation of cortexolone (c). Subsequently, molecular dynamics simulations with an oxy-ferrous model of P450BM3 variants revealed that the glycine mutations of residues that are repulsive to the substrate allow for more stable exposure of the substrate above Fe=O. Finally, the developed P450 variants were employed to construct efficient Escherichia coli catalytic systems, which further achieved 11 alpha/beta-hydrocortisone (f/e) production in one pot from 1 g/L PG at a molar conversion rate of 81 and 84% (912 and 955 mg/L), respectively. Thus, this study provides feasible strategies for simplifying the biosynthetic steps and biocatalysts for steroidal pharmaceutical production.