posted on 2024-03-04, 12:39authored byQihang Chen, Zikai Chao, Ke Wang, Xinglong Wang, Hao Meng, Xirong Liu, Xiaoyu Shan, Jingwen Zhou
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 C16β,
C17α, C21, and C17α/21 oxidation of PG and C11α
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
FeO. Finally, the developed P450 variants were employed to
construct efficient Escherichia coli catalytic systems, which further achieved 11α/β-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.