Leveraging Oxidative Stress to Regulate Redox Balance-Based, In Vivo Growth Selections for Oxygenase Engineering

Directed evolution methods based on high-throughput growth selection enable efficient discovery of enzymes with improved function in vivo. High-throughput selection is particularly useful when engineering oxygenases, which are sensitive to structural perturbations and prone to uncoupled activity. In this work, we combine the principle that reactive oxygen species (ROS) produced by uncoupled oxygenase activity are detrimental to cell fitness with a redox balance-based growth selection method for oxygenase engineering that enables concurrent advancement in catalytic activity and coupling efficiency. As a proof-of-concept, we engineered P450-BM3 for degradation of acenaphthene (ACN), a recalcitrant environmental pollutant. Selection of site-saturation mutagenesis libraries in E. coli strain MX203 identified P450-BM3 variants GVQ-AL and GVQ-D222N, which have both improved coupling efficiency and catalytic activity compared to the starting variant. Computational modeling indicates that the discovered mutations cooperatively optimize binding pocket shape complementarity to ACN, and shift the protein's conformational dynamics to favor the lid-closed, catalytically competent state. We further demonstrated that the selective pressure on coupling efficiency can be tuned by modulating cellular ROS defense mechanisms.