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

ABSTRACT: 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. col

SUBMITTER: Maxel S 

PROVIDER: S-EPMC10441625 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature