(671e) Directed Evolution of Oxygenases In Vivo Using a High-Throughput, Growth-Based Selection Platform

Oxygenases activate inert C-H bond with high specificity, which is difficult in chemical synthesis. However, engineering oxygenases for desired activity has been challenging mainly because of their highly complex catalytic mechanisms, which hampered the elucidation of structure-function relationship. We report the first growth-dependent selection platform to engineer both NADH- and NADPH-dependent monooxygenases in vivo with an unprecedentedly high throughput (~10⁷ per selection). Briefly, the selection scheme is based on metabolically engineered Escherichia coli strains which cannot recycle reduced cofactors under aerobic condition and thus rely on heterologous NAD(P)H-consuming enzymes to grow. We demonstrate the efficiency and universality of this selection platform by using it to improve the thermostability of a Baeyer–Villiger monooxygenase, to enhance the coupling efficiency of a cytochrome P450, and to alter the substrate specificity of a p-hydroxybenzoate hydroxylase through directed evolution. We envision the tools report here to greatly accelerate the development of catalysts in biomanufacturing.