A Flow Cytometry-Based Ultrahigh-Throughput Screening Method for Directed Evolution of Oxidases

Oxidases are of interest to chemical and pharmaceutical industries because they catalyze highly selective oxidations. However, oxidases found in nature often need to be re-engineered for synthetic applications. Herein, we developed a versatile and robust flow cytometry-based screening platform “FlOxi” for directed oxidase evolution. FlOxi utilizes hydrogen peroxide produced by oxidases expressed in E. coli to oxidize Fe²⁺ to Fe³⁺ (Fenton reaction). Fe³⁺ mediates the immobilization of a His₆-tagged eGFP (eGFP^His) on the E. coli cell surface, ensuring the identification of beneficial oxidase variants by flow cytometry. FlOxi was validated with two oxidases—a galactose oxidase (GalOx) and a D-amino acid oxidase (D-AAO)—yielding a GalOx variant (T521A) with a 4.4-fold lower K_m value and a D-AAO variant (L86M/G14/A48/T205) with a 4.2-fold higher k_cat than their wildtypes. Thus, FlOxi can be used for the evolution of hydrogen peroxide-producing oxidases and applied for non-fluorescent substrates.