A Flow Cytometry-Based Ultrahigh-Throughput Screening Method for Directed Evolution of Oxidases
Lilin Feng, Liang Gao, Volkan Besirlioglu, Khalil Essani, Wittwer Malte, Tetiana Kurkina, Yu Ji, and Ulrich Schwaneberg
A versatile and robust flow cytometry-based screening system for the evolution of hydrogen peroxide-producing 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 the 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 (eGFPHis) 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 Km value and a D-AAO variant (L86M/G14/A48/T205) with a 4.2-fold higher kcat than their wildtypes. Thus, FlOxi can be used for the evolution of hydrogen peroxide-producing oxidases and applied for non-fluorescence substrates.
Lilin Feng and Liang Gao are financially supported by a Ph.D. scholarship from the China Scholarship Council (CSC No.201708330279; No.201708330280).
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Lilin Feng, Liang Gao, Volkan Besirlioglu, Khalil Essani, Wittwer Malte, Tetiana Kurkina, Yu Ji, and Ulrich Schwaneberg, Angewandte Chemie International Edition, doi.org/10.1002/ange.202214999
An ultrahigh-throughput screening platform “FlOxi” was developed by integrating oxidation/Fenton reaction cascade, Fe3+ mediated eGFPHis decoration, and fluorescence-activated-cell-sorting into a general strategy. FlOxi was validated in two evolution campaigns targeting two oxidases, exemplifying the suitability of the platform for screening oxidase variants in an ultrahigh-throughput manner.