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

Angewandte Chemie International Edition, vol. 62, pp. e202214999

Abstract

Abstract 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 Fe 2+ to Fe 3+ (Fenton reaction). Fe 3+ mediates the immobilization of a His 6 ‐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.

Authors 8

1. Lilin Feng Aachen Lehrstuhl für Biotechnologie

   DWI – Leibniz Institute for Interactive Materials · RWTH Aachen University

   Affiliation as printed

   DWI—Leibniz Institut für Interaktive Materialien Forckenbeckstraße 50 52074 Aachen Germany

   Lehrstuhl für Biotechnologie RWTH Aachen University Worringerweg 3 52074 Aachen Germany

2. Liang Gao Aachen Lehrstuhl für Biotechnologie

   RWTH Aachen University

   Affiliation as printed

   Lehrstuhl für Biotechnologie RWTH Aachen University Worringerweg 3 52074 Aachen Germany

3. Volkan Besirlioglu Aachen Lehrstuhl für Biotechnologie

   RWTH Aachen University

   Affiliation as printed

   Lehrstuhl für Biotechnologie RWTH Aachen University Worringerweg 3 52074 Aachen Germany

4. Khalil Essani Aachen Lehrstuhl für Biotechnologie

   RWTH Aachen University

   Affiliation as printed

   Lehrstuhl für Biotechnologie RWTH Aachen University Worringerweg 3 52074 Aachen Germany

5. Malte Wittwer Aachen Lehrstuhl für Biotechnologie

   RWTH Aachen University

   Affiliation as printed

   Lehrstuhl für Biotechnologie RWTH Aachen University Worringerweg 3 52074 Aachen Germany

6. Tetiana Kurkina Aachen Lehrstuhl für Biotechnologie

   RWTH Aachen University

   Affiliation as printed

   Lehrstuhl für Biotechnologie RWTH Aachen University Worringerweg 3 52074 Aachen Germany

7. Yu Ji Aachen Lehrstuhl für Biotechnologie

   RWTH Aachen University

   Affiliation as printed

   Lehrstuhl für Biotechnologie RWTH Aachen University Worringerweg 3 52074 Aachen Germany

8. Ulrich Schwaneberg corresponding Aachen Lehrstuhl für Biotechnologie Lehrstuhl fuer Biotechnologie

   DWI – Leibniz Institute for Interactive Materials · RWTH Aachen University

   Affiliation as printed

   DWI—Leibniz Institut für Interaktive Materialien Forckenbeckstraße 50 52074 Aachen Germany

   Lehrstuhl für Biotechnologie RWTH Aachen University Worringerweg 3 52074 Aachen Germany

   RWTH Aachen University, Lehrstuhl fuer Biotechnologie, Worringer Weg 3, 52074 Aachen, GERMANY