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

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 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‐fluorescent substrates.

In Situ Glycan Analysis and Editing in Living Systems

“…Birmingham et al improved the activity and specificity of GAO to oxidize 5-hydroxymethylfurfural, an important renewable feedstock . Also recently, Feng et al developed a directed evolution screening platform on the basis of flow cytometry and screened for a mutant GAO variant (T521A) with a K m value 4.4-fold lower than that of the wild-type GAO . In addition to regulating enzyme specificity and activity, spatiotemporal control of enzyme activity is important for improving the specificity of glycan labeling.…”

“… 62 Also recently, Feng et al developed a directed evolution screening platform on the basis of flow cytometry and screened for a mutant GAO variant (T521A) with a K m value 4.4-fold lower than that of the wild-type GAO. 63 In addition to regulating enzyme specificity and activity, spatiotemporal control of enzyme activity is important for improving the specificity of glycan labeling. Ding and Ju et al has developed a series of strategies to modulate GAO activity via metal–organic framework (MOF) ( Figure 2 e) 64 or polymer 65 , 66 caging.…”