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 (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.

Advances in ultrahigh-throughput screening for directed enzyme evolution.

Enzymes are versatile catalysts and their synthetic potential has been recognized for a long time. In order to exploit their full potential, enzymes often need to be re-engineered or optimized for a given application. (Semi-) rational design has emerged as a powerful means to engineer proteins, but requires detailed knowledge about structure function relationships. In turn, directed evolution methodologies, which consist of iterative rounds of diversity generation and screening, can improve an enzyme's properties with virtually no structural knowledge. Current diversity generation methods grant us access to a vast sequence space (libraries of >1012 enzyme variants) that may hide yet unexplored catalytic activities and selectivity. However, the time investment for conventional agar plate or microtiter plate-based screening assays represents a major bottleneck in directed evolution and limits the improvements that are obtainable in reasonable time. Ultrahigh-throughput screening (uHTS) methods dramatically increase the number of screening events per time, which is crucial to speed up biocatalyst design, and to widen our knowledge about sequence function relationships. In this review, we summarize recent advances in uHTS for directed enzyme evolution. We shed light on the importance of compartmentalization to preserve the essential link between genotype and phenotype and discuss how cells and biomimetic compartments can be applied to serve this function. Finally, we discuss how uHTS can inspire novel functional metagenomics approaches to identify natural biocatalysts for novel chemical transformations.