Winning the numbers game in enzyme evolution – fast screening methods for improved biotechnology proteins

[Display omitted] •Overview of (ultra)high-throughput screening assay technologies.•Focus on sample throughput for measuring enzyme activity, substrate scope, and stability to process conditions.•Functional screening methods for biocatalysts from various enzyme classes.•Emphasis on development of universal assay strategies.•Novel screening protocols for microfluidic cell or droplet sorting. The booming demand for environmentally benign industrial processes relies on the ability to quickly find or engineer a biocatalyst suitable to ideal process conditions. Both metagenomic approaches and directed evolution involve the screening of huge libraries of protein variants, which can only be managed reasonably by flexible platforms for (ultra)high-throughput profiling against the desired criteria. Here, we review the most recent additions toward a growing toolbox of versatile assays using fluorescence, absorbance and mass spectrometry readouts. While conventional solution based high-throughput screening in microtiter plate formats is still important, the implementation of novel screening protocols for microfluidic cell or droplet sorting systems supports technological advances for ultra-high-frequency screening that now can dramatically reduce the timescale of engineering projects. We discuss practical issues of scope, scalability, sensitivity and stereoselectivity for the improvement of biotechnologically relevant enzymes from different classes.