Reverse Engineering Enzymes with High-Throughput Sequence-Function Mapping

High-throughput functional characterization can be paired with next-generation DNA sequencing technology to generate rich data sets of the mapping from protein sequence to function. The resulting sequence-function data sets can be used to explore the molecular basis of protein function in a comprehensive and unbiased manner. However, current high-throughput functional assays are restricted to a subset of protein functions that can be coupled to cellular growth or protein binding, severely limiting the types of proteins that can be analyzed. Here, we present a general method for characterizing millions of enzyme variants that utilizes droplet-based microfluidic screening technology. We apply this method to map the local sequence space of a glycosidase enzyme. Large sequence-function data sets allowed us to discover new sites within the enzyme that play important functional roles and to identify mutations that confer enhanced properties. Our results demonstrate the power of combining droplet microfluidic screening with next generation sequencing for understanding and engineering enzyme function.