Reshaping of a Glycoside Hydrolase Active Site through Expression-Compensated Droplet-Based Microfluidic Screening...

The glycosylation of proteins endows them with distinct biophysical properties and allows them to play fundamental roles in cellular communication. Much of our understanding of glycoproteins has derived from the ability to enzymatically manipulate glycan structures. In particular, selective cleavage of glycans from proteins simplifies the analysis of glycoproteins and the determination of structure-activity relationships. However, limited enzymatic tools are available for the study of mucin-type O-glycans. To address this, we carried out the directed evolution of a glycoside hydrolase to increase its ability to cleave the sialyl T-antigen, a ubiquitous O-glycan structure in humans. We employed ultrahigh-throughput droplet-based microfluidics to rapidly screen vast libraries of variants in pL-sized droplets, thus minimizing the quantities of complex substrate required. Furthermore, by use of fluorescent protein-fusion and ratiometric gating during droplet sorting we could account for varying expression levels and identify highly active hits that could have been overlooked due to lower expression levels. Within just two rounds of screening, we uncovered variants with 840-fold enhancements in activity and new specificities compared to those of the WT enzyme. This campaign highlights the versatility of glycoside hydrolases and provides a broadly applicable strategy to engineer enzymatic tools for glycomics through microfluidic screening.