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Abstract

Mucin-type O-glycosylation (O-glycosylation) is a relatively common post-translational modification that can confer proteins with new complexity and function. In Nature, O-glycosylation occurs in a non-templated manner and as such there can be considerable heterogeneity. As a result, the roles of O-glycans are often difficult to understand. This thesis focuses on the discovery and engineering of enzymes to manipulate glycoproteins to better determine the roles of different structures and to aid in their analysis. To discover and engineer these enzymes, I employed and developed a number of different high-throughput and ultrahigh-throughput screening methodologies. Ultrahigh-throughput screens are of particular interest as they are capable of screening >10⁵ clones/h and thus provide deep explorations of sequence space. Initially I was focused upon the development of glycosidases to catalyze the cleavage of a common O-glycan known as the sialyl T-antigen (STAg). Prior to this thesis, no enzyme was known to cleave this structure despite its ubiquity. First, via functional metagenomic screening I discovered that STAg hydrolase activity was present within a known family of glycosidases. I then applied ultrahigh-throughput droplet-based microfluidic screening to evolve one of these enzymes to better carry out this reaction. The most improved mutant from this screen was able to carry out STAg hydrolysis 140-fold more efficiently than did the wild-type enzyme. I then shifted my focus towards the development of screening methodologies for the engineering of diverse enzymes that act on O-glycoproteins. These methods utilize metabolically engineered E. coli that can synthesize mammalian glycoproteins to enable screening with highly complex substrates that would be difficult to access otherwise. Of note is that these assays also provide new means to engineer and analyse O-glycopeptidases. O-glycopeptidases are a class of enzyme for which characterization had been limited due to the inaccessibility of the substrates. However, with these newly developed methodologies I was able to readily characterize and evolve their activities. As a whole, this work provides new enzymatic tools for the deciphering of the roles of glycans as well as new methods for the development of additional enzymatic tools.