GlycoSNAP: A High-Throughput Screening Methodology for Engineering Designer Glycosylation Enzymes

The Campylobacter jejuni protein glycosylation locus (pgl) encodes enzymes for asparagine-linked (N-linked) glycosylation and serves as the prototype for N-glycosylation in bacteria. This pathway has been functionally transferred into Escherichia coli, thereby enabling efficient N-linked glycosylation of acceptor proteins with the C. jejuni heptasaccharide in this genetically tractable host. However, expansion of this bacterial system to encompass the vast biological diversity of potential glycans and target proteins often requires engineering of the non-native enzymes to isolate variants with optimal or altered functions. Here, we describe a genetic assay named glycoSNAP (glycosylation of secreted N-linked acceptor proteins) that facilitates engineering of glycosylation enzymes directly in E. coli. To date, this method has been successfully applied to screen (1) combinatorial libraries of bacterial oligosaccharyltransferases (OSTs) for relaxed substrate specificity and (2) acceptor site libraries to identify sequons recognized by natural OSTs as well as engineered variants.