Engineered Biosynthesis of Glycoproteins with Eukaryotic N-Glycans In Escherichia Coli

Asparagine-linked (N-linked) glycosylation pathways have recently been discovered in bacteria. However, bacterial N-glycans are structurally distinct from their eukaryotic counterparts, rendering bacteria marginally useful for therapeutic applications. Here we report the engineering of a eukaryotic protein glycosylation pathway in the Gram-negative bacterium Escherichia coli. This involved the heterologous expression of four eukaryotic proteins, including yeast uridine diphosphate-N-acetylglucosamine transferases (Alg13, Alg14) and mannosyltransferases (Alg1, Alg2), and a bacterial oligosaccharyltransferase (PglB). The resulting synthetic pathway enabled a laboratory strain of E. coli to produce secretory glycoproteins bearing asparagine-linked eukaryotic trimannosyl core N-glycans (mannose₃-N-acetylglucosamine₂). This development opens the door for the production of therapeutic glycoproteins in E. coli and the biosynthesis of novel N-glycan structures.