(48e) Glycans-by-Design: Bottom-up Engineering of a Eukaryotic Protein Glycosylation Pathway In Escherichia Coli

Until recently, asparagine-linked (N-linked) protein glycosylation was thought to be the exclusive domain of eukaryotes. However, studies over the past decade have firmly established that certain bacteria also perform this important post-translational modification. However, bacterial N-glycans are structurally distinct from their eukaryotic counterparts, severely limiting the use of the bacterial N-glycosylation system for the production of therapeutic N-glycoproteins. Here we report the bottom-up engineering of a eukaryotic protein glycosylation pathway in the bacterium Escherichia coli. This involved the heterologous expression of four eukaryotic glycosyltransferases, including yeast uridine diphosphate-N-acetylglucosamine transferases (Alg13, Alg14) and mannosyltransferases (Alg1, Alg2), and a bacterial oligosaccharyltransferase (PglB). The resulting synthetic pathway enabled E. coli to produce secretory glycoproteins bearing N-linked eukaryotic trimannosyl core glycans (mannose₃-N-acetylglucosamine₂). Overall, the engineering of defined glycosylation pathways in E. coli sets the stage for further engineering of this host for the production of vaccines and therapeutics with even more structurally complex human-like glycans. Moreover, glycoengineered E. coli has the potential to serve as a model genetic system for deciphering the “glycosylation code” which governs the non-template driven synthesis of diverse glycans and their specific attachment to proteins.