Engineering orthogonal human O-linked glycoprotein biosynthesis in bacteria

A major objective of synthetic glycobiology is to re-engineer existing cellular glycosylation pathways from the top down or construct non-natural ones from the bottom up for new and useful purposes. Here, we have developed a set of orthogonal pathways for eukaryotic O -linked protein glycosylation in Escherichia coli that installed the cancer-associated mucin-type glycans Tn, T, sialyl-Tn and sialyl-T onto serine residues in acceptor motifs derived from different human O- glycoproteins. These same glycoengineered bacteria were used to supply crude cell extracts enriched with glycosylation machinery that permitted cell-free construction of O- glycoproteins in a one-pot reaction. In addition, O -glycosylation-competent bacteria were able to generate an antigenically authentic Tn-MUC1 glycoform that exhibited reactivity with antibody 5E5, which specifically recognizes cancer-associated glycoforms of MUC1. We anticipate that the orthogonal glycoprotein biosynthesis pathways developed here will provide facile access to structurally diverse O- glycoforms for a range of important scientific and therapeutic applications. An orthogonal O -glycan biosynthesis system was engineered in Escherichia coli to support the production of glycoproteins displaying human mucin O -glycans, including Tn antigens, in living bacteria and in cell-free extracts.