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 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 Oglycoproteins. 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.
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