Bioprospecting for bacterial oligosaccharyltransferases with relaxed acceptor site specificity

N-linked glycosylation is an important post-translational modification of proteins and is known to occur in all domains of life. As the central enzyme of all N-linked glycosylation pathways, the oligosaccharyltransferase (OST) is responsible for transferring an oligosaccharide from a lipid carrier in the membrane onto a target consensus sequence within a protein. A major difference between eukaryotic and bacterial glycosylation is that all bacterial OSTs studied so far (e.g., PglB from Campylobacter jejuni) glycosylate the motif D/E-X₁-N-X₂-S/T (where X₁ and X₂ are any amino acids except proline), whereas eukaryotic OSTs do not require aspartic or glutamic acid at the -2 position. We hypothesized that some bacteria may encode OSTs whose acceptor site specificity differs from that of prototypical bacterial OSTs such as PglB from C. jejuni or C. lari. To test this, we characterized 24 bacterial OSTs for their ability to transfer glycans onto a model glycoprotein containing either a canonical bacterial acceptor site (DQNAT) or an acceptor site lacking the D residue in the -2 position (AQNAT). Remarkably, four OSTs were identified that efficiently catalyzed glycan attachment to the AQNAT site. Some of these OSTs transferred glycans to an even wider array of acceptor sites including the native glycan acceptor site in human Fc domains. Taken together, these data confirm the existence of naturally occurring bacterial OSTs with greatly relaxed acceptor site specificity. By breaking the “minus 2” rule, these flexible biocatalysts pave the way for using glycoengineered bacteria to make therapeutic glycoproteins such as authentic human antibodies.