A rationally designed PglB mutant with increased in vivo O86-reactive AcrA signal for glycoprotein production.

Protein N-glycosylation is a fundamental post-translational modification in biology, biotechnology and vaccinology. The bacterial oligosaccharyltransferase PglB is used in one of the common approaches for the biosynthesis of antibody-drug conjugates and conjugate vaccines, catalyzing the transfer of glycans to specific asparagine residues in acceptor proteins. Despite its wide application, the practical use of PglB has long been constrained by limited in vivo productivity. Here, we report a rational engineering strategy to enhance Campylobacter jejuni PglB in vivo glycosylation efficiency by targeting its loop regions using a fragment-replacement approach previously developed in our laboratory. By redesigning flexible segments without perturbing the conserved catalytic core, we generated a mutant library of eight PglB variants. Functional evaluation was performed through an in vivo glycosylation assay in Escherichia coli O86:K61:B7 ΔwaaL::FRT, using AcrA as the acceptor protein. Remarkably, two variants (G7 and G8) produced stronger O86-reactive AcrA signals than wild-type PglB in this cellular assay. These results suggest that rational modulation of loop regions can modulate in vivo glycoprotein production. Our work provides an initial framework for exploring the performance of glycotransferases and expands the potential of PglB for glycoprotein production.