Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli

Campylobacter jejuni has a general N-linked protein glycosylation system that can be functionally transferred to Escherichia coli. In this study, we engineered E. coli cells in a way that two different pathways, protein N-glycosylation and lipopolysaccharide (LIPS) biosynthesis, converge at the step in which PgIB, the key enzyme of the C jejuni N-glycosylation system, transfers Opolysaccharide from a lipid carrier (undecaprenyl pyrophosphate) to an acceptor protein. PgIB was the only protein of the bacterial N-glycosylation machinery both necessary and sufficient for the transfer. The relaxed specificity of the PgIB oligosaccharyltransferase toward the glycan structure was exploited to create novel N-glycan structures containing two distinct E. coli or Pseudomonas aeruginosa O antigens. PgIB-mediated transfer of polysaccharides might be valuable for in vivo production of O polysaccharides-protein conjugates for use as antibacterial vaccines.