Engineered bacteria are provided that produce modified lipid A and a polypeptide or polysaccharide antigens. In some aspects, immunogenic compositions are provided comprising a modified a lipid A and a polypeptide or polysaccharide antigen.

Disclosed are methods, systems, components, and compositions for cell-free synthesis of glycosylated proteins. The glycosylated proteins may be utilized in vaccines, including anti-bacterial vaccines. The glycosylated proteins may include a bacterial polysaccharide conjugated to a carrier, which may be utilized to generate an immune response in an immunized host against the polysaccharide conjugated to the carrier. The glycosylated proteins may be synthesized in cell-free glycoprotein synthesis (CFGpS) systems using prokaryote cell lysates that are enriched in components for glycoprotein synthesis such as oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs) including OSTs and LLOs associated with synthesis of bacterial O antigens.

Disclosed are protocols for preparing cell-free extracts preparation protocols that are enriched in membrane vesicles and use of the disclosed extract in cell-free glycoprotein synthesis methods and platforms for increasing glycoprotein yields.

The present disclosure provides mutated PglB oligosaccharyltransferase enzymes that have the ability to efficiently catalyze the transfer of a saccharide from a lipid carrier to an asparagine reissue in a glycosylation motif on a protein. Also provided are polynucleotides encoding the mutated PglB oligosaccharyltransferase enzymes, host cells capable of expressing the engineered PglB oligosaccharyltransferase enzymes, and methods of using the engineered PglB oligosaccharyltransferase enzymes to make N-glycosylated proteins. Also provided are N-glycosylated proteins that are made using the engineered PglB oligosaccharyltransferase enzymes.