Biosynthesis of a Polysaccharide-based Glycoconjugate Vaccine against Neonatal Meningitis-Causing <i> <i>Escherichia coli</i> </i> O45

Extraintestinal pathogenic <i><i>Escherichia coli</i></i> O45 is an emerging multidrug-resistant serotype. Herein, we developed a glycoengineered <i><i>E. coli</i></i> strain for efficient biosynthesis of O45 <i>O</i>-polysaccharide (OPS45) and the OPS45-based glycoconjugate vaccine. Systematic optimization enhanced the production of OPS45, a polysaccharide containing the rare sugars 6-deoxy-l-talose (6d-l-Tal) and <i>N</i>-acetylfucosamine (FucNAc), with structure confirmed by NMR. Using oligosaccharyltransferase-mediated conjugation in this glyco-optimized chassis strain, we generated a homogeneous cholera toxin B subunit (CTB)-OPS45 glycoconjugate with enhanced antigen loading yield (11.31 ± 0.59 mg/L vs 8.24 ± 0.075 mg/L). LC-MS/MS verified site-specific glycosylation on CTB. Immunization in mice elicited strong O45-specific IgG responses and conferred 90% protection against lethal neonatal meningitis-causing <i><i>Escherichia coli</i></i> (NMEC) infection, with a nearly 80% reduction in bacterial burden. These results demonstrate that our integrated biosynthesis and conjugation approach enables rapid and efficient production of a well-defined glycoconjugate vaccine, showing strong potential for combatting resistant NMEC and O45 infections.