Engineering Escherichia coli to biosynthesize O-polysaccharide-derived recombinant glycoconjugate vaccines against...

The development of effective vaccines against extraintestinal pathogenic Escherichia coli (ExPEC) serotypes O8 and O9a remains an unmet medical need. We established a sustainable biomanufacturing platform for glycoconjugate vaccines by engineering an E. coli chassis strain with optimized nucleotide sugar precursor supply and enhanced O-polysaccharide (OPS) biosynthesis. Genes related to competitive and catabolic pathways (gnd-rfbB, wcaM-wcaJ and wcaI-wza, glgC, ushA, pfkA/B) were systematically deleted using CRISPR-Cas9 and λ-RED genome editing, while the phosphotransferase system gene ptsA was overexpressed, yielding chassis strain EG01. This rational metabolic engineering increased O8- and O9a-OPS yields by 2.11-fold and 2.4-fold, respectively. Co-expression of Neisseria meningitidis glycosyltransferase PglL and carrier protein CTB enabled efficient protein glycosylation, improving conjugate yields by 2.59-fold (O8) and 4.18-fold (O9a) versus wild-type. Mass spectrometry confirmed site-specific O-glycosylation at CTB Thr19 with preserved OPS repeat-unit structure. The vaccine candidate demonstrated a favorable safety profile in mice, inducing only transient cytokine responses without systemic toxicity. Immunization elicited robust, durable Th1-biased IgG responses, conferring 80-90% protection against lethal challenge and significantly reducing bacterial loads in vivo. This work establishes an efficient, scalable glycoconjugate vaccine production platform, highlighting a promising metabolic engineering strategy to combat ExPEC infections.