Engineering Escherichia coli to produce Bordetella pertussis oligosaccharide with multiple trisaccharide units

The immunogenicity of the pertussis vaccine can be significantly improved by adding Bordetella pertussis oligosaccharide with multiple trisaccharide units. The more trisaccharide units there are, the better the efficiency of the immune response induction. However, natural B. pertussis oligosaccharides usually contain only a single terminal trisaccharide unit. In addition, B. pertussis is pathogenic, and there are potential safety hazards when preparing oligosaccharides from B. pertussis. In this study, Escherichia coli MG1655 was engineered to produce B. pertussis oligosaccharides containing multiple trisaccharide units. Fifty-nine genes relevant to the biosynthesis of the O-antigen and core oligosaccharide of lipopolysaccharide, enterobacterial common antigen, and colanic acid were deleted in MG1655, resulting in strain MDCO020. Then, 25 genes relevant to the biosynthesis of the oligosaccharide antigen in B. pertussis and 3 genes relevant to the repeating trisaccharide unit in Pseudomonas aeruginosa PAO1 were overexpressed in MDCO020, resulting in the recombinant E. coli MDCO020/pWpBpD5. The production of B. pertussis oligosaccharide with multiple trisaccharide units by MDCO020/pWpBpD5 was confirmed by SDS-PAGE and 1H NMR analyses, and its immune response-stimulating activity was confirmed by using rabbit anti-pertussis serum.