Methods of producing bioconjugates of e. coli o-antigen polysaccharides, compositions thereof, and methods of use thereof

A recombinant host cell for preparing a bioconjugate of an E. coli Ox-antigen polysaccharide covalently linked to a carrier protein, the recombinant host cell comprising:

• a. a nucleotide sequence of an rfb gene cluster for the Ox-antigen polysaccharide;
• b. a nucleotide sequence encoding the carrier protein comprising at least one glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO:
• 1;
• and
• c. a nucleotide sequence encoding an oligosaccharyl transferase PglBy
• wherein:
• the PglBy comprises the amino acid mutation N311V relative to a wild-type PglB having the amino acid sequence of SEQ ID NO:
• 6, and
• the Ox-antigen is O1A antigen polysaccharide, glucosylated O4 antigen polysaccharide, O6A antigen polysaccharide, O15 antigen polysaccharide, O16 antigen polysaccharide, or O75 antigen polysaccharide,
• and when the Ox-antigen is glucosylated O4 antigen polysaccharide, the recombinant host cell further comprises a sequence encoding a glucosyltransferase GtrS that modifies an E. coli O4 antigen polysaccharide by addition of glucose to produce the E. coli glucosylated O4 antigen polysaccharide, and nucleotide sequences encoding a translocase GtrA and a glycosyltransferase GtrB, wherein the translocase GtrA translocates bactoprenol linked glucose and the glycosyltransferase GtrB glucosylates bactoprenol.