Modified host cells and hybrid oligosaccharides for use in bioconjugate production

A E. coli strain W3110 comprising a deletion of the waaL, wherein said E. coli strain comprises an expression vector comprising nucleic acids for expression in the host cell:

• a) nucleic acids that encode (i) glycosyltransferases that produce an oligosaccharide or polysaccharide repeat unit, wherein said repeat unit does not comprise a hexose at the reducing end, and wherein said oligosaccharide or polysaccharide repeat unit is derived from a donor oligosaccharide or polysaccharide repeat unit that comprises a hexose at the reducing end, and (ii) heterologous glycosyltransferases sufficient for synthesis of the donor oligosaccharide or polysaccharide repeat unit wherein the donor oligosaccharide or polysaccharide is that of S. pneumoniae;
• and
• b) a nucleic acid that encodes a carrier protein comprising an N-glycosylation consensus sequence;
• and
• c) a nucleic acid that encodes an oligosaccharyl transferase;
• d) a nucleic acid that encodes a wzy polymerase, wherein the polymerase catalyzes the production of a hybrid oligosaccharide or polysaccharide, wherein the hybrid oligosaccharide or polysaccharide (i) comprises a hexose monosaccharide derivative at the reducing end of the first repeat unit and (ii) comprises hexose monosaccharides at the reducing end of all other repeat units;
• e) nucleic acid that encodes a heterologous flippase of the donor polysaccharide or oligosaccharide gene cluster;
• f) nucleic acid that encodes RcsA;
• and
• g) nucleic acid that encodes an epimerase (in the instant the hybrid oligosaccharide or polysaccharide comprises a hexose monosaccharide derivative at the reducing end of the first repeat unit in addition to the hexose monosaccharide normally present at the reducing end of the first repeat unit of the donor oligosaccharide or polysaccharide).