Compositions and methods for rapid in vitro synthesis of bioconjugate vaccines in vitro via production and N-glycosylation of protein carriers in detoxified prokaryotic cell lysates

A method for synthesizing an N-glycosylated carrier protein for a polysaccharide via coordinated transcription, translation, and N-glycosylation in vitro, in a single vessel, the method comprising:

• transcribing and translating a carrier protein in a cell-free protein synthesis reaction, the carrier protein comprising an inserted consensus sequence, D/E-X1-N-X2-SIT or N-X-S/T, wherein X, X1 and X2 may be any natural or unnatural amino acid except proline;
• (ii) glycosylating the carrier protein in the cell-free protein synthesis reaction with at least one polysaccharide, wherein the at least one polysaccharide is at least one bacterial O-antigen;
• wherein the single vessel comprises a template encoding the carrier protein, an exogenous lipid-linked oligosaccharide (LLO) comprising the bacterial O-antigen, and one or more Escherichia coli (E. coli) cell lysates from engineered E. coli strains, wherein the engineered E. coli strains comprise:
• (a) a nucleic acid encoding an orthogonal or heterologous oligosaccharyltransferase (OST) which is expressed in the engineered E. coli strains;
• (b) one or both of:
• (1) a mutation of the endogenous lpxM gene wherein the mutation results in the reduced expression or and/or activity of the encoded myristoyltransferase;
• (2) a nucleic acid comprising an orthogonal or heterologous LpxE gene and encoding a lipidA 1-phosphatase which is expressed in the engineered E. coli strains;
• (c) a mutation in the endogenous waaL gene, wherein the mutation results in the reduced expression and/or activity of the encoded O-antigen ligase;
• (d) prokaryotic transcription and translation machinery.