Described herein are oligosaccharyl transferases for use in N-glycosylating proteins of interest in vitro and in host cells. Methods for using such oligosaccharyl transferases, nucleic acids encoding such oligosaccharyl transferases, and host cells comprising such oligosaccharyl transferases are also provided herein. Glycoconjugates generated by using such oligosaccharyl transferases are also provided herein.

Disclosed are methods, systems, components, and compositions for cell-free synthesis of glycosylated carrier proteins. The glycosylated carrier proteins may be utilized in vaccines, including anti-bacterial vaccines. The glycosylated carrier proteins may include a bacterial polysaccharide conjugated to a carrier, which may be utilized to generate an immune response in an immunized host against the polysaccharide conjugated to the carrier. The glycosylated carrier proteins may be synthesized in cell-free glycoprotein synthesis (CFGpS) systems using prokaryote cell lysates that are enriched in components for glycoprotein synthesis such as oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs) including OSTs and LLOs associated with synthesis of bacterial O antigens.

The present invention is in the technical field of synthetic biology and metabolic engineering. More particularly, the present invention is in the technical field of fermentation of metabolically engineered host cells. The present invention describes a method of making sialylated oligosaccharide by fermentation with a genetically modified cell, as well as to the genetically modified cell used in the method. The genetically modified cell comprises at least one nucleic acid sequence coding for an enzyme involved in sialylated oligosaccharide synthesis and at least one nucleic acid expressing a membrane protein.

Disclosed are methods, systems, components, and compositions for cell-free synthesis of glycosylated proteins. The glycosylated proteins may be utilized in vaccines, including anti-bacterial vaccines. The glycosylated proteins may include a bacterial polysaccharide conjugated to a carrier, which may be utilized to generate an immune response in an immunized host against the polysaccharide conjugated to the carrier. The glycosylated proteins may be synthesized in cell-free glycoprotein synthesis (CFGpS) systems using prokaryote cell lysates that are enriched in components for glycoprotein synthesis such as oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs) including OSTs and LLOs associated with synthesis of bacterial O antigens.

Methods for recombinant production of steviol glycoside and compositions containing steviol glycosides are provided by this invention.

Disclosed are methods, systems, components, and compositions for cell-free synthesis of glycosylated carrier proteins. The glycosylated carrier proteins may be utilized in vaccines, including anti-bacterial vaccines. The glycosylated carrier proteins may include a bacterial polysaccharide conjugated to a carrier, which may be utilized to generate an immune response in an immunized host against the polysaccharide conjugated to the carrier. The glycosylated carrier proteins may be synthesized in cell-free glycoprotein synthesis (CFGpS) systems using prokaryote cell lysates that are enriched in components for glycoprotein synthesis such as oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs) including OSTs and LLOs associated with synthesis of bacterial O antigens.

Disclosed are protocols for preparing cell-free extracts preparation protocols that are enriched in membrane vesicles and use of the disclosed extract in cell-free glycoprotein synthesis methods and platforms for increasing glycoprotein yields.

Provided herein are enzymatic compositions for protein O-glycosylation and sialylation, methods and systems associated therewith. In particular, the composition for in vivo sialylation of therapeutic proteins. The composition comprises a polypeptide N-acetylgalactosaminyltransferase; a β-1,3-galactosyltransferase; an UDP-Glc/GlcNAc 4-epimerase; a disulfide bond isomerase; and an α-2,3-sialyltransferase or an α-2,6-sialyltransferase. Furthermore, provided herein are compositions for efficient and complete O-glycosylation and di-sialylation of therapeutic proteins.

The present application provides methods and uses of O-oligosaccharyltransferase (O-OTases) for generating vaccines. In particular, the present application provides a method of synthesizing a glycoprotein comprising glycosylation of pilin-like protein ComP using a Pg1L.sub.ComP O-OTase. Uses of glycoproteins synthesized by glycosylating ComP using Pg1L.sub.ComP O-OTase, particularly for the preparation of vaccines and the like, including a vaccine to Streptococcus, is also provided.

Methods of producing bioconjugates of O-antigen polysaccharides covalently linked to a carrier protein using recombinant host cells are provided. The recombinant host cells used in the methods described herein encode a particular oligosaccharyl transferase enzyme depending on the O-antigen polysaccharide bioconjugate to be produced. The oligosaccharyl transferase enzymes can be PglB oligosaccharyl transferase or variants thereof. Also provided are compositions containing the bioconjugates, and methods of using the bioconjugates and compositions described herein to vaccinate a subject against extra-intestinal pathogenic E. coli. (ExPEC).