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 herein is a rapid genetics-free method for eliciting and detecting cryptic metabolites using an imaging mass spectrometry-based approach. An organism of choice is challenged with elicitors from a small molecule library. The molecules elicited are then imaged by mass spec, which allows for rapid identification of cryptic metabolites. These are then isolated and characterized. Employing the disclosed approach activated production of cryptic glycopeptides from an actinomycete bacterium. The molecules that result, the keratinimicins and keratinicyclins, are metabolites with important structural features. At least two of these, keratinimicins B and C, are highly bioactive against several pathogenic strains. This approach will allow for rapid activation and identification of cryptic metabolites from diverse microorganisms in the future.

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.

Culture media comprising manganese and methods of culturing cells to improve sialylation and glycosylation of glycoproteins are provided.

This disclosure is in the technical field of synthetic biology and metabolic engineering. The disclosure provides engineered viable bacteria having a reduced or abolished synthesis of poly-N-acetyl-glucosamine (PNAG), Enterobacterial Common Antigen (ECA), cellulose, colanic acid, core oligosaccharides, Osmoregulated Periplasmic Glucans and Glucosylglycerol (O), glycan, and trebalose. The disclosure further provides methods for the production of bioproduct by the viable bacteria and uses thereof. Furthermore, the disclosure is in the technical field of fermentation of metabolically engineered microorganisms producing bioproduct.

The present disclosure provides mutated PglB oligosaccharyltransferase enzymes that have the ability to efficiently catalyze the transfer of a saccharide from a lipid carrier to an asparagine reissue in a glycosylation motif on a protein. Also provided are polynucleotides encoding the mutated PglB oligosaccharyltransferase enzymes, host cells capable of expressing the engineered PglB oligosaccharyltransferase enzymes, and methods of using the engineered PglB oligosaccharyltransferase enzymes to make N-glycosylated proteins. Also provided are N-glycosylated proteins that are made using the engineered PglB oligosaccharyltransferase enzymes.

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 disclosure is in the technical field of synthetic biology and metabolic engineering. The disclosure provides engineered viable bacteria. In particular, the disclosure provides viable bacteria with reduced cell wall biosynthesis additionally modified for production of glycosylated product. The disclosure further provides methods of generating viable bacteria and uses thereof. Furthermore, the disclosure in the technical field of fermentation of metabolically engineered microorganisms producing glycosylated product.

The present invention relates to an enzyme-catalyzed process for producing UDP-galactose from low-cost substrates uridine monophosphate and D-galactose in a single reaction mixture. Said process can be operated (semi)continuously or in batch mode. Said process can be extended to uridine as starting material instead of uridine monophosphate. Further, said process can be adapted to produce galactosylated molecules and biomolecules including saccharides, proteins, peptides, glycoproteins or glycopeptides, particularly human milk oligosaccharides (HMO) and (monoclonal) antibodies.

The present invention relates to an enzyme-catalyzed process for producing UDP-N-acetyl-α-D-glucosamine (UDP-GlcNAc) from low-cost substrates uridine monophosphate and N-acetyl-D glucosamine in a single reaction mixture with immobilized or preferably co-immobilized enzymes. Uridine may be used as starting material instead of uridine monophosphate as well. Further, said process may be adapted to produce GlcNAcylated molecules and biomolecules including saccharides, particularly human milk oligosaccharides (HMO), proteins, peptides, glycoproteins, particularly antibodies, or glycopeptides, and bioconjugates, particularly carbohydrate conjugate vaccines and antibody-drug conjugates.