Methods for producing oligosaccharide products such as difucosylated oligosaccharides are disclosed. The methods include culturing recombinant cells in a cell culture medium comprising L-fucose, an oligosaccharide acceptor, and a carbon source. The cells are cultured under conditions in which a first fucosyltransferase polypeptide having a first substrate selectivity (e.g., an 1-2-fucosyltransferase polypeptide), a second fucosyltransferase polypeptide having a second substrate specificity (e.g., an 1-3-fucosyltransferase polypeptide), a nucleotide sugar pyrophosphorylase polypeptide, a lactose transporter polypeptide, and an L-fucose transporter polypeptide are expressed, and in which the oligosaccharide acceptor is converted to the difucosylated oligosaccharide. Recombinant cells for use in the methods are also described.

The present invention relates inter alia to methods of biosynthetic production of QS-21, precursors and variants thereof, and to related aspects.

The present invention relates to primarily genetically modified microorganisms for in vivo synthesis of lacto-N-tetrose (LNT) and lacto-N-neotetrose (LNnT), and their fucosylated derivatives, and to uses of such microorganisms in methods of producing lacto-N-tetrose and lacto-N-neotetrose, and their fucosylated derivatives.

The invention provides the production of sugar-nucleotides and the isolation of sugar-nucleotides. In some embodiments the production or isolation are accomplished under acidic conditions. The production is a cell-free synthesis using enzymes, including immobilized enzymes. They may be accomplished using a one-pot reaction protocol. The synthesis may be used as a highly customizable and highly efficient cell-free manufacturing process. In some embodiments, the sugar-nucleotides are used to prepare UDP-Gal, lactose derivatives, and human milk oligosaccharides (HMOs).