A Bacterial β1-3-Galactosyltransferase Enables Multigram-Scale Synthesis of Human Milk Lacto-N-tetraose (LNT) and Its Fucosides

beta 1-3-Linked galactosides such as Gal beta 1-3G1cNAc beta OR are common carbohydrate motifs found in human milk oligosaccharides (HMOSs), glycolipids, and glycoproteins. Efficient and scalable enzymatic syntheses of these structures have proven challenging because of the lack of access to a highly active beta 1-3-galactosyltransferase (beta 3Ga1T) in large amounts. Previously reported E. coli beta 3GalT (EcWbgO) has been identified as a limiting factor for producing a beta 1-3-galactose-terminated human milk oligo-saccharide lacto-N-tetraose (LNT) by fermentation. Here, we report the identification of an EcWbgO homologue from C. violaceum (Cv beta 3GalT) which showed a high efficiency in catalyzing the formation of LNT from lacto-N-triose (LNT II). With the highly active Cv beta 3GalT, multigram-scale (>10 g) synthesis of LNT from lactose was achieved using a sequential one-pot multienzyme (OPME) glycosylation process. The access to Cv beta 3GalT enabled enzymatic synthesis of several fucosylated HMOSs with or without further sialylation, including LNFP II, S-LNF II, LNDFH I, LNFP V, and DiFuc-LNT. Among these, LNFP V and DiFuc-LNT would not be accessible by enzymatic synthesis if an active beta 3GalT were not available.