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

β1–3-Linked galactosides such as Galβ1–3GlcNAcβOR 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 β1–3-galactosyltransferase (β3GalT) in large amounts. Previously reported E. coli β3GalT (EcWbgO) has been identified as a limiting factor for producing a β1–3-galactose-terminated human milk oligosaccharide lacto- N -tetraose (LNT) by fermentation. Here, we report the identification of an EcWbgO homologue from C. violaceum (Cvβ3GalT) which showed a high efficiency in catalyzing the formation of LNT from lacto- N -triose (LNT II). With the highly active Cvβ3GalT, multigram-scale (>10 g) synthesis of LNT from lactose was achieved using a sequential one-pot multienzyme (OPME) glycosylation process. The access to Cvβ3GalT 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 β3GalT were not available.