Biosynthesis of human milk oligosaccharides (HMOs) in glycoengineered human cells.

Human milk oligosaccharides (HMOs) are unconjugated and structurally diverse glycans synthesized in the lactating mammary gland through the stepwise action of glycosyltransferases that extend a free lactose core. Several HMOs are capped with sialic acids, including 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL), that promote early-life microbiota development and contribute to immune system and neuronal functions. These health-promoting properties make sialylated HMOs attractive biomolecules for incorporation in infant nutrition and functional food products. Mammalian cell lines lack endogenous HMO production, limiting mechanistic studies of HMO biosynthesis and constraining production strategies based on human cells. Here, we developed a human cell-based strategy for the production of the two common sialyllactose isomers 3'-SL and 6'-SL in glycoengineered human embryonic kidney (HEK293) cells. We co-expressed LALBA and B4GALT1, that together form the lactose synthase complex, to introduce free lactose biosynthesis capacity into a genetically engineered human cell line without sialylation (HEK293ΔSia). Stable expression of either ST3GAL or ST6GAL isoenzymes in HEK293ΔSia cells revealed that ST3GAL3/4/5, and especially ST3GAL5, efficiently convert lactose into 3'-SL while ST6GAL1 and ST6GAL2 produce the 6'-SL isomer. These results provide insights into the in vivo ability of sialyltransferase isoenzymes to use lactose as substrate. Establishing HMOs biosynthesis pathways into controllable human cell systems offers an alternative strategy for production of HMOs and provides a starting point to unlock biosynthesis of more complex HMOs in human cells.