Human milk oligosaccharides (HMOs) are naturally occurring, non-digestible sugars found in human milk. They have recently become a popular target for industrial synthesis due to their positive effects on the developing gut microbiome and immune system of infants. Microbial synthesis has shown great promise in driving down the cost of these sugars and making them more available for consumers and researchers. The application of common metabolic engineering techniques such as gene knockouts, gene overexpression, and expression of exogenous genes has enabled the rational design of whole-cell biocatalysts which can produce increasingly complex HMOs. Herein, we discuss how these strategies have been applied to produce a variety of sugars from sialylated to complex fucosylated HMOs. With increased availability of HMOs, more research can be done to understand their beneficial effects.

A human sialyltransferase ST3GAL II (hST3GAL II) was successfully expressed in Escherichia coli as an active soluble fusion protein with an N-terminal maltose-binding protein (MBP) and a C-terminal hexa-histidine tag. It was used as an efficient catalyst in a one-pot multienzyme (OPME) sialylation system for high-yield production of the glycans of ganglioside GM1b and highly sialylated brain gangliosides GD1a and GT1b. Further sialylation of GM1b and GD1a glycans using a bacterial α2-8-sialyltransferase in another OPME sialylation reaction led to the formation of the glycans of GD1c and brain ganglioside GT1a, respectively. The lower reverse glycosylation activity of the recombinant hST3GAL II compared to its bacterial sialyltransferase counterpart simplifies the handling of enzymatic synthetic reactions and has an advantage for future use in automated chemoenzymatic synthetic processes.