LAUSR

3'-Sialyllactose (3'-SL) is one of the most important and simplest sialylated human milk oligosaccharides. In this study, a plasmid-based pathway optimization along with chromosomal integration strategies was applied for 3'-SL production. Specifically, the precursor CMP-Neu5Ac synthesis pathway genes and α2,3-sialyltransferase-encoding gene were introduced into Escherichia coli BL21(DE3)ΔlacZ to realize 3'-SL synthesis. Genes nanA and nanK involved in Neu5Ac catabolism were further deleted to reduce the metabolic flux of competitive pathway. Several α2,3-sialyltransferases from different species were selected to evaluate the sialylation effect. The precursor pools were balanced and improved by optimizing key enzyme expression involved in the UDP-GlcNAc and CMP-Neu5Ac synthesis pathway. Finally, an additional α2,3-sialyltransferase expression cassette was integrated into chromosome to maximize 3'-SL synthesis, and 4.5 g/L extracellular 3'-SL was produced at a shake-flask level. The extracellular 3'-SL concentration was raised to 23.1 g/L in a 5 L bioreactor fermentation, which represents the highest extracellular value ever reported.