Highly Efficient In Vivo Production of Sialyllacto-N-tetraose C via Screening of Beneficial β1,4-galactosyltransferase and α2,6-sialyltransferase

Biological production of human milk oligosaccharides (HMOs) using metabolically engineered strains is a research hotspot in food biotechnology, but less effort has been made on the biological production of sialylated complex HMOs. Sialyllacto-N-tetraose c is the only monosialylated complex HMO in the top 15 HMOs. In this study, the metabolic pathway of LST c was constructed in Escherichia coli BL21(DE3) by introducing three sequential glycosyltransferases: β1,3-N-acetylglucosaminyltransferase, β1,4-galactosyltransferase, and α2,6-sialyltransferase. The cytidine 5'-monophospho (CMP)-N-acetylneuraminic acid (Neu5Ac) pathway was enhanced to improve LST c production. The β1,4-galactosyltransferase from Helicobacter pylori J99 (HpGalT) and α2,6-sialyltransferase from Vespertiliibacter pulmonis (ED6ST) were screened as a pair of key glycosyltransferases for enhancing LST c production. The final engineered strain could produce 1.718 and 9.745 g/L LST c by shake-flask and fed-batch cultivation, respectively, indicating the feasibility of efficient biosynthesis of complex sialylated HMOs.