Efficient Biosynthesisof Sialyllacto‑<i>N</i>‑tetraose a by a MetabolicallyEngineered <i>Escherichia coli</i> BL21(DE3) Strain

Recently, the construction of metabolically engineered strains for the microbial synthesis of human milk oligosaccharides (HMOs) has attracted increasing attention. However, fewer efforts were made in the in vivo biosynthesis of complex HMOs, especially sialylated complex HMOs. In this study, we engineered Escherichia coli BL21(DE3) to efficiently produce sialyllacto-<i>N</i>-tetraose a (LST-a) efficiently. Three sequential glycosylation steps were introduced to construct the LST-a pathway, catalyzed by β1,3-<i>N</i>-acetylglucosaminylation, β1,3-galactosylation, and α2,3-sialylation. Pathway genes for cytidine 5′-monophospho (CMP)-<i>N</i>-acetylneuraminic acid (Neu5Ac) were introduced to support the sialylation donor supply. Production of LST-a was improved by deleting competitive genes of CMP-Neu5Ac synthesis, screening a more efficient α2,3-sialyltransferase, and combinatorial optimization of pathway gene expression. LST-a was finally produced with the titer of 1.235 and 4.85 g/L by shake-flask and fed-batch cultivation, respectively, demonstrating the feasibility of efficient microbial production of complex sialylated HMOs.