Enhanced Biosynthesis of 6’-Sialyllactose in Escherichia coli via Systematic Metabolic Engineering

6’-Sialyllactose (6’-SL), a sialylated oligosaccharide belonging to human milk oligosaccharides (HMOs), finds extensive applications in food, pharmaceuticals, cosmetics, and particularly in infant nutrition and dietary supplements. In this study, Escherichia coli K12 MG1655 was subjected to metabolic engineering to enhance 6’-SL biosynthesis. Initial pathway optimization involved the deletion of competing metabolic pathway genes (<i>nanATEK</i>, <i>nagAB</i>, and <i>lacZ</i>). Following overexpression of NmCSS and Pd2,6ST, an initial titer of 0.98 g/L was achieved. Subsequent combinatorial expression of heterologous enzymes from diverse microbial sources increased the yield to 1.26 g/L. Through systematic optimization of promoter strength and ribosome binding site (RBS) elements, the 6’-SL titer was further elevated to 2.41 g/L. Implementation of structure-guided mutagenesis on both key enzymes resulted in a significant yield improvement to 4.27 g/L. Final engineering strategies included overexpression of rate-limiting enzymes in the CTP biosynthesis pathway. The engineered strain demonstrated a production capacity of 4.92 g/L in shake-flask cultures. We transferred the strain to a 5-L fermenter and achieved a final 6’-SL production of 12.82 g/L after 76 h of cultivation, demonstrating a 13-fold enhancement compared to the initial shake-flask culture.