Enhanced Biosynthesis of 6’-Sialyllactose in <i>Escherichia coli</i> 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, <i>Escherichia coli</i> 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.