A Cellular Coupling Strategy Leveraging Pathway Modularization and Cofactor Regeneration for the Biosynthesis of 3′-Sialyllactose

3'-Sialyllactose is a major sialylated human milk oligosaccharide that plays an important role in promoting infant development and supporting various physiological functions critical for early life. In this study, <i>N</i>-acetylneuraminic acid and 3'-sialyllactose were successfully synthesized utilizing four genes across three modules through a whole-cell catalysis. Among these, Module III was employed to facilitate the conversion of cytidine-5'-monophosphate to cytidine 5'-triphosphate utilizing the intrinsic enzyme system of <i>Saccharomyces cerevisiae</i>. This study presents the inaugural report demonstrating the synergistic synthesis of 3'-sialyllactose through three modules derived from <i>Escherichia coli</i> and <i>S. cerevisiae</i>, utilizing pyruvate, <i>N</i>-acetylglucosamine, lactose, and cytidine-5'-monophosphate as substrates. The induction and the whole-cell catalytic substrate dosage of the engineered <i>E. coli</i> JM109(DE3) were optimized, leading to a 3'-sialyllactose titer of 71.62 g/L. Ultimately, the strain underwent culture and whole-cell catalysis in a 5 L bioreactor, achieving a maximum titer of 78.03 g/L for 3'-sialyllactose.