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, N -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 Saccharomyces cerevisiae . This study presents the inaugural report demonstrating the synergistic synthesis of 3′-sialyllactose through three modules derived from Escherichia coli and S. cerevisiae, utilizing pyruvate, N -acetylglucosamine, lactose, and cytidine-5′-monophosphate as substrates. The induction and the whole-cell catalytic substrate dosage of the engineered E. coli 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.