Efficient Production of 3′-Sialyllactose Using <i>Escherichia coli</i>

3'-Sialyllactose (3'-SL), a key component of human milk oligosaccharides, provides significant health benefits and immune modulation, and is increasingly used in infant formula and dietary supplements. This study presents a novel approach for the efficient biosynthesis of 3'-SL using <i>Escherichia coli</i> BL21star(DE3)Δ<i>lacZ</i> through genomic integration. We first addressed the issue of metabolic competition by deleting crucial genes, <i>nanA</i>, <i>nanK</i>, <i>nanE</i>, and <i>nanT</i>, that are involved in the degradation of <i>N</i>-acetylneuraminic acid. This strategic gene knockout minimized the flux through competing pathways. The engineered <i>Escherichia coli</i> strain was subsequently transformed with the exogenous genes <i>neuBCA</i> and <i>nST</i>, enabling the <i>de novo</i> synthesis of 3'-SL. A modular metabolic engineering strategy was utilized to optimize the expression of key enzymes within the MSU module, enhancing and balancing the carbon flux distribution. Additionally, a cofactor regeneration strategy was implemented to increase CTP availability, which improved cofactor recycling and fine-tuned the metabolic pathway for maximal 3'-SL production. Transport protein screening was incorporated to further increase the extracellular concentration of 3'-SL, resulting in an unprecedented yield of 56.8 g/L in a 5L bioreactor fermentation, setting a new benchmark in the field.