Efficient Production of 3′-Sialyllactose Using Escherichia coli

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 Escherichia coli BL21star(DE3)ΔlacZ through genomic integration. We first addressed the issue of metabolic competition by deleting crucial genes, nanA, nanK, nanE, and nanT, that are involved in the degradation of N-acetylneuraminic acid. This strategic gene knockout minimized the flux through competing pathways. The engineered Escherichia coli strain was subsequently transformed with the exogenous genes neuBCA and nST, enabling the de novo 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.