Engineered Escherichia coli as a Microbial Cell Factory for De Novo 6′-Sialyllactose Biosynthesis

6′-Sialyllactose (6′-SL), one of the most abundant and structurally simplest sialyllactoses in human milk, represents a critical target for biomanufacturing. The development of high-performance microbial cell factories offers a promising approach for industrial-scale biosynthesis. In this study, we first established a de novo 6′-SL pathway by coexpressing key synthetic genes (neuB, neuC, neuA, and α2,6-SiaT) through a single-plasmid system in Escherichia coli. Subsequent inactivation of the nanA, nanK, nanE, and nanT genes in DH5α generated strain DAT01, demonstrating shake flask production titers of 1.36 g/L. Through systematic evaluation of α2,6-sias from diverse microbial sources and comparative analyses across multiple engineered E. coli chassis strains, we identified bst* as the optimal variant, culminating in strain DAT03 with 1.67 g/L productivity. We further investigated the physiological consequences of pfkA, pfkB, and murQ gene deletions on biomass accumulation and product synthesis. Combinatorial optimization of promoter strength and ribosome binding sites achieved enhanced α2,6-SiaT expression, resulting in strain DAT07 that produced 3.42 g/L of 6′-SL in shake flasks. Scale-up fermentation in a 5 L bioreactor yielded 30.18 g/L at 85 h, corresponding to a volumetric productivity of 0.36 g/L/h. This work validates the efficacy of our modular metabolic engineering strategy and establishes a robust platform for 6′-SL bioproduction.