Engineering Escherichia coli for the efficient biosynthesis of 6′-sialyllactose

6′-Sialyllactose (6′-SL), an abundant sialylated human milk oligosaccharide, has attracted considerable attention for its potential applications in nutritional healthcare and pharmaceutical utilization. In this work, a whole-cell biotransformation method was constructed for high-titer 6′-SL synthesis in E. coli BL21 (DE3). Genes neuB, neuC and neuA were initially introduced to enable CMP-Neu5Ac biosynthesis, and pst6-224, lacY were additionally transferred into 6′-SL metabolic pathway to gain the initiating strain BZ1, yielding 0.51 g/L of 6′-SL. Subsequently, the titer of 6′-SL was significantly maximized with modular metabolic engineering, optimization of efficacious availability of precursors UDP-GlcNAc and CMP-Neu5Ac, knockdown of metabolic bypass genes lacZ, nanA, pfkA, nagB, and screening of a highly efficient a2,6-sialyltransferase for functional replacement. We obtained a high-yielding 6′-SL strain BZAPN3 with titer of 3.98 g/L, 7.80-fold higher than the original strain BZ1. In 3-L bioreactor, fed-batch fermentation yielded 22.85 g/L of 6′-SL, with a specific yield and lactose conversion of 0.31 g/L/h and 0.494 mol 6′-SL/mol, respectively. This work offers a strategy for the efficient production of 6′-SL in E. coli.