Systematic pathway engineering of Escherichia coli for enhanced 3'-sialyllactose production.

3'-Sialyllactose (3'-SL), the simplest sialylated human milk oligosaccharide (HMO), is widely incorporated into infant formula due to its roles in shaping the gut microbiota, inhibiting pathogen adhesion, and supporting neurodevelopment. In this study, we developed an efficient microbial cell factory for 3'-SL production by enhancing carbon utilization and CTP regeneration. Following the identification of a highly active α-2,3-sialyltransferase, metabolic flux was redirected through pfkA deletion and fbaA overexpression. The precursor pool was strengthened by optimizing the expression of key enzymes involved in UDP-GlcNAc and CMP-Neu5Ac biosynthesis. To further improve pathway balance and stability, promoter engineering was applied to optimize the expression of four key genes (neuA, neuB, neuC and ST). The optimized multigene cassette was subsequently integrated into the chromosome using a one-step multicopy integration strategy, generating a plasmid-free and antibiotic marker-free production strain. The resulting strain achieved 3'-SL titers of 7.75 g L-1 in shake flasks and 102.18 g L-1 in a 5-L fed-batch bioreactor, representing, to the best of our knowledge, the highest reported level to date. This work demonstrates a robust strategy combining metabolic rewiring, adaptive gene expression tuning and multicopy genome integration, providing a versatile platform for the sustainable production of 3'-SL and other high-value HMOs.