Harnessing 3-Fucosyllactose: Dynamic Regulation Unlocks Efficient l -fucose Production in Escherichia coli

l-fucose, a key component of human milk oligosaccharides, which affects the intestinal health and immune development of infants and is widely used in infant formula food, functional food and nutritional supplements. To enable efficient and sustainable biosynthesis, we developed a novel synthetic route using 3-fucosyllactose as a precursor and employed Escherichia coli BL21star (DE3) as the host. A complete l-fucose synthesis system was established by introducing α-1,3-fucosyltransferase (FutM2) from Bacteroides gallinaceum and α-l-fucosidase (BiafcB) from Bifidobacterium longum subsp. We knocked out competing pathways and balanced the expression of modular enzymes. Additionally, a temperature-coupled mixed carbon source strategy and dynamic metabolic control were employed to decouple cell growth from product synthesis, thereby directing metabolic flux toward l-fucose production. In a 5 L fed-batch system, the engineered strain achieved an l-fucose titer of 86.22 g/L within 89 h. This stable, plasmid-free and antibiotic-free system provides a new way for the safe and large-scale production of l-fucose.