Efficient de novo synthesis of 3-fucosyllactose in Escherichia coli via enzyme engineering and pathway refinement

3-Fucosyllactose (3-FL), a functional oligosaccharide in human milk, holds great promise due to its health benefits. However, its microbial production is limited by the low activity of α-1,3-fucosyltransferase and the limited GDP-l-fucose supply. This study combined structure-based modeling and evolutionary analysis to identify five double-site mutants with enhanced 3-FL production. When the best-performing mutant N199V/K301P was expressed in double-copy, the 3-FL titer increased by 35 % (4.92 g/L) compared to the wild-type (3.65 g/L). To improve carbon flux and GDP-l-fucose supply, the engineered Escherichia coli strain L (BL21(DE3)ΔlacZΔwcaJΔnudDΔlon) was further modified by deleting mtlD, wcaE, and wcaI—the latter two functionally validated for the first time in 3-FL biosynthesis. Through the synergistic optimization of these strategies, the best strains l-MEI carrying double-copy S98R/D340E or N199V/K301P achieved 3-FL titers of 53.88 g/L and 54.64 g/L in 3 L fed-batch fermentations, which are at relatively high levels among currently reported titers.