Multidimensional Engineering of MG1655 for the Efficient Biosynthesis of Difucosyllactose.

Difucosyllactose (DFL), a representative bisfucosylated oligosaccharide found in human milk, has garnered significant attention due to its immense health benefits. To date, several plasmid-based engineered strains have been established for DFL synthesis. However, these strains face challenges such as antibiotic dependence and plasmid instability, which limit their commercial application in the food industry. For this, plasmid-free MG1655 strains were established by integrating multicopy numbers of and into the genome and overexpressing key genes involved in the GDP-l-fucose pathway. To enhance the catalytic efficiency, Fut3Bc was mutated based on AlphaFold 3, obtaining a beneficial mutant Fut3Bc (F24Y). The optimized plasmid-free strain, MGA2S-5, containing 2 copies of and 4 copies of (F24Y) in the genome was obtained. Eventually, strain MGA2S-5 synthesized 35.04 g/L DFL in a 7 L bioreactor by fed-batch cultivation, with no intermediate products remaining in the medium.