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