Regulation of Metabolic Pathways to Enhance Difucosyllactose Biosynthesis in <i>Escherichia coli</i>

Difucosyllactose (DFL), an important kind of fucosylated human milk oligosaccharides (HMOs), has garnered considerable attention due to its excellent physiological activities in infants. Previously, we achieved <i>de novo</i> biosynthesis of DFL; however, substantial residual intermediates of fucosyllactoses (FL) were detected. In this study, DFL biosynthesis was optimized, and residual FL were reduced by regulating metabolic pathways. Different plasmid combinations were used to regulate gene expression, achieving an optimal flux balance between 2'-FL and DFL. The expression level of key enzyme α-1,3-fucosyltransferase (α-1,3-FT, FucTa) was then enhanced by increasing plasmid copy number and integrating <i>fucTa</i> gene into the chromosome. Exocytosis of 2'-FL was reduced by deleting the sugar efflux transporter <i>setA</i> gene, thereby minimizing residual FL. Finally, strain BSF41 produced 55.3 g/L of DFL with only 2.59 g/L of residual FL in a 5 L fermentor, representing the highest reported titer to date. This study provides an important foundation for advancing the biosynthesis of fucosylated HMOs.