High-Level Difucosyllactose Biosynthesis via Metabolic and Transporter Engineering

Difucosyllactose (DFL) is a complex human milk oligosaccharide with significant prebiotic potential. In this study, we engineered Escherichia coli for high-titer biosynthesis of DFL via the 2′-fucosyllactose (2′-FL) intermediate pathway. By screening glycosyltransferases, we identified WbgL (α-1,2-fucosyltransferase) and FutM1 (α-1,3-fucosyltransferase) as the optimal enzyme pair and maximized their catalytic efficiency by optimizing gene copy numbers. To enhance precursor availability, we disrupted competitive pathways ( wcaI, wcaH, and nudK ) and redirected carbon flux by deleting pykA . Furthermore, transporter engineering enhanced synthesis efficiency through the deletion of emrE to prevent intermediate efflux and the overexpression of ompF to facilitate DFL secretion. Finally, implementing a continuous low-concentration lactose feeding strategy in a 5-L bioreactor achieved a record-breaking DFL titer of 110.3 g/L, the highest production level reported to date. This study demonstrates that integrating systematic metabolic engineering with refined process control provides a robust platform for the industrial production of complex oligosaccharides.