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.