Enhanced production of 2-fucosylgalactose from renewable substrates via GDP-L-fucose pathway optimization in Escherichia coli

2-Fucosylgalactose (2-FG), a structural analog of fucosyl-oligosaccharides (FOSs) that represent a major component of human milk oligosaccharides, holds promise as both a neuronal growth enhancer and prebiotic. In this study, 2-FG was biosynthesized from renewable substrates—glycerol (a biodiesel byproduct) and galactose (a monosaccharide that can be obtained from red macroalgae)—using engineered Escherichia coli strain DF, which co-expresses the de novo GDP-L-fucose biosynthetic pathway and an α-1,2-fucosyltransferase. To improve GDP-L-fucose accumulation, four chromosomal genes were deleted in strain DF: pfkA and pgi, which modulate central carbon flux, and wcaJ and waaF, which divert GDP-L-fucose into competing pathways. The resulting strain, ΔPWWP_DF, achieved a 2-FG titer of 31.8 g/L in fed-batch fermentation—a 2.27-fold improvement over that of the parental DF strain. These findings demonstrate that strategic metabolic rewiring can enhance GDP-L-fucose availability and then 2-FG production, offering a robust microbial platform for the sustainable biosynthesis of FOSs.