Module-Guided Metabolic Rewiring for Fucosyllactose Biosynthesis in Engineered Escherichia coli with Lactose De Novo Pathway

Fucosyllactose (FL) has garnered considerable attention for its benefits on infant health. In this study, we report an efficient E. coli cell factory to produce 2′/3-fucosyllactose (2′/3-FL) with lactose de novo pathway through metabolic network remodeling, including (1) modification of the PTSGlc system to enhance glucose internalization efficiency; (2) screening for β-1,4-galactosyltransferase (β-1,4-GalT) and introduction of lactose synthesis pathway; (3) eliminating inhibition of byproduct pathways; (4) constructing antibiotic-free and inducer-free FL strains; and (5) up-regulating the expression of genes in the GDP-l-fucose module. The final engineered strains BP10-3 and BP11-3 produced 4.36 g/L for 2′-FL and 3.23 g/L for 3-FL in shake flasks. In 3 L bioreactors, fed-batch cultivations of the two strains produced 40.44 g/L for 2′-FL and 30.42 g/L for 3-FL, yielding 0.63 and 0.69 g/g glucose, respectively. The strategy described in this work will help to engineer E. coli as a safe chassis for other lactose-independent HMOs production.