LAUSR

Fucosyllactoses (FL), including 2′‐fucosyllactose (2′‐FL) and 3‐fucosyllactose (3‐FL), have garnered considerable interest for their value in newborn formula and pharmaceuticals. In this study, an engineered Escherichia coli was developed for high‐titer FL biosynthesis by introducing multi‐level metabolic engineering strategies, including (1) individual construction of the 2′/3‐FL‐producing strains through gene combination optimization of the GDP‐L‐fucose module; (2) screening of rate‐limiting enzymes (α‐1,2‐fucosyltransferase and α‐1,3‐fucosyltransferase); (3) analysis of critical intermediates and inactivation of competing pathways to redirect carbon fluxes to FL biosynthesis; (4) enhancement of the catalytic performance of rate‐limiting enzymes by the RBS screening, fusion peptides and multi‐copy gene cloning. The final strains EC49 and EM47 produced 9.36 g/L for 2′‐FL and 6.28 g/L for 3‐FL in shake flasks with a modified‐M9CA medium. Fed‐batch cultivations of the two strains generated 64.62 g/L of 2′‐FL and 40.68 g/L of 3‐FL in the 3‐L bioreactors, with yields of 0.65 mol 2′‐FL/mol lactose and 0.67 mol 3‐FL/mol lactose, respectively. This research provides a viable platform for other high‐value‐added compounds production in microbial cell factories.