High-Level De Novo Biosynthesis of 2′-Fucosyllactose by Metabolically Engineered Escherichia coli

2′-Fucosyllactose (2′-FL) is the most abundant oligosaccharide in human milk. In this study, a highly efficient biosynthetic route for 2′-FL production was designed via the de novo pathway of GDP-l-fucose using engineered Escherichia coli BL21­(DE3). Specifically, plasmid-based strains with previously deleted lacZ and wcaJ were further reconstructed by introducing de novo pathway genes and α1,2-fucosyltransferase-encoding wbgL to realize 2′-FL synthesis. The 2′-FL titer was enhanced to 3.92 g/L by further introducing rcsA and rcsB. Subsequently, the additional wbgL expression cassette was chromosomally integrated into recA locus to strengthen fucosylation reaction and a strong constitutive promoter (PJ23119) was used to replace the original promoters of manC-manB and gmd-wcaG to improve 2′-FL synthesis. The maximal 2′-FL titer reached 9.06 and 79.23 g/L in shake-flask and fed-batch cultivation, respectively. The 2′-FL productivity reached 1.45 g/L/h, showing remarkable production potential in large-scale industrial application.