High-Level Production of 2′-Fucosyllactose in Escherichia coli by Systematic Metabolic Engineering

2′-Fucosyllactose (2′-FL), one of the primary human milk oligosaccharides (HMOs), exerts a pivotal influence on early human development as well as health benefits on other life stages. In this study, a high-yielding strain for 2′-FL production was constructed in Escherichia coli BL21star(DE3) using a systematic metabolic engineering strategy. The α-1,2-fucosyltransferase BKHT from Helicobacter sp. 13S00401-1 was selected as the optimal enzyme for 2′-FL biosynthesis, and the best variant F304W of BKHT was obtained to improve 2′-FL biosynthesis using semirational enzyme modifications. A “push–pull” strategy was implemented to optimize the carbon flux within the 2′-FL biosynthetic pathways. After the enhancement of the availability of cofactor GTP and NADPH, the 2′-FL titer reached 13.71 g/L in a shake flask. Finally, the engineered strain EFL54 produced 136.71 g/L 2′-FL with a productivity of 1.85 g/(L h) in a 5 L fermentor. This work provides a concrete foundation for the industrial-scale production of 2′-FL.