Efficient production of 3-fucosyllactose in a plasmid-free engineered Escherichia coli through strengthening of the GDP-fucose pathway

Human milk oligosaccharides (HMOs) are bioactive oligosaccharides with characterization of complexity and indigestion, which support the progress of immune system, gut microenvironment, and brain in infants. In this work, 3-fucosyllactose (3-FL) was successfully synthesized in recombinant plasmid-free Escherichia coli by strengthening GDP-L-fucose pathway. For this, the native promoters of manB, manC, gmd, and wcaG in chromosome of E. coli were replaced by PJ23119, a constitutive promoter. To further increase the 3-FL production, multiple copies of fut3Bc (encoding α1,3-fucosyltransferase from Neobacillus cucumis) were integrated into the genome of engineered strains to replace the native genes arsB, ldhA, poxB, and manXYZ, respectively. The titer of 3-FL was boosted to 3.17 g/L via overexpressing manA with promoter PJ23119 in chromosome of the engineered hosts. Ultimately, in a 5 L bioreactor, the titer of 3-FL achieved 28.13 g/L by fed-batch culture at 60 h, with a productivity of 0.54 g/L/h and a yield of 0.95 mol/mol lactose, without using plasmids and antibiotics. Importantly, only 3-FL could be synthesized in fut3Bc-based strains without producing other HMOs, showing great potential in either the purity or productivity.