Abstract GDP-l-fucose, the key substrate for fucosyloligosaccharide biosynthesis, has been synthesized via a de novo pathway in bacteria. In the present study, genes for GDP-l-fucose biosynthesis were cloned into the expression vector pET-28a (+) to construct five E. coli strains, with recombinant enzymes being purified by using Ni–NTA chromatography. Following optimization of the 3-step reaction, Glk, ManB and ManC were added to the reaction mixture, after which Gmd and WcaG were added to overcome feedback inhibition from the end-product to produce GDP-l-fucose at 178.6 mg/l, with a yield of 14.1%. Our studies provide the basis for using cell-free enzyme production of GDP-l-fucose.
