Combinatorial Modular Pathway Engineering for Guanosine 5′-Diphosphate- l -fucose Production in Recombinant Escherichia coli

Guanosine 5′-diphosphate (GDP)- l -fucose is an important nucleotide sugar involved in the synthesis of fucosylated oligosaccharides, such as fucosylated human milk oligosaccharides, which play important roles in physiological and pathological processes. Here, a combinatorial modular pathway engineering strategy was implemented to efficiently increase the intracellular titers of GDP- l -fucose in engineered Escherichia coli . The de novo GDP- l -fucose synthesis pathway was partitioned into two modules and fine-tuned at both transcriptional and translational levels, which remarkably improved the GDP- l -fucose production. In addition, the gene encoding the UDP-glucose lipid carrier transferase (WcaJ) was inactivated to eliminate the competing metabolite pathway from GDP- l -fucose to colanic acid. Furthermore, cofactors were regenerated to promote biocatalysis. Taken together, the final engineered strain EWL37, which could achieve a titer of 18.33 mg/L in shake-flask cultivation, showed 106.21 mg/L intracellular GDP- l -fucose accumulation and a DCW-specific GDP- l -fucose content of 4.28 mg/g through fed-batch cultivation. In general, this study demonstrated that the utilization of combinatorial modular pathway engineering significantly improved the de novo synthesis of GDP- l -fucose in engineered E. coli .