Combinatorial modular pathway engineering for guanosine 5′-diphosphate-L-fucose production in recombinant Escherichia coli

Background: Guanosine 5′-diphosphate (GDP)-L-fucose is a vital nucleotide sugar involved in the synthesis of fucosylated oligosaccharides, such as fucosylated human milk oligosaccharides, which play important roles in physiological and pathological processes. Results: : In this study, 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 in 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 regeneration was underpinned to promote biocatalysis. Taken together, the final engineered strain EWL37, which could achieve the titers of 18.33 mg/L in shake-flask cultivation, was able to produce 106.21 mg/L (4.28 mg/g DCW) GDP-L-fucose through fed-batch cultivation. Conclusion: To date, this is the first utilization of a modular pathway optimization approach to increase the production potential of the de novo synthesized GDP-L-fucose. In general, this study manifests that via combinatorial modular pathway engineering, the GDP-L-fucose biosynthesis can be significantly improved.