Enhancing GDP-fucose production in recombinant Escherichia coli by metabolic pathway engineering

• The salvage pathway for GDP-fucose biosynthesis from Bacterioides fragilis was introduced into Escherichia coli.
• The biosynthesis of guanosine 5′-triphosphate (GTP), an essential substrate for GDP-fucose biosynthesis, was enhanced.
• The production capacities of metabolically engineered strains bearing different combinations of recombinant enzymes were compared.
• The recombinant strain containing enzymes Fkp, Gpt, Gmk and Ndk have a better productivity.
• The established schemes in this work also have the advantage to be a potential avenue to GDP-fucose analogs.

Guanosine 5′-diphosphate (GDP)-fucose is the indispensible donor substrate for fucosyltransferase-catalyzed synthesis of fucose-containing biomolecules, which have been found involving in various biological functions. In this work, the salvage pathway for GDP-fucose biosynthesis from Bacterioides fragilis was introduced into Escherichia coli. Besides, the biosynthesis of guanosine 5′-triphosphate (GTP), an essential substrate for GDP-fucose biosynthesis, was enhanced via overexpression of enzymes involved in the salvage pathway of GTP biosynthesis. The production capacities of metabolically engineered strains bearing different combinations of recombinant enzymes were compared. The shake flask fermentation of the strain expressing Fkp, Gpt, Gmk and Ndk obtained the maximum GDP-fucose content of 4.6 ± 0.22 μmol/g (dry cell mass), which is 4.2 fold that of the strain only expressing Fkp. Through fed-batch fermentation, the GDP-fucose content further rose to 6.6 ± 0.14 μmol/g (dry cell mass). In addition to a better productivity than previous fermentation processes based on the de novo pathway for GDP-fucose biosynthesis, the established schemes in this work also have the advantage to be a potential avenue to GDP-fucose analogs encompassing chemical modification on the fucose residue.