High-Level Production of l-Fucose by Plasmid-Free or Antibiotic-Independent Metabolically Engineered Escherichia coli Strains

l-Fucose is an important monosaccharide unit that exists in various biomasses, especially in microalgae. Microbial l-fucose using metabolically engineered strains has attracted attention due to its high yield and industrial feasibility. Previously, we engineered Escherichia coli MG1655 to efficiently produce 2′-fucosyllactose by genomic integration. Herein, this plasmid-free strain was further engineered to produce l-fucose by integrating a specific α-l-fucosidase gene and deleting the l-fucose degradation pathway. Its effectiveness of l-fucose biosynthesis by plasmid-free and inducer-free fermentation was demonstrated by both shake-flask and fed-batch cultivation with titers of 2.74 and 21.15 g/L, respectively. The precursor GDP-l-fucose supply was strengthened to obviously enhance l-fucose biosynthesis by introducing a single plasmid expressing four pathway genes. The hok/sok system was introduced to promote the plasmid stabilization without antibiotic. The final engineered strain efficiently could produce l-fucose without antibiotics, with titers of 6.83 and 35.68 g/L by shake-flask and fed-cultivation cultivation, respectively.