Metabolic Engineering of Genomically Integrated Escherichia coli MG1655 for High-Titer Production of Lacto- N -fucopentaose I

Lacto- N -fucopentaose I (LNFP I) is a representative oligosaccharide present in human milk and ranks as the second most abundant fucosylated oligosaccharide after 2′-fucosyllactose (2′-FL). LNFP I exhibits valuable physiological functions, including antiviral activity, immunomodulatory properties, and the ability to modulate the gut microbiota. It has been approved as a functional ingredient for incorporation into infant formula and a range of dietary supplements. The construction of genomically integrated engineered strains via metabolic engineering strategies offers a feasible approach for the industrial-scale production of LNFP I without relying on antibiotics or inducers. Building upon a previously developed plasmid-free engineered strain for lacto- N -tetraose (LNT) synthesis, this study further enhanced uridine 5′-diphosphate galactose (UDP-Gal) supply through chromosomal integration or promoter strengthening of the key genes galE and galU . Combined with additional integration of an efflux gene and copy number optimization of lgtA (encoding β1,3- N -acetylglucosaminyltransferase) and wbgO (encoding β1,3-galactosyltransferase), these modifications resulted in a 102.4% increase in LNT production. On this basis, the highly specific α1,2-fucosyltransferase gene teSA was introduced and its copy number optimized, while key genes ( cpsG – cpsB and gmd – fcl ) in the guanosine 5′-diphosphate fucose (GDP-Fuc) synthesis pathway were reinforced. This led to the construction of an efficient LNFP I-producing engineered strain, which achieved a shake-flask yield of 4.34 g/L. In a 5 L bioreactor, the LNFP I titer reached 37.44 g/L, with the maximum productivity and conversion ratio from LNT to LNFP I reaching 0.83 g/L/h and 90.73%, respectively.