Enhanced Biosynthesis of Lacto-N-Fucopentaose I in <i>Escherichia coli</i> through Spatial Organization of Enzyme Complexes and Modular Pathway Engineering

Lacto-N-fucopentaose I (LNFP I; Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc), the second most abundant fucosylated human milk oligosaccharide, accounts for approximately 8% of total HMOs and exhibits significant nutritional and biofunctional properties. Here, we constructed a <i>de novo</i> biosynthetic pathway for LNFP I in <i>Escherichia coli</i> using a three-plasmid system. Screening identified an α-1,2-fucosyltransferase from <i>Sulfuriflexus mobilis</i> that catalyzed LNFP I formation without detectable byproducts. The pathway was partitioned into three modular units, and optimal expression was achieved by screening plasmid combinations with different copy numbers. Implementation of the RIDD-RIAD self-assembling peptide system, involving peptide fusions at the N- and C-termini of the glycosyltransferases WbdO and Smob, yielded the high-performing strain zLNP22. After shake-flask optimization and scale-up to a 5 L bioreactor, this strain produced 2.37 g/L LNFP I in flasks and 11.6 g/L in fed-batch fermentation, achieving a productivity rate of 0.34 g/L/h and an LNT-to-LNFP I conversion efficiency of 86.70%.