Multimodular Metabolic Engineering Strategy Enables High-Efficiency Synthesis of Lacto-<i>N</i>-fucopentaose I in Engineered <i>Escherichia coli</i>

Lacto-<i>N</i>-fucopentaose I (LNFP I), a fucosylated neutral human milk oligosaccharide (HMO) with diverse biological functions, was biosynthesized through metabolic engineering in <i>Escherichia coli</i> BL21star (DE3). A <i>de novo</i> pathway was constructed by chromosomal integration of three key enzymes: <i>lgtA</i> (β-1,3-<i>N</i>-acetylglucosaminyltransferase), <i>wbdO</i> (β-1,3-galactosyltransferase), and <i>galE</i> (UDP-galactose-4-epimerase), generating a plasmid-free strain that achieved a lacto-<i>N</i>-tetraose (LNT) titer of 109.80 g/L in a 5 L bioreactor, the highest yield reported to date. Subsequent screening identified α-1,2-fucosyltransferase (FutC) from <i>Helicobacter pylori</i> as the optimal catalyst for LNFP I biosynthesis. Multidimensional optimization strategies were systematically implemented, including copy number balancing of rate-limiting transferases, promoter-RBS engineering, enhanced intracellular cofactor regeneration, and knockout of competing pathways. Fed-batch fermentation under optimized conditions yielded 77 g/L LNFP I with 93.05% LNT-to-LNFP I conversion efficiency, representing both the highest reported titer and precursor utilization efficiency for LNFP I.