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

This is the authors' abstract. We don't add key points for this paper.

Lacto- N -fucopentaose I (LNFP I), a fucosylated neutral human milk oligosaccharide (HMO) with diverse biological functions, was biosynthesized through metabolic engineering in Escherichia coli BL21star (DE3). A de novo pathway was constructed by chromosomal integration of three key enzymes: lgtA (β-1,3- N -acetylglucosaminyltransferase), wbdO (β-1,3-galactosyltransferase), and galE (UDP-galactose-4-epimerase), generating a plasmid-free strain that achieved a lacto- N -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 Helicobacter pylori 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.