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

作者：Jin Wang, Caiwen Lao, Jinyong Wu, Lixia Yuan, Zheng Lei, Xiangsong Chen, Jianming Yao · 发表于：Journal of Agricultural and Food Chemistry · 年份：2025 · DOI：10.1021/acs.jafc.5c03851 · 被引用次数：10 · 研究领域：Infant Nutrition and Health、Digestive system and related health、Microbial Metabolic Engineering and Bioproduction

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.