Abstract

2′-Fucosyllactose (2′-FL), the most abundant human milk oligosaccharide (HMO) with well-recognized health benefits, can be efficiently synthesized by microbial fermentation. However, most high-titer strains reported still rely on plasmid expression systems, which introduces risks of genetic instability and antibiotic contamination. In contrast, achieving high-level 2′-FL production in plasmid-free strains remains challenging due to insufficient pathway flux and limited catalytic activity of α-1,2-fucosyltransferase (α1,2-FucT). Herein, we engineered a plasmid-free, antibiotic-free, and inducer-free Escherichia coli strain for efficient 2′-FL biosynthesis. A modular metabolic engineering strategy was employed to enhance precursor supply by improving lactose utilization and GDP- l -fucose biosynthesis. To eliminate the undesirable byproduct difucosyllactose (DFL), a highly efficient α1,2-FucT (WPfutC) was identified, and its catalytic performance was further improved through rational truncation of the membrane-interacting region. Additionally, the expression of critical pathway genes and cofactor regeneration were optimized by constructing artificial multi-enzyme complexes. The final strain, WPL, achieved 25.6 g/L of 2′-FL in shake flask cultivation and 108.3 g/L in fed-batch fermentation with a productivity of 1.18 g/L/h, reaching the highest reported titer among plasmid-free strains. This work provides a promising strategy for sustainable industrial production of 2′-FL and offers insights into the rational design of efficient plasmid-free production strains for other HMOs.