Engineering Probiotic Escherichia coli Nissle 1917 for 2′-Fucosyllactose and 3-Fucosyllactose Biosynthesis

ABSTRACT: Fucosylated human milk oligosaccharides are valuable targets for microbial biomanufacturing. Here we engineered the probiotic Escherichia coli Nissle 1917 as a versatile chassis for synthesizing both 2′-fucosyllactose and 3-fucosyllactose. We implemented lactose preservation and blocked colanic acid precursor consumption while reinforcing the de novo GDP-L-fucose pathway. This core module was coupled with either WbgL or FutM2 to drive terminal fucosylation. Initial screening yielded a 2′-fucosyllactose titer of approximately 1.21 g/L. Integrating an additional chromosomal wbgL cassette increased the titer to approximately 1.58 g/L, whereas subsequent genomic modifications did not further improve the 2′-FL titer. Cultivation with 30 g/L glycerol and 5 g/L yeast extract significantly improved productivity, enabling the strain to produce approximately 7.71 g/L of 2′-fucosyllactose in shake-flask cultures. Conversely, the FutM2 strain produced 0.38 g/L 3-fucosyllactose without yeast extract and nutrient supplementation unexpectedly suppressed product accumulation despite promoting biomass formation. The work provides a probiotic-chassis proof of concept and identifies product-module physiology as a priority for further engineering.