High-Level De Novo Biosynthesis of Difucosyllactose in <i>Escherichia coli</i> through Application and Engineering of a Novel α-1,3-Fucosyltransferase

Difucosyllactose (DFL) is a difucosylated oligosaccharide that has garnered significant attention for its numerous health benefits. In this study, glycerol was utilized as the carbon source to construct a chassis strain by overexpressing GDP-l-fucose pathway genes, along with α-1,2-fucosyltransferase and α-1,3-fucosyltransferase. A novel α-1,3-fucosyltransferase derived from <i>Bacillus fengqiuensis</i> was discovered and employed as a key catalyst. Semirational enzyme engineering was achieved by sequence alignment-guided point-directed mutations and HotSpot Wizard 3 prediction-guided catalytic center adjacent residue saturation mutations. Combined with the high-throughput screening platform for reducing sugar by 3,5-dinitrosalicylic acid colorimetry, five-point cooperative mutant MM5 (F22Y/N25K/T98K/E99Q/N276D) was finally obtained. The MM5-expressing engineering strain BZL01-26 achieved a DFL yield of 65.12 g/L and a productivity of 0.83 g/(L h) in 3-L fed-batch fermentation, representing the highest titer reported to date. This study provides a novel α-1,3-fucosyltransferase and efficient biomanufacturing strategy for industrial production of DFL.