Enhanced bioproduction of fucosylated oligosaccharide 3-fucosyllactose in engineered Escherichia coli with an improved de novo pathway.

3-fucosyllactose (3-FL) and 2'-fucosyllactose (2'-FL), are two important fucosylated oligosaccharides in human milk. Extensive studies on 2'-FL enabled its official approval for use in infant formula. However, development of 3-FL has been somewhat sluggish due to its low content in human milk and poor yield in enlarged production. Here, an α-1,3-fucosyltransferase mutant was introduced into an engineered Escherichia coli (E. coli) capable of producing GDP-L-fucose, leading to a promising 3-FL titer in a 5.0-L bioreactor. To increase the availability of cofactors (NADPH and GTP) for optimized 3-FL production, zwf, pntAB, and gsk genes were successively overexpressed, finally resulting in a higher 3-FL level with a titer of 35.72 g/L and a yield of 0.82 mol 3-FL/mol lactose. Unexpectedly, the deletion of pfkA gene led to a much lower performance of 3-FL production than the control strain. Still, our strategy achieved the highest 3-FL level in E. coli to date.