Combinatorial engineering of microbial cell factory for 2′-fucosyllactose biosynthesis

2’-fucosyllactose (2’-FL), a predominant human milk oligosaccharide (HMOs), has showed significant potential in infant health applications including intestinal protection, cognitive development, and immune system modulation. Therefore, to enhance microbial biosynthesis of 2’-FL, we engineered a de novo synthesis pathway through deleting key enzymes gene: wecB (encoding UDP-N-acetyl glucosamine-2-epimerase), lacZ (encoding β-galactosidase), wcaJ (encoding UDP-glucose lipid carrier transferase), which could increase the 2’-FL titer and productivity to 2.25 g/L and 0.03 g/L/h, respectively. Furthermore, optimization of induction time further improved production performance to 4.06 g/L and 0.06 g/L/h. Through systematic gene deletion analysis, we identified lacA and iclR as critical metabolic nodes. Their knockout negatively impacted 2’-FL biosynthesis by reducing lactose availability and disturbing metabolic flux. Finally, the 2’-FL was furthermore improved to 4.75 g/L through expressing gsk and zwf genes for NADPH and GTP regeneration. This study can effectively increase the production of 2’-fucosyllactose, which can provide a research basis for the large-scale bioproduction of 2’-fucosyllactose in the industrial factory.