Efficient production of 2'-fucosyllactose in through metabolic engineering and constructing an orthogonal energy supply system.

2'-fucosyllactose (2'-FL) holds significant role in the infants' nutrition. While microbial production of 2'-FL has predominantly utilized and , the potential of , renowned for its robust NADPH regeneration capability, remains underexplored. Herein, we systematically engineered the metabolism of to develop an efficient 2'-FL-producing cell factory. We first constructed the biosynthesis pathway for 2'-FL in , achieving an initial titer of 0.143 g/L. By optimizing enzyme selection and solubility of α-1,2-fucosyltransferase (FutC), 2'-FL production was enhanced by nearly ten folds. Subsequently, engineering NADPH supply further increased the 2'-FL production by 170 %. Furthermore, we enhanced energy supply by incorporating an orthogonal energy module based on the methanol dissimilation pathway and increasing GTP availability, resulting in a 32 % improvement in 2'-FL production. Finally, through the optimization of fermentation condition, we realized the production titer of 2'-FL to 3.50 g/L in shake-flask, representing the highest titer in . These findings highlight the potential of as a chassis to produce chemicals by providing abundant NADPH and utilizing methanol as co-substrate to supply sufficient energy.