Efficient production of 2'-fucosyllactose in Pichia pastoris 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 Escherichia coli and Saccharomyces cerevisiae, the potential of Pichia pastoris, renowned for its robust NADPH regeneration capability, remains underexplored. Herein, we systematically engineered the metabolism of P. pastoris to develop an efficient 2'-FL-producing cell factory. We first constructed the de novo biosynthesis pathway for 2'-FL in P. pastoris, 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 P. pastoris. These findings highlight the potential of P. pastoris as a chassis to produce chemicals by providing abundant NADPH and utilizing methanol as co-substrate to supply sufficient energy.