Engineering Pichia pastoris for Efficient <i>De Novo</i> Synthesis of 2′-Fucosyllactose

2′-Fucosyllactose (2′-FL), the most abundant in human milk oligosaccharides (HMOs), is a nutrient of great importance. As a safe organism widely used in industries, Pichia pastoris was tested here for 2′-FL production. The <i>de novo</i> biosynthesis pathway of 2′-FL was constructed using genome-editing technology based on CRISPR-Cas9 with an initial titer of 1.01 g/L. Introducing N-terminal SUMO or Ub tag to FucT2 and the transporter CDT2 from Neurospora crassa into P. pastoris was found to improve 2′-FL production. Then, modular metabolic engineering was conducted to improve 2′-FL production, enhancing the GTP supply module, NADPH regeneration module, and precursor supply module. Subsequently, the key enzyme FucT2 was semirationally designed to further increase 2′-FL production. Finally, the 2′-FL production by engineered P. pastoris was scaled up to the 3 L fermenter in fed-batch mode, resulting in a titer of 22.35 g/L that is the highest by P. pastoris. The results prove the effectiveness of the metabolic engineering strategies and demonstrate that P. pastoris could be a potential chassis to produce HMOs.