Engineering <i>Pichia pastoris</i> 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, <i>Pichia pastoris</i> 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 <i>Neurospora crassa</i> into <i>P. pastoris</i> 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 <i>P. pastoris</i> 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 <i>P. pastoris</i>. The results prove the effectiveness of the metabolic engineering strategies and demonstrate that <i>P. pastoris</i> could be a potential chassis to produce HMOs.