Humanization of N-glycan-dependent protein quality-control system in Kluyveromyces marxianus promotes glycoprotein secretion.

Human N-glycoproteins represent a market worth hundreds of billions of dollars, yet their production in yeast is often limited by misfolding and degradation. However, few strategies have addressed this limitation by targeting differences between human and yeast N-glycan-dependent protein quality control (QC), including the absence of the UGGT-mediated reglucosylation-refolding cycle and the simpler glycoprotein degradation pathway in yeast. Here, we engineered the glycoprotein QC system of Kluyveromyces marxianus by introducing key human components and modifying native pathways. Human UGGT1 or UGGT2 enhanced soluble and secretory glycoprotein production in an activity-dependent manner, with further improvements achieved by co-expressing the human cochaperone SEP15 and reducing native glucosidase II trimming. Human EDEM2 delayed endoplasmic reticulum-associated degradation and increased secretion. Combining these strategies enhanced the production of diverse N-glycoproteins, including Fc, γ-glutamyl hydrolase, fungal xylanase, and Fc-fusion therapeutics, by up to ∼12-fold, demonstrating an effective strategy for engineering human-like glycoprotein QC in yeast to improve glycoprotein production.