Optimization of Multiple Combinations of Early Secretion Pathways to Enhance Extracellular Protein Secretion in Komagataella phaffii

The methylotrophic yeast Komagataella phaffii (K. phaffii) is a pivotal heterologous protein production platform, yet inefficient secretory trafficking hinders its industrial scalability. We addressed this limitation through the systematic engineering of early secretory pathways to enhance extracellular yields for three phylogenetically distinct proteins: fungal α-amylase (AmyA), bacterial β-glucanase (BgA), and human serum albumin (HSA). By integrating secretory peptide engineering with 3′ untranslated region (3′ UTR) optimization, cotranslational ER translocation efficiency improved, elevating extracellular activity by 1.3–3.4-fold. Subsequent multitiered interventions targeted ER membrane expansion (to boost secretory capacity), ER-associated degradation (ERAD) modulation, and vesicular transport enhancement, collectively increasing extracellular protein levels by 1.8–4.0-fold. Protein-specific optimization combinations achieved cumulative improvements of 5.2–6.4-fold over the baseline. Notably, optimal secretion strategies varied across proteins, underscoring the need for tailored engineering. This modular approach establishes a universal framework to enhance secretory efficiency in K. phaffii, broadly applicable to food industrial enzymes and therapeutic biologics.