Enhancing the Secretion Efficiency of EGFP in Pichia pastoris Through N-Glycosylation and SOD1-Mediated Redox Regulation.

Pichia pastoris has emerged as a highly efficient heterologous protein expression system with significant potential for secretory production. However, its secretion efficiency is influenced by multiple factors, among which intracellular redox homeostasis plays a critical role. In this study, differential EGFP-secreting strains (GS115-High/Low) were constructed via genomic integration of site-directed mutations at N-glycosylation sites in the α-mating factor signal peptide of P. pastoris. Notably, the expression of the superoxide dismutase (SOD1) gene was significantly upregulated in the high-secretion strain GS115-High. To further investigate the regulatory role of SOD1 in protein secretion, the SOD1 gene was overexpressed in both high- and low-secretion strains. Experimental results demonstrated that SOD1 overexpression markedly enhanced EGFP secretion in the high-secretion strain (p ≤ 0.05), but had no significant effect on the low-secretion strain. Transcriptomic analysis revealed that amino acid metabolism pathways and oxidoreductase activity were significantly enhanced in the high-secretion strain, suggesting a synergistic optimization of secretion efficiency through metabolic resource reallocation and antioxidant capacity. This study demonstrates that glycosylation modification and SOD1-mediated redox regulation both contribute to enhanced EGFP secretion, providing a basis for a "glycosylation-antioxidant" dual-track optimization strategy for the rational design of industrial P. pastoris strains and high-efficiency protein production.