Optimizing expression of vesicle trafficking components and protein folding factors to further enhance secretory glucose oxidase production in a hyperproducing Pichia pastoris

Glucose oxidase (GOD) is an important oxidoreductase widely applied in food, pharmaceutical, biofuel, and environmental industries. Previously, we developed a GOD-hyperproducing strain, G1EeSe, by optimizing protein properties, expression cassette, and vesicle trafficking proteins EES and SEC22. However, further enhancement of GOD secretion remained challenging. Here, we developed an effective strategy to boost GOD secretion in G1EeSe by fine-tuning the expression of vesicle trafficking and protein-folding factors. Systematic replacement of AOX1 promoter and terminator regulating EES and SEC22 with various promoter–terminator combinations increased extracellular GOD production by 16% and reduced the repetitive gene sequences. Screening of endoplasmic-reticulum export factors and protein-folding genes identified HAC1, JEM1, and ERO1 as positive regulators of GOD secretion. Moderate overexpression under the P ADH2 promoter proved more effective than the strong P FDH1 promoter. Notably, ERO1 overexpression driven by P ADH2 in strain G6-AERO achieved the highest reported GOD production in shake-flask culture—72,743.9 U/g DCW and 625.5 U/mL—representing 1.5- and 1.4-fold improvements over G1EeSe, respectively. This corresponds to an extracellular GOD concentration of approximately 2.4 g/L derived from the semi-quantitative densitometry. Our work provides a practical approach for enhancing enzyme secretion in a hyperproducing yeast strain and underscores the importance of balanced gene expression when engineering protein folding and trafficking.