Hac1p-based inverse secretory pathway engineering (Hi-SPE) of Pichia pastoris for improved glucose oxidase production

Secretion and folding are common bottlenecks in protein expression using eukaryotic systems, and engineering the secretory pathway to enhance host cell capabilities is a key strategy for improving protein secretion. However, secretion is a very complex process, making the identification of likely targets for engineering a formidable task. In this study, using glucose oxidase (GOX) expression in Pichia pastoris (Komagataella phaffii) as a model, we introduce a strategy called Hac1p-based inverse secretory pathway engineering (Hi-SPE). This strategy leverages Hac1p, the actuator of the unfolded protein response, which is a naturally evolved mechanism to cope with protein overload in endoplasmic reticulum (ER) of eukaryotic cells. When combined with comparative transcriptomics, Hi-SPE narrows down the target from several hundred genes in traditional approaches to 20 secretion-related protein genes. Results showed that overexpression of six out of seven selected genes improved GOX secretion, including the co-chaperone, JEM1, which increased GOX expression per OD₆₀₀ by 147.6 %. Further optimization through combinatorial expression of secretion-related proteins led to a strain co-expressing JEM1, KAR2, and CNE1, achieving a GOX titer of 1903.2 U/mL in 1-L fed-batch fermentation. Additionally, transcriptomic analysis revealed the physiological effects of JEM1 overexpression on P. pastoris. This study highlights Hi-SPE as a powerful strategy for improving protein secretion in eukaryotic systems.