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

Abstract

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.