Alleviation of ER stress via targeted genetic engineering enhances recombinant ovalbumin secretion in Saccharomyces cerevisiae.

Enhancing the secretion of heterologous proteins in Saccharomyces cerevisiae is often hindered by endoplasmic reticulum (ER) stress and inefficiencies in intracellular trafficking. To address these limitations, we systematically evaluated 14 genetic modifications associated with the secretory pathway to improve recombinant ovalbumin (OVA) production. Deletion of PAH1, a negative regulator of phospholipid biosynthesis, resulted in the highest OVA secretion (5.68 mg/L), representing a 74% improvement over the background strain-likely due to increased ER membrane biogenesis. Similarly, knockout of GOS1, a Golgi-to-ER SNARE protein, enhanced secretion, possibly by reducing retrograde trafficking and limiting ER retention of secretory cargo. In contrast, disruption of endosome-to-Golgi transport via VPS5 deletion resulted in intracellular accumulation of OVA and complete secretion failure. These findings suggest that alleviating ER stress and modulating vesicular trafficking are key strategies for enhancing protein secretion in yeast, offering a functional basis for rational host strain engineering. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-025-02044-1.