Harnessing the PpHog1 Regulatory Network to Engineer a UPR-Adaptive Komagataella phaffii Chassis for High-Copy Secretory Protein Production

High-copy secretory expression genes were first confirmed to adversely affect yeast growth fitness, a phenotype consistent with DTT-induced unfolded protein response (UPR). Screening of a kinase knockout library established that UPR signaling in K. phaffii is modulated by the PpHog1-mediated MAPK pathway. Further comparison of the PpHog1 interactome with versus without DTT treatment, combined with follow-up genetic knockout/knockdown screens, leads to the identification of two critical UPR regulators: components 0498 and 0120. Subsequently, an engineered UPR-adaptive chassis (UPR-Ad+) was constructed by overexpressing these three key regulators. Using Human Serum Albumin (HSA) and alpha-amylase as reporters, UPR-Ad+ enhanced the production of high-copy secretory proteins by 50% and 23%, respectively. Collectively, this work advances the understanding of UPR regulation in K. phaffii and provides a strategic basis for designing high-yield strains.