Abstract

Komagataella phaffii is one of the most widely used eukaryotic protein expression systems. Increasing the copy number of exogenous genes is a common method to enhance expression efficiency. However, at extremely high copy numbers, the resulting endoplasmic reticulum stress (ERS) can hinder further improvement in the expression efficiency of secreted proteins. And elucidating the underlying molecular mechanisms serves as the foundation for engineering and constructing stress-adapted strains. High-copy secretory expression was 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 signalling in K. phaffii was modulated by the PpHog1-mediated MAPK pathway. PpHog1, the K. phaffii ortholog of the well-characterized Saccharomyces cerevisiae Hog1 (67% sequence identity), is traditionally known for its roles in osmotic stress and cell wall integrity. Notably, while ScHog1 has also been implicated in ERS regulation, the downstream pathway remains obscure. 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: PpLRR-0498 and PpPINT-0120. Subsequently, an engineered UPR-adaptive chassis (UPR-Ad + ) was constructed by over-expressing these three key regulators. Using human serum albumin (HSA), human calcium-regulated actin binding protein LCP1, and α-amylase as reporters, UPR-Ad + enhanced the production of high-copy secretory proteins by 30%–46%. Collectively, this work advances the understanding of UPR regulation in K. phaffii and provides a strategic basis for designing high-yield strains. Graphical Increasing gene copy number is a common strategy to boost heterologous expression, but an excessively high copy number of genes encoding secretory proteins can cause ER stress, leading to growth defect