Rational Design and Model Predictions for Optimized Elastase Production in <i>Saccharomyces cerevisiae</i>

<i>Pseudomonas aeruginosa</i> elastase is a metalloprotease with significant industrial potential but is challenging to produce due to its pathogenic origin and folding complexities. In this study, we applied rational design to engineer nonfunctional regions of elastase within <i>Saccharomyces cerevisiae</i>, specifically targeting propeptide and signal peptide cleavage sites, and N-glycosylation in the propeptide. This led to the development of several improved elastase variants. Integrating the yeast protein secretory model pcSecYeast with protease production characteristics, a total of 75 targets were identified and validated, comprising both model-predicted and production-feature-based targets. Notably, overexpression of <i>POS5</i> enhanced protease activity to 2.43-fold that of the control, while knockout of <i>TES1</i> or <i>VPS10</i> further optimized production. This work demonstrates the potential of systems biology in creating yeast cell factories for protease production and highlights <i>S. cerevisiae</i> as a versatile host for biotechnological applications.