Abstract
Efficient secretion of heterologous proteases in S. cerevisiae remains a major bottleneck due to host cytotoxicity and secretory stress. In this study, we developed a yeast chassis strain through three rounds of iterative evolution, combining ultraviolet (UV) and atmospheric and room temperature plasma (ARTP) mutagenesis with a fluorescence-based screening pipeline. The final strain, MA10Ah121, exhibited protease production nearly three times that of its parental strain LB184M and maintained genetic stability across passages. Importantly, MA10Ah121 supported enhanced expression of two structurally distinct collagenases, ColG (Class I) and ColH (Class II), demonstrating its broad utility for recombinant protease production. Whole-genome sequencing revealed substantial chromosomal remodeling, including reversion of a 151 kb duplication enriched in genes related to energy metabolism and protein synthesis, along with widespread structural variations. These changes reflect a genome-wide adaptation that rebalances biosynthetic capacity and stress tolerance. This work provides a robust and versatile S. cerevisiae chassis for the biosynthesis of proteases and other challenging recombinant enzymes in industrial biotechnology applications.