Mechanistic insights into atmospheric and room-temperature plasma (ARTP)-induced genomic alterations and phenotypic evolution in Saccharomyces cerevisiae

Atmospheric and room-temperature plasma (ARTP) is a potent tool for microbial strain engineering; however, the mechanistic basis of its genome-wide mutagenic effects remains poorly understood. Utilizing Saccharomyces cerevisiae as a model, we performed a comprehensive characterization of the mutational landscape and DNA repair pathways elicited by ARTP. Under high-lethality conditions (~99% mortality), ARTP treatment induced a substantial mutational burden, with SNVs and InDels rates increasing 25.2- and 36.7-fold relative to untreated controls, respectively. These mutations exhibited a pronounced bias toward C-T/G-A and A-T/T-A substitutions and InDels within AT-rich regions. Remarkably, the frequency of loss of heterozygosity (LOH) surged 57.4-fold, suggesting a potent capacity for inducing DNA breaks. Through sectored colony assays, we demonstrate that ARTP primarily inflicts pre-replicative double-strand breaks (DSBs), with reciprocal crossovers serving as the predominant driver of terminal LOH. The distribution and pattern of LOH were also altered in ARTP-treated cells. Mechanistically, our study establishes that the translesion synthesis polymerases Rev1 and Pol ζ are indispensable for converting ARTP-induced lesions—likely pyrimidine dimers—into fixed point mutations. In contrast, Rad30 facilitates high-fidelity bypass across these dimers. The base excision repair pathway appears largely dispensable for the processing of these lesions. In contrast, the nucleotide excision repair pathway plays a predominant role in mitigating ARTP-induced mutations. Finally, the strategic application of iterative ARTP mutagenesis facilitated the rapid evolution of acetate-tolerant strains. Our findings provide fundamental insights into the molecular signatures of ARTP mutagenesis, offering a framework for its optimized application in directed evolution.

IMPORTANCE

While ARTP is a cornerstone of non-thermal mutagenesis in industrial biotechnology, its genetic impact has long been treated as a “black box.” This study provides a high-resolution molecular dissection of ARTP-induced genomic alterations using a diploid yeast model. We demonstrate that ARTP is not merely a source of point mutations but a potent catalyst for pre-replicative double-strand breaks and extensive mitotic recombination. By elucidating the pivotal roles of specific DNA repair pathways in mutation fixation, we transition ARTP from a purely empirical screening tool to a mechanistically grounded platform for directed evolution. Furthermore, we demonstrate the practical utility of this platform by evolving acetate-tolerant strains and identifying the underlying genetic drivers of their resilience.