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. 2025 Jan 30:25:foaf062.
doi: 10.1093/femsyr/foaf062.

Evolutionary engineering and molecular characterization of an antimycin A-resistant Saccharomyces cerevisiae strain: the key role of pleiotropic drug resistance (PDR1)

Alican Topaloğlu  1   2 , Can Holyavkin  1   2 , Ömer Esen  1   2 , Ogün Morkoç  2   3 , Karl Persson  4 , Cecilia Geijer  4 , Zeynep Petek Çakar  1   2
Affiliations

Evolutionary engineering and molecular characterization of an antimycin A-resistant Saccharomyces cerevisiae strain: the key role of pleiotropic drug resistance (PDR1)

Alican Topaloğlu et al. FEMS Yeast Res. .

Abstract

Antimycin A, an antifungal agent that inhibits mitochondrial respiration, provides a useful model for studying resistance mechanisms. Antifungal resistance is an escalating clinical concern with limited treatment options available. To understand the molecular mechanisms of antimycin A resistance, a genetically stable, antimycin A-resistant Saccharomyces cerevisiae strain was successfully developed for the first time through an evolutionary engineering strategy, based on long-term systematic application of gradually increasing antimycin A stress in repetitive batch cultures without prior chemical mutagenesis. Comparative whole genome resequencing analysis of the evolved strain ant905-9 revealed two missense mutations in PDR1 and PRP8 genes involved in pleiotropic drug resistance and RNA splicing, respectively. Using CRISPR/Cas9 genome editing tools, the identified mutations were introduced individually and together into the reference strain, and it was confirmed that the Pdr1p.M732R mutation alone confers antimycin A-resistance in S. cerevisiae. Comparative transcriptomic analysis of the reverse-engineered Pdr1p.M732R strain showed alterations in PDR (pleiotropic drug resistance), transmembrane transport, vesicular trafficking, and autophagy pathways. Our results highlight the potential key role of PDR1 in antifungal drug resistance. This study provides new insights into mitochondrial drug resistance and the adaptive potential of yeast under respiratory stress.

Keywords: CRISPR/Cas9; adaptive laboratory evolution; antifungal drug resistance; antimycin A; evolutionary engineering; pleiotropic drug resistance (PDR1).

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Conflict of interest statement

None declared.

Figures

Figure 1.
Figure 1.
Antimycin A-resistance of selected evolved strains (ant905-1 to ant905-12), the reference strain and the final passage (final population) of selection (52nd Pass.) assessed by using the spot assay in YNBE agar supplemented with 2.5 and 15 nM antimycin A. Images were taken at the 72nd hour of incubation at 30°C.
Figure 2.
Figure 2.
Spot assay results of the antimycin A-resistant ant905-9 evolved strain and the reference strain, indicating their resistance and sensitivities against diverse stressors. Control indicates YMM plates without any stressor. Images were taken at the 72nd h of incubation at 30°C.
Figure 3.
Figure 3.
Growth and metabolite profiles of the evolved strain ant905-9 and the reference strain under (A) respiratory and (B) fermentative growth conditions, and final trehalose and glycogen amounts (mg/g cell dry weight) of the cultures measured at the end of the cultivations.
Figure 4.
Figure 4.
Lyticase sensitivity of ant905-9 and the reference strain (Ref) in the presence and absence of 1 nM antimycin A-stress. Lyticase sensitivity was assessed as the % decrease in lyticase resistance, where the initial value was 100%.
Figure 5.
Figure 5.
Spot assay results of the reference (unevolved) strain and its single transformants PDR1M732R, PRP8V2218 L, and the double transformant (PDR1M732R and PRP8V2218L) obtained by reverse engineering, indicating their resistance and sensitivities against antimycin A and other stressors. Control indicates YMM plates without any stressors. Images were taken at the 72nd h of incubation at 30°C. The PDR1M732R transformant strains were resistant to antimycin A, caffeine, propolis, coniferyl aldehyde, and cycloheximide, just as the evolved strain ant905-9 (shown in a frame).
Figure 6.
Figure 6.
The differentially expressed genes in the reverse engineered, Pdr1p.M732R mutant strain (at least 2-fold change and adjusted P-values < .05), compared to the reference strain. Genes with increased transcript levels are shown in red, and the genes with decreased transcript levels are shown in violet.
Figure 7.
Figure 7.
Major cellular processes that are differentially regulated in the reverse engineered, Pdr1p.M732R mutant strain under nonstress conditions, based on the transcriptomic analysis results. Green and orange boxes indicate up- and downregulated genes, respectively.
See this image and copyright information in PMC

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