Molecular characterization of an antimycin a-resistant saccharomyces cerevisiae strain obtained by inverse metabolic engineering

Abstract

Antifungal drug resistance is a growing global concern due to its increasing clinical prevalence. Elucidation of the molecular mechanisms underlying resistance to natural antifungal agents, such as antimycin A, can provide insights into the regulatory and metabolic reprogramming mechanisms that occur in eukaryotic microorganisms and also contribute to antifungal drug development studies. Antimycin A is a well-characterized, natural inhibitor of the mitochondrial complex III, where it binds to the Qi site of the cytochrome bc1 complex and interferes with proton translocation, leading to a collapse in mitochondrial respiration and severe growth inhibition under non-fermentative conditions. However, microorganisms can be engineered and evolved in order to compensate for the inhibitory effects of antimycin A by complex adaptive responses. In this study, a previously developed and genetically stable, antimycin A-resistant Saccharomyces cerevisiae strain (ant905-9) was used. It had been developed by using an evolutionary engineering strategy, without prior mutagenic treatment and by applying a systematic batch selection approach under respirative growth conditions with gradually increasing antimycin A concentrations. The evolved strain, ant905-9, could tolerate antimycin A concentrations higher than 25 nM in liquid respiratory (yeast nitrogen base-ethanol) medium, whereas the reference strain was unable to survive concentrations higher than 1 nM. Remarkably, the evolved strain maintained its growth rate without an extended lag phase under both fermentative and respiratory growth conditions supplemented with 1 nM antimycin A, indicating that antimycin A resistance did not impair the growth of the evolved strain. Additionally, stress cross-resistance analyses showed that ant905-9 strain was resistant to several pleiotropic drug-related stressors including caffeine, coniferyl aldehyde, propolis extract, and cycloheximide, in addition to antimycin A. Nevertheless, it became more susceptible to some heavy metals, such as cobalt and aluminum, indicating the complex regulatory effects of antimycin A-resistance. Growth and metabolite profiling under respiratory growth conditions showed a decrease in acetate accumulation and an increase in glycerol production possibly due to a metabolic shift to preserve redox balance under conditions of impaired mitochondrial electron transport. Additionally, the evolved strain ant905-9 showed significant alterations in storage carbohydrate metabolism. Trehalose and glycogen are the reserve carbohydrates in yeast and are typically upregulated in response to environmental stresses. These carbohydrate levels were found to be elevated in the evolved strain, even under fermentative conditions. In comparison to the reference strain, the evolved strain retained greater amounts of both carbohydrates under respiratory and antimycin A-stress conditions. These findings imply that the evolved strain improved stress tolerance by reprogramming its carbohydrate metabolism. Another critical determinant of yeast stress tolerance, the cell wall integrity, was also examined in the evolved strain. Findings from the lyticase susceptibility experiments suggested that the evolved strain ant905-9 has a stronger cell wall than the reference strain, regardless of antimycin A stress. To elucidate the genomic changes that cause antimycin A-resistance, comparative whole-genome resequencing analysis was conducted between the evolved ant905-9 strain and the reference strain. Interestingly, only two single-nucleotide variants (SNVs), PDR1M732R and PRP8V2218L, were detected, which also resulted in amino acid substitutions. To analyse their individual contributions, both point mutations were introduced separately and together into the prototrophic background reference strain, using CRISPR/Cas9 genome editing strategy. Cross-resistance analysis of the resulting mutants showed that the PDR1M732R mutation alone was sufficient to confer high-level resistance to antimycin A, as well as to the other pleiotropic drugs. These findings confirmed that Pdr1p, a key transcription factor, may contribute significantly to the acquired resistance and indicated the complex regulatory mechanisms of the pleiotropic drug resistance (PDR) network. To determine the molecular consequences specifically caused by the PDR1M732R mutation, comparative transcriptomic analysis was performed between the reverse-engineered Pdr1p.M732R mutant strain and the reference strain, under non-stress conditions using RNA sequencing. The results revealed a global activation of the PDR1-dependent ABC transporter system, with the most strongly upregulated genes PDR5 (6.31-fold), PDR10 (2.35-fold), and YOR1 (2.57-fold). Furthermore, genes involved in vesicular trafficking (VPS3, TRS33), endoplasmic reticulum organization (OST2, OST5, PER33), and oxidative stress management (GPX2) were significantly upregulated, indicating increased intravesicular trafficcing and cellular demand for protein folding, as well as membrane organization. Notably, while some autophagy-related genes (ATG12, TRS33, and VPS3) were upregulated, ATG16, another autophagy-related gene, was downregulated. Similarly, some genes related to structural cytoskeletal components, ARP10 and SPC110, and a gene that encodes an endosomal protein involved in the turnover of plasma membrane proteins COS8, were also downregulated. Gene Ontology (GO) enrichment and KEGG pathway analyses further highlighted that "ABC transporter activity," "transmembrane transport," "siderophore transmembrane transport," "xenobiotic detoxification," and "autophagy" were the most significantly enriched biological processes in the upregulated gene set. These transcriptomic alterations align with the phenotypic observations of multidrug resistance, support the central role of the PDR pathway in the adaptive mechanism and also suggest a selective autophagic pathway for membrane turnover and organelle maintenance. Taken together, these findings demonstrate that evolutionary engineering serves as an effective strategy to develop complex traits such as antifungal drug resistance. Owing to the combined physiological, genomic and transcriptomic analyses and reverse engineering using CRISPR/Cas9, the roles of the identified point mutations and their molecular effects can be verified and analysed in detail. The reverse-engineered strain Pdr1p.M732R and the previously evolved S. cerevisiae strains resistant to propolis (FD11), coniferyl aldehyde (BH13) and caffeine (caf905-2) stress highlight the key role of the PDR1 gene in pleiotropic drug resistance and suggest that the mechanism underlying the PDR1M732R mutation likely involves multiple, overlapping pathways that remain to be fully elucidated. As a future work, a comprehensive study focusing on targeted deletions of PDR1-related genes and their combined physiological, bioinformatic, and proteomic analyses would shed light on the specific contributions of individual transporters and regulatory networks to the observed resistant phenotypes.