Evolutionary engineering and physiological analysis of antimycin-a resistant Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae is a well known eukaryotic model organism in terms of its clearly known genetic and metabolic basis and the ease of cultivation. Its genome was fully sequenced at 1996 and it was the first eukaryotic organisms which the genome was fully sequenced. Additionally, S. cerevisiae genome has high homology genes with the human genome. Furthermore, it is also a good model organism to study mitochondrial functions because of its special properties. Most of the discoveries about mitochondria were found on S. cerevisiae model organism. Most importantly, it is a facultative microorganism which can survive under mitochondrial dysfunction and it has several survival mechanisms against mitochondrial DNA mutations. Mitochondria is an organelle which has the main function of producing energy by oxidative phosphorylation. In addition to this function, it has also other responsibilities like thermogenesis, apoptosis, lipid and sterol synthesis and calcium storage. It is a double-membrane organelle and includes its own mitochondrial DNA (mtDNA) and ribosomes which are structuraly different than cytoplasmic ones. As its structure and functions, it is more likely to prokaryotic microorganism. Functional disorders of mitochondria might cause mitochondrial diseases which are observed in the most energy-requiring systems, mainly nervous and muscle system. Those disorders typically cause cardiovascular and neurodegenerative disorders, vision and hearing loss, diabetes, kidney and liver failures. Although many discoveries were made on the pathogenesis of mitochondrial disorders in the last two decades, there are still no effective treatments to cure those diseases. Most of the genetic and functional information about mitochondria was obtained by the studies with S. cerevisiae as the model organism. Today, most of the mitochondrial diseases caused by genetic mutations are researched on S. cerevisiae models by using recombinant DNA technologies and rational metabolic engineering techniques. However, such experimental design has some limitations, such as requiring detailed genetic background knowledge, rejection of recombination by the organism and obtaining unexpected phenotypic results. Because of these limitations, inverse metabolic engineering has been introduced as an alternative approach. In light of this information, in the present study, it was aimed to obtain antimycin-A resistant S. cerevisiae mutants by evolutionary engineering, an inverse metabolic engineering strategy, and to complete physiological analyses that are the first steps to enlighten the molecular basis of this resistance. Antimycin-A is a mitochondrial complex III inhibitor which binds to bc1 complex and inhibits electron transfer. It is the analog of quinone and binds Qi site of the bc1 region (Quinone binding site) Binding of antimycin-A to this site blocks further electron transfer from Qo site and electron transfer to complex IV is stalls. As a result, cells cannot produce enough energy via mitochondrial oxidation and are obliged to find alternative metabolicpathways to survive. Additionally, antimycin-A cause increased Reactive Oxygen Species (ROS) generation by disrupting redox reactions on the mitochondria. Other than antimycin-A, there are various mitochondrial inhibitors such as atpenin, oligomycin, stigmatellin or cyanide which are widely used in research. While deciding the mitochondrial inhibitor among others, many factors were considered. Thus, considering the high affinity to bind mitochondrial complex III, the power of inhibition at low concentrations and safety of use, it was decided to use antimycin-A as the mitochondrial inhbitior in this study. In this study, antimycin-A resistant mutants were selected successfully from both reference strain and EMS-mutagenized reference strain population. As the selection method, gradually increasing stress level application protocol was used in succesive batch cultures under respirative conditions to make cells dependent on mitochondrial functioning. After 52 passages, antimycin-A concentration was increased to 6.6 nM and mutant individuals were randomly picked from the final populations. Twelve mutants were chosen from the reference strain selection and 15 mutants were chosen from the EMS-mutagenized reference strain population. After those mutants were screened at different antimycin-A concentrations, two mutants with the highest resistance levels were chosen. It was observed that mutant individuals obtained from the reference strain selection were resistant to 15 nM antimycin-A, while mutants obtained from the EMS-mutagenized reference strain population were resistant to 50 nM antimycin-A. This study, based on the literature information, is the first one where antimycin-A resistant S. cerevisiae mutants were obtained by evolutionary engineering strategy. To determine whether the gained antimycin-A resistance was permanent or not, genetic stability test was applied. Except for one mutant individual obtained from the reference strain selection, all resistant mutants tested were genetically stable. Additionally, antimycin-A resistant mutant population obtained from the reference strain selection might indicate possible mutagenic effects of antimycin-A. Within the frame of physiological characterization experiments, cross-resistance analyses results revealed that, antimycin-A resistant mutants were also cross-resistant to caffeine, propolis, coniferyl aldehyde and ethanol. Similarly, mutants resistant to these chemicals except for the ethanol-resistant mutant were also found to be cross-resistant to antimycin-A in a previous study. This implies some similar molecular stress resistance mechanisms between these mutants. Resistance to ethanol is also an expected result, because the selection procedure was applied by using ethanol as the sole carbon source. In this study, antimycin-A resistant S. cerevisiae mutants were obtained by evolutionary engineering strategy and the physiological characterization experiments were performed. For further studies, genomic and transcriptomic analyses could be performed to identify alternative pathways which by-pass the effects of the nonfunctional mitochondrial complex, and possible pharmaceutical targets could be identified.