Browsing by Author "Kisakesen, Halil Ibrahim"

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Evolutionary Engineering and Molecular Characterization of a Sulfur Dioxide-Stress Saccharomyces Cerevisiae Strain
(MDPI, 2025) Kisakesen, Halil Ibrahim; Canbay, Zeynep Basak; Korkmaz, Aziz Kaan; Topaloglu, Alican; Esen, Omer; Arslan, Mevlut; Cakar, Zeynep Petek
Sulfiting agents are common preservatives in the food and beverage industry to inhibit spoilage microorganisms. Sulfite produced by the dissolution of sulfur dioxide (SO2) in water is used as a microbial inhibitor and antioxidant during winemaking. Thus, sulfite resistance is a desirable trait for wine yeasts. However, consumer health concerns regarding SO2 exposure require a better understanding of the molecular basis of sulfite resistance/response. In this study, we have developed a highly SO2-stress-resistant Saccharomyces cerevisiae strain (F3) using evolutionary engineering by repeated batch selection at gradually increased potassium metabisulfite (K2S2O5) levels. F3 was resistant to 1.1 mM K2S2O5 stress, which was strongly inhibitory to the reference strain, and cross-resistant to oxidative, heat, and freeze-thaw stresses. F3 also had enhanced cell wall integrity and altered carbon metabolism, indicating its potential for industrial applications, including winemaking. Comparative whole genome sequencing revealed point mutations in SSU1 and FZF1 that are related to SO2 transport; ATG14, related to autophagy; and other genes involved in vacuolar protein sorting. Comparative transcriptomic analysis showed significant upregulation of SSU1 and differential expression of genes related to transport and carbohydrate metabolism. These findings may shed light on the molecular mechanisms contributing to SO2 resistance and industrial robustness in S. cerevisiae.
Genomic, Transcriptomic and Physiological Analyses of Silver-Resistantsaccharomyces Cerevisiaeobtained by Evolutionary Engineering
(Wiley, 2020) Terzioglu, Ergi; Alkim, Ceren; Arslan, Mevlut; Balaban, Berrak Gulcin; Holyavkin, Can; Kisakesen, Halil Ibrahim; Cakar, Zeynep Petek
Silver is a non-essential metal used in medical applications as an antimicrobial agent, but it is also toxic for biological systems. To investigate the molecular basis of silver resistance in yeast, we employed evolutionary engineering using successive batch cultures at gradually increased silver stress levels up to 0.25-mM AgNO(3)in 29 populations and obtained highly silver-resistant and genetically stableSaccharomyces cerevisiaestrains. Cross-resistance analysis results indicated that the silver-resistant mutants also gained resistance against copper and oxidative stress. Growth physiological analysis results revealed that the highly silver-resistant evolved strain 2E was not significantly inhibited by silver stress, unlike the reference strain. Genomic and transcriptomic analysis results revealed that there were mutations and/or significant changes in the expression levels of the genes involved in cell wall integrity, cellular respiration, oxidative metabolism, copper homeostasis, endocytosis and vesicular transport activities. Particularly the missense mutation in theRLM1gene encoding a transcription factor involved in the maintenance of cell wall integrity and with 707 potential gene targets might have a key role in the high silver resistance of 2E, along with its improved cell wall integrity, as confirmed by the lyticase sensitivity assay results. In conclusion, the comparative physiological, transcriptomic and genomic analysis results of the silver-resistantS. cerevisiaestrain revealed potential key factors that will help understand the complex molecular mechanisms of silver resistance in yeast.
Physiological and Transcriptomic Analysis of a Chronologically Long-Lived Saccharomyces Cerevisiae Strain Obtained by Evolutionary Engineering
(Springernature, 2018) Arslan, Mevlut; Holyavkin, Can; Kisakesen, Halil Ibrahim; Topaloglu, Alican; Surmeli, Yusuf; Cakar, Zeynep Petek
High-throughput aging studies with yeast as a model organism involve transposon-mutagenesis and yeast knockout collection, which have been pivotal strategies for understanding the complex cellular aging process. In this study, a chronologically long-lived Saccharomyces cerevisiae mutant was successfully obtained by using another high-throughput approach, evolutionary engineering, based on systematic selection in successive batch cultures under gradually increasing levels of caloric restriction. Detailed comparative physiological and transcriptomic analyses of the chronologically long-lived mutant and the reference strain revealed enhanced levels of respiratory metabolism, upregulation of genes related to carbohydrate metabolic processes, glycogen-trehalose pathways, stress response, and repression of protein synthesis-related genes in the long-lived mutant SRM11, already in the absence of caloric restriction. Interestingly, SRM11 had also significantly higher resistance to copper stress, and higher resistance to silver, ethanol, and 2-phenylethanol stresses than the reference strain. It also had lower ethanol production levels and an enhanced ethanol catabolism. To conclude, evolutionary engineering is another powerful high-throughput method for aging research, in addition to its widespread use in industrial strain development. Additionally, the interesting results revealed by this study about the potential relationship between longevity and various cellular properties are yet to be investigated further at molecular level.