Browsing by Author "Holyavkin, C."

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Evolutionary Engineering and Molecular Characterization of a Caffeine-Resistant Saccharomyces Cerevisiae Strain
(Springer Netherlands, 2019) Sürmeli, Y.; Holyavkin, C.; Topaloğlu, A.; Arslan, M.; Kısakesen, H.İ.; Çakar, Z.P.
Abstract: Caffeine is a naturally occurring alkaloid, where its major consumption occurs with beverages such as coffee, soft drinks and tea. Despite a variety of reports on the effects of caffeine on diverse organisms including yeast, the complex molecular basis of caffeine resistance and response has yet to be understood. In this study, a caffeine-hyperresistant and genetically stable Saccharomyces cerevisiae mutant was obtained for the first time by evolutionary engineering, using batch selection in the presence of gradually increased caffeine stress levels and without any mutagenesis of the initial population prior to selection. The selected mutant could resist up to 50 mM caffeine, a level, to our knowledge, that has not been reported for S. cerevisiae so far. The mutant was also resistant to the cell wall-damaging agent lyticase, and it showed cross-resistance against various compounds such as rapamycin, antimycin, coniferyl aldehyde and cycloheximide. Comparative transcriptomic analysis results revealed that the genes involved in the energy conservation and production pathways, and pleiotropic drug resistance were overexpressed. Whole genome re-sequencing identified single nucleotide polymorphisms in only three genes of the caffeine-hyperresistant mutant; PDR1, PDR5 and RIM8, which may play a potential role in caffeine-hyperresistance. Graphic abstract: [Figure not available: see fulltext.]. © 2019, Springer Nature B.V.
Evolutionary Engineering and Molecular Characterization of Cobalt-Resistant Rhodobacter Sphaeroides
(Frontiers Media SA, 2024) Atay, G.; Holyavkin, C.; Can, H.; Arslan, M.; Topaloğlu, A.; Trotta, M.; Çakar, Z.P.
With its versatile metabolism including aerobic and anaerobic respiration, photosynthesis, photo-fermentation and nitrogen fixation, Rhodobacter sphaeroides can adapt to diverse environmental and nutritional conditions, including the presence of various stressors such as heavy metals. Thus, it is an important microorganism to study the molecular mechanisms of bacterial stress response and resistance, and to be used as a microbial cell factory for biotechnological applications or bioremediation. In this study, a highly cobalt-resistant and genetically stable R. sphaeroides strain was obtained by evolutionary engineering, also known as adaptive laboratory evolution (ALE), a powerful strategy to improve and characterize genetically complex, desired microbial phenotypes, such as stress resistance. For this purpose, successive batch selection was performed in the presence of gradually increased cobalt stress levels between 0.1–15 mM CoCl2 for 64 passages and without any mutagenesis of the initial population prior to selection. The mutant individuals were randomly chosen from the last population and analyzed in detail. Among these, a highly cobalt-resistant and genetically stable evolved strain called G7 showed significant cross-resistance against various stressors such as iron, magnesium, nickel, aluminum, and NaCl. Growth profiles and flame atomic absorption spectrometry analysis results revealed that in the presence of 4 mM CoCl2 that significantly inhibited growth of the reference strain, the growth of the evolved strain was unaffected, and higher levels of cobalt ions were associated with G7 cells than the reference strain. This may imply that cobalt ions accumulated in or on G7 cells, indicating the potential of G7 for cobalt bioremediation. Whole genome sequencing of the evolved strain identified 23 single nucleotide polymorphisms in various genes that are associated with transcriptional regulators, NifB family-FeMo cofactor biosynthesis, putative virulence factors, TRAP-T family transporter, sodium/proton antiporter, and also in genes with unknown functions, which may have a potential role in the cobalt resistance of R. sphaeroides. Copyright © 2024 Atay, Holyavkin, Can, Arslan, Topaloğlu, Trotta and Çakar.