Tersine metabolik mühendislik yöntemi ile aluminyuma dirençli Saccharomyces cerevisiae eldesi

Although Al3+ is non-essential and toxic to life, it is commonly encountered in water, foods, medicines and beverages. Al3+ has been reported to interact with organic molecules in vitro, however, molecular mechanisms of aluminium toxicity/tolerance are not well-known. Aluminium is also related with human neurodegenerative diseases such as Alzheimer and Parkinson. The yeast Saccharomyces cerevisiae is a model organism which has been used widely in molecular biology and biotechnology for a long time. In addition to research, it has also been used for many industrial applications. It can make fermentation which is very important in baking and wine industry. S.cerevisiae can be in haploid and diploid form and its genome has a high degree of homology with those of higher eukaryotes. This makes it important in understanding molecular mechanisms of human-related diseases. In this study, S. cerevisiae was used as a eukaryotic model organism to investigate aluminium tolerance mechanisms. An evolutionary engineering strategy based on batch selection at increasing Al3+ concentrations was employed to obtain aluminium-resistant S. cerevisiae mutants: initially, S. cerevisiae CEN.PK 113-7D wild type strain was chemically mutagenized to increase the genetic diversity of the initial population for selection. 905 (Wild Type) and 906 (EMS mutagenized wild type) were screened under different aluminium levels such as; 0.05, 0.1, 0.4, 0.6, 1, 2, 5, 10, 25, 50 mM to determine the initial aluminium stress level to be used during increasing stress level selections. A batch selection strategy was then applied to obtain aluminium-resistant S. cerevisiae mutants by gradually increasing aluminium-stress levels from 0.5 to 21.5 mM AlCl3 through 43 passages. The 43th last population was spread on plates and 12 individual mutants were randomly chosen. They were tested for their aluminium resistance by using spot assay procedure. According to spot assay results, 4 highly resistant mutants (named as Alu9, Alu10, Alu11, Alu12) were chosen for further analysis. In order to quantify the aluminium resistances of the mutants in detail Most Probable Number method was applied to those 4 mutant individuals. They were then tested for the genetic stability of the Al-resistance.Results showed that the mutants are genetically stable. Cross-resistance tests were also applied using a variety of other metal and non-metal stress types to test if the aluminium-resistant mutants developed cross-resistance or sensitivities to other stress types. For this purpose, the following stresses were tested: 8%(v/v) Ethanol, 0.2 mM NiCl2, 0.7 mM H2O2 , 0.25 mM CuSO4, 1 ml/L phenyl ethanol, 1.3 M MgCl2, 2.5 mM CrCl3, 10 mM MnCl2, 30 mM FeSO4, 80 mM H3BO3, 300 mg/ml Propolis, 0.5 M CaCl2, 2 mM GaNO3, 75 µM AgNO3, 0.20 NiCl2, 10 mM NaCl, 3 mM ZnCl2, 1 mM CoCl2, 2 mM LiCl, pH 4, 25 mM FeCl2. However, no significant cross-resistance or sensitivities to other stress types were detected, except for a slight level of LiCl,GaNO3 and ethanol tolerance, and a slight sensitivity to AgNO3. According to the experimental results, the best individual mutant (Alu10) was chosen for further detailed analyses at physiological and molecular levels.