Evrimsel mühendislik yöntemiyle elde edilmiş tuza dirençli bir Saccharomyces cerevisiae mutantının moleküler ve fizyolojik karakterizasyonu

Yeast is an excellent model system for molecular biology, cell morphogenesis, chromosome stability , and even aging. Saccharomyces cerevisiae CEN.PK 113-7D is widely used for metabolic engineering and systems biology research in industry and academia. A consortium of German yeast researchers developed the isogenic family of CEN.PK strains by crossing different laboratory strains of S.cerevisiae in the 1990s. The haploid MATa strain CEN.PK113-7D, has been extremely popular for studies in systems biology and is a preferred laboratory strain for industrial biotechnology research. Maintenance of alkali cation homeostasis is a key process in most types of cells, including yeasts. Sodium chloride is very important for human biology. Na+ and K+ are essential for human nutrition and also Li+ is an essential nutrient in rodents and goats. The budding yeast S. cerevisiae is able to grow in the presence of a broad range of external concentrations of K+ (10 mM–2.5 M) and Na+ (1.5 M). The aim of this study was to investigate the effects of NaCl stress at physiological and transcriptomic level. S.cerevisiae was first mutagenized by ethyl methanesulfonate (EMS) and then exposed to salt stress (NaCl) by Sezgin T. (2010). An individual was chosen from the population and compared to wild type to investigate how it gained resistance to salt stress. Specific growth curves of wild type and "T8" in YMM were calculated by growing these strains in 5 mM LiCl, 0.5 M NaCl, 0.7 M NaCl and 0.9 M NaCl. In all of these conditions, T8 grew better than the wild type. Spot test results of T8 and wild type (CENPK-7D) showed that resistance to sodium and lithium are obvious, however potassium resistance was not observed. NaCl resistant mutant "T8" was resistant to sodium and lithium, while the wild type as well as T8 grew in the presence of potassium. It couldbe inferred that "T8" had resistance to cation toxicity, but not to osmotic stress. Flame Atomic Absorption Spectrometry (FAAS) results showed that high extracellular Na+ and Li+ concentrations increase intracellular Na+ and decrease intracellular K+ , in both wild type and T8. Apperantly, cells maintain the total monovalent cation content low after K+, Na+ and Li+ stress. NaCl resistant mutant "T8" did not exhibit significantly increased lithium accumulation, compared to the wild type strain. The lithium resistance of the wild type was not caused by increased lithium efflux. "T8" accumulated slightly more Li+ than the wild type in double stress conditions. FAAS results showed that T8 has higher K+/Na+ ratio at every stress condition compared to the wild type. Difference of K+/Na+ ratio between T8 and the wildtype is obvious at 5 mM LiCl. K+/Na+ ratio of T8 was 4.21 and of wildtype was 1.97 at 5 mM LiCl stress. ENA6 and NHA1 genes' expression were lower at LiCl stress, compared to NaCl stress. The reason why K+/Na+ ratio is higher at LiCl stress can be the lower expression of these efflux genes so that T8 may not have active efflux at LiCl stress as at NaCl stress. Glycogen and trehalose are two important glucose stores of the yeast S. cerevisiae, and production of them change in response to a number of environmental stress conditions. The disaccharide trehalose accumulates during salt adaptation. Assessment of glycogen and trehalose content showed that amount of trehalose and glycogen in T8 are prominently high compared to the wild type without stress conditions. Lithium triggers the amount of glycogen and trehalose more than sodium. It could be explained by higher toxicity of lithium. Trehalose accumulation is higher in T8 than glycogen without and with sodium and lithium stress. In our NaCl-resistant evolved S. cerevisiae strain "T8", production of glycerol is higher and production of ethanol is reduced compared to the wildtype. The metabolism of the evolved strain is redirected toward glycerol and away from ethanol production. The cross-resistance of T8 was assessed by growth on LiCl, ZnCl2, H2O2, CoCl2, KCl, ethanol, CuCl2, MnCl2, CrCl3, AlCl3YPD and YMM plates.T8 has significantly high cross resistance to lithium.T8 has slight resistance to Co+2, Mn+2 Ni+2, Zn+2 and is sensitive to aluminum. The S. cerevisiae genome contains three genes encoding alkali metal cation/H antiporters (Nha1p, Nhx1p, Kha1p) that differ in cell localization, substrate specificity and physiological function. Sequencing of NHX1, NHA1, and KHA1 were checked to be compared with mutant strain "T8". Sequencing results showed that EMS mutagenesis and NaCl stress did not affect the sequences of NHX1, NHA1, and KHA1 genes. The transcriptional response to high NaCl concentration stress in the yeast S. cerevisiae was analyzed by using DNA microarray to understand yeast strains showing tolerance to high salt stress. In microarray results, several hundred genes were identified associated with NaCl tolerance involving a broad range of functional categories including energy; cell rescue, defense and virulence; metabolism; cellular transport, transport facilities and transport routes; cellular communication/signal transduction mechanism. FLO11 gene encoding GPI-anchored cell surface glycoprotein(flocculin) which is required for flocculation and biofilms was the most highly expressed gene in T8. In "T8" microarray results, HOG pathway is not induced. "T8" had slower glucose uptake and did not accumulate high levels of succinate and acetate. In microarray results,T8 has induced ENA6 expression, however NHA1 is not induced. In Real-time PCR results, both ENA6 and NHA1 genes are induced. This may result from the differences in sensitivities of microarray and real-time PCR, the latter being more sensitive. Microarray results indicated that "T8" has many genes overexpressed compared to the wild type even in the absence of NaCl stress. These upregulated and downregulated genes adapted this mutant to high sodium and lithium stresses. Nha1 was cloned to be overproduced and it is checked by complementation assays. Overexpression of Nha1p showed that this protein complements better at pH 3.5. Specific single site mutations [G372S, D266H, D267A, D266H/D267A, D266N/D267N, N176, D177N ] on Nha1 were applied to understand these influence of this mutations on pH. In this study it was shown that under S. cerevisiae strains with enhanced glycerol and reduced ethanol yields could be obtained by evolutionary engineering for NaCl-stress resistance.This study is the first one that uses EMS and increasing NaCl stress levels for the selection of S. cerevisiae strains by evolutionary engineering. Additionally, the results of this thesis work suggest that FLO11 may have an important role in high NaCl-resistance in S.cerevisiae.