Project description:Prolonged cultivation (>25 generations) of Saccharomyces cerevisiae in aerobic, maltose-limited chemostat cultures led to profound physiological changes. Maltose hypersensitivity was observed when cells from prolonged cultivations were suddenly exposed to excess maltose. This substrate hypersensitivity was evident from massive cell lysis and loss of viability. During prolonged cultivation at a fixed specific growth rate, the affinity for the growth-limiting nutrient (i.e., maltose) increased, as evident from a decreasing residual maltose concentration. Furthermore, the capacity of maltose-dependent proton uptake increased up to 2.5-fold during prolonged cultivation. Genome-wide transcriptome analysis showed that the increased maltose transport capacity was not primarily due to increased transcript levels of maltose-permease genes upon prolonged cultivation. We propose that selection for improved substrate affinity (ratio of maximum substrate consumption rate and substrate saturation constant) in maltose-limited cultures leads to selection for cells with an increased capacity for maltose uptake. At the same time, the accumulative nature of maltose-proton symport in S. cerevisiae leads to unrestricted uptake when maltose-adapted cells are exposed to a substrate excess. These changes were retained after isolation of individual cell lines from the chemostat cultures and nonselective cultivation, indicating that mutations were involved. The observed trade-off between substrate affinity and substrate tolerance may be relevant for metabolic engineering and strain selection for utilization of substrates that are taken up by proton symport. Keywords: Evolution
Project description:Gene expression of a vital, stained and sorted subpopulation of Saccharomyces cerevisiae with high affinity to glucose, harvested at a dilution rate of D=0.160 h-1, and of cells from the whole population without further treatment grown at the same dilution rate were analysed. The isolation of RNA was accomplished by using lyticase to lyse the cells and the Qiagen RNeasy Mini Kit with some modifications within the manufacturers´ protocol. 6 µg of the total RNA per sample was used for each microarray experiments. The indirect labelling by the tyramide-signal-amplification method (MicromaxTM TSATM labelling and detection Kit from Perkin Elmer life sciences) was used to increase the Cy3 and Cy5 signals of microarray detection. Each cDNA containing Biotin- and Fluorescein-nucleotides respectively was purified with a QIAquick PCR purification kit and suspended in 11 µl of the formamide containing hybridization buffer. The slides were hybridized at 42°C over night under a cover slip. The microarrays were scanned by the Axon 4000B scanner; image intensity data were extracted and analysed with GenePix® Pro 6.0 software. Data from different scans of Dye-swap experiment were extracted by GenePix Pro 6.0 software, normalized and united. An outliertest has been applied in order to find outliers amongst the gene replicats. Subsequently, a t-test (1% and 5% probability of error) has been used in order to find regulated genes. Keywords: sorted yeast cells