Michael C. Walsh

Physiological and Transcriptional Responses of Saccharomyces cerevisiae to Zinc Limitation in Chemostat Cultures

ABSTRACT Transcriptional responses of the yeast Saccharomyces cerevisiae to Zn availability were investigated at a fixed specific growth rate under limiting and abundant Zn concentrations in chemostat culture. To investigate the context dependency of this transcriptional response and eliminate growth rate-dependent variations in transcription, yeast was grown under several chemostat regimens, resulting in various carbon (glucose), nitrogen (ammonium), zinc, and oxygen supplies. A robust set of genes that responded consistently to Zn limitation was identified, and the set enabled the definition of the Zn-specific Zap1p regulon, comprised of 26 genes and characterized by a broader zinc-responsive element consensus (MHHAACCBYNMRGGT) than so far described. Most surprising was the Zn-dependent regulation of genes involved in storage carbohydrate metabolism. Their concerted down-regulation was physiologically relevant as revealed by a substantial decrease in glycogen and trehalose cellular content under Zn limitation. An unexpectedly large number of genes were synergistically or antagonistically regulated by oxygen and Zn availability. This combinatorial regulation suggested a more prominent involvement of Zn in mitochondrial biogenesis and function than hitherto identified.

Control of the Glycolytic Flux in Saccharomyces cerevisiae Grown at Low Temperature A MULTI-LEVEL ANALYSIS IN ANAEROBIC CHEMOSTAT CULTURES

Growth temperature has a profound impact on the kinetic properties of enzymes in microbial metabolic networks. Activities of glycolytic enzymes in Saccharomyces cerevisiae were up to 7.5-fold lower when assayed at 12 °C than at 30 °C. Nevertheless, the in vivo glycolytic flux in chemostat cultures (dilution rate: 0.03 h–1) grown at these two temperatures was essentially the same. To investigate how yeast maintained a constant glycolytic flux despite the kinetic challenge imposed by a lower growth temperature, a systems approach was applied that involved metabolic flux analysis, transcript analysis, enzyme activity assays, and metabolite analysis. Expression of hexose-transporter genes was affected by the growth temperature, as indicated by differential transcription of five HXT genes and changed zero trans-influx kinetics of [14C]glucose transport. No such significant changes in gene expression were observed for any of the glycolytic enzymes. Fermentative capacity (assayed off-line at 30 °C), which was 2-fold higher in cells grown at 12 °C, was therefore probably controlled predominantly by glucose transport. Massive differences in the intracellular concentrations of nucleotides (resulting in an increased adenylate energy charge at low temperature) and glycolytic intermediates indicated a dominant role of metabolic control as opposed to gene expression in the adaptation of glycolytic enzyme activity to different temperatures. In evolutionary terms, this predominant reliance on metabolic control of a central pathway, which represents a significant fraction of the cellular protein of the organism, may be advantageous to limit the need for protein synthesis and degradation during adaptation to diurnal temperature cycles.

Selection from Industrial Lager Yeast Strains of Variants with Improved Fermentation Performance in Very-High-Gravity Worts

ABSTRACT There are economic and other advantages if the fermentable sugar concentration in industrial brewery fermentations can be increased from that of currently used high-gravity (ca. 14 to 17°P [degrees Plato]) worts into the very-high-gravity (VHG; 18 to 25°P) range. Many industrial strains of brewers yeast perform poorly in VHG worts, exhibiting decreased growth, slow and incomplete fermentations, and low viability of the yeast cropped for recycling into subsequent fermentations. A new and efficient method for selecting variant cells with improved performance in VHG worts is described. In this new method, mutagenized industrial yeast was put through a VHG wort fermentation and then incubated anaerobically in the resulting beer while maintaining the α-glucoside concentration at about 10 to 20 g·liter−1 by slowly feeding the yeast maltose or maltotriose until most of the cells had died. When survival rates fell to 1 to 10 cells per 106 original cells, a high proportion (up to 30%) of survivors fermented VHG worts 10 to 30% faster and more completely (residual sugars lower by 2 to 8 g·liter−1) than the parent strains, but the sedimentation behavior and profiles of yeast-derived flavor compounds of the survivors were similar to those of the parent strains.

Effect of Nutrient Starvation on the Cellular Composition and Metabolic Capacity of Saccharomyces cerevisiae

ABSTRACT This investigation addresses the following question: what are the important factors for maintenance of a high catabolic capacity under various starvation conditions? Saccharomyces cerevisiae was cultured in aerobic batch cultures, and during the diauxic shift cells were transferred and subjected to 24 h of starvation. The following conditions were used: carbon starvation, nitrogen starvation in the presence of glucose or ethanol, and both carbon starvation and nitrogen starvation. During the starvation period changes in biomass composition (including protein, carbohydrate, lipid, and nucleic acid contents), metabolic activity, sugar transport kinetics, and the levels of selected enzymes were recorded. Subsequent to the starvation period the remaining catabolic capacity was measured by addition of 50 mM glucose. The results showed that the glucose transport capacity is a key factor for maintenance of high metabolic capacity in many, but not all, cases. The results for cells starved of carbon, carbon and nitrogen, or nitrogen in the presence of glucose all indicated that the metabolic capacity was indeed controlled by the glucose transport ability, perhaps with some influence of hexokinase, phosphofructokinase, aldolase, and enolase levels. However, it was also demonstrated that there was no such correlation when nitrogen starvation occurred in the presence of ethanol instead of glucose.

Identity of the growth-limiting nutrient strongly affects storage carbohydrate accumulation in anaerobic chemostat cultures of Saccharomyces cerevisiae.

ABSTRACT Accumulation of glycogen and trehalose in nutrient-limited cultures of Saccharomyces cerevisiae is negatively correlated with the specific growth rate. Additionally, glucose-excess conditions (i.e., growth limitation by nutrients other than glucose) are often implicated in high-level accumulation of these storage carbohydrates. The present study investigates how the identity of the growth-limiting nutrient affects accumulation of storage carbohydrates in cultures grown at a fixed specific growth rate. In anaerobic chemostat cultures (dilution rate, 0.10 h−1) of S. cerevisiae, the identity of the growth-limiting nutrient (glucose, ammonia, sulfate, phosphate, or zinc) strongly affected storage carbohydrate accumulation. The glycogen contents of the biomass from glucose- and ammonia-limited cultures were 10- to 14-fold higher than those of the biomass from cultures grown under the other three glucose-excess regimens. Trehalose levels were specifically higher under nitrogen-limited conditions. These results demonstrate that storage carbohydrate accumulation in nutrient-limited cultures of S. cerevisiae is not a generic response to excess glucose but instead is strongly dependent on the identity of the growth-limiting nutrient. While transcriptome analysis of wild-type and msn2Δ msn4Δ strains confirmed that transcriptional upregulation of glycogen and trehalose biosynthesis genes is mediated by Msn2p/Msn4p, transcriptional regulation could not quantitatively account for the drastic changes in storage carbohydrate accumulation. The results of assays of glycogen synthase and glycogen phosphorylase activities supported involvement of posttranscriptional regulation. Consistent with the high glycogen levels in ammonia-limited cultures, the ratio of glycogen synthase to glycogen phosphorylase in these cultures was up to eightfold higher than the ratio in the other glucose-excess cultures.

Involvement of Vacuolar Sequestration and Active Transport in Tolerance of Saccharomyces cerevisiae to Hop Iso-α-Acids

ABSTRACT The hop plant, Humuluslupulus L., has an exceptionally high content of secondary metabolites, the hop α-acids, which possess a range of beneficial properties, including antiseptic action. Studies performed on the mode of action of hop iso-α-acids have hitherto been restricted to lactic acid bacteria. The present study investigated molecular mechanisms of hop iso-α-acid resistance in the model eukaryote Saccharomycescerevisiae. Growth inhibition occurred at concentrations of hop iso-α-acids that were an order of magnitude higher than those found with hop-tolerant prokaryotes. Chemostat-based transcriptome analysis and phenotype screening of the S. cerevisiae haploid gene deletion collection were used as complementary methods to screen for genes involved in hop iso-α-acid detoxification and tolerance. This screening and further analysis of deletion mutants confirmed that yeast tolerance to hop iso-α-acids involves three major processes, active proton pumping into the vacuole by the vacuolar-type ATPase to enable vacuolar sequestration of iso-α-acids and alteration of cell wall structure and, to a lesser extent, active export of iso-α-acids across the plasma membrane. Furthermore, iso-α-acids were shown to affect cellular metal homeostasis by acting as strong zinc and iron chelators.