Tony D'Amore

The involvement of trehalose in yeast stress tolerance

SummaryA total of 12 yeast strains from various genera were examined for their ability to produce ethanol in the presence of high concentrations of glucose. From these studies, the yeastsTorulaspora delbrueckii andZygosaccharomyces rouxii were observed to the most osmotolerant. These osmotolerant yeast strains were also observed to possess high concentrations of intracellular trehalose. Futhermore, these strains were found to be tolerant to long-term storage at −20°C and to storage at 4°C in beer containing 5% (v/v) ethanol. Cells containing high trehalose levels at the time of freezing or cold storage exhibited the highest cell viabilities. Trehalose concentration was observed to increase during growth on glucose, reaching a maximum after 24–48 h. Increasing the incubation temperature from 21 to 40°C also resulted in an increase in intracellular trehalose content. These results suggest that trehalose plays a role in enhancing yeast survival under environmentally stressful conditions.

Alterations in fatty acid composition and trehalose concentration ofSaccharomyces brewing strains in response to heat and ethanol shock

SummaryThe effects of heat and ethanol shock on fatty acid composition and intracellular trehalose concentration of lager and ale brewing yeasts were examined. Exposure of cells to heat shock at 37°C or 10% (v/v) ethanol for 60 min resulted in a significant increase in the ratio of the total unsaturated to saturated fatty acyl residues and the intracellular trehalose concentration of cells. A similar increase in the amount of unsaturated fatty acids was observed in cells after 24 h of fermentation of 16°P (degree Plato) or 25°P wort, at which time more than 2% (v/v) ethanol was present in the growth medium. These results suggest that unsaturated fatty acids and high concentrations of intracellular trehalose may protect the cells from the inhibitory effects of heat and ethanol shock.

Effects of heat shock and ethanol stress on the viability of aSaccharomyces uvarum (carlsbergensis) brewing yeast strain during fermentation of high gravity wort

SummaryThe effects of heat shock and ethanol stress on the viability of a lager brewing yeast strain during fermentation of high gravity wort were studied. These stress effects resulted in reduced cell viability and inhibition of cell growth during fermentation. Cells were observed to be less tolerant to heat shock during the fermentation of 25°P (degree Plato) wort than cells fermenting 16°P wort. Degree Plato (oP) is the weight of extract (sugar) equivalent to the weight of sucrose in a 100 g solution at 20°C. Relieving the stress effects of ethanol by washing the cells free of culture medium, improved their tolerance to heat shock. Cellular changes in yeast protein composition were observed after 24 h of fermentation at which time more than 2% (v/v) ethanol was present in the growth medium. The synthesis of these proteins was either induced by ethanol or was the result of the transition of cells from exponential phase to stationary phase of growth. No differences were observed in the protein composition of cells fermenting 16°P wort compared to those fermenting 25°P wort. Thus, the differences in the tolerance of these cells to heat shock may be due to the higher ethanol concentration produced in 25°P wort which enhanced their sensitivity to heat shock.

Transport kinetics of maltotriose in strains ofSaccharomyces

SummaryMaltotriose transport was studied in two brewers yeast strains, an ale strain 3001 and a lager strain 3021, using laboratory-synthesized14C-maltotriose. The maltotriose transport systems preferred a lower pH (pH 4.3) to a higher pH (pH 6.6). Two maltotriose transport affinity systems have been indentified. The high affinity system hasKm values of 1.3 mM for strain 3021 and 1.4 mM for strain 3001. The low affinity competitively inhibited by maltose and glucose withKi values of 58 mM and 177 mM. respectively, for strain 3021, and 55 mM and 147 mM, respectively, for strain 3001. Cells grown in maltotriose and maltose had higher maltotriose and maltose transport rates, and cells grown in glucose had lower maltortriose and maltose transport rates. Early-logarithmic phase cells transported glucose faster than either maltose or maltotriose. Cells harvested later in the growth phase had increased maltotriose and maltose transport activity. Neither strain exhibited significant differences with respect to maltose and maltotriose transport activity.

2-Deoxy-D-glucose resistant yeast with altered sugar transport activity.

The transport of glucose and maltose in Saccharomyces cerevisiae was observed to occur by both high and low affinity transport systems. A spontaneously isolated 2‐deoxy‐D‐glucose resistant mutant was observed to transport glucose and maltose only by the high affinity transport systems. Associated with this was an increase in the V max values, indicating derepression of the high affinity transport systems. The low affinity transport systems could not be detected. This mutant will be important in examining the repression regulatory and sugar transport mechanisms in yeast.

Changes in protein composition ofSaccharomyces brewing strains in response to heat shock and ethanol stress

SummaryHeat shock and ethanol stress of brewing yeast strains resulted in the induction of a set of proteins referred to as heat shock proteins (HSPs). At least six strongly induced HSPs were identified in a lager brewing strain and four HSPs in an ale brewing strain. Four of these HSPs with molecular masses of approximately 70, 38, 26 and 23 kDa were also identified in two laboratory strains ofSaccharomyces cerevisiae. The appearance of HSPs correlated with increased survival of strains at elevated temperatures and high concentrations of ethanol. These results suggest that HSPs may play a role in the ethanol and thermotolerance of yeasts. The properties of these proteins and membrane fatty acids in relation to heat and ethanol shock are being investigated.

Sugar uptake in a 2-deoxy-d-glucose resistant mutant ofSaccharomyces cerevisiae

SummaryThe non-metabolizable and toxic glucose analogue 2-deoxy-d-glucose (2-DOG) has been widely employed to screen for regulatory mutants which lack catabolite repression. A number of yeast mutants resistant to 2-DOG have recently been isolated in this laboratory. One such mutant, derived from aSaccharomyces cerevisiae haploid strain, was demonstrated to be derepressed for maltose, galactose and sucrose uptake. Furthermore, kinetic analysis of glucose transport suggested that the high affinity glucose transport system was also derepressed in the mutant strain. In addition, the mutant had an increased intracellular concentration of trehalose relative to the parental strain. These results indicate that the 2-DOG resistant mutant is defective in general glucose repression.

Regulation of glucose and maltose transport in strains ofSaccharomyces

SummaryGrowth of yeast cells on glucose resulted in complete inactivation of maltose transport and repression of the high affinity glucose transport system. When the cells were grown on maltose or subjected to substrate starvation, an increase in glucose and maltose transport was observed in both brewing and non-brewing yeast strains. The concentration of glucose employed in the growth medium was also observed to affect sugar transport activity. The higher the glucose concentration, the more pronounced the repressive effect. In addition, the time of growth of yeast on glucose or maltose also intermining the rate of sugar transport. These results are consistent with the repressive effect of glucose on the high affinity glucose and maltose transport systems.

Characterization of sugar transport in 2-deoxy-d-glucose resistant mutants of yeast

SummaryA number of 2-deoxy-d-glucose (2-DOG) resistant mutants exhibiting resistance to glucose repression were isolated from variousSaccharomyces yeast strains. Most of the mutants isolated were observed to have improved maltose uptake ability in the presence of glucose. Fermentation studies indicated that maltose was taken up at a faster rate and glucose taken up at a slower rate in the mutant strains compared to the parental strains, when these sugars were fermented together. When these sugars were fermented separately, only the 2-DOG resistant mutant obtained fromSaccharomyces cerevisiae strain 1190 exhibited alterations in glucose and maltose uptake compared to the parental strain. Kinetic analysis of sugar transport employing radiolabelled glucose and maltose indicated that both glucose and maltose were transported with higher rates in the mutant strain. These results suggested that the high affinity glucose transport system was regulated by glucose repression in the parental strain but was derepressed in the mutant.

The reaction of 4-deoxy-4-fluoro-D-glucose with an outer membrane protein of Pseudomonas putida

Glucose-grown or cell-free extracts of Pseudomonas putida metabolise 3FG to 3FGA and 3F2KGA [I]. These fluorinated metabolites are produced by the enzymes glucose oxidase (EC 1.1.3.4) and gluconate dehydrogenase (EC 1.1.99.3) which are considered to be located on the outer surface of the cytoplasmic membrane [2,3]. Using membrane vesicles prepared from glucose or succinate grown P. putida we have shown that 3FG is transported by the same active-transport system as glucose [4]. 3FG [5] and 4FG act as catabolite repressors in lactose-grown Escherichia coli 161. In the above studies with 3FG or 4FG the carbon-fluorine bond remains intact. Here, we wish to report that: (i) Unlike 3FG, the isomeric 4FG is not transported or oxidized by whole cells of glucose grown P. putida; instead an extensive release of Foccurs. 2.1. Chemicals Crystalline 4FG 4-deoxy-D-glucose and a-Me4FG were synthesized as in [8]. NEM, DTT, chloramphenicol, lysozyme, ribonuclease A, deoxyribonuclease I, DCIP, PMS and all other carbohydrates, chemicals and reagents were obtained from Sigma (St Louis MO).