Growth and growth estimation of Saccharomyces cerevisiae in solid-state ethanol fermentation

The anaerobic growth of a respiration-deficient mutant of Saccharomyces cerevisiae on solid medium was estimated by the CO₂ evolution rate (CER). The cell growth and ethanol production were calculated by a growth-model associated with CER. The estimated cell growth agreed with the observed data. The calculation and the observed CER suggested that the maximum ethanol production and maximum cell groth are restricted by the initial moisture content of the solid medium.     

Global gene expression during short-term ethanol stress in Saccharomyces cerevisiae

DNA microarrays were used to investigate the expression profile of yeast genes in response to ethanol. Up to 3.1% of the genes encoded in the yeast genome were up-regulated by at least a factor of three after 30 min ethanol stress (7% v/v). Concomitantly, 3.2% of the genes were down-regulated by a factor of three. Of the genes up-regulated in response to ethanol 49.4% belong to the environmental stress response and 14.2% belong to the stress gene family. Our data show that in addition to the previously identified ethanol-induced genes, a very large number of genes involved in ionic homeostasis, heat protection, trehalose synthesis and antioxidant defence also respond to ethanol stress. It appears that a large number of the up-regulated genes are involved in energy metabolism. Thus, 'management' of the energy pool (especially ATP) seems to constitute an ethanol stress response and to involve different mechanisms.     

Influence of acetate on growth kinetics and production control of Saccharomyces cerevisiae on glucose and ethanol

Summary The production and assimilation of acetate during the growth of Saccharomyces cerevisiae on glucose and ethanol have been studied. Acetate inhibits growth and causes decreased yields on both substrates. The usual respiratory quotient based policy for fed batch control cannot be used for highly acetate producing yeast because of compensation between O₂ over-consumption (due to acetate) and CO₂ over-production (due to ethanol) in case of glucose over-feeding.     

Xylose isomerase improves growth and ethanol production rates from biomass sugars for both Saccharomyces pastorianus and Saccharomyces cerevisiae

The demand for biofuel ethanol made from clean, renewable nonfood sources is growing. Cellulosic biomass, such as switch grass (Panicum virgatum L.), is an alternative feedstock for ethanol production; however, cellulosic feedstock hydrolysates contain high levels of xylose, which needs to be converted to ethanol to meet economic feasibility. In this study, the effects of xylose isomerase on cell growth and ethanol production from biomass sugars representative of switch grass were investigated using low cell density cultures. The lager yeast species Saccharomyces pastorianus was grown with immobilized xylose isomerase in the fermentation step to determine the impact of the glucose and xylose concentrations on the ethanol production rates. Ethanol production rates were improved due to xylose isomerase; however, the positive effect was not due solely to the conversion of xylose to xylulose. Xylose isomerase also has glucose isomerase activity, so to better understand the impact of the xylose isomerase on S. pastorianus, growth and ethanol production were examined in cultures provided fructose as the sole carbon. It was observed that growth and ethanol production rates were higher for the fructose cultures with xylose isomerase even in the absence of xylose. To determine whether the positive effects of xylose isomerase extended to other yeast species, a side-by-side comparison of S. pastorianus and Saccharomyces cerevisiae was conducted. These comparisons demonstrated that the xylose isomerase increased ethanol productivity for both the yeast species by increasing the glucose consumption rate. These results suggest that xylose isomerase can contribute to improved ethanol productivity, even without significant xylose conversion.     

The ethanol stress response and ethanol tolerance of Saccharomyces cerevisiae

Saccharomyces cerevisiae is traditionally used for alcoholic beverage and bioethanol production; however, its performance during fermentation is compromised by the impact of ethanol accumulation on cell vitality. This article reviews studies into the molecular basis of the ethanol stress response and ethanol tolerance of S. cerevisiae; such knowledge can facilitate the development of genetic engineering strategies for improving cell performance during ethanol stress. Previous studies have used a variety of strains and conditions, which is problematic, because the impact of ethanol stress on gene expression is influenced by the environment. There is however some commonality in Gene Ontology categories affected by ethanol assault that suggests that the ethanol stress response of S. cerevisiae is compromised by constraints on energy production, leading to increased expression of genes associated with glycolysis and mitochondrial function, and decreased gene expression in energy‐demanding growth‐related processes. Studies using genome‐wide screens suggest that the maintenance of vacuole function is important for ethanol tolerance, possibly because of the roles of this organelle in protein turnover and maintaining ion homoeostasis. Accumulation of Asr1 and Rat8 in the nucleus specifically during ethanol stress suggests S. cerevisiae has a specific response to ethanol stress although this supposition remains controversial.     

Polyphosphates as an energy source for growth of Saccharomyces cerevisiae

Cells of the yeast Saccharomyces cerevisiae with a low content of polyphosphates (polyP) are characterized by disturbance of growth in medium with 0.5% glucose. The parent strain with polyP level reduced by phosphate starvation had a longer lag phase. The growth rate of strains with genetically determined low content of polyP due to their enhanced hydrolysis (CRN/pMB1_PPN1 Sc is a superproducer of exopolyphosphatase PPN1) or reduced synthesis (the BY4741 vma2Δ mutant with impaired vacuolar membrane energization) was lower in the exponential phase. The growth of cells with high content of polyP was accompanied by polyP consumption. In cells of strains with low content of polyP, CRN/pMB1_PPN1 Sc and BY4741 vma2Δ, their consumption was insignificant. These findings provide more evidence indicating the use of polyP as an extra energy source for maintaining high growth rate.     

Role of growth phase and ethanol in freeze-thaw stress resistance of Saccharomyces cerevisiae.

The freeze-thaw tolerance of Saccharomyces cerevisiae was examined throughout growth in aerobic batch culture. Minimum tolerance to rapid freezing (immersion in liquid nitrogen; cooling rate, approximately 200 degrees C min-1) was associated with respirofermentative (exponential) growth on glucose. However, maximum tolerance occurred not during the stationary phase but during active respiratory growth on ethanol accumulated during respirofermentative growth on glucose. The peak in tolerance occurred several hours after entry into the respiratory growth phase and did not correspond to a transient accumulation of trehalose which occurred at the point of glucose exhaustion. Substitution of ethanol with other carbon sources which permit high levels of respiration (acetate and galactose) also induced high freeze-thaw tolerance, and the peak did not occur in cells shifted directly from fermentative growth to starvation conditions or in two respiratorily incompetent mutants. These results imply a direct link with respiration, rather than exhaustion of glucose. The role of ethanol as a cryoprotectant per se was also investigated, and under conditions of rapid freezing (cooling rate, approximately 200 degrees C min-1), ethanol demonstrated a significant cryoprotective effect. Under the same freezing conditions, glycerol had little effect at high concentrations and acted as a cryosensitizer at low concentrations. Conversely, under slow-freezing conditions (step freezing at -20, -70, and then -196 degrees C; initial cooling rate, approximately 3 degrees C min-1), glycerol acted as a cryoprotectant while ethanol lost this ability. Ethanol may thus have two effects on the cryotolerance of baker's yeast, as a respirable carbon source and as a cryoprotectant under rapid-freezing conditions.     

Comprehensive phenotypic analysis for identification of genes affecting growth under ethanol stress in Saccharomyces cerevisiae

We quantified the growth behavior of all available single gene deletion strains of budding yeast under ethanol stress. Genome-wide analyses enabled the extraction of the genes and determination of the functional categories required for growth under this condition. Statistical analyses revealed that the growth of 446 deletion strains under stress induced by 8% ethanol was defective. We classified these deleted genes into known functional categories, and found that many were important for growth under ethanol stress including several categories that have not been characterized, such as peroxisome. We also performed genome-wide screening under osmotic stress and identified 329 osmotic-sensitive strains. We excluded these strains from the 446 ethanol-sensitive strains to extract the genes whose deletion caused sensitivity to ethanol-specific (359 genes), osmotic-specific (242 genes), and both stresses (87 genes). We also extracted the functional categories that are specifically important for growth under ethanol stress. The genes and functional categories identified in the analysis might provide clues to improving ethanol stress tolerance among yeast cells.     

Saccharomyces cerevisiae mutants, producers of lysine on an ethanol medium

Using the sensitivity of Saccharomyces cerevisiae to S-(2-aminoethyl)-L-cysteine (AEC) depending on different carbons, the regulatory resistant mutants producing lysine were isolated. By the further selection of the mutant resistant to monoiodine acetate (MIA) (an inhibitor of the alcohol dehydrogenase) the production of lysine was increased. The mutants resistant to AEC and MIC excreting 0.7 g/l of lysine when grown on the medium with ethanol were prepared.     

Heterologous expression of mammalian Na/H antiporters in Saccharomyces cerevisiae

Na+/H+ antiporters, integral membrane proteins that exchange protons for alkali metal cations, play multiple roles in probably all living organisms (preventing cells from excessive amounts of alkali metal cations, regulating intracellular pH and cell volume). In this work, we studied the functionality of rat plasma membrane NHE1-3 exchangers upon their heterologous expression in alkali-metal-cation sensitive Saccharomyces cerevisiae, and searched for conditions that would increase their level in the plasma membrane and improve their functionality. Though three tested exchangers were partially localized to the plasma membrane (and two of them (NHE2 and NHE3) in an active form), the bulk of the synthesized proteins were arrested along the secretory pathway, mainly in the ER. To increase the level of exchangers in the yeast plasma membrane several approaches (truncation of C-terminal regulatory sequences, expression in mutant yeast strains, construction of rat/yeast protein chimeras, various growth conditions and chemical chaperones) were tested. The only increase in the amount of NHE exchangers in the plasma membrane was obtained upon expression in a strain with the npi1 mutation, which significantly lowers the level of Rsp5 ubiquitin ligase in cells. This mutation helped to stabilize proteins in the plasma membrane.     

Regulation of sugar and ethanol metabolism in Saccharomyces cerevisiae

This review briefly surveys the literature on the nature, regulation, genetics, and molecular biology of the major energy-yielding pathways in yeasts, with emphasis on Saccharomyces cerevisiae. While sugar metabolism has received the lion's share of attention from workers in this field because of its bearing on the production of ethanol and other metabolites, more attention is now being paid to ethanol metabolism and the regulation of aerobic metabolism by fermentable and nonfermentable substrates. The utility of yeast as a highly manipulable organism and the discovery that yeast metabolic pathways are subject to the same types of control as those of higher cells open up many opportunities in such diverse areas as molecular evolution and cancer research.     

Intracellular acidification does not account for inhibition of Saccharomyces cerevisiae growth in the presence of ethanol

Abstract Intracellular acidification has been considered one of a number of mechanisms underlying the inhibition of growth and fermentation by ethanol in yeast. However, most of the studies on the effect of ethanol on yeast intracellular pH (pH_i) were carried out by using unadapted cells to which ethanol was added. In this paper we show that the pH_i of exponential cells of Saccharomyces cerevisiae IGC 3507 III grown in a medium with glucose and inhibitory concentrations of ethanol only decreased to values below those in unstressed cells (6.9) for concentrations equal to or above 7% (v/v). Only at these supracritical levels (7–10% (v/v)) was pH homeostasis in ethanol-adapted yeast affected. This is consistent with the significant increase of plasma membrane permeability and decrease of plasma membrane H⁺-ATPase in comparison with the corresponding values in unstressed cells. These deleterious effects were only observed with those high concentrations of toxin. These results indicate that intracellular acidification does not account for inhibition of yeast growth in the presence of ethanol. In fact, growth was inhibited by ethanol concentrations (3–6% (v/v)) that did not lead to the decrease of pH_i. Furthermore, even for supracritical concentrations, close to the maximal that allowed growth (10% (v/v)), the dedrease of pH_i was not important reaching, at the most, values of 6.5–6.6.     

Saccharomyces cerevisiae membrane sterol modifications in response to growth in the presence of ethanol

Membranes isolated from yeasts grown in the presence of ethanol do not display the thermally induced transition in diphenylhexatriene anisotropy that is seen in control cells when they are exposed to ethanol in vitro. The total sterol content of the cells that were exposed to ethanol during growth is reduced, with no steryl esters being detected. A greater proportion of the total sterol pool is ergosterol in cells grown in the presence of alcohol. The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase is reduced by ethanol in vitro. Ethanol-exposed cells take up more exogenous sterol under aerobic conditions than do control cells. The presence of ethanol during growth reduces the activity of the plasma membrane enzyme, chitin synthase, as well as increasing the thermosensitivity of this enzyme.