Characterization of maltotriose transporters from the Saccharomyces eubayanus subgenome of the hybrid Saccharomyces pastorianus lager brewing yeast strain Weihenstephan 34/70

The genome from the Saccharomyces pastorianus industrial lager brewing strain Weihenstephan 34/70, a natural Saccharomyces cerevisiae/Saccharomyces eubayanus hybrid, indicated the presence of two different maltotriose transporter genes: a new gene in the S. eubayanus subgenome with 81% of homology to the AGT1 permease from S. cerevisiae, and an amplification of the S. eubayanus MTY1 maltotriose permease previously identified in S. pastorianus yeasts. To characterize these S. eubayanus transporter genes, we used a S. cerevisiae strain deleted in the AGT1 permease and introduced the desired permease gene(s) into this locus through homologous recombination. Our results indicate that both the MTY1 and AGT1 genes from the S. eubayanus subgenome encode functional maltotriose transporters that allow fermentation of this sugar by yeast cells, despite their apparent differences in the kinetics of maltotriose‐H⁺ symport activity. The presence of two maltotriose transporters in the S. eubayanus subgenome not only highlights the importance of sugar transport for efficient maltotriose utilization by industrial yeasts, but these new genes can be used in breeding and/or selection programs aimed at increasing yeast fitness for the efficient fermentation of brewer's wort.     

Production of ethanol by Saccharomyces cerevisiae under high-pressure conditions

A research project was initiated to examine the possibility of using supercritical carbon dioxide for in situ recovery of ethanol during its production by yeast Saccharomyces cerevisiae. As a preliminary step, it was necessary to study the behavior of ethanol production under high-pressure conditions, up to 7 MPa (1000 psi). The results show that pressure has a significant inhibiting effect on the production of ethanol. There is a significant decrease in the initial rate of production as well as in the final ethanol concentration as pressure is increased. This decrease is more significant when carbon dioxide is used to pressurize the fermentor. The pressure affects the ability of the cells to produce ethanol in a reversible way. When the fermentor is returned to atmospheric conditions, the reaction resumes its normal fermentation rate.     

Effect of L-proline on sake brewing and ethanol stress in Saccharomyces cerevisiae

During the fermentation of sake, cells of Saccharomyces cerevisiae are exposed to high concentrations of ethanol, thereby damaging the cell membrane and functional proteins. L-proline protects yeast cells from damage caused by freezing or oxidative stress. In this study, we evaluated the role of intracellular L-proline in cells of S. cerevisiae grown under ethanol stress. An L-proline-accumulating laboratory strain carries a mutant allele of PRO1, pro1(D154N), which encodes the Asp154Asn mutant gamma-glutamyl kinase. This mutation increases the activity of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase, which catalyze the first two steps of L-proline synthesis and which together may form a complex in vivo. When cultured in liquid medium in the presence of 9% and 18% ethanol under static conditions, the cell viability of the L-proline-accumulating laboratory strain is greater than the cell viability of the parent strain. This result suggests that intracellular accumulation of L-proline may confer tolerance to ethanol stress. We constructed a novel sake yeast strain by disrupting the PUT1 gene, which is required for L-proline utilization, and replacing the wild-type PRO1 allele with the pro1(D154N) allele. The resultant strain accumulated L-proline and was more tolerant to ethanol stress than was the control strain. We used the strain that could accumulate L-proline to brew sake containing five times more L-proline than what is found in sake brewed with the control strain, without affecting the fermentation profiles.     

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

Summary Heat 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.     

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.     

Continuous cultivation of Saccharomyces cerevisiae. I. Growth on ethanol under steady-state conditions

Growth of Saccharomyces cerevisiae LBG H 1022 on ethanol under steady-state conditions was studied. As a cultivation device, an aerated Chemap fermentor combined with continuously working gas analyzers for oxygen and carbon dioxide was used. Dry matter, substrate concentration, yield, specific oxygen uptake, specific carbon dioxide release, and respiration quotient, as well as nitrogen, carbon, phosphorus, hydrogen, and protein content of the cells were measured in dependence on the dilution rate. Cell size distribution, as a function of the specific growth rate, was determined with the aid of a Celloscope 202. A fair agreement with the theory of continuous culture for all metabolic curves could be established. An increased turnover rate resulted from the addition of glutamic acid to the synthetic growth medium. The primary effect of this supplement could be a rise in the flow rate of the tricarboxylic acid cycle.     

Continuous cultivation of Saccharomyces cerevisiae. II. Growth on ethanol under transient-state conditions

Growth of Saccharomyces cerevisiae LBG H 1022 on ethanol under transient-state conditions was studied. As a cultivation device, an aerated Chemap fermentor combined with continuously working gas analyzers for oxygen and carbon dioxide was used. Yeast cell dry matter, substrate concentration, specific oxygen uptake, specific carbon dioxide release, and respiration quotient were measured during the different transient states. Depending on which range of the dilution rate the initial steady state was found, we obtain different responses to the shift experiment. For the lower range, up to D = 0.07, we deal with damped oscillations ranging above and below the steady-state values. For the higher specific growth rates, the rate of damping is strongly enhanced and the shape of the curves becomes an asymptotic approach to the final steady states.     

Rapid response to artificial selection on flower size in Phlox

Quantitative characters are often said to evolve rather slowly, taking many generations to exhibit appreciable differences among populations. We tested this notion experimentally by performing bi-directional selection on corolla diameter of plants from a wild population of Phlox drummondii for three generations. By monitoring flower size, tube length and stigma-anther proximity of flowers, we obtained the direct and indirect responses to selection, and calculated genetic correlations, realized and narrow sense heritabilities using offspring-mother regression. Realized heritability of flower size was high (0.83), whereas genetic correlations among traits were weak or not significant. The per-generation average of the response in corolla diameter was about 5%. We found that P. drummondii has a great capacity to respond rapidly to selection, and this capacity may be in part responsible for the observed high degree of differentiation within the species. We also concluded that rapid evolution of morphological floral traits is possible.     

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.     

Mining the pig genome to investigate the domestication process

Pig domestication began around 9000 YBP in the Fertile Crescent and Far East, involving marked morphological and genetic changes that occurred in a relatively short window of time. Identifying the alleles that drove the behavioural and physiological transformation of wild boars into pigs through artificial selection constitutes a formidable challenge that can only be faced from an interdisciplinary perspective. Indeed, although basic facts regarding the demography of pig domestication and dispersal have been uncovered, the biological substrate of these processes remains enigmatic. Considerable hope has been placed on new approaches, based on next-generation sequencing, which allow whole-genome variation to be analyzed at the population level. In this review, we provide an outline of the current knowledge on pig domestication by considering both archaeological and genetic data. Moreover, we discuss several potential scenarios of genome evolution under the complex mixture of demography and selection forces at play during domestication. Finally, we highlight several technical and methodological approaches that may represent significant advances in resolving the conundrum of livestock domestication.     

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

Summary The 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.     

Application of oscillation for efficiency improvement of continuous ethanol fermentation with Saccharomyces cerevisiae under very-high-gravity conditions

Compared with steady state, oscillation in continuous very-high-gravity ethanol fermentation with Saccharomyces cerevisiae improved process productivity, which was thus introduced for the fermentation system composed of a tank fermentor followed by four-stage packed tubular bioreactors. When the very-high-gravity medium containing 280 g l⁻¹ glucose was fed at the dilution rate of 0.04 h⁻¹, the average ethanol of 15.8% (v/v) and residual glucose of 1.5 g l⁻¹ were achieved under the oscillatory state, with an average ethanol productivity of 2.14 g h⁻¹ l⁻¹. By contrast, only 14.8% (v/v) ethanol was achieved under the steady state at the same dilution rate, and the residual glucose was as high as 17.1 g l⁻¹, with an ethanol productivity of 2.00 g h⁻¹ l⁻¹, indicating a 7% improvement under the oscillatory state. When the fermentation system was operated under the steady state at the dilution rate of 0.027 h⁻¹ to extend the average fermentation time to 88 h from 59 h, the ethanol concentration increased slightly to 15.4% (v/v) and residual glucose decreased to 7.3 g l⁻¹, correspondingly, but the ethanol productivity was decreased drastically to 1.43 g h⁻¹ l⁻¹, indicating a 48% improvement under the oscillatory state at the dilution rate of 0.04 h⁻¹.     

Rapid response to intraclonal selection in the pea aphid (Acyrthosiphon pisum)

Summary Pea aphids show intraclonal variability in antipredator behaviour. Among the offspring of a single parthenogenetically reproducing female, some individuals drop from the plant in response to alarm pheromone while others remain on the plant. We demonstrate that this intraclonal behavioural variability can be altered by selection. The proportion of aphids dropping in response to alarm pheromone was significantly greater in lines in which this behaviour was selected than in clonally identical lines in which the opposite phenotype was favoured. This change occurred within one generation and could not be attributed to grand-maternal effects, nor to environmental effects. These results demonstrate the ability of clonal aphids to adapt to changes in the environment within a single generation.     

Vacuolar morphology of Saccharomyces cerevisiae during the process of wine making and Japanese sake brewing

Although ethanol and osmotic stress affect the vacuolar morphology of Saccharomyces cerevisiae, little information is available about changes in vacuolar morphology during the processes of wine making and Japanese sake (rice wine) brewing. Here, we elucidated changes in the morphology of yeast vacuoles using Zrc1p-GFP, a vacuolar membrane protein, so as to better understand yeast physiology during the brewing process. Wine yeast cells (OC-2 and EC1118) contained highly fragmented vacuoles in the sake mash (moromi) as well as in the grape must. Although sake yeast cells (Kyokai no. 9 and no. 10) also contained highly fragmented vacuoles during the wine-making process, they showed quite a distinct vacuolar morphology during sake brewing. Since the environment surrounding sake yeast cells in the sake mash did not differ much from that surrounding wine yeast cells, the difference in vacuolar morphology during sake brewing between wine yeast and sake yeast was likely caused by innate characters.     

Differences in response of Zymomonas mobilis and Saccharomyces cerevisiae to change in extracellular ethanol concentration

In high cell density batch fermentations, Zymomonas mobilis produced 91 g L(-1) ethanol in 90 min but culture viability fell significantly. Similar viability losses in rapid fermentations by yeast have recently been shown to be attributable in part to the high rate of change of the extracellular ethanol concentration. However, in simulated rapid fermentations in which ethanol was pumped continuously to low cell density Z. mobilis suspensions, increases in the rate of change of ethanol concentration in the range 21-83 g L(-1) h(-1) did not lead to accelerated viability losses. The lag phase of Zymomonas cultures exposed to a 30-g L(-1) step change in ethanol concentration was much shorter than that of Saccharomyces cerevisiae, providing evidence that the comparative insensitivity of Zymomonas to high rates of change of ethanol concentration is due to its ability to adapt to changes in ethanol concentration more rapidly than yeast. (c) 1994 John Wiley & Sons, Inc.     

Genetic engineering of brewing yeast to reduce the content of ethanol in beer

The GPD1 gene encoding the glycerol-3-phosphate dehydrogenase was overexpressed in an industrial lager brewing yeast (Saccharomyces cerevisiae ssp. carlsbergensis) to reduce the content of ethanol in beer. The amount of glycerol produced by the GPD1-overexpressing yeast in fermentation experiments simulating brewing conditions was increased 5.6 times and ethanol was decreased by 18% when compared to the wild-type. Overexpression of GPD1 does not affect the consumption of wort sugars. Only minor changes in the concentration of higher alcohols, esters and fatty acids could be observed in beer produced by the GPD1-overexpressing brewing yeast. However, the concentrations of several other by-products, particularly acetoin, diacetyl and acetaldehyde, were considerably increased.     

Engineering a Saccharomyces cerevisiae wine yeast that exhibits reduced ethanol production during fermentation under controlled microoxygenation conditions

We recently showed that expressing an H(2)O-NADH oxidase in Saccharomyces cerevisiae drastically reduces the intracellular NADH concentration and substantially alters the distribution of metabolic fluxes in the cell. Although the engineered strain produces a reduced amount of ethanol, a high level of acetaldehyde accumulates early in the process (1 g/liter), impairing growth and fermentation performance. To overcome these undesirable effects, we carried out a comprehensive analysis of the impact of oxygen on the metabolic network of the same NADH oxidase-expressing strain. While reducing the oxygen transfer rate led to a gradual recovery of the growth and fermentation performance, its impact on the ethanol yield was negligible. In contrast, supplying oxygen only during the stationary phase resulted in a 7% reduction in the ethanol yield, but without affecting growth and fermentation. This approach thus represents an effective strategy for producing wine with reduced levels of alcohol. Importantly, our data also point to a significant role for NAD(+) reoxidation in controlling the glycolytic flux, indicating that engineered yeast strains expressing an NADH oxidase can be used as a powerful tool for gaining insight into redox metabolism in yeast.     

The yeast Saccharomyces cerevisiae- the main character in beer brewing

Historically, mankind and yeast developed a relationship that led to the discovery of fermented beverages. Numerous inventions have led to improved technologies and capabilities to optimize fermentation technology on an industrial scale. The role of brewing yeast in the beer-making process is reviewed and its importance as the main character is highlighted. On considering the various outcomes of functions in a brewery, it has been found that these functions are focused on supporting the supply of yeast requirements for fermentation and ultimately to maintain the integrity of the product. The functions/processes include: nutrient supply to the yeast (raw material supply for brewhouse wort production); utilities (supply of water, heat and cooling); quality assurance practices (hygiene practices, microbiological integrity measures and other specifications); plant automation (vessels, pipes, pumps, valves, sensors, stirrers and centrifuges); filtration and packaging (product preservation until consumption); distribution (consumer supply); and marketing (consumer awareness). Considering this value chain of beer production and the 'bottle neck' during production, the spotlight falls on fermentation, the age-old process where yeast transforms wort into beer.