The ability to adapt to altered availability of free water is a fundamental property of living cells. The principles underlying osmoadaptation are well conserved. The yeast Saccharomyces cerevisiae is an excellent model system with which to study the molecular biology and physiology of osmoadaptation. Upon a shift to high osmolarity, yeast cells rapidly stimulate a mitogen-activated protein (MAP) kinase cascade, the high-osmolarity glycerol (HOG) pathway, which orchestrates part of the transcriptional response. The dynamic operation of the HOG pathway has been well studied, and similar osmosensing pathways exist in other eukaryotes. Protein kinase A, which seems to mediate a response to diverse stress conditions, is also involved in the transcriptional response program. Expression changes after a shift to high osmolarity aim at adjusting metabolism and the production of cellular protectants. Accumulation of the osmolyte glycerol, which is also controlled by altering transmembrane glycerol transport, is of central importance. Upon a shift from high to low osmolarity, yeast cells stimulate a different MAP kinase cascade, the cell integrity pathway. The transcriptional program upon hypo-osmotic shock seems to aim at adjusting cell surface properties. Rapid export of glycerol is an important event in adaptation to low osmolarity. Osmoadaptation, adjustment of cell surface properties, and the control of cell morphogenesis, growth, and proliferation are highly coordinated processes. The Skn7p response regulator may be involved in coordinating these events. An integrated understanding of osmoadaptation requires not only knowledge of the function of many uncharacterized genes but also further insight into the time line of events, their interdependence, their dynamics, and their spatial organization as well as the importance of subtle effects.

Osmotic Stress Signaling and Osmoadaptation in Yeasts

Summary: The traditional use of the yeast Saccharomyces cerevisiae in alcoholic fermentation has, over time, resulted in substantial accumulated knowledge concerning genetics, physiology, and biochemistry as well as genetic engineering and fermentation technologies. S. cerevisiae has become a platform organism for developing metabolic engineering strategies, methods, and tools. The current review discusses the relevance of several engineering strategies, such as rational and inverse metabolic engineering, evolutionary engineering, and global transcription machinery engineering, in yeast strain improvement. It also summarizes existing tools for fine-tuning and regulating enzyme activities and thus metabolic pathways. Recent examples of yeast metabolic engineering for food, beverage, and industrial biotechnology (bioethanol and bulk and fine chemicals) follow. S. cerevisiae currently enjoys increasing popularity as a production organism in industrial (“white”) biotechnology due to its inherent tolerance of low pH values and high ethanol and inhibitor concentrations and its ability to grow anaerobically. Attention is paid to utilizing lignocellulosic biomass as a potential substrate.

Progress in Metabolic Engineering of Saccharomyces cerevisiae

Methylglyoxal levels in plants under salinity stress are dependent on glyoxalase I and glutathione.

In industrial fermentation processes, the yeast Saccharomyces cerevisiae is commonly used for ethanol production. However, it lacks the ability to ferment pentose sugars like d-xylose and l-arabinose. Heterologous expression of a xylose isomerase (XI) would enable yeast cells to metabolize xylose. However, many attempts to express a prokaryotic XI with high activity in S. cerevisiae have failed so far. We have screened nucleic acid databases for sequences encoding putative XIs and finally were able to clone and successfully express a highly active new kind of XI from the anaerobic bacterium Clostridium phytofermentans in S. cerevisiae. Heterologous expression of this enzyme confers on the yeast cells the ability to metabolize d-xylose and to use it as the sole carbon and energy source. The new enzyme has low sequence similarities to the XIs from Piromyces sp. strain E2 and Thermus thermophilus, which were the only two XIs previously functionally expressed in S. cerevisiae. The activity and kinetic parameters of the new enzyme are comparable to those of the Piromyces XI. Importantly, the new enzyme is far less inhibited by xylitol, which accrues as a side product during xylose fermentation. Furthermore, expression of the gene could be improved by adapting its codon usage to that of the highly expressed glycolytic genes of S. cerevisiae. Expression of the bacterial XI in an industrially employed yeast strain enabled it to grow on xylose and to ferment xylose to ethanol. Thus, our findings provide an excellent starting point for further improvement of xylose fermentation in industrial yeast strains.

Functional expression of a bacterial xylose isomerase in Saccharomyces cerevisiae.

The production of bioethanol from lignocellulose hydrolysates requires a robust, D-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. The purpose of the present work was to develop such a strain from a prime industrial yeast strain, Ethanol Red, used for bioethanol production. An expression cassette containing 13 genes including Clostridium phytofermentans XylA, encoding D-xylose isomerase (XI), and enzymes of the pentose phosphate pathway was inserted in two copies in the genome of Ethanol Red. Subsequent EMS mutagenesis, genome shuffling and selection in D-xylose-enriched lignocellulose hydrolysate, followed by multiple rounds of evolutionary engineering in complex medium with D-xylose, gradually established efficient D-xylose fermentation. The best-performing strain, GS1.11-26, showed a maximum specific D-xylose consumption rate of 1.1 g/g DW/h in synthetic medium, with complete attenuation of 35 g/L D-xylose in about 17 h. In separate hydrolysis and fermentation of lignocellulose hydrolysates of Arundo donax (giant reed), spruce and a wheat straw/hay mixture, the maximum specific D-xylose consumption rate was 0.36, 0.23 and 1.1 g/g DW inoculum/h, and the final ethanol titer was 4.2, 3.9 and 5.8% (v/v), respectively. In simultaneous saccharification and fermentation of Arundo hydrolysate, GS1.11-26 produced 32% more ethanol than the parent strain Ethanol Red, due to efficient D-xylose utilization. The high D-xylose fermentation capacity was stable after extended growth in glucose. Cell extracts of strain GS1.11-26 displayed 17-fold higher XI activity compared to the parent strain, but overexpression of XI alone was not enough to establish D-xylose fermentation. The high D-xylose consumption rate was due to synergistic interaction between the high XI activity and one or more mutations in the genome. The GS1.11-26 had a partial respiratory defect causing a reduced aerobic growth rate. An industrial yeast strain for bioethanol production with lignocellulose hydrolysates has been developed in the genetic background of a strain widely used for commercial bioethanol production. The strain uses glucose and D-xylose with high consumption rates and partial cofermentation in various lignocellulose hydrolysates with very high ethanol yield. The GS1.11-26 strain shows highly promising potential for further development of an all-round robust yeast strain for efficient fermentation of various lignocellulose hydrolysates.

/pdf/development-of-a-d-xylose-fermenting-and-inhibitor-tolerant-17uaaksbvm.pdf

Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering

The response of yeast cells to sudden temperature downshifts has received little attention compared with other stress conditions. Like other organisms, both prokaryotes and eukaryotes, in Saccharomyces cerevisiae a decrease in temperature induces the expression of many genes involved in transcription and translation, some of which display a cold-sensitivity phenotype. However, little is known about the role played by many cold-responsive genes, the sensing and regulatory mechanisms that control this response or the biochemical adaptations at or near 0°C. This review focuses on the physiological significance of cold-shock responses, emphasizing the molecular mechanisms that generate and transmit cold signals. There is now enough experimental evidence to conclude that exposure to low temperature protects yeast cells against freeze injury through the cold-induced accumulation of trehalose, glycerol and heat-shock proteins. Recent results also show that changes in membrane fluidity are the primary signal triggering the cold-shock response. Notably, this signal is transduced and regulated through classical stress pathways and transcriptional factors, the high-osmolarity glycerol mitogen-activated protein kinase pathway and Msn2/4p. Alternative cold-stress generators and transducers will also be presented and discussed.

http://digital.csic.es/bitstream/10261/114800/1/FEMS%20Microbiology%20Reviews-Aguilera.pdf

Cold response in Saccharomyces cerevisiae: new functions for old mechanisms

The response of yeast cells to sudden temperature downshifts has received little attention compared with other stress conditions. Like other organisms, both prokaryotes and eukaryotes, in Saccharomyces cerevisiae a decrease in temperature induces the expression of many genes involved in transcription and translation, some of which display a cold-sensitivity phenotype. However, little is known about the role played by many cold-responsive genes, the sensing and regulatory mechanisms that control this response or the biochemical adaptations at or near 0 degrees C. This review focuses on the physiological significance of cold-shock responses, emphasizing the molecular mechanisms that generate and transmit cold signals. There is now enough experimental evidence to conclude that exposure to low temperature protects yeast cells against freeze injury through the cold-induced accumulation of trehalose, glycerol and heat-shock proteins. Recent results also show that changes in membrane fluidity are the primary signal triggering the cold-shock response. Notably, this signal is transduced and regulated through classical stress pathways and transcriptional factors, the high-osmolarity glycerol mitogen-activated protein kinase pathway and Msn2/4p. Alternative cold-stress generators and transducers will also be presented and discussed.

Cold response in Saccharomyces cerevisiae: New functions for old mechanisms. FEMS Microbiol Rev

The enzyme aldose reductase plays an important role in the osmo-protection mechanism of diverse organisms. Here, we show that yeast aldose reductase is encoded by the GRE3 gene. Expression of GRE3 is carbon-source independent and up-regulated by different stress conditions, such as NaCl, H2O2, 39 °C and carbon starvation. Measurements of enzyme activity and intracellular sorbitol in wild-type cells also indicate that yeast aldose reductase is stress-regulated. Overexpression of GRE3 increases methylglyoxal tolerance in Saccharomyces cerevisiae. Furthermore, high expression of GRE3 complements the deficiency of the glyoxalase system of a glo1Δ mutant strain. Consistent with this, in vitro and in vivo assays of yeast aldose reductase activity indicate that methylglyoxal is an endogenous substrate of aldose reductase. Furthermore, addition of NaCl or H2O2 to exponential-phase cells triggers an initial transient increase in the intracellular level of methylglyoxal, which is dependent on the Gre3p and Glo1p function. These observations indicate that the metabolism of methylglyoxal is stimulated under stress conditions; and they support a methylglyoxal degradative pathway, in which this compound is metabolised by the action of aldose reductase.

The Saccharomyces cerevisiae aldose reductase is implied in the metabolism of methylglyoxal in response to stress conditions.

A sudden overaccumulation of methylglyoxal (MG) induces, in Saccharomyces cerevisiae, the expression of MG-protective genes, including GPD1, GLO1 and GRE3. The response is partially dependent on the transcriptional factors Msn2p/Msn4p, but unrelated with the general stress response mechanism. Here, we show that the high-osmolarity glycerol (HOG)-pathway controls the genetic response to MG and determines the yeast growth capacity upon MG exposure. Strains lacking the MAPK Hog1p, the upstream component Ssk1p or the HOG-dependent nuclear factor Msn1p, showed a reduction in the mRNA accumulation of MG-responsive genes after MG addition. Moreover, hyperactivation of Hog1p by deletion of protein phosphatase PTP2 enhanced the response, while blocking the pathway by deletion of the MAPKK PBS2 had a negative effect. In addition, the activity of Hog1p affected the basal level of GPD1 mRNA under non-inducing conditions. These effects had a great influence on MG resistance, as hog1Delta and other HOG-pathway mutants with impaired MG-specific expression displayed MG sensitivity, whereas those with enhanced expression exhibited MG resistance as compared with the wild-type. However, MG does not trigger the overphosphorylation of Hog1p or its nuclear import in the parental strain. Moreover, dual phosphorylation of Hog1p appears to be dispensable in the triggering of the transcriptional response, although a phosphorylable form of Hog1p is fundamental for the transcriptional activity. Overall, our results suggest that the basal activity of the HOG-pathway serves to amplify the expression of MG-responsive genes under non-inducing and inducing conditions, ensuring cell protection against this toxic glycolytic by-product.

The HOG MAP kinase pathway is required for the induction of methylglyoxal-responsive genes and determines methylglyoxal resistance in Saccharomyces cerevisiae

Methylglyoxal (MG), a glycolytic by-product, is an extremely toxic compound. This fact suggests that its synthesis and degradation should be tightly controlled. However, little is known about the mechanisms that protect yeast cells against MG toxicity. Here, we show that in Saccharomyces cerevisiae, MG exposure increased the internal MG content and activated the expression of GLO1 and GRE3, two genes involved in MG detoxification; GPD1, the gene for glycerol synthesis; and TPS1 and TPS2, the trehalose pathway genes. This response was specific as demonstrated by the analysis of marker genes and effectors of the general stress response. Physiological experiments with MG-treated cells showed that this compound triggers the overproduction of glycerol. Furthermore, a gpd1 gpd2 double mutant showed enhanced MG contents compared with the wild-type. Overall, these results appeared to indicate that up-variations in the intracellular content of the toxic compound are perceived by the cell as a primary signal to trigger the transcriptional response. In agreement with this, MG-instigated GPD1 activation was enhanced in strains lacking GLO1, and this effect correlated with the internal MG content. Finally, induction of GPD1, TPS1 and GRE3, and enhanced MG contents were also observed in low-glucose-growing cells subjected to a sudden increase in glucose availability. The implications of this regulatory mechanism on protection against MG are discussed.

Jaime Aguilera

Papers

Cold response in Saccharomyces cerevisiae: new functions for old mechanisms

Cold response in Saccharomyces cerevisiae: New functions for old mechanisms. FEMS Microbiol Rev

The Saccharomyces cerevisiae aldose reductase is implied in the metabolism of methylglyoxal in response to stress conditions.

The HOG MAP kinase pathway is required for the induction of methylglyoxal-responsive genes and determines methylglyoxal resistance in Saccharomyces cerevisiae

Yeast cells display a regulatory mechanism in response to methylglyoxal