Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Saccharomyces cerevisiae producing carotenoid cells

ABSTRACT: To obtain insight in the genome-wide response of heterologous carotenoid production in Saccharomyces cerevisiae, we have analyzed the transcriptome of S. cerevisiae strains overexpressing carotenogenic genes from the yeast Xanthophyllomyces dendrorhous. For this purpose, two strains producing different levels of carotenoids were grown in carbon-limited continuous cultures and genome-wide expression was analyzed. The strain producing low carotenoid levels did not exhibit a clear genome-wide transcriptional response, suggesting that low carotenoid levels do not result in cellular stress. Transcriptome analysis of a strain producing high carotenoid levels resulted in specific induction of genes involved in pleiotropic drug resistance (PDR). These genes encode ATP-binding cassette (ABC) type transporters and major facilitator transporters which are involved in secretion of toxic compounds out of cells. Our results suggest that production of high amounts of carotenoids in S. cerevisiae lead to toxicity and that these cells are prone to secrete carotenoids out of the cell. Indeed, secretion of ï¢-carotene into sunflower oil was observed upon addition of this hydrophobic solvent to the growth medium. Finally, it was observed that deletion of the ABC transporter pdr10, one of the induced PDR transporters, highly decreased the transformation efficiency of an episomal vector containing carotenogenic genes. The few colored transformants that were obtained had decreased growth rates and lower carotenoid production levels compared to control strains transformed with the same carotenogenic genes. These results indicate that Pdr10 might be specifically involved in carotenoid tolerance in S. cerevisiae strains. Experiment Overall Design: The genome wide transcriptional response of S. cerevisiae cells that heterologously produce carotenoids might provide information concerning the impact of carotenoid production on yeast physiology and might identify bottlenecks relevant for the production of these compounds. DNA microarray experiments have been proven to be a powerful tool to study the genome wide transcriptional response of S. cerevisiae to changes of the physiological state and the environment (for example 3,. Genomics approaches on cells producing heterologous metabolites to study their impact on yeast physiology have not been reported yet for S. cerevisiae. Additionally, most transcriptome studies with S. cerevisiae have been performed with cells grown in shake flasks cultures. The main drawback of shake flask cultivation is that the environment is continuously changing, which may be of high influence on carotenoid production, and interpretation of transcriptome data . Chemostat cultivation offers advantages for studies with DNA microarrays because it enables cultivation of microorganisms under tightly defined environmental conditions. An interlaboratory comparison of transcriptome data obtained in chemostat cultures has indeed demonstrated that the accuracy and reproducibility of this approach are superior to those obtained in previous studies with shake-flask cultures .

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Jean-Marc Daran

PROVIDER: E-GEOD-8451 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The hop plant, Humulus lupulus L., contains an exceptionally high content of secondary metabolites, the hop iso-Î±-acids, which possess a range of beneficial properties including antiseptic action. Studies performed on the mode of action of hop iso-Î±-acids have hitherto been restricted to lactic acid bacteria. The present study investigates molecular mechanisms of hop iso-Î±-acid resistance in the model eukaryote Saccharomyces cerevisiae. Growth inhibition occurred at concentrations of hop iso-Î±-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-Î±-acids detoxification and tolerance. Further analysis of deletion mutants confirmed that yeast tolerance to hop iso-Î±-acids involves two major processes: active export of iso-Î±-acids across the plasma membrane and active proton pumping into the vacuole by the V-ATPase to enable vacuolar sequestration of iso-Î±-acids. Furthermore, iso-Î±-acids were shown to affect cellular metal homeostasis by acting as strong zinc and iron chelator. Experiment Overall Design: Two complementary genome-wide approaches were employed to investigate cellular responses of S. cerevisiae to hop extracts enriched in iso-Î±-acids. Microarray transcriptome analysis was performed on chemostat cultures of an S. cerevisiae reference strain grown in the presence and absence of iso-Î±-acids. In addition, screening of the nearly complete set of yeast open reading frame (ORF) haploid knock-outs generated by the Saccharomyces Genome Deletion Project (SGDP) (Open Biosystems) identified the mutants with increased hop sensitivity. Subsequently, involvement of selected genes and cellular processes in hop acid sensitivity and tolerance was analyzed by construction and detailed analysis of selected mutant strains.

Project description:Raw expression values (CHP data) for transcriptional profiling of the response of Saccharomyces cerevisiae to challenges with lactic acid at pH 3 and pH 5. Experiment Overall Design: The laboratory reference strain CEN.PK 113-7D (MATa) was grown at 30 Â°C in 1.5-L chemostat fermentors (Applikon, Schiedam, The Netherlands) with a working volume of 1-L using an electronic level sensor to maintain a constant volume. All cultures, including the reference, were fed with minimal medium as described by Verduyn et al. (1992) with 25 g L-1 glucose as the limiting nutrient and 0.15 ml L-1 silicone antifoam (BDH, Poole, England) to prevent excessive foaming. The dilution rate was set to 0.10 h-1 and the pH was controlled at 5.0 with the automatic addition (ADI 1031 bio controller, Applikon) of 2 M KOH. The stirrer speed was set at 800 RPM and anaerobicity was maintained by sparging the fermentor with N2 gas at 500 ml min-1. To prevent diffusion of oxygen, the fermentor was equipped with Norprene tubing and Viton O-rings and the medium vessel was also flushed with N2 gas. A comparable degree of weak acid uncoupling was ensured by decreasing the biomass yield to approximately 50% of the reference condition (no organic acids added) with the addition of the appropriate concentration of lactic acid to the reservoir media. Experiment Overall Design: Sampling of chemostat cultures, probe preparation and hybridization to Affymetrix GeneChip microarrays was performed as described previously (Piper et al., 2002), but with the following modifications. Double-stranded cDNA synthesis was carried out using 15 Î¼g of total RNA and the components of the One Cycle cDNA Synthesis Kit (Affymetrix). The double-stranded cDNA was purified (Genechip Sample Cleanup Module, Qiagen) before in vitro transcription and labeling (GeneChip IVT Labeling Kit, Affymetrix). Finally, labeled cRNA was purified (GeneChip Sample Cleanup Module) prior to fragmentation and hybridization of 15 Î¼g of biotinylated cRNA. Experiment Overall Design: Data acquisition was performed using the Affymetrix scanner 3000, quantification of array images and data filtering were performed with the Affymetrix software packages Microarray Suite v5.0, MicroDB v3.0 and Data Mining Tool v3.0.

Project description:Sucrose is a major carbon source for industrial bioethanol production by Saccharomyces cerevisiae. In yeasts, two modes of sucrose metabolism occur: (i) extracellular hydrolysis by invertase, followed by uptake and metabolism of glucose and fructose, and (ii) uptake via sucrose-H+ symport followed by intracellular hydrolysis and metabolism. Although alternative start codons in the SUC2 gene enable synthesis of extracellular and intracellular invertase isoforms, sucrose hydrolysis in S. cerevisiae predominantly occurs extracellularly. In anaerobic cultures, intracellular hydrolysis theoretically enables a 9 % higher ethanol yield than extracellular hydrolysis, due to energy costs of sucrose-proton symport. This prediction was tested by engineering the promoter and 5’ coding sequences of SUC2, resulting in relocation of invertase to the cytosol. In anaerobic sucrose-limited chemostats, this iSUC2-strain showed an only 4% increased ethanol yield and high residual sucrose concentrations indicated suboptimal sucrose-transport kinetics. To improve sucrose-uptake affinity, it was subjected to 95 generations of anaerobic, sucrose-limited chemostat cultivation, resulting in a 20-fold decrease of residual sucrose concentrations and a 10-fold increase of the sucrose-transport capacity. A single-cell isolate showed an 11 % higher ethanol yield on sucrose in chemostat and batch cultures than an isogenic SUC2 reference strain, while transcriptome analysis revealed elevated expression of AGT1, encoding a disaccharide-proton symporter, and other maltose-related genes. Deletion of AGT1, which had been duplicated during laboratory evolution, restored the growth characteristics of the unevolved iSUC2 strain. This study demonstrates that engineering the topology of sucrose metabolism is an attractive strategy to improve ethanol yields in industrial processes. The goal of the present study was to investigate whether a relocation of sucrose hydrolysis to the cytosol can be used to improve ethanol yields on sucrose and which additional steps may be required to improve sucrose utilization by strains that only express intracellular invertase. To this end, the SUC2 gene was modified to cause an exclusive intracellular localization. Growth and product formation by the engineered strain were compared with that of the parental strain in anaerobic sucrose-limited chemostat cultures. Subsequently, evolutionary engineering was used to improve sucrose uptake kinetics and an evolved strain was characterized for growth and product formation in chemostat cultures. Transcriptome analysis and gene deletion studies were used to identify genetic changes in the evolved strain that contribute to its improved sucrose-uptake kinetics.

Project description:Raw expression values (CHP data) for transcriptional profiling of the response of Saccharomyces cerevisiae to challenges with various weak organic acids Experiment Overall Design: The laboratory reference strain CEN.PK 113-7D (MATa) was grown at 30 Â°C in 2-L chemostat fermentors (Applikon, Schiedam, The Netherlands) with a working volume of 1-L using an electronic level sensor to maintain a constant volume. All cultures, including the reference, were fed with minimal medium as described by Verduyn et al. (1992) with 25 g L-1 glucose as the limiting nutrient and 0.15 ml L-1 silicone antifoam (BDH, Poole, England) to prevent excessive foaming. The dilution rate was set to 0.10 h-1 and the pH was controlled at 5.0 with the automatic addition (ADI 1031 bio controller, Applikon) of 2 M KOH. The stirrer speed was set at 800 RPM and anaerobicity was maintained by sparging the fermentor with N2 gas at 500 ml min-1. To prevent diffusion of oxygen, the fermentor was equipped with Norprene tubing and Viton O-rings and the medium vessel was also flushed with N2 gas. A comparable degree of weak acid uncoupling was ensured by decreasing the biomass yield to approximately 50% of the reference condition (no organic acids added) with the addition of the appropriate concentration of acetic acid, sodium benzoate, propionic acid or potassium sorbate to the reservoir media. Triplicate cultivations and microarrays were performed for each condition. Experiment Overall Design: Sampling of chemostat cultures, probe preparation and hybridization to Affymetrix GeneChip microarrays was performed as described previously (Piper et al., 2002), but with the following modifications. Double-stranded cDNA synthesis was carried out using 15 Î¼g of total RNA and the components of the One Cycle cDNA Synthesis Kit (Affymetrix). The double-stranded cDNA was purified (Genechip Sample Cleanup Module, Qiagen) before in vitro transcription and labeling (GeneChip IVT Labeling Kit, Affymetrix). Finally, labeled cRNA was purified (GeneChip Sample Cleanup Module) prior to fragmentation and hybridization of 15 Î¼g of biotinylated cRNA. Experiment Overall Design: Data acquisition was performed using the Affymetrix scanner 3000, quantification of array images and data filtering were performed with the Affymetrix software packages Microarray Suite v5.0, MicroDB v3.0 and Data Mining Tool v3.0. **note 2014-04-02: file GSM137686.CEL is truncated.

Project description:Prolonged cultivation (>25 generations) of Saccharomyces cerevisiae in aerobic, maltose-limited chemostat cultures led to profound physiological changes. Maltose hypersensitivity was observed when cells from prolonged cultivations were suddenly exposed to excess maltose. This substrate hypersensitivity was evident from massive cell lysis and loss of viability. During prolonged cultivation at a fixed specific growth rate, the affinity for the growth-limiting nutrient (i.e., maltose) increased, as evident from a decreasing residual maltose concentration. Furthermore, the capacity of maltose-dependent proton uptake increased up to 2.5-fold during prolonged cultivation. Genome-wide transcriptome analysis showed that the increased maltose transport capacity was not primarily due to increased transcript levels of maltose-permease genes upon prolonged cultivation. We propose that selection for improved substrate affinity (ratio of maximum substrate consumption rate and substrate saturation constant) in maltose-limited cultures leads to selection for cells with an increased capacity for maltose uptake. At the same time, the accumulative nature of maltose-proton symport in S. cerevisiae leads to unrestricted uptake when maltose-adapted cells are exposed to a substrate excess. These changes were retained after isolation of individual cell lines from the chemostat cultures and nonselective cultivation, indicating that mutations were involved. The observed trade-off between substrate affinity and substrate tolerance may be relevant for metabolic engineering and strain selection for utilization of substrates that are taken up by proton symport. Experiment Overall Design: In a recent study (25) we analyzed glucose efflux upon exposure of S. cerevisiae to excess maltose, with yeast cells originating from âyoungâ chemostat cultures (<20 generations). In these experiments no cell lysis was observed upon exposure to excess maltose. However, in further work on this subject, we observed an apparent effect of chemostat culture age on transport capacity. The aim of the present study was to further investigate the effect of prolonged maltose-limited chemostat cultivation on the physiology of S. cerevisiae. To this end we monitored the affinity for maltose, genome-wide transcript levels, activities of key enzymes, and physiological responses to maltose excess during long-term cultivation in maltose-limited chemostat cultures. Experiment Overall Design: Jansen, M. L. A., J. H. de Winde, and J. T. Pronk. 2002. Hxt-carrier-mediated glucose efflux upon exposure of Saccharomyces cerevisiaeSaccharomyces cerevisiae to excess maltose. Appl. Environ. Microbiol. 68:4259-4265.

Project description:The global transcriptional response of Saccharomyces cerevisiae was investigated in low temperature chemostat cultures grown in carbon or nitrogen limitation. During steady state chemostats, the growth rates and in vivo fluxes were kept constant however the growth-limiting nutrient was significantly higher at 12oC than at 30oC and had significant effects on transcriptional responses. Growth at 12oC resulted in a rearrangement of transporters for the limiting nutrient, where hexose transporters (HXTs) and ammonium permeases (MEPs) were differentially expressed in cultures grown at 30oC in carbon and nitrogen limitations, respectively. In addition, we found repression of genes encoding proteins in reserve carbohydrates metabolism and metabolism of alternative carbon or nitrogen sources other than glucose or ammonia. However, there were also similar responses when the transcriptional response was evaluated regardless of the growth-limiting nutrient. In particular, induction of ribosome biogenesis genes emphasizes the significance of transcription and translational adaptation at low temperature. In contrast, genes encoding proteins during stress response were downregulated. This down-regulation of stress elements better known as environmental stress response (ESR) is in contradiction with previous low temperature transcriptome analyses. During continuous steady state low temperature cultivation, ESR no longer plays an integral role in S. cerevisiaeM-bM-^@M-^Ys response to temperature change. Similarly, trehalose accumulation, consistent with its gene expression, was not indispensable for growth at 12oC. This response, however, does not exclude that ESR may be required for transition phase in low temperature growth when cells are transferred from one temperature to another. Keywords: chemostat temperature 12 degree celsuis 30 degree celsius The global transcriptional response of Saccharomyces cerevisiae was investigated in low temperature chemostat cultures grown in carbon or nitrogen limitation at a dilution rate of 0.03h-1

Project description:In contrast to batch cultivation, chemostat cultivation allows the identification of carbon source responses without interference by carbon-catabolite repression, accumulation of toxic products, and differences in specific growth rate. This study focuses on the yeast Saccharomyces cerevisiae, grown in aerobic, carbon-limited chemostat cultures. Genome-wide transcript levels and in vivo fluxes were compared for growth on two sugars, glucose and maltose, and for two C2-compounds, ethanol and acetate. In contrast to previous reports on batch cultures, few genes (180 genes) responded to changes of the carbon source by a changed transcript level. Very few transcript levels were changed when glucose as the growth-limiting nutrient was compared with maltose (33 transcripts), or when acetate was compared with ethanol (16 transcripts). Although metabolic flux analysis using a stoichiometric model revealed major changes in the central carbon metabolism, only 117 genes exhibited a significantly different transcript level when sugars and C2-compounds were provided as the growthlimiting nutrient. Despite the extensive knowledge on carbon source regulation in yeast, many of the carbon source-responsive genes encoded proteins with unknown or incompletely characterized biological functions. In silico promoter analysis of carbon source-responsive genes confirmed the involvement of several known transcriptional regulators and suggested the involvement of additional regulators. Transcripts involved in the glyoxylate cycle and gluconeogenesis showed a good correlation with in vivo fluxes. This correlation was, however, not observed for other important pathways, including the pentose-phosphate pathway, tricarboxylic acid cycle, and, in particular, glycolysis. These results indicate that in vivo fluxes in the central carbon metabolism of S. cerevisiae grown in steadystate, carbon-limited chemostat cultures are controlled to a large extent via post-transcriptional mechanisms. Experiment Overall Design: Cultivation of microorganisms in chemostats offers numerous advantages for studying the structure and regulation of metabolic networks (11). In chemostat cultures, individual culture parameters can be changed, while keeping other relevant physical and chemical culture parameters (composition of synthetic medium, pH, temperature, aeration, etc.) constant. An especially important parameter in this respect is the specific growth rate, which, in a chemostat, is equal to the dilution rate, which can be accurately controlled. This allows the experimenter to investigate the effects of environmental changes or genetic interventions at a fixed specific growth rate, even if these changes result in different specific growth rates in batch cultures. In a chemostat, growth can be limited by a single, selected nutrient. The very low residual concentrations of this growth-limiting nutrient in chemostat cultures alleviate effects of catabolite repression and inactivation. Furthermore, these low residual substrate concentrations prevent substrate toxicity, which, for example, occurs when S. cerevisiae is grown on ethanol or acetate as the carbon source in batch cultures . Experiment Overall Design: The central goal of the present study is to assess to what extent carbon source-dependent regulation of fluxes through central carbon metabolism in S. cerevisiae is regulated at the level of transcription. To this end, we compare the transcriptome of carbon-limited, aerobic chemostat cultures grown on four different carbon sources: glucose, maltose, ethanol, and acetate. Data from the transcriptome analysis are compared with flux distribution profiles calculated with a stoichiometric metabolic network model. Questions that will be addressed are as follows: (i) does glucose-limited aerobic cultivation lead to a complete alleviation of glucose-catabolite repression; (ii) how (in)complete is our understanding of the genes involved in the transcriptional response of S. cerevisiae to four of the most common carbon sources for this yeast; and (iii) to what extent do transcriptome analyses with microarrays provide a reliable indication of flux distribution in metabolic networks?

Project description:Zinc is indispensable for the catalytic activity and structural stability of many proteins, and its deficiency can have severe consequences for microbial growth in natural and industrial environments. For example, Zn depletion in wort negatively affects beer fermentation and quality. Several studies have investigated yeast adaptation to low Zn supply, but were all performed in batch cultures, where specific growth rate depends on Zn availability. The transcriptional responses to growth-rate and Zn availability are then intertwined, which obscures result interpretation. In the present study, transcriptional responses of Saccharomyces cerevisiae to Zn availability were investigated at a fixed specific growth rate under Zn limitation and excess in chemostat culture. To investigate the context-dependency of this transcriptional response, yeast was grown under several chemostat regimes resulting in various carbon (glucose), nitrogen (ammonium) and oxygen supplies. A robust set of genes that responded consistently to Zn limitation was identified and enabled the definition of a Zn-specific Zap1 regulon comprising of 26 genes and characterized by a broader ZRE 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 amount 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 Experiment Overall Design: Zinc is indispensable for the catalytic activity and structural stability of many proteins, and its deficiency can have severe consequences for microbial growth in natural and industrial environments. For example, Zn depletion in wort negatively affects beer fermentation and quality. Several studies have investigated yeast adaptation to low Zn supply, but were all performed in batch cultures, where specific growth rate depends on Zn availability. The transcriptional responses to growth-rate and Zn availability are then intertwined, which obscures result interpretation. In the present study, transcriptional responses of Saccharomyces cerevisiae to Zn availability were investigated at a fixed specific growth rate under Zn limitation and excess in chemostat culture. To investigate the context-dependency of this transcriptional response, yeast was grown under several chemostat regimes resulting in various carbon (glucose), nitrogen (ammonium) and oxygen supplies. A robust set of genes that responded consistently to Zn limitation was identified and enabled the definition of a Zn-specific Zap1 regulon comprising of 26 genes and characterized by a broader ZRE 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 amount 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

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Transcription profiling of Saccharomyces cerevisiae producing carotenoid cells

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