scholarly journals Prolonged Maltose-Limited Cultivation of Saccharomyces cerevisiae Selects for Cells with Improved Maltose Affinity and Hypersensitivity

2004 ◽  
Vol 70 (4) ◽  
pp. 1956-1963 ◽  
Author(s):  
Mickel L. A. Jansen ◽  
Pascale Daran-Lapujade ◽  
Johannes H. de Winde ◽  
Matthew D. W. Piper ◽  
Jack T. Pronk
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Substrate Affinity ◽  
Maltose Permease ◽  
Prolonged Cultivation ◽  
Chemostat Cultures ◽  
Proton Symport ◽  
Limiting Nutrient ◽  
Substrate Saturation ◽  
Selection For ◽  
Substrate Tolerance

ABSTRACT 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.

scholarly journals Identity of the Growth-Limiting Nutrient Strongly Affects Storage Carbohydrate Accumulation in Anaerobic Chemostat Cultures of Saccharomyces cerevisiae

2009 ◽  
Vol 75 (21) ◽  
pp. 6876-6885 ◽  
Author(s):  
Lucie A. Hazelwood ◽  
Michael C. Walsh ◽  
Marijke A. H. Luttik ◽  
Pascale Daran-Lapujade ◽  
Jack T. Pronk ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Glycogen Phosphorylase ◽  
Specific Growth ◽  
Glycogen Synthase ◽  
The Other ◽  
Carbohydrate Accumulation ◽  
Storage Carbohydrate ◽  
Chemostat Cultures ◽  
Limiting Nutrient

ABSTRACT Accumulation of glycogen and trehalose in nutrient-limited cultures of Saccharomyces cerevisiae is negatively correlated with the specific growth rate. Additionally, glucose-excess conditions (i.e., growth limitation by nutrients other than glucose) are often implicated in high-level accumulation of these storage carbohydrates. The present study investigates how the identity of the growth-limiting nutrient affects accumulation of storage carbohydrates in cultures grown at a fixed specific growth rate. In anaerobic chemostat cultures (dilution rate, 0.10 h−1) of S. cerevisiae, the identity of the growth-limiting nutrient (glucose, ammonia, sulfate, phosphate, or zinc) strongly affected storage carbohydrate accumulation. The glycogen contents of the biomass from glucose- and ammonia-limited cultures were 10- to 14-fold higher than those of the biomass from cultures grown under the other three glucose-excess regimens. Trehalose levels were specifically higher under nitrogen-limited conditions. These results demonstrate that storage carbohydrate accumulation in nutrient-limited cultures of S. cerevisiae is not a generic response to excess glucose but instead is strongly dependent on the identity of the growth-limiting nutrient. While transcriptome analysis of wild-type and msn2Δ msn4Δ strains confirmed that transcriptional upregulation of glycogen and trehalose biosynthesis genes is mediated by Msn2p/Msn4p, transcriptional regulation could not quantitatively account for the drastic changes in storage carbohydrate accumulation. The results of assays of glycogen synthase and glycogen phosphorylase activities supported involvement of posttranscriptional regulation. Consistent with the high glycogen levels in ammonia-limited cultures, the ratio of glycogen synthase to glycogen phosphorylase in these cultures was up to eightfold higher than the ratio in the other glucose-excess cultures.

scholarly journals Two Distinct Pathways for Trehalose Assimilation in the Yeast Saccharomyces cerevisiae

2004 ◽  
Vol 70 (5) ◽  
pp. 2771-2778 ◽  
Author(s):  
Matthieu Jules ◽  
Vincent Guillou ◽  
Jean François ◽  
Jean-Luc Parrou
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Saccharomyces Cerevisiae ◽  
Maltose Permease ◽  
Batch Cultures ◽  
Whole Cells ◽  
Neutral Trehalase ◽  
Acid Trehalase ◽  
Glucose Flux ◽  
Chemostat Cultures ◽  
Hydrolysis Of

ABSTRACT The yeast Saccharomyces cerevisiae can synthesize trehalose and also use this disaccharide as a carbon source for growth. However, the molecular mechanism by which extracellular trehalose can be transported to the vacuole and degraded by the acid trehalase Ath1p is not clear. By using an adaptation of the assay of invertase on whole cells with NaF, we showed that more than 90% of the activity of Ath1p is extracellular, splitting of the disaccharide into glucose. We also found that Agt1p-mediated trehalose transport and the hydrolysis of the disaccharide by the cytosolic neutral trehalase Nth1p are coupled and represent a second, independent pathway, although there are several constraints on this alternative route. First, the AGT1/MAL11 gene is controlled by the MAL system, and Agt1p was active in neither non-maltose-fermenting nor maltose-inducible strains. Second, Agt1p rapidly lost activity during growth on trehalose, by a mechanism similar to the sugar-induced inactivation of the maltose permease. Finally, both pathways are highly pH sensitive and effective growth on trehalose occurred only when the medium was buffered at around pH 5.0. The catabolism of trehalose was purely oxidative, and since levels of Ath1p limit the glucose flux in the cells, batch cultures on trehalose may provide a useful alternative to glucose-limited chemostat cultures for investigation of metabolic responses in yeast.

scholarly journals Molecular Basis for Anaerobic Growth of Saccharomyces cerevisiae on Xylose, Investigated by Global Gene Expression and Metabolic Flux Analysis

2004 ◽  
Vol 70 (4) ◽  
pp. 2307-2317 ◽  
Author(s):  
Marco Sonderegger ◽  
Marie Jeppsson ◽  
Bärbel Hahn-Hägerdal ◽  
Uwe Sauer
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Global Gene Expression ◽  
Anaerobic Growth ◽  
Flux Analysis ◽  
Atp Production ◽  
Redox Imbalance ◽  
Chemostat Cultures

ABSTRACT Yeast xylose metabolism is generally considered to be restricted to respirative conditions because the two-step oxidoreductase reactions from xylose to xylulose impose an anaerobic redox imbalance. We have recently developed, however, a Saccharomyces cerevisiae strain that is at present the only known yeast capable of anaerobic growth on xylose alone. Using transcriptome analysis of aerobic chemostat cultures grown on xylose-glucose mixtures and xylose alone, as well as a combination of global gene expression and metabolic flux analysis of anaerobic chemostat cultures grown on xylose-glucose mixtures, we identified the distinguishing characteristics of this unique phenotype. First, the transcript levels and metabolic fluxes throughout central carbon metabolism were significantly higher than those in the parent strain, and they were most pronounced in the xylose-specific, pentose phosphate, and glycerol pathways. Second, differential expression of many genes involved in redox metabolism indicates that increased cytosolic NADPH formation and NADH consumption enable a higher flux through the two-step oxidoreductase reaction of xylose to xylulose in the mutant. Redox balancing is apparently still a problem in this strain, since anaerobic growth on xylose could be improved further by providing acetoin as an external NADH sink. This improved growth was accompanied by an increased ATP production rate and was not accompanied by higher rates of xylose uptake or cytosolic NADPH production. We concluded that anaerobic growth of the yeast on xylose is ultimately limited by the rate of ATP production and not by the redox balance per se, although the redox imbalance, in turn, limits ATP production.

scholarly journals Microhomology Selection for Microhomology Mediated End Joining in Saccharomyces cerevisiae

Genes ◽  
2019 ◽  
Vol 10 (4) ◽  
pp. 284 ◽  
Author(s):  
Kihoon Lee ◽  
Jae-Hoon Ji ◽  
Kihoon Yoon ◽  
Jun Che ◽  
Ja-Hwan Seol ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Budding Yeast ◽  
Product Formation ◽  
Dna Breaks ◽  
End Joining ◽  
Base Pairs ◽  
Circular Chromosome ◽  
Proximal Side ◽  
Selection For ◽  
Insight Into

Microhomology-mediated end joining (MMEJ) anneals short, imperfect microhomologies flanking DNA breaks, producing repair products with deletions in a Ku- and RAD52-independent fashion. Puzzlingly, MMEJ preferentially selects certain microhomologies over others, even when multiple microhomologies are available. To define rules and parameters for microhomology selection, we altered the length, the position, and the level of mismatches to the microhomologies flanking homothallic switching (HO) endonuclease-induced breaks and assessed their effect on MMEJ frequency and the types of repair product formation. We found that microhomology of eight to 20 base pairs carrying no more than 20% mismatches efficiently induced MMEJ. Deletion of MSH6 did not impact MMEJ frequency. MMEJ preferentially chose a microhomology pair that was more proximal from the break. Interestingly, MMEJ events preferentially retained the centromere proximal side of the HO break, while the sequences proximal to the telomere were frequently deleted. The asymmetry in the deletional profile among MMEJ products was reduced when HO was induced on the circular chromosome. The results provide insight into how cells search and select microhomologies for MMEJ in budding yeast.

scholarly journals Mitochondrial-nuclear coadaptation revealed through mtDNA replacements in Saccharomyces cerevisiae

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Tuc H. M. Nguyen ◽  
Sargunvir Sondhi ◽  
Andrew Ziesel ◽  
Swati Paliwal ◽  
Heather L. Fiumera
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Natural Variation ◽  
Epistatic Effect ◽  
Genetic Distances ◽  
Genetic Interactions ◽  
Effect Sizes ◽  
Ecological Niches ◽  
Environmentally Sensitive ◽  
Selection For ◽  
Growth Differences

Abstract Background Mitochondrial function requires numerous genetic interactions between mitochondrial- and nuclear- encoded genes. While selection for optimal mitonuclear interactions should result in coevolution between both genomes, evidence for mitonuclear coadaptation is challenging to document. Genetic models where mitonuclear interactions can be explored are needed. Results We systematically exchanged mtDNAs between 15 Saccharomyces cerevisiae isolates from a variety of ecological niches to create 225 unique mitochondrial-nuclear genotypes. Analysis of phenotypic profiles confirmed that environmentally-sensitive interactions between mitochondrial and nuclear genotype contributed to growth differences. Exchanges of mtDNAs between strains of the same or different clades were just as likely to demonstrate mitonuclear epistasis although epistatic effect sizes increased with genetic distances. Strains with their original mtDNAs were more fit than strains with synthetic mitonuclear combinations when grown in media that resembled isolation habitats. Conclusions This study shows that natural variation in mitonuclear interactions contributes to fitness landscapes. Multiple examples of coadapted mitochondrial-nuclear genotypes suggest that selection for mitonuclear interactions may play a role in helping yeasts adapt to novel environments and promote coevolution.

scholarly journals Intracellular Maltose Is Sufficient To Induce MAL Gene Expression in Saccharomyces cerevisiae

2002 ◽  
Vol 1 (5) ◽  
pp. 696-703 ◽  
Author(s):  
Xin Wang ◽  
Mehtap Bali ◽  
Igor Medintz ◽  
Corinne A. Michels
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Plantago Major ◽  
Null Mutant ◽  
Transport Activity ◽  
Maltose Permease ◽  
Maltose Transport ◽  
Constitutive Overexpression ◽  
Reduced Rate

ABSTRACT The presence of maltose induces MAL gene expression in Saccharomyces cells, but little is known about how maltose is sensed. Strains with all maltose permease genes deleted are unable to induce MAL gene expression. In this study, we examined the role of maltose permease in maltose sensing by substituting a heterologous transporter for the native maltose permease. PmSUC2 encodes a sucrose transporter from the dicot plant Plantago major that exhibits no significant sequence homology to maltose permease. When expressed in Saccharomyces cerevisiae, PmSUC2 is capable of transporting maltose, albeit at a reduced rate. We showed that introduction of PmSUC2 restores maltose-inducible MAL gene expression to a maltose permease-null mutant and that this induction requires the MAL activator. These data indicate that intracellular maltose is sufficient to induce MAL gene expression independently of the mechanism of maltose transport. By using strains expressing defective mal61 mutant alleles, we demonstrated a correlation between the rate of maltose transport and the level of the induction, which is particularly evident in medium containing very limiting concentrations of maltose. Moreover, our results indicate that a rather low concentration of intracellular maltose is needed to trigger MAL gene expression. We also showed that constitutive overexpression of either MAL61 maltose permease or PmSUC2 suppresses the noninducible phenotype of a defective mal13 MAL-activator allele, suggesting that this suppression is solely a function of maltose transport activity and is not specific to the sequence of the permease. Our studies indicate that maltose permease does not function as the maltose sensor in S. cerevisiae.

scholarly journals New integrative computational approaches unveil the Saccharomyces cerevisiae pheno-metabolomic fermentative profile and allow strain selection for winemaking

2016 ◽  
Vol 211 ◽  
pp. 509-520 ◽  
Author(s):  
Ricardo Franco-Duarte ◽  
Lan Umek ◽  
Inês Mendes ◽  
Cristiana C. Castro ◽  
Nuno Fonseca ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Strain Selection ◽  
Computational Approaches ◽  
Selection For

Inactivation of maltose permease and maltase in sporulating Saccharomyces cerevisiae

2000 ◽  
Vol 46 (4) ◽  
pp. 383-386 ◽  
Author(s):  
Julio C. Ferreira ◽  
Anita D. Panek ◽  
Pedro S. de Araujo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Maltose Permease

scholarly journals Overproduction of Threonine Aldolase Circumvents the Biosynthetic Role of Pyruvate Decarboxylase in Glucose-Limited Chemostat Cultures of Saccharomyces cerevisiae

2003 ◽  
Vol 69 (4) ◽  
pp. 2094-2099 ◽  
Author(s):  
Antonius J. A. van Maris ◽  
Marijke A. H. Luttik ◽  
Aaron A. Winkler ◽  
Johannes P. van Dijken ◽  
Jack T. Pronk
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Alternative Pathway ◽  
Pyruvate Decarboxylase ◽  
Lysine Biosynthesis ◽  
Carbon Limitation ◽  
Growth Media ◽  
Nutritional Requirement ◽  
Acetyl Coa ◽  
Threonine Aldolase ◽  
Chemostat Cultures

ABSTRACT Pyruvate decarboxylase-negative (Pdc−) mutants of Saccharomyces cerevisiae require small amounts of ethanol or acetate to sustain aerobic, glucose-limited growth. This nutritional requirement has been proposed to originate from (i) a need for cytosolic acetyl coenzyme A (acetyl-CoA) for lipid and lysine biosynthesis and (ii) an inability to export mitochondrial acetyl-CoA to the cytosol. To test this hypothesis and to eliminate the C2 requirement of Pdc− S. cerevisiae, we attempted to introduce an alternative pathway for the synthesis of cytosolic acetyl-CoA. The addition of l-carnitine to growth media did not restore growth of a Pdc− strain on glucose, indicating that the C2 requirement was not solely due to the inability of S. cerevisiae to synthesize this compound. The S. cerevisiae GLY1 gene encodes threonine aldolase (EC 4.1.2.5), which catalyzes the cleavage of threonine to glycine and acetaldehyde. Overexpression of GLY1 enabled a Pdc− strain to grow under conditions of carbon limitation in chemostat cultures on glucose as the sole carbon source, indicating that acetaldehyde formed by threonine aldolase served as a precursor for the synthesis of cytosolic acetyl-CoA. Fractionation studies revealed a cytosolic localization of threonine aldolase. The absence of glycine in these cultures indicates that all glycine produced by threonine aldolase was either dissimilated or assimilated. These results confirm the involvement of pyruvate decarboxylase in cytosolic acetyl-CoA synthesis. The Pdc− GLY1 overexpressing strain was still glucose sensitive with respect to growth in batch cultivations. Like any other Pdc− strain, it failed to grow on excess glucose in batch cultures and excreted pyruvate when transferred from glucose limitation to glucose excess.

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