Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of S. cerevisiae reveaks increasing nadh oxidation reduces overflow metabolism

ABSTRACT: When the yeast Saccharomyces cerevisiae is subjected to increasing glycolytic fluxes under aerobic conditions, there is a threshold value of the glucose uptake rate at which the metabolism shifts from being purely respiratory to mixed respiratory and fermentative. This shift is characterized by ethanol production, a phenomenon known as the Crabtree effect due to its analogy with lactate overflow in cancer cells. It is well known that at high glycolytic fluxes there is glucose repression of respiratory pathways resulting in a decrease in the respiratory capacity. Despite many years of detailed studies on this subject, it is not known whether the onset of the Crabtree effect (or overflow metabolism) is due to a limited respiratory capacity or caused by glucose-mediated repression of respiration. We addressed this issue by increasing respiration in S. cerevisiae by introducing a heterologous alternative oxidase, and observed reduced aerobic ethanol formation. In contrast, increasing non-respiratory NADH oxidation by overexpression of a water-forming NADH oxidase reduced aerobic glycerol formation. The metabolic response to elevated alternative oxidase occurred predominantly in the mitochondria, while NADH oxidase affected genes that catalyze cytosolic reactions. Moreover, NADH oxidase restored the deficiency of cytosolic NADH dehydrogenases in S. cerevisiae. These results indicate that NADH oxidase localizes in the cytosol, while alternative oxidase is directed to the mitochondria. The onset of aerobic ethanol formation is demonstrated to be a consequence of an imbalance in mitochondrial redox balancing. In addition to answering fundamental physiological questions, our findings are relevant for all biomass derived applications of S. cerevisiae. Experiment Overall Design: Heterologous gene expression in chemostats using Affymetrix Yeast Genome 2.0 arrays. Total RNA extraction and sample preparation, hybridization was done according to the manufacturer's protocol.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Goutham Vemuri

PROVIDER: E-GEOD-6277 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae.

Vemuri G N GN Eiteman M A MA McEwen J E JE Olsson L L Nielsen J J

Proceedings of the National Academy of Sciences of the United States of America 20070207 7

Respiratory metabolism plays an important role in energy production in the form of ATP in all aerobically growing cells. However, a limitation in respiratory capacity results in overflow metabolism, leading to the formation of byproducts, a phenomenon known as "overflow metabolism" or "the Crabtree effect." The yeast Saccharomyces cerevisiae has served as an important model organism for studying the Crabtree effect. When subjected to increasing glycolytic fluxes under aerobic conditions, there i ...[more]

PMID: 17287356

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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:Metabolic cofactors such as NADH and ATP play important roles in a large number of cellular reactions and it is of great interest to dissect the role of these cofactors in different aspects of metabolism. Towards this goal, we overexpressed NADH oxidase and the soluble F1-ATPase in Escherichia coli to lower the level of NADH and ATP, respectively. We used a systems biology approach to study the response to these perturbations by measuring global transcription profiles, metabolic fluxes and the metabolite levels. We integrated information from the different measurements using network-based methods to identify high-scoring networks in a global interaction map that included protein interactions, transcriptional regulation and metabolism. The results revealed that the action of many global transcription factors such as ArcA, Fnr, CRP and IHF commonly involved both NADH and ATP while others were influential only in one of the pertubations. In general, overexpressing NADH oxidase invokes response in widespread aspects of metabolism involving the redox cofactors (NADH and NADPH) while ATPase has a more focused response to restore ATP level by enhancing proton translocation mechanisms and repressing biosynthesis. Interestingly, NADPH played a key role in restoring redox homeostasis through the concerted activity of isocitrate dehydrogenase and UdhA transhydrogenase. We present a reconciled network of regulation that illustrates the overlapping and distinct aspects of metabolism controlled by NADH and ATP. Our study contributes to the general understanding of redox and energy metabolism and should help in developing metabolic engineering strategies in E. coli. The experimental design involves measuring transcriptome in three strains (in triplicates) of E. coli during mid-exponential phase of growth on MOPS media supplemented with glucose. The three strains are: 1. REF: MG1655/pAK80 (MG1655 transformed with a plasmid with no insert) 2. NOX: MG1655/pAC06 (MG1655 transformed with a plasmid containing NADH oxidase) 3. ATPase: MG1655/pCP41 (MG1655 transformed with a plasmid containing soluble ATPase) RNA was extracted using Qiagen RNeasy kit and processes according to AffymetrixÂ® guidelines. The quality of RNA was verified using BioAnalyzer (Agilent BioSystems) and the electropherograms are shown in the file âRNA quality.pdfâ. Samples 1-3 are REF, Samples 4-6 are NOX and Samples 7-9 are ATPase. The fragmented cRNA was hybridized to E. coli Genome 2.0 chips.

Dataset Information

Transcription profiling of S. cerevisiae reveaks increasing nadh oxidation reduces overflow metabolism

Publications

Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae.

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