Exometabolic and transcriptional response in relation to phenotype and gene copy number in respiration-related deletion mutants of S. cerevisiae.

The transcriptional and metabolic impact of deleting one or both copies of a respiration-related gene has been studied in glucose-limited chemostats. Integration of literature information on phenotype with our exometabolome and transcriptome data enabled the identification of novel relationships between gene copy number, transcriptional regulation and phenotype. We found that the effect of complete respiratory deficiency on transcription was limited to downregulation of genes involved in oxidoreductase activity and iron assimilation. Partial respiratory deficiency had no significant impact on gene transcription. Changes in the copy number of two transcription-factor genes, HAP4 and MIG1, had a major impact on genes involved in mitochondrial function. Regulation of respiratory chain components encoded in the nucleus and mitochondria appears to be divided between Hap4p and Oxa1p, respectively. Similarly, repression of respiration may be imposed by the action of Mig1p and Mba1p on nuclear and mitochondrial gene expression, respectively. However, it is not clear whether Oxa1p and Mba1p regulate mitochondrial gene expression via their interaction with mitochondrial ribosomes or by some indirect means. The phenotype of nuclear petite mutants may not simply be due to the absence of respiration; e.g. Oxa1p or Bcs1p may play a role in the regulation of ribosome assembly in the nucleolus. Integration between respiration and cell growth may also result from the action of a single transcription factor. Thus, Hap4p targets genes that are required for respiration and for fitness in nutrient-limited conditions. This suggests that Hap4p may enable cells to adapt to nutrient limitation as well as diauxy.

Integration of metabolic modeling and phenotypic data in evaluation and improvement of ethanol production using respiration-deficient mutants of Saccharomyces cerevisiae.

Flux balance analysis and phenotypic data were used to provide clues to the relationships between the activities of gene products and the phenotypes resulting from the deletion of genes involved in respiratory function in Saccharomyces cerevisiae. The effect of partial or complete respiratory deficiency on the ethanol production and growth characteristics of hap4Delta/hap4Delta, mig1Delta/mig1Delta, qdr3Delta/qdr3Delta, pdr3Delta/pdr3Delta, qcr7Delta/qcr7Delta, cyt1Delta/cyt1Delta, and rip1Delta/rip1Delta mutants grown in microaerated chemostats was investigated. The study provided additional evidence for the importance of the selection of a physiologically relevant objective function, and it may improve quantitative predictions of exchange fluxes, as well as qualitative estimations of changes in intracellular fluxes. Ethanol production was successfully predicted by flux balance analysis in the case of the qdr3Delta/qdr3Delta mutant, with maximization of ethanol production as the objective function, suggesting an additional role for Qdr3p in respiration. The absence of similar changes in estimated intracellular fluxes in the qcr7Delta/qcr7Delta mutant compared to the rip1Delta/rip1Delta and cyt1Delta/cyt1Delta mutants indicated that the effect of the deletion of this subunit of complex III was somehow compensated for. Analysis of predicted flux distributions indicated self-organization of intracellular fluxes to avoid NAD(+)/NADH imbalance in rip1Delta/rip1Delta and cyt1Delta/cyt1Delta mutants, but not the qcr7Delta/qcr7Delta mutant. The flux through the glycerol efflux channel, Fps1p, was estimated to be zero in all strains under the investigated conditions. This indicates that previous strategies for improving ethanol production, such as the overexpression of the glutamate synthase gene GLT1 in a GDH1 deletion background or deletion of the glycerol efflux channel gene FPS1 and overexpression of GLT1, are unnecessary in a respiration-deficient background.

Effect of carbon source perturbations on transcriptional regulation of metabolic fluxes in Saccharomyces cerevisiae.

BACKGROUND: Control effective flux (CEF) of a reaction is the weighted sum of all fluxes through that reaction, derived from elementary flux modes (EFM) of a metabolic network. Change in CEFs under different environmental conditions has earlier been proven to be correlated with the corresponding changes in the transcriptome. Here we use this to investigate the degree of transcriptional regulation of fluxes in the metabolism of Saccharomyces cerevisiae. We do this by quantifying correlations between changes in CEFs and changes in transcript levels for shifts in carbon source, i.e. between the fermentative carbon source glucose and nonfermentative carbon sources like ethanol, acetate, and lactate. The CEF analysis is based on a simple stoichiometric model that includes reactions of the central carbon metabolism and the amino acid metabolism. RESULTS: The effect of the carbon shift on the metabolic fluxes was investigated for both batch and chemostat cultures. For growth on glucose in batch (respiro-fermentative) cultures, EFMs with no by-product formation were removed from the analysis of the CEFs, whereas those including any by-products (ethanol, glycerol, acetate, succinate) were omitted in the analysis of growth on glucose in chemostat (respiratory) cultures. This resulted in improved correlations between CEF changes and transcript levels. A regression correlation coefficient of 0.60 was obtained between CEF changes and gene expression changes in the central carbon metabolism for the analysis of 5 different perturbations. Out of 45 data points there were no more than 6 data points deviating from the correlation. Additionally, up- or down-regulation of at least 75% of the genes were in qualitative agreement with the CEF changes for all perturbations studied. CONCLUSION: The analysis indicates that changes in carbon source are associated with a high degree of hierarchical regulation of metabolic fluxes in the central carbon metabolism as the change in fluxes are correlating directly with the change in transcript levels of genes encoding their corresponding enzymes. For amino acid biosynthesis there was, however, not found to exist a similar correlation, and this may point to either post-transcriptional and/or metabolic regulation, or be due to the absence of a direct perturbation on the amino acid pathways in these experiments.

Understanding signaling in yeast: Insights from network analysis.

Reconstruction of protein interaction networks that represent groups of proteins contributing to the same cellular function is a key step towards quantitative studies of signal transduction pathways. Here we present a novel approach to reconstruct a highly correlated protein interaction network and to identify previously unknown components of a signaling pathway through integration of protein-protein interaction data, gene expression data, and Gene Ontology annotations. A novel algorithm is designed to reconstruct a highly correlated protein interaction network which is composed of the candidate proteins for signal transduction mechanisms in yeast Saccharomyces cerevisiae. The high efficiency of the reconstruction process is proved by a Receiver Operating Characteristic curve analysis. Identification and scoring of the possible linear pathways enables reconstruction of specific sub-networks for glucose-induction signaling and high osmolarity MAPK signaling in S. cerevisiae. All of the known components of these pathways are identified together with several new "candidate" proteins, indicating the successful reconstructions of two model pathways involved in S. cerevisiae. The integrated approach is hence shown useful for (i) prediction of new signaling pathways, (ii) identification of unknown members of documented pathways, and (iii) identification of network modules consisting of a group of related components that often incorporate the same functional mechanism.