Transcriptional regulation of respiration in yeast metabolizing differently repressive carbon substrates

BACKGROUND: Depending on the carbon source, Saccharomyces cerevisiae displays various degrees of respiration. These range from complete respiration as in the case of ethanol, to almost complete fermentation, and thus very low degrees of respiration on glucose. While many key regulators are known for these extreme cases, we focus here on regulators that are relevant at intermediate levels of respiration. RESULTS: We address this question by linking the functional degree of respiration to transcriptional regulation via enzyme abundances. Specifically, we investigated aerobic batch cultures with the differently repressive carbon sources glucose, mannose, galactose and pyruvate. Based on 13C flux analysis, we found that the respiratory contribution to cellular energy production was largely absent on glucose and mannose, intermediate on galactose and highest on pyruvate. In vivo abundances of 40 respiratory enzymes were quantified by GFP-fusions under each condition. During growth on the partly and fully respired substrates galactose and pyruvate, several TCA cycle and respiratory chain enzymes were significantly up-regulated. From these enzyme levels and the known regulatory network structure, we determined the probability for a given transcription factor to cause the coordinated expression changes. The most probable transcription factors to regulate the different degrees of respiration were Gcr1p, Cat8p, the Rtg-proteins and the Hap-complex. For the latter three ones we confirmed their importance for respiration by quantifying the degree of respiration and biomass yields in the corresponding deletion strains. CONCLUSIONS: Cat8p is required for wild-type like respiration, independent of its known activation of gluconeogenic genes. The Rtg-proteins and the Hap-complex are essential for wild-type like respiration under partially respiratory conditions. Under fully respiratory conditions, the Hap-complex, but not the Rtg-proteins are essential for respiration

Unraveling condition-dependent networks of transcription factors that control metabolic pathway activity in yeast

While typically many expression levels change in transcription factor mutants, only few of these changes lead to functional changes. The predictive capability of expression and DNA binding data for such functional changes in metabolism is very limited.Large-scale 13C-flux data reveal the condition specificity of transcriptional control of metabolic function.Transcription control in yeast focuses on the switch between respiration and fermentation.Follow-up modeling on the basis of transcriptomics and proteomics data suggest the newly discovered Gcn4 control of respiration to be mediated via PKA and Snf1

Long‑Term Adaptation to Galactose as a Sole Carbon Source Selects for Mutations Outside the Canonical GAL Pathway

Galactose is a secondary fermentable sugar that requires specific regulatory and structural genes for its assimilation, which are under catabolite repression by glucose. When glucose is absent, the catabolic repression is attenuated, and the structural GAL genes are fully activated. In Saccharomyces cerevisiae, the GAL pathway is under selection in environments where galactose is present. However, it is unclear the adaptive strategies in response to long-term propagation in galactose as a sole carbon source in laboratory evolution experiments. Here, we performed a 4,000-generation evolution experiment using 48 diploid Saccharomyces cerevisiae populations to study adaptation in galactose. We show that fitness gains were greater in the galactose-evolved population than in identically evolved populations with glucose as a sole carbon source. Whole-genome sequencing of 96 evolved clones revealed recurrent de novo single nucleotide mutations in candidate targets of selection, copy number variations, and ploidy changes. We find that most mutations that improve fitness in galactose lie outside of the canonical GAL pathway. Reconstruction of specific evolved alleles in candidate target of selection, SEC23 and IRA1, showed a significant increase in fitness in galactose compared to glucose. In addition, most of our evolved populations (28/46; 61%) fixed aneuploidies on Chromosome VIII, suggesting a parallel adaptive amplification. Finally, we show greater loss of extrachromosomal elements in our glucose-evolved lineages compared with previous glucose evolution. Broadly, these data further our understanding of the evolutionary pressures that drive adaptation to less-preferred carbon sources

NF-Y activates genes of metabolic pathways altered in cancer cells

The trimeric transcription factor NF-Y binds to the CCAAT box, an element enriched in promoters of genes overexpressed in tumors. Previous studies on the NF-Y regulome identified the general term metabolism as significantly enriched. We dissect here in detail the targeting of metabolic genes by integrating analysis of NF-Y genomic binding and profilings after inactivation of NF-Y subunits in different cell types. NF-Y controls de novo biosynthetic pathways of lipids, teaming up with the master SREBPs regulators. It activates glycolytic genes, but, surprisingly, is neutral or represses mitochondrial respiratory genes. NF-Y targets the SOCG (Serine, One Carbon, Glycine) and Glutamine pathways, as well as genes involved in the biosynthesis of polyamines and purines. Specific cancer-driving nodes are generally under NF-Y control. Altogether, these data delineate a coherent strategy to promote expression of metabolic genes fuelling anaerobic energy production and other anabolic pathways commonly altered in cancer cells

Compartmentation of glycogen metabolism revealed from 13C isotopologue distributions

Background: Stable isotope tracers are used to assess metabolic flux profiles in living cells. The existing methods of measurement average out the isotopic isomer distribution in metabolites throughout the cell, whereas the knowledge of compartmental organization of analyzed pathways is crucial for the evaluation of true fluxes. That is why we accepted a challenge to create a software tool that allows deciphering the compartmentation of metabolites based on the analysis of average isotopic isomer distribution. Results: The software Isodyn, which simulates the dynamics of isotopic isomer distribution in central metabolic pathways, was supplemented by algorithms facilitating the transition between various analyzed metabolic schemes, and by the tools for model discrimination. It simulated 13C isotope distributions in glucose, lactate, glutamate and glycogen, measured by mass spectrometry after incubation of hepatocytes in the presence of only labeled glucose or glucose and lactate together (with label either in glucose or lactate). The simulations assumed either a single intracellular hexose phosphate pool, or also channeling of hexose phosphates resulting in a different isotopic composition of glycogen. Model discrimination test was applied to check the consistency of both models with experimental data. Metabolic flux profiles, evaluated with the accepted model that assumes channeling, revealed the range of changes in metabolic fluxes in liver cells. Conclusions: The analysis of compartmentation of metabolic networks based on the measured 13C distribution was included in Isodyn as a routine procedure. The advantage of this implementation is that, being a part of evaluation of metabolic fluxes, it does not require additional experiments to study metabolic compartmentation. The analysis of experimental data revealed that the distribution of measured 13C-labeled glucose metabolites is inconsistent with the idea of perfect mixing of hexose phosphates in cytosol. In contrast, the observed distribution indicates the presence of a separate pool of hexose phosphates that is channeled towards glycogen synthesis

Production efficiency of the bacterial non-ribosomal peptide indigoidine relies on the respiratory metabolic state in <i>S. cerevisiae</i>

Abstract Background Beyond pathway engineering, the metabolic state of the production host is critical in maintaining the efficiency of cellular production. The biotechnologically important yeast Saccharomyces cerevisiae adjusts its energy metabolism based on the availability of oxygen and carbon sources. This transition between respiratory and non-respiratory metabolic state is accompanied by substantial modifications of central carbon metabolism, which impact the efficiency of metabolic pathways and the corresponding final product titers. Non-ribosomal peptide synthetases (NRPS) are an important class of biocatalysts that provide access to a wide array of secondary metabolites. Indigoidine, a blue pigment, is a representative NRP that is valuable by itself as a renewably produced pigment. Results Saccharomyces cerevisiae was engineered to express a bacterial NRPS that converts glutamine to indigoidine. We characterize carbon source use and production dynamics, and demonstrate that indigoidine is solely produced during respiratory cell growth. Production of indigoidine is abolished during non-respiratory growth even under aerobic conditions. By promoting respiratory conditions via controlled feeding, we scaled the production to a 2 L bioreactor scale, reaching a maximum titer of 980 mg/L. Conclusions This study represents the first use of the Streptomyces lavendulae NRPS (BpsA) in a fungal host and its scale-up. The final product indigoidine is linked to the activity of the TCA cycle and serves as a reporter for the respiratory state of S. cerevisiae. Our approach can be broadly applied to investigate diversion of flux from central carbon metabolism for NRPS and other heterologous pathway engineering, or to follow a population switch between respiratory and non-respiratory modes

Chemical and Synthetic Genetic Array Analysis Identifies Genes that Suppress Xylose Utilization and Fermentation in Saccharomyces cerevisiae

Though highly efficient at fermenting hexose sugars, Saccharomyces cerevisiae has limited ability to ferment five-carbon sugars. As a significant portion of sugars found in cellulosic biomass is the five-carbon sugar xylose, S. cerevisiae must be engineered to metabolize pentose sugars, commonly by the addition of exogenous genes from xylose fermenting fungi. However, these recombinant strains grow poorly on xylose and require further improvement through rational engineering or evolutionary adaptation. To identify unknown genes that contribute to improved xylose fermentation in these recombinant S. cerevisiae, we performed genome-wide synthetic interaction screens to identify deletion mutants that impact xylose utilization of strains expressing the xylose isomerase gene XYLA from Piromyces sp. E2 alone or with an additional copy of the endogenous xylulokinase gene XKS1. We also screened the deletion mutant array to identify mutants whose growth is affected by xylose. Our genetic network reveals that more than 80 nonessential genes from a diverse range of cellular processes impact xylose utilization. Surprisingly, we identified four genes, ALP1, ISC1, RPL20B, and BUD21, that when individually deleted improved xylose utilization of both S. cerevisiae S288C and CEN.PK strains. We further characterized BUD21 deletion mutant cells in batch fermentations and found that they produce ethanol even the absence of exogenous XYLA. We have demonstrated that the ability of laboratory strains of S. cerevisiae to utilize xylose as a sole carbon source is suppressed, which implies that S. cerevisiae may not require the addition of exogenous genes for efficient xylose fermentation

Importance of S. Cerevisiae Rcf1 and Rcf2 Proteins for the Mitochondrial Protonmotive Force Generation

Mitochondria are the site of oxidative phosphorylation (OXPHOS) pathway, which can supply majority of energy in a eukaryotic cell. OXPHOS enzyme activities generate electrochemical gradient known as mitochondrial protonmotive force (PMF). PMF coordinates OXPHOS enzyme activities and supports essential cell survival functions such as transport of proteins and metabolites in and out of mitochondria. PMF is maintained despite variations in cellular energy demand and oxygen availability.Mitochondrial proteins belonging to the conserved hypoxia induced gene domain (HIGD) family improve cell survival during the hypoxic and hypoglycemic stress. Their molecular function is not fully understood but they seem to act through regulating OXPHOS enzyme cytochrome c oxidase (complex IV). Complex IV activity is important for PMF generation. Using yeast as a model organism, this study addresses function of HIGD proteins Rcf1 and Rcf2. The data presented here indicate that Rcf1 and Rcf2 support complex IV PMF generation and/or prevent proton leak across the inner membrane of the mitochondria. Deletion of Rcf1 (Δrcf1) causes lower complex IV steady state levels and electron transfer activity. Deletion of Rcf2 in Δrcf1 strain (Δrcf1;Δrcf2) does not further decrease complex IV steady state levels and electron transfer activity, yet strongly impairs respiratory growth. Analyses of single mutant strains Δrcf1 and Δrcf2 indicated that deletion of Rcf1 or Rcf2 lowers OXPHOS efficiency and decreases PMF. These defects become more severe when both Rcf1 and Rcf2 are deleted. The inability to maintain PMF and PMF-dependent functions is proposed to underlie the strong respiratory growth deficiency of Δrcf1;Δrcf2 mutant. In addition to this new role of Rcf1 and Rcf2 in PMF maintenance, several pleiotropic phenotypes of Δrcf1;Δrcf2 mutant – such as decreased ATP synthase levels, abnormal mitochondrial morphology – suggest that these proteins may have wider impact on OXPHOS function