Global transcriptional response of Saccharomyces cerevisiae to the deletion of SDH3

<p>Abstract</p> <p>Background</p> <p>Mitochondrial respiration is an important and widely conserved cellular function in eukaryotic cells. The succinate dehydrogenase complex (Sdhp) plays an important role in respiration as it connects the mitochondrial respiratory chain to the tricarboxylic acid (TCA) cycle where it catalyzes the oxidation of succinate to fumarate. Cellular response to the Sdhp dysfunction (i.e. impaired respiration) thus has important implications not only for biotechnological applications but also for understanding cellular physiology underlying metabolic diseases such as diabetes. We therefore explored the physiological and transcriptional response of <it>Saccharomyces cerevisiae </it>to the deletion of <it>SDH3</it>, that codes for an essential subunit of the Sdhp.</p> <p>Results</p> <p>Although the Sdhp has no direct role in transcriptional regulation and the flux through the corresponding reaction under the studied conditions is very low, deletion of <it>SDH3 </it>resulted in significant changes in the expression of several genes involved in various cellular processes ranging from metabolism to the cell-cycle. By using various bioinformatics tools we explored the organization of these transcriptional changes in the metabolic and other cellular functional interaction networks.</p> <p>Conclusion</p> <p>Our results show that the transcriptional regulatory response resulting from the impaired respiratory function is linked to several different parts of the metabolism, including fatty acid and sterol metabolism.</p

Global transcriptional response of Saccharomyces cerevisiae to the deletion of SDH3

<p>Abstract</p> <p>Background</p> <p>Mitochondrial respiration is an important and widely conserved cellular function in eukaryotic cells. The succinate dehydrogenase complex (Sdhp) plays an important role in respiration as it connects the mitochondrial respiratory chain to the tricarboxylic acid (TCA) cycle where it catalyzes the oxidation of succinate to fumarate. Cellular response to the Sdhp dysfunction (i.e. impaired respiration) thus has important implications not only for biotechnological applications but also for understanding cellular physiology underlying metabolic diseases such as diabetes. We therefore explored the physiological and transcriptional response of <it>Saccharomyces cerevisiae </it>to the deletion of <it>SDH3</it>, that codes for an essential subunit of the Sdhp.</p> <p>Results</p> <p>Although the Sdhp has no direct role in transcriptional regulation and the flux through the corresponding reaction under the studied conditions is very low, deletion of <it>SDH3 </it>resulted in significant changes in the expression of several genes involved in various cellular processes ranging from metabolism to the cell-cycle. By using various bioinformatics tools we explored the organization of these transcriptional changes in the metabolic and other cellular functional interaction networks.</p> <p>Conclusion</p> <p>Our results show that the transcriptional regulatory response resulting from the impaired respiratory function is linked to several different parts of the metabolism, including fatty acid and sterol metabolism.</p

Industrial Systems Biology of Saccharomyces cerevisiae Enables Novel Succinic Acid Cell Factory.

Saccharomyces cerevisiae is the most well characterized eukaryote, the preferred microbial cell factory for the largest industrial biotechnology product (bioethanol), and a robust commerically compatible scaffold to be exploitted for diverse chemical production. Succinic acid is a highly sought after added-value chemical for which there is no native pre-disposition for production and accmulation in S. cerevisiae. The genome-scale metabolic network reconstruction of S. cerevisiae enabled in silico gene deletion predictions using an evolutionary programming method to couple biomass and succinate production. Glycine and serine, both essential amino acids required for biomass formation, are formed from both glycolytic and TCA cycle intermediates. Succinate formation results from the isocitrate lyase catalyzed conversion of isocitrate, and from the alpha-keto-glutarate dehydrogenase catalyzed conversion of alpha-keto-glutarate. Succinate is subsequently depleted by the succinate dehydrogenase complex. The metabolic engineering strategy identified included deletion of the primary succinate consuming reaction, Sdh3p, and interruption of glycolysis derived serine by deletion of 3-phosphoglycerate dehydrogenase, Ser3p/Ser33p. Pursuing these targets, a multi-gene deletion strain was constructed, and directed evolution with selection used to identify a succinate producing mutant. Physiological characterization coupled with integrated data analysis of transcriptome data in the metabolically engineered strain were used to identify 2nd-round metabolic engineering targets. The resulting strain represents a 30-fold improvement in succinate titer, and a 43-fold improvement in succinate yield on biomass, with only a 2.8-fold decrease in the specific growth rate compared to the reference strain. Intuitive genetic targets for either over-expression or interruption of succinate producing or consuming pathways, respectively, do not lead to increased succinate. Rather, we demonstrate how systems biology tools coupled with directed evolution and selection allows non-intuitive, rapid and substantial re-direction of carbon fluxes in S. cerevisiae, and hence show proof of concept that this is a potentially attractive cell factory for over-producing different platform chemicals

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

The S. Cerevisiae HAP Complex, a Key Regulator of Mitochondrial Function, Coordinates Nuclear and Mitochondrial Gene Expression

We have compared Saccharomyces cerevisiae global gene expression in wild-type and mutants (Δhap2 and Δhap4) of the HAP transcriptional complex, which has been shown to be necessary for growth on respiratory substrates. Several hundred ORFs are under positive or negative control of this complex and we analyse here in detail the effect of HAP on mitochondria. We found that most of the genes upregulated in the wild-type strain were involved in organelle functions, but practically none of the downregulated ones. Nuclear genes encoding the different subunits of the respiratory chain complexes figure in the genes more expressed in the wild-type than in the mutants, as expected, but in this group we also found key components of the mitochondrial translation apparatus. This control of mitochondrial translation may be one of the means of coordinating mitochondrial and nuclear gene expression in elaborating the respiratory chain. In addition, HAP controls the nuclear genes involved in several other mitochondrial processes (import, mitochondrial division) that define the metabolic state of the cell, but not mitochondrial DNA replication and transcription. In most cases, a putative CCAAT-binding site is present upstream of the ORF, while in others no such sites are present, suggesting the control to be indirect. The large number of genes regulated by the HAP complex, as well as the fact that HAP also regulates some putative transcriptional activators of unknown function, place this complex at a hierarchically high position in the global transcriptional regulation of the cell

Differential proteomic analysis by SWATH-MS unravels the most dominant mechanisms underlying yeast adaptation to non-optimal temperatures under anaerobic conditions

Elucidation of temperature tolerance mechanisms in yeast is essential for enhancing cellular robustness of strains, providing more economically and sustainable processes. We investigated the differential responses of three distinct Saccharomyces cerevisiae strains, an industrial wine strain, ADY5, a laboratory strain, CEN.PK113-7D and an industrial bioethanol strain, Ethanol Red, grown at sub- and supra-optimal temperatures under chemostat conditions. We employed anaerobic conditions, mimicking the industrial processes. The proteomic profile of these strains in all conditions was performed by sequential window acquisition of all theoretical spectra-mass spectrometry (SWATH-MS), allowing the quantification of 997 proteins, data available via ProteomeXchange (PXD016567). Our analysis demonstrated that temperature responses differ between the strains; however, we also found some common responsive proteins, revealing that the response to temperature involves general stress and specific mechanisms. Overall, sub-optimal temperature conditions involved a higher remodeling of the proteome. The proteomic data evidenced that the cold response involves strong repression of translation-related proteins as well as induction of amino acid metabolism, together with components related to protein folding and degradation while, the high temperature response mainly recruits amino acid metabolism. Our study provides a global and thorough insight into how growth temperature affects the yeast proteome, which can be a step forward in the comprehension and improvement of yeast thermotolerance.Financial support is acknowledged to Project ERA-IB “YeastTempTation” (ERA-IB-2-6/0001/2014), and FCT for the strategic funding of UIDB/04469/2020 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), and BioTecNorte operation (NORTE-01-0145-FEDER-000004). Lallemand Ibéria, SA is acknowledged for the supply of yeast strains. Te proteomic analysis was carried out in the SCSIE University of Valencia Proteomics Unit, a member of the ISCIII ProteoRed Proteomics Platform. Authors thank to Luz Valero, researcher of the Proteomic unit, for her valuable support.info:eu-repo/semantics/publishedVersio

Doctor of Philosophy

dissertationSuccinate dehydrogenase (SDH, also known as complex II) is a protein complex located in the inner mitochondrial membrane. SDH has dual roles as a part of both the citric acid cycle and the electron transport chain required for aerobic respiration in eukaryotes. In the past two decades, the structure of SDH has been extensively studied. The tetrameric complex consists of SDHA, SDHB, SDHC and SDHD (Sdh1, Sdh2, Sdh3 and Sdh4 in yeast) and contains five cofactors: one flavin adenine dinucleotide, three Fe- S clusters and one heme b. Mutations in SDH subunit genes are associated with various human diseases. Recently, interest has been refocused on SDH by discovery of SDH assembly factors, SDHAF1 and SDHAF2. However, the mechanism for SDH assembly was still poorly understood. The fact that SDH consists of four subunits and five cofactors supports the idea that maturation of SDH would require several assembly factors, as is the case for other electron transport chain complex I, III and IV. SDHdeficient gastrointestinal stromal tumors and neuroblastomas associated with SDH deficiency without mutations in genes encoding SDH subunits or two aforementioned assembly factors also suggest additional unknown SDH assembly factors. In this study, we focus on understanding the mechanism for maturation of the Fe- S cluster subunit of SDH, Sdh2. We discovered a novel SDH assembly factor, Sdh7, and characterized its function. We also revealed the molecular function of Sdh6, a yeast ortholog of human SDHAF1, whose mutations were shown to result in SDH deficiency iv through an unknown mechanism. We demonstrate that Sdh6 and Sdh7 impart protection for the Fe-S cluster subunit of SDH against reactive oxygen species under oxidative stress conditions during SDH assembly. We also propose that a sequence variant of SDHAF3, a yeast ortholog of Sdh7, could be a pathogenic allele that enhances predisposition to endocrine-related tumors. Lastly, we elucidate the function of Nfu1 in Fe-S cluster delivery to target proteins including Sdh2

An evolutionary and functional assessment of regulatory network motifs.

BackgroundCellular functions are regulated by complex webs of interactions that might be schematically represented as networks. Two major examples are transcriptional regulatory networks, describing the interactions among transcription factors and their targets, and protein-protein interaction networks. Some patterns, dubbed motifs, have been found to be statistically over-represented when biological networks are compared to randomized versions thereof. Their function in vitro has been analyzed both experimentally and theoretically, but their functional role in vivo, that is, within the full network, and the resulting evolutionary pressures remain largely to be examined.ResultsWe investigated an integrated network of the yeast Saccharomyces cerevisiae comprising transcriptional and protein-protein interaction data. A comparative analysis was performed with respect to Candida glabrata, Kluyveromyces lactis, Debaryomyces hansenii and Yarrowia lipolytica, which belong to the same class of hemiascomycetes as S. cerevisiae but span a broad evolutionary range. Phylogenetic profiles of genes within different forms of the motifs show that they are not subject to any particular evolutionary pressure to preserve the corresponding interaction patterns. The functional role in vivo of the motifs was examined for those instances where enough biological information is available. In each case, the regulatory processes for the biological function under consideration were found to hinge on post-transcriptional regulatory mechanisms, rather than on the transcriptional regulation by network motifs.ConclusionThe overabundance of the network motifs does not have any immediate functional or evolutionary counterpart. A likely reason is that motifs within the networks are not isolated, that is, they strongly aggregate and have important edge and/or node sharing with the rest of the network

The Role of the GATA Transcription Factor Gaf1 in Nutrient Responses and Cellular Ageing

The discovery of the biological bases of ageing continues to be one of the most fascinating challenges in modern science. Current efforts have narrowed the complexity of such task by focusing on mechanisms used by the cell to couple its physiology with environmental stimuli as they are often involved in the regulation of ageing. The Target of Rapamycin (TOR) have been proved to be a rheostat of nutritional status orchestrating cellular growth and homeostasis mainly through the regulation of transcriptional responses that remain to be understood. Recent studies unveiled novel functions of the evolutionarily conserved GATA transcription factor Gaf1 in nutrient sensing pathways and potentially in cellular ageing by regulating transcription downstream of TOR signalling. To elucidate these questions, the robust model organism Schizosaccharomyces pombe was used in this study due to its relevant similarity with higher eukaryotes and thoroughly described genetics. The experimental settings involved a combination of in silico analyses, fitness assessments, revivability assays, transcriptomics, mutagenesis, chemical-genetics, and interactome to further characterise functions of Gaf1. This study also contributed to the identification of candidate genes that promote longevity and mediate the resistance of mutant cells depleted of gaf1 gene to the TOR-kinase inhibitor torin1. The results indicate that upon TOR complex 1 (TORC1) inhibition, Gaf1 represses genes that induce protein translation (anabolism) and upregulates genes required for survival (catabolism) under adverse nutritional conditions downstream of TORC1