| 1. |
- Chen, Yun, 1978, et al.
(författare)
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Profiling of Cytosolic and Peroxisomal Acetyl-CoA Metabolism in Saccharomyces cerevisiae
- 2012
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 7:8, s. e42475-
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Tidskriftsartikel (refereegranskat)abstract
- As a key intracellular metabolite, acetyl-coenzyme A (acetyl-CoA) plays a major role in various metabolic pathways that link anabolism and catabolism. In the yeast Saccharomyces cerevisiae, acetyl-CoA involving metabolism is compartmentalized, and may vary with the nutrient supply of a cell. Membranes separating intracellular compartments are impermeable to acetyl-CoA and no direct transport between the compartments occurs. Thus, without carnitine supply the glyoxylate shunt is the sole possible route for transferring acetyl-CoA from the cytosol or the peroxisomes into the mitochondria. Here, we investigate the physiological profiling of different deletion mutants of ACS1, ACS2, CIT2 and MLS1 individually or in combination under alternative carbon sources, and study how various mutations alter carbon distribution. Based on our results a detailed model of carbon distribution about cytosolic and peroxisomal acetyl-CoA metabolism in yeast is suggested. This will be useful to further develop yeast as a cell factory for the biosynthesis of acetyl-CoA-derived products.
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| 2. |
- Feizi, Amir, 1980, et al.
(författare)
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Genome-Scale Modeling of the Protein Secretory Machinery in Yeast
- 2013
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 8:5
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Tidskriftsartikel (refereegranskat)abstract
- The protein secretory machinery in Eukarya is involved in post-translational modification (PTMs) and sorting of the secretory and many transmembrane proteins. While the secretory machinery has been well-studied using classic reductionist approaches, a holistic view of its complex nature is lacking. Here, we present the first genome-scale model for the yeast secretory machinery which captures the knowledge generated through more than 50 years of research. The model is based on the concept of a Protein Specific Information Matrix (PSIM: characterized by seven PTMs features). An algorithm was developed which mimics secretory machinery and assigns each secretory protein to a particular secretory class that determines the set of PTMs and transport steps specific to each protein. Protein abundances were integrated with the model in order to gain system level estimation of the metabolic demands associated with the processing of each specific protein as well as a quantitative estimation of the activity of each component of the secretory machinery.
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| 3. |
- Lindqvist, Madelene, 1982, et al.
(författare)
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Unraveling molecular signatures of immunostimulatory adjuvants in the female genital tract through systems biology
- 2011
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:6
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Tidskriftsartikel (refereegranskat)abstract
- Sexually transmitted infections (STIs) unequivocally represent a major public health concern in both industrialized and developing countries. Previous efforts to develop vaccines for systemic immunization against a large number of STIs in humans have been unsuccessful. There is currently a drive to develop mucosal vaccines and adjuvants for delivery through the genital tract to confer protective immunity against STIs. Identification of molecular signatures that can be used as biomarkers for adjuvant potency can inform rational development of potent mucosal adjuvants. Here, we used systems biology to study global gene expression and signature molecules and pathways in the mouse vagina after treatment with two classes of experimental adjuvants. The Toll-like receptor 9 agonist CpG ODN and the invariant natural killer T cell agonist alpha-galactosylceramide, which we previously identified as equally potent vaginal adjuvants, were selected for this study. Our integrated analysis of genome-wide transcriptome data determined which signature pathways, processes and networks are shared by or otherwise exclusive to these 2 classes of experimental vaginal adjuvants in the mouse vagina. To our knowledge, this is the first integrated genome-wide transcriptome analysis of the effects of immunomodulatory adjuvants on the female genital tract of a mammal. These results could inform rational development of effective mucosal adjuvants for vaccination against STIs.
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| 4. |
- Madsen, Karina M., et al.
(författare)
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Linking Genotype and Phenotype of Saccharomyces cerevisiae Strains Reveals Metabolic Engineering Targets and Leads to Triterpene Hyper-Producers
- 2011
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 6:3, s. e14763-
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundMetabolic engineering is an attractive approach in order to improve the microbial production of drugs. Triterpenes is a chemically diverse class of compounds and many among them are of interest from a human health perspective. A systematic experimental or computational survey of all feasible gene modifications to determine the genotype yielding the optimal triterpene production phenotype is a laborious and time-consuming process.Methodology/Principal FindingsBased on the recent genome-wide sequencing of Saccharomyces cerevisiae CEN.PK 113-7D and its phenotypic differences with the S288C strain, we implemented a strategy for the construction of a β-amyrin production platform. The genes Erg8, Erg9 and HFA1 contained non-silent SNPs that were computationally analyzed to evaluate the changes that cause in the respective protein structures. Subsequently, Erg8, Erg9 and HFA1 were correlated with the increased levels of ergosterol and fatty acids in CEN.PK 113-7D and single, double, and triple gene over-expression strains were constructed.ConclusionsThe six out of seven gene over-expression constructs had a considerable impact on both ergosterol and β-amyrin production. In the case of β-amyrin formation the triple over-expression construct exhibited a nearly 500% increase over the control strain making our metabolic engineering strategy the most successful design of triterpene microbial producers.
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| 5. |
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| 6. |
- Olivares Hernandez, Roberto, 1975, et al.
(författare)
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Combining Substrate Specificity Analysis with Support Vector Classifiers Reveals Feruloyl Esterase as a Phylogenetically Informative Protein Group
- 2010
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 5:9, s. Article Number: e12781-11
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Tidskriftsartikel (refereegranskat)abstract
- Background: Our understanding of how fungi evolved to develop a variety of ecological niches, is limited but of fundamental biological importance. Specifically, the evolution of enzymes affects how well species can adapt to new environmental conditions. Feruloyl esterases (FAEs) are enzymes able to hydrolyze the ester bonds linking ferulic acid to plant cell wall polysaccharides. The diversity of substrate specificities found in the FAE family shows that this family is old enough to have experienced the emergence and loss of many activities. Methodology/Principal Findings: In this study we evaluate the relative activity of FAEs against a variety of model substrates as a novel predictive tool for Ascomycota taxonomic classification. Our approach consists of two analytical steps; (1) an initial unsupervised analysis to cluster the FAEs substrate specificity data which were generated by cultivation of 34 Ascomycota strains and then an analysis of the produced enzyme cocktail against 10 substituted cinnamate and phenylalkanoate methyl esters, (2) a second, supervised analysis for training a predictor built on these substrate activities. By applying both linear and non-linear models we were able to correctly predict the taxonomic Class (similar to 86% correct classification), Order (similar to 88% correct classification) and Family (similar to 88% correct classification) that the 34 Ascomycota belong to, using the activity profiles of the FAEs. Conclusion/Significance: The good correlation with the FAEs substrate specificities that we have defined via our phylogenetic analysis not only suggests that FAEs are phylogenetically informative proteins but it is also a considerable step towards improved FAEs functional prediction.
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| 7. |
- Otero, José Manuel, 1979, et al.
(författare)
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Industrial Systems Biology of Saccharomyces cerevisiae Enables Novel Succinic Acid Cell Factory
- 2013
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Otero, José Manuel, 1979, et al.
(författare)
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Yeast Biological Networks Unfold the Interplay of Antioxidants, Genome and Phenotype, and Reveal a Novel Regulator of the Oxidative Stress Response
- 2010
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 5:10, s. e13606-
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundIdentifying causative biological networks associated with relevant phenotypes is essential in the field of systems biology. We used ferulic acid (FA) as a model antioxidant to characterize the global expression programs triggered by this small molecule and decipher the transcriptional network controlling the phenotypic adaptation of the yeast Saccharomyces cerevisiae.Methodology/Principal FindingsBy employing a strict cut off value during gene expression data analysis, 106 genes were found to be involved in the cell response to FA, independent of aerobic or anaerobic conditions. Network analysis of the system guided us to a key target node, the FMP43 protein, that when deleted resulted in marked acceleration of cellular growth (~15% in both minimal and rich media). To extend our findings to human cells and identify proteins that could serve as drug targets, we replaced the yeast FMP43 protein with its human ortholog BRP44 in the genetic background of the yeast strain Δfmp43. The conservation of the two proteins was phenotypically evident, with BRP44 restoring the normal specific growth rate of the wild type. We also applied homology modeling to predict the 3D structure of the FMP43 and BRP44 proteins. The binding sites in the homology models of FMP43 and BRP44 were computationally predicted, and further docking studies were performed using FA as the ligand. The docking studies demonstrated the affinity of FA towards both FMP43 and BRP44.ConclusionsThis study proposes a hypothesis on the mechanisms yeast employs to respond to antioxidant molecules, while demonstrating how phenome and metabolome yeast data can serve as biomarkers for nutraceutical discovery and development. Additionally, we provide evidence for a putative therapeutic target, revealed by replacing the FMP43 protein with its human ortholog BRP44, a brain protein, and functionally characterizing the relevant mutant strain.
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| 9. |
- Partow, Siavash, 1979, et al.
(författare)
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Reconstruction and Evaluation of the Synthetic Bacterial MEP Pathway in Saccharomyces cerevisiae
- 2012
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 7:12, s. Article Number: e52498 -
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Tidskriftsartikel (refereegranskat)abstract
- Isoprenoids, which are a large group of natural and chemical compounds with a variety of applications as e.g. fragrances, pharmaceuticals and potential biofuels, are produced via two different metabolic pathways, the mevalonate (MVA) pathway and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we attempted to replace the endogenous MVA pathway in Saccharomyces cerevisiae by a synthetic bacterial MEP pathway integrated into the genome to benefit from its superior properties in terms of energy consumption and productivity at defined growth conditions. It was shown that the growth of a MVA pathway deficient S. cerevisiae strain could not be restored by the heterologous MEP pathway even when accompanied by the co-expression of genes erpA, hISCA1 and CpIscA involved in the Fe-S trafficking routes leading to maturation of IspG and IspH and E. coli genes fldA and fpr encoding flavodoxin and flavodoxin reductase believed to be responsible for electron transfer to IspG and IspH.
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