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| 2. |
- Anasontzis, George E, 1980, et al.
(författare)
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Investigating the Yet-Unknown Biomass Degrading and Modifying Enzymes of Aspergillus oryzae
- 2012
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Ingår i: Science and Technology Day, 27 March 2012, Göteborg, Sweden.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The association of plant cell wall components with A. oryzae’s extracellular enzyme machinery was investigated by analyzing the transcriptome profile in relation to the chemical structure of the carbon sources. Strain RIB40 of A. oryzae was cultured on various carbon sources, namely cellohexaose, mannohexaose, xylopentaose, arabinoheptaose, glucohexaose, glucosyl maltotriosyl maltotriose, galactosyl mannotriose, turanose and sophorose, and the transcribed genes were determined with DNA microarrays. The statistically significant genes were selected and potential novel hydrolases were identified.Presently, we aim at the heterologous expression and characterization of seven different hypothetical and non classified proteins of A. oryzae, which could prove to be useful tools in the wood biomass separation and modification process. This work represents a novel way of integrating computational chemical biology and classical enzyme research for improving lignocellulose bioconversion.
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| 3. |
- Anasontzis, George E, 1980, et al.
(författare)
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Investigating the yet-unknown biomass degrading and modifying enzymes of Aspergillus oryzae
- 2011
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Ingår i: WWSC Workshop, 29 November - 1 December, Södertuna, Gnesta, Sweden.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The associations of plant cell wall components with A. oryzae’s extracellular enzyme machinery were investigated from a chemical perspective using an integrated analysis of the transcriptome profile. We were able to identify novel hydrolases, which we heterologous expressed for subsequent characterization. This work represents a novel way of integrating computational chemical biology and classical enzyme research for improving lignocellulose bioconversion. In this work, we aim at the heterologous expression and characterization of seven different hypothetical and non classified proteins of A. oryzae, which could prove to be useful tools in the wood biomass separation and modification process.
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| 4. |
- Kouskoumvekaki, Irene, et al.
(författare)
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Identification of biomarkers for genotyping Aspergilli using non-linear methods for clustering and classification
- 2008
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Ingår i: BMC Bioinformatics. - 1471-2105. ; 9:59
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Tidskriftsartikel (refereegranskat)abstract
- Background: In the present investigation, we have used an exhaustive metabolite profiling approach to search for biomarkers in recombinant Aspergillus nidulans (mutants that produce the 6-methyl salicylic acid polyketide molecule) for application in metabolic engineering. Results: More than 450 metabolites were detected and subsequently used in the analysis. Our approach consists of two analytical steps of the metabolic profiling data, an initial non-linear unsupervised analysis with Self-Organizing Maps (SOM) to identify similarities and differences among the metabolic profiles of the studied strains, followed by a second, supervised analysis for training a classifier based on the selected biomarkers. Our analysis identified seven putative biomarkers that were able to cluster the samples according to their genotypes. A Support Vector Machine was subsequently employed to construct a predictive model based on the seven biomarkers, capable of distinguishing correctly 14 out of the 16 samples of the different A. nidulans strains. Conclusion: Our study demonstrates that it is possible to use metabolite profiling for the classification of filamentous fungi as well as for the identification of metabolic engineering targets and draws the attention towards the development of a common database for storage of metabolomics data.
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| 5. |
- 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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| 6. |
- 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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| 7. |
- Panagiotou, Gianni, 1974, et al.
(författare)
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Fermentation characteristics of Fusarium oxysporum grown on acetate
- 2008
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Ingår i: Bioresource Technology. - : Elsevier BV. - 0960-8524 .- 1873-2976. ; :99, s. 7397-7401
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Tidskriftsartikel (refereegranskat)abstract
- In this study, the growth characteristics of Fusarium oxysporum were evaluated in minimal medium using acetate or different mixtures of acetate and glucose as carbon source. The minimum inhibitory concentration (MIC) of acetic acid that F. oxysporum cells could tolerate was 0.8% w/v while glucose was consumed preferentially to acetate. The activity of isocitrate lyase was high when cells were grown on acetate and acetate plus glucose indicating an activation of the glyoxylate cycle. Investigation of the metabolic fingerprinting and footprinting revealed higher levels of intracellular and extracellular TCA cycle intermediates when F. oxysporum cells were grown on mixtures of acetate and glucose compared to growth on only glycose. Our data support the hypothesis that a higher flux through TCA cycle during acetate consumption could significantly increase the pool of NADH, resulting in the activation of succinate-propionate pathway which consumes reducing power (NADH) via conversion of succinate to propionyl-CoA and produce propionate.
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| 8. |
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| 9. |
- Panagiotou, Gianni, 1974, et al.
(författare)
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Identification of NADH kinase activity in filamentous fungi and structural modelling of the novel enzyme from Fusarium oxysporum
- 2008
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Ingår i: Process Biochemistry. - : Elsevier BV. - 1359-5113 .- 1873-3298. ; :43, s. 1114-1120
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Tidskriftsartikel (refereegranskat)abstract
- ATP-NADH kinase phosphorylates NADH to produce NADPH at the expense of ATP. The present study describes Fusarium oxysporum NADH kinase (ATP:NADH 2'-phosphotransferase, EC 2.7.1.86), a novel fungal enzyme capable of synthesizing NADPH using NADH as the preferred diphosphonicotinamide (diphosphopyridine) nucleotide donor. NADH kinase was highly purified (~66-fold) and the enzyme was found to be a homodimeric with a subunit of Mr 72,000. Isoelectric focusing in the pH range of 3.0-9.5 of the purified NADH kinase yielded a pI value of about 5.6. The Km values of NADH kinase for NADH and ATP were found to be 0.13 and 2.59 mM, respectively. Prediction of the secondary structure of the protein was performed in the PSIPRED server while modelling the three-dimensional (3D) structure was accomplished by the use of the HH 3D-structure prediction server.
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| 10. |
- Panagiotou, Gianni, 1974, et al.
(författare)
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Overexpression of a novel endogenous NADH kinase in Aspergillus nidulans enhances growth
- 2009
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; :11, s. 31-39
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Tidskriftsartikel (refereegranskat)abstract
- The complete genome sequence of the filamentous fungi Aspergillus nidulans has paved the way for fundamental research on this industrially important species. To the best of your knowledge, this is the first time a gene encoding for ATP-dependent NADH kinase (ATP:NADH 2´-phosphotransferase, EC 2.7.1.86) has been identified. The enzyme has a predicted molecular weight of 49 kDa. We characterised the role of this NADH kinase by genomic integration of the putative gene AN8837.2 under a strong constitutive promoter. The physiological effects of overexpressed NADH kinase in combination with defferent aeration rates were studied in well-controlled glucose batch fermentations. Metabolite profiling and metabolic network analysis with [1-13C] glucose were used for characterisation of the strains, and the results demonstrated that NADH kinase activity has paramount influence on growth physiology. Biomass yield on glucose and the maximum specific growth rate increased from 0.47 g/g and 0.22 h-1 (wild type) to 0.54 g/g and 0.26 h-1 (NADH kinase overexpressed), respectively. The results suggest that over-expression of NADH kinase improves the growth efficiency of the cell by increasing the access to NADPH. Our findings indicate that A. nidulans is not optimised for growth in nutrient-rich conditions typically found in laboratory and industrial fermentors. This conclusion may impact the design of new strains capable of generating reducing power in the form of NADPH, which is crucial for efficient production of many industrially important metabolities and enzymes.
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