| 1. |
- Meijer, S. L., et al.
(författare)
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Gene deletion of cytosolic ATP: citrate lyase leads to altered organic acid production in Aspergillus niger
- 2009
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Ingår i: Journal of Industrial Microbiology and Biotechnology. - : Oxford University Press (OUP). - 1367-5435 .- 1476-5535. ; 36:10, s. 1275-1280
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Tidskriftsartikel (refereegranskat)abstract
- With the availability of the genome sequence of the filamentous fungus Aspergillus niger, the use of targeted genetic modifications has become feasible. This, together with the fact that A. niger is well established industrially, makes this fungus an attractive micro-organism for creating a cell factory platform for production of chemicals. Using molecular biology techniques, this study focused on metabolic engineering of A. niger to manipulate its organic acid production in the direction of succinic acid. The gene target for complete gene deletion was cytosolic ATP: citrate lyase (acl), which had previously been identified by using genome-scale stoichiometric metabolic model simulations. The acl gene was deleted using the bipartite gene-targeting method, and the mutant was characterized in batch cultivation. It was found that the succinic acid yield was increased threefold by deleting the acl gene. Additionally, the total amount of organic acids produced in the deletion strain was significantly increased. Genome-scale stoichiometric metabolic predictions can be used for identifying gene targets. Deletion of the acl led to increased succinic acid production by A. niger.
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| 2. |
- de Jongh, Willem A., et al.
(författare)
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The Roles of Galactitol, Galactose-1-Phosphate, and Phosphoglucomutase in Galactose-Induced Toxicity in Saccharomyces cerevisiae
- 2008
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Ingår i: Biotechnology and Bioengineering. - 0006-3592 .- 1097-0290. ; 101:2, s. 317-326
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Tidskriftsartikel (refereegranskat)abstract
- The uptake and catabolism of galactose by the yeast Saccharomyces cerevisiae is much lower than for glucose and fructose, and in applications of this yeast for utilization of complex substrates that contain galactose, for example, lignocellulose and raffinose, this causes prolonged fermentations. Galactose is metabolized via the Leloir pathway, and besides the industrial interest in improving the flux through this pathway it is also of medical relevance to study the Leloir pathway. Thus, genetic disorders in the genes encoding galactose-1-phosphate uridylyltransferase or galactokinase result in galactose toxicity both in patients with galactosemia and in yeast. In order to elucidate galactose related toxicity, which may explain the low uptake and catabolic rates of S. cerevisiae, we have studied the physiological characteristics and intracellular metabolite profiles of recombinant S. cerevisiae strains with improved or impaired growth on galactose. Aerobic batch cultivations on galactose of strains with different combiantions of overexpression of the genes GAL1, GAL2, GAL7, and GAL10, which encode proteins that together convert extracellular galactose into glucose-1-phosphate, revealed a decrease in the maximum specific growth rate when compared to the reference strain. The hypothesized toxic intermediate galactose-1-phosphate cannot be the sole cause of galactose related toxicity, but indications were found that galactose-1-phosphate might cause a negative effect through inhibition of phosphoglucomutase. Furtermore, we show that galactitol is formed in S. cerevisiae, and that the combination of elevated intracellular galactitol concentration, and the ratio between galactose-1-phosphate concentration and phosphoglucomutase activity seems to be important for galactose related toxicity causing decreased growth rates.
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| 3. |
- Grotkjær, Thomas, et al.
(författare)
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Comparative metabolic network analysis of two xylose fermenting recombinant Saccharomyces cerevisiae strains
- 2005
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Ingår i: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 7:5-6, s. 437-444
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Tidskriftsartikel (refereegranskat)abstract
- The recombinant xylose fermenting strain Saccharomyces cerevisiae TMB3001 can grow on xylose, but the xylose utilisation rate is low. One important reason for the inefficient fermentation of xylose to ethanol is believed to be the imbalance of redox co-factors. In the present study, a metabolic flux model was constructed for two recombinant S. cerevisiae strains: TMB3001 and CPB.CR4 which in addition to xylose metabolism have a modulated redox metabolism, i.e. ammonia assimilation was shifted from being NADPH to NADH dependent by deletion of gdh1 and over-expression of GDH2. The intracellular fluxes were estimated for both strains in anaerobic continuous cultivations when the growth limiting feed consisted of glucose (2.5 g L−1) and xylose (13 g L−1). The metabolic network analysis with 13C labelled glucose showed that there was a shift in the specific xylose reductase activity towards use of NADH as co-factor rather than NADPH. This shift is beneficial for solving the redox imbalance and it can therefore partly explain the 25% increase in the ethanol yield observed for CPB.CR4. Furthermore, the analysis indicated that the glyoxylate cycle was activated in CPB.CR4.
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| 4. |
- Mapelli, Valeria, 1978, et al.
(författare)
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Metabolic footprinting in microbiology: methods and applications in functional genomics and biotechnology
- 2008
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Ingår i: Trends in Biotechnology. ; 26:9, s. 490-497
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Tidskriftsartikel (refereegranskat)abstract
- Metabolomics embraces several strategies that aim to quantify cell metabolites in order to increase our understanding of how metabolite levels and interactions influence phenotypes. Metabolic footprinting represents a niche within metabolomics, because it focuses on the analysis of extracellular metabolites. Although metabolic footprinting represents only a fraction of the entire metabolome, it provides important information for functional genomics and strain characterization, and it can also provide scientists with a key understanding of cell communication mechanisms, metabolic engineering and industrial biotechnological processes. Due to the tight and convoluted relationship between intracellular metabolism and metabolic footprinting, metabolic footprinting can provide precious information about the intracellular metabolic status. Hereby, we state that integrative information from metabolic footprinting can assist in further interpretation of metabolic networks.
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| 5. |
- Meijer, S. L., et al.
(författare)
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Physiological characterisation of acuB deletion in Aspergillus niger
- 2009
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 84:1, s. 157-167
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Tidskriftsartikel (refereegranskat)abstract
- The acuB gene of Aspergillus niger is an ortholog of facB in Aspergillus nidulans. Under carbon-repression conditions, facB is repressed, thereby preventing acetate metabolism when the repressing carbon source is present. Even though facB is reported to be repressed directly by CreA, it is believed that a basal level of FacB activity exists under glucose-repressive conditions. In the present study, the effect of deletion of acuB on the physiology of A. niger was assessed. Differences in organic acid and acetate production, enzyme activities and extracellular amino and non-amino organic acid production were determined under glucose-repressing and -derepressing conditions. Furthermore, consumption of alternative carbon sources (e.g. xylose, citrate, lactate and succinate) was investigated. It was shown that AcuB has pleiotropic effects on the physiology of A. niger. The results indicate that metabolic pathways that are not directly involved in acetate metabolism are influenced by acuB deletion. Clear differences in organic acid consumption and production were detected between the a dagger acuB and reference strain. However, the hypothesis that AcuB is responsible for basal AcuA activity necessary for activation of acetate metabolic pathways, even during growth on glucose, could not be confirmed. The experiments demonstrated that also when acuB was deleted, no acetate was formed. Therefore, AcuB cannot be the only activator of AcuA, and another control mechanism has to be available for activating AcuA.
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| 6. |
- Meijer, S, et al.
(författare)
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Overexpression of isocitrate lyase-glyoxylate bypass influence on matabolism in Aspergillus niger
- 2009
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; :11, s. 107-116
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Tidskriftsartikel (refereegranskat)abstract
- In order to improve the production of succinate and malate by the filamentous fungus Aspergillus niger the activity of the glyoxylate bypass pathway was increased by over-expression of the isocitrate lyase (icl) gene. The hypothesis was that when isocitrate lyase was up-regulated the flux towards glyoxylate would increase, leading to excess formation of malate and succinate compared to the wild-type. However, metabolic network analysis showed that an increased icl expression did not result in an increased glyoxylate bypass flux. The analysis did show a global response with respect to gene expression, leading to an increased flux through the oxidative part of the TCA cycle. Instead of an increased production of succinate and malate, a major increase in fumarate production was observed. The effect of malonate, a competative inhibitor of succinate dehydrogenase (SDH), on the physiological behaviour of the cells was investigated. Inhibition of SDH was expected to lead to succinate production, but this was not observed. There was an increase in citrate and oxalate production in the wild-type strain. Furthermore, in the strain with over-expression of icl the organic acid production shifted from fumarate towards malate production when malonate was added to the cultivation medium. Overall, the icl over-expression and malonate addition had a significant impact on metabolism and on organic acid production profiles. Although the expected succiante and malate formation was not observed, a distinct and interesting production of fumarate and malate was found.
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| 7. |
- Panagiotou, Gianni, et al.
(författare)
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Intracellular metabolite profiling of Fusarium oxysporum converting glucose to ethanol
- 2005
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 115:4, s. 425-434
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Tidskriftsartikel (refereegranskat)abstract
- The filamentous fungus Fusarium oxysporum is known for its ability to produce ethanol by simultaneous saccharification and fermentation (SSF) of cellulose. However, the conversion rate is low and significant amounts of acetic acid are produced as a by-product. In this study, the growth characteristics of F. oxysporum were evaluated in a minimal medium using glucose as the sole carbon source in aerobic, anaerobic and oxygen-limited batch cultivations. Under aerobic conditions the maximum specific growth rate was found to be 0.043 h−1, and the highest ethanol yield (1.66 mol/mol) was found under anaerobic conditions. During the different phases of the cultivations, the intracellular profiles were determined under aerobic and anaerobic conditions. The profiles of the phosphorylated intermediates indicated that there was a high glycolytic flux at anaerobic growth conditions, characterized by high efflux of glyceraldehyde-3-phosphate (G3P) and fructose-6-phosphate (F6P) from the pentose phosphate pathway (PPP) to the Embden–Meyerhof–Parnas (EMP) pathway, resulting in the highest ethanol production under these conditions. The amino acid profile clearly suggests that the TCA cycle was primarily active under aerobic cultivation. On the other hand, the presence of high levels of γ-amino-n-butyric acid (GABA) under anaerobic conditions suggests a functional GABA bypass and a possible block in the TCA cycle at these conditions.
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| 8. |
- Panagiotou, Gianni, 1974, et al.
(författare)
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Studies of the Production of Fungal Polyketides in Aspergillus nidulans by Using Systems Biology Tools
- 2009
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Ingår i: Applied and Environmental Microbiology. - 1098-5336 .- 0099-2240. ; 75:7, s. 2212-2220
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Tidskriftsartikel (refereegranskat)abstract
- Many filamentous fungi produce polyketide molecules with great significance as human pharmaceuticals; these molecules include the cholesterol-lowering compound lovastatin, which was originally isolated from Aspergillus terreus. The chemical diversity and potential uses of these compounds are virtually unlimited, and it is thus of great interest to develop a well-described microbial production platform for polyketides. Using genetic engineering tools available for the model organism Aspergillus nidulans, we constructed two recombinant strains, one expressing the Penicillium grisefulvum 6-methylsalicylic acid (6-MSA) synthase gene and one expressing the 6-MSA syntase gene and overexpressing the native xylulose-5-phosphate phosphoketolase gene (xpkA) for increasing the pool of polyketide precursor levels. The physiology of the recombinant strains and that of a reference wild-type strain were characterized on glucose, xylose, glycerol, and ethanol media in controlled bioreactors. Glucose was found to be the preferred carbon source for 6-MSA production, and 6-MSA concentrations up to 455 mg/liter were obtained for the recombinant strain harboring the 6-MSA gene. Our findings indicate that overexpression of xpkA does not directly improve 6-MSA production on glucose, but it is possible, if the metabolic flux through the lower part of glycolysis is reduced, to obtain quite high yields for conversion of sugar to 6-MSA. Systems biology tools were employed for in-depth analysis of the metabolic processes. Transcriptome analysis of 6-MSA-producing strains grown on glucose and xylose in the presence and absence of xpkA overexpression, combined with flux and phsyiology data, enabled us to propose an xpkA-msaS interaction model describing the competition between biomass formation and 6-MSA production for the available acetyl coenzyme A.
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| 9. |
- Panagiotou, Gianni, 1974, et al.
(författare)
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Systems Analysis Unfolds the Relationship between the Phosphoketolase Pathway and Growth in Aspergillus nidulans
- 2008
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203 .- 1932-6203. ; 3:12
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Tidskriftsartikel (refereegranskat)abstract
- Background: Aspergillus nidulans is an important model organism for studies on fundamental eukaryotic cell biology and on industrial processes due to its close relation to A. niger and A. oryzae. Here we identified the gene coding for a novel metabolic pathway in A. nidulans, namely the phosphoketolase pathway, and investigated the role of an increased phosphoketolase activity. Methodology/Principal findings: Over-expression of the phosphoketolase gene (phk) improved the specific growth rate on xylose, glycerol and ethanol. Transcriptome analysis showed that a total of 1,222 genes were significantly affected by over-expression of the phk, while more than half of the affected genes were carbon source specific. During growth on glucose medium, the transcriptome analysis showed that the response to phk over-expression is targeted to neutralize the effect of the over-expression by regulating the acetate metabolism and initiate a growth dampening response. Conclusions/Significance: Metabolic flux analysis using 13C-labelled glucose, showed that over-expression of phosphoketolase added flexibility to the central metabolism. Our findings further suggests that A. nidulans is not optimized for growth on xylose, glycerol or ethanol as the sole carbon sources.
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| 10. |
- Piddocke, Maya P, et al.
(författare)
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Physiological characterization of brewer’s yeast in high-gravity beer fermentations with glucose or maltose syrups as adjuncts
- 2009
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 84:3, s. 453-464
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Tidskriftsartikel (refereegranskat)abstract
- High-gravity brewing, which can decrease production costs by increasing brewery yields, has become an attractive alternative to traditional brewing methods. However, as higher sugar concentration is required, the yeast is exposed to various stresses during fermentation. We evaluated the influence of high-gravity brewing on the fermentation performance of the brewer’s yeast under model brewing conditions. The lager brewer’s strain Weihenstephan 34/70 strain was characterized at three different gravities by adding either glucose or maltose syrups to the basic wort. We observed that increased gravity resulted in a lower specific growth rate, a longer lag phase before initiation of ethanol production, incomplete sugar utilization, and an increase in the concentrations of ethyl acetate and isoamyl acetate in the final beer. Increasing the gravity by adding maltose syrup as opposed to glucose syrup resulted in more balanced fermentation performance in terms of higher cell numbers, respectively, higher wort fermentability and a more favorable flavor profile of the final beer. Our study underlines the effects of the various stress factors on brewer’s yeast metabolism and the influence of the type of sugar syrups on the fermentation performance and the flavor profile of the final beer.
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