| 1. |
- Asadollahi, M. A., et al.
(författare)
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Enhancing sesquiterpene production in Saccharomyces cerevisiae through in silico driven metabolic engineering
- 2009
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 11:6, s. 328-334
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Tidskriftsartikel (refereegranskat)abstract
- A genome-scale metabolic model was used to identify new target genes for enhanced biosynthesis of sesquiterpenes in the yeast Saccharomyces cerevisiae. The effect of gene deletions on the flux distributions in the metabolic model of S. cerevisiae was assessed using Opt Gene as the modeling framework and minimization of metabolic adjustments (MOMA) as objective function. Deletion of NADPH-dependent glutamate dehydrogenase encoded by GDH1 was identified as the best target gene for the improvement of sesquiterpene biosynthesis in yeast. Deletion of this gene enhances the available NADPH in the cytosol for other NADPH requiring enzymes, including HMG-CoA reductase. However, since disruption of GDH1 impairs the ammonia utilization, simultaneous over-expression of the NADH-dependent glutamate dehydrogenase en coded by GDH2 was also considered in this study. Deletion of GDH1 led to an approximately 85% increase in the final cubebol titer. However, deletion of this gene also caused a significant decrease in the maximum specific growth rate. Over-expression of GDH2 did not show a further effect on the final cubebol titer but this alteration significantly improved the growth rate compared to the GDH1 deleted strain. (C) 2009 Elsevier Inc. All rights reserved.
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| 2. |
- 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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| 3. |
- Headman Van Vleet, Jennifer, et al.
(författare)
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Deleting the para-nitrophenyl phosphatase (pNPPase), PHO13, in recombinant Saccharomyces cerevisiae improves growth and ethanol production on D-xylose
- 2008
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Ingår i: Metabolic Engineering. - 1096-7176 .- 1096-7184. ; :10, s. 360-369
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Tidskriftsartikel (refereegranskat)abstract
- Overexpression of D-xylulokinase in Saccharomyces cerevisiae engineered for assimilation of xylose results in growth inhibition that is more pronounced at higher xylose concentrations. Mutants deficient in the para-nitrophenyl phosphatase, PHO13, resist growth inhibition on xylose. We studied this inhibition under aerobic growth conditions in well-controlled bioreactors using engineered S. cerevisiae CEN.PK. Growth on glucose was not significantly affected in pho13∆ mutants, but acetate production increased by 75%. Cell growth, ethanol production, and xylose consumption all increased markedly in pho13∆ mutants. The specific growth rate and rate of specific xylose uptake were approximately 1.5 times higher in the deletion strain than in the parental strain when growing on glucose-xylose mixtures and up to 10-fold higher when growing on xylose alone. In addition to showing higher acetate levels, pho13∆ mutants also produced less glycerol on xylose, suggesting that deletion of Pho13p could improve growth by altering redox levels when cells are grown on xylose.
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| 4. |
- Hofmann, G., et al.
(författare)
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Recombinant bacterial hemoglobin alters metabolism of Aspergillus niger
- 2009
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 11:1, s. 8-12
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Tidskriftsartikel (refereegranskat)abstract
- The filamentous fungus Aspergillus niger is used extensively for the production of enzymes and organic acids. A major problem in industrial fermentations with this fungus is to ensure sufficient supply of oxygen required for respiratory metabolism of the fungus. In case of oxygen limitation, the fungus will produce various by-products like organic acids and polyols. In order to circumvent this problem we here study the effects of the expression of a bacterial hemoglobin protein on the metabolism of A. niger. We integrated the vgb gene from Vitreoscilla sp. into the genome at the pyrA locus behind the strong gpdA promoter from Aspergillus nidulans. Analysis of secreted metabolites, oxygen uptake, CO2 evolution and biomass formation points towards a relief of stress in the mutant expressing VHB when it is exposed to oxygen limitation. Our findings therefore point to an interesting strategy to attenuate unwanted side effects resulting from oxygen limitation during industrial fermentations with A. niger. (C) 2008 Elsevier Inc. All rights reserved.
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| 5. |
- Hou, J., et al.
(författare)
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Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae
- 2009
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 11:4-5, s. 253-261
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Tidskriftsartikel (refereegranskat)abstract
- Redox cofactors play a pivotal role in coupling catabolism with anabolism and energy generation during metabolism. There exists a delicate balance in the intracellular level of these cofactors to ascertain an optimal metabolic output. Therefore, cofactors are emerging to be attractive targets to induce widespread changes in metabolism. We present a detailed analysis of the impact of perturbations in redox cofactors in the cytosol or mitochondria on glucose and energy metabolism in Saccharomyces cerevisiae to aid metabolic engineering decisions that involve cofactor engineering. We enhanced NADH oxidation by introducing NADH oxidase or alternative oxidase, its ATP-mediated conversion to NADPH using NADH kinase as well as the interconversion of NADH and NADPH independent of ATP by the soluble, non-proton-translocating bacterial transhydrogenase. Decreasing cytosolic NADH level lowered glycerol production, while decreasing mitochondrial NADH lowered ethanol production. However, when these reactions were coupled with NADPH production, the metabolic changes were more moderated. The direct consequence of these perturbations could be seen in the shift of the intracellular concentrations of the cofactors. The changes in product profile and intracellular metabolite levels were closely linked to the ATP requirement for biomass synthesis and the efficiency of oxidative phosphorylation, as estimated from a simple stoichiometric model. The results presented here will provide valuable insights for a quantitative understanding and prediction of cellular response to redox-based perturbations for metabolic engineering applications. (C) 2009 Elsevier Inc. All rights reserved.
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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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Engineering of the redox imbalance of Fusarium oxysporum enables anaerobic growth on xylose
- 2006
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Ingår i: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 8:5, s. 474-482
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Tidskriftsartikel (refereegranskat)abstract
- Dissimilatory nitrate reduction metabolism, of the natural xylose-fermenting fungus Fusarium oxysporum, was used as a strategy to achieve anaerobic growth and ethanol production from xylose. Beneficial alterations of the redox fluxes and thereby of the xylose metabolism were obtained by taking advantage of the regeneration of the cofactor NAD+ during the denitrification process. In batch cultivations, nitrate sustained growth under anaerobic conditions (1.21 g L−1 biomass) and simultaneously a maximum yield of 0.55 moles of ethanol per mole of xylose was achieved, whereas substitution of nitrate with ammonium limited the growth significantly (0.15 g L−1 biomass). Using nitrate, the maximum acetate yield was 0.21 moles per mole of xylose and no xylitol excretion was observed. Furthermore, the network structure in the central carbon metabolism of F. oxysporum was characterized in steady state. F. oxysporum grew anaerobically on [1-13C] labelled glucose and unlabelled xylose in chemostat cultivation with nitrate as nitrogen source. The use of labelled substrate allowed the precise determination of the glucose and xylose contribution to the carbon fluxes in the central metabolism of this poorly described microorganism. It was demonstrated that dissimilatory nitrate reduction allows F. oxysporum to exhibit typical respiratory metabolic behaviour with a highly active TCA cycle and a large demand for NADPH.
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| 8. |
- 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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| 9. |
- Siewers, Verena, 1976, et al.
(författare)
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Heterologous production of non-ribosomal peptide LLD-ACV in Saccharomyces cerevisiae
- 2009
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 11:6, s. 391-397
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Tidskriftsartikel (refereegranskat)abstract
- Non-ribosomal peptides (NRPs) are a diverse family of secondary metabolites with a broad range of biological activities. We started to develop an eukaryotic microbial platform based on the yeast Saccharomyces cerevisiae for heterologous production of NRPs using delta-(L-alpha-aminoadipyl)-L-cysteinyl-D- valine (ACV) as a model NRP. The Penicillium chrysogenum gene pcbAB encoding ACV synthetase was expressed in S. cerevisiae from a high-copy plasmid together with phosphopantetheinyl transferase (PPTase) encoding genes from Aspergillus nidulans, P. chrysogenum and Bacillus subtilis, and in all the three cases production of ACV was observed. To improve ACV synthesis, several factors were investigated. Codon optimization of the 50 end of pcbAB did not significantly increase ACV production. However, a 30-fold enhancement was achieved by lowering the cultivation temperature from 30 to 20 degrees C. When ACVS and PPT a seen coding genes were integrated into the yeast genome, a 6-fold decrease in ACV production was observed indicating that gene copy number was one of the rate-limiting factors for ACV production in yeast. (C) 2009 Elsevier Inc. All rights reserved.
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