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- Anasontzis, George E, 1980, et al.
(författare)
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Constitutive homologous expression of phosphoglucomutase and transaldolase increases the metabolic flux of Fusarium oxysporum
- 2014
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Fusarium oxysporum is among the few filamentous fungi that have been reported of being able to directly ferment biomass to ethanol in a consolidated bioprocess. Understanding its metabolic pathways and their limitations can provide some insights on the genetic modifications required to enhance its growth and subsequent fermentation capability. In this study, we investigated the hypothesis reported previously that phosphoglucomutase and transaldolase are metabolic bottlenecks in the glycolysis and pentose phosphate pathway of the F. oxysporum metabolism.Results: Both enzymes were homologously overexpressed in F. oxysporum F3 using the gpdA promoter of Aspergillus nidulans for constitutive expression. Transformants were screened for their phosphoglucomutase and transaldolase genes expression levels with northern blot. The selected transformant exhibited high mRNA levels for both genes, as well as higher specific activities of the corresponding enzymes, compared to the wild type. It also displayed more than 20 and 15% higher specific growth rate upon aerobic growth on glucose and xylose, respectively, as carbon sources and 30% higher biomass to xylose yield. The determination of the relative intracellular amino and non-amino organic acid concentrations at the end of growth on glucose revealed higher abundance of most determined metabolites between 1.5- and 3-times in the recombinant strain compared to the wild type. Lower abundance of the determined metabolites of the Krebs cycle and an 68-fold more glutamate were observed at the end of the cultivation, when xylose was used as carbon source.Conclusions: Homologous overexpression of phosphoglucomutase and transaldolase in F. oxysporum was shown to enhance the growth characteristics of the strain in both xylose and glucose in aerobic conditions. The intracellular metabolites profile indicated how the changes in the metabolome could have resulted in the observed growth characteristics. © 2014 Anasontzis et al.; licensee BioMed Central Ltd.
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| 2. |
- Anasontzis, George E, 1980, et al.
(författare)
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Challenges in ethanol production with fusarium oxysporum through consolidated bioprocessing
- 2014
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Ingår i: Bioengineered Bugs. - : Informa UK Limited. - 1949-1018 .- 1949-1026 .- 2165-5979 .- 2165-5987. ; 5:6, s. 393-395
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Tidskriftsartikel (refereegranskat)abstract
- Fusarium oxysporum has been reported as being able to both produce the enzymes necessary to degrade lignocellulosic biomass to sugars and also ferment the monosaccharides to ethanol under anaerobic or microaerobic conditions. However, in order to become an economically feasible alternative to other ethanol-producing microorganisms, a better understanding of its physiology, metabolic pathways, and bottlenecks is required, together with an improvement in its efficiency and robustness. In this report, we describe the challenges for the future and give additional justification for our recent publication.
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| 3. |
- Anasontzis, George E, 1980, et al.
(författare)
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Effects of temperature and glycerol and methanol-feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris
- 2014
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Ingår i: Biotechnology Progress. - : Wiley. - 1520-6033 .- 8756-7938. ; 30:3, s. 728-735
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Tidskriftsartikel (refereegranskat)abstract
- Optimization of protein production from methanol-induced Pichia pastoris cultures is necessary to ensure high productivity rates and high yields of recombinant proteins. We investigated the effects of temperature and different linear or exponential methanol-feeding rates on the production of recombinant Fusarium graminearum galactose oxidase (EC 1.1.3.9) in a P. pastoris Mut+ strain, under regulation of the AOX1 promoter. We found that low exponential methanol feeding led to 1.5-fold higher volumetric productivity compared to high exponential feeding rates. The duration of glycerol feeding did not affect the subsequent product yield, but longer glycerol feeding led to higher initial biomass concentration, which would reduce the oxygen demand and generate less heat during induction. A linear and a low exponential feeding profile led to productivities in the same range, but the latter was characterized by intense fluctuations in the titers of galactose oxidase and total protein. An exponential feeding profile that has been adapted to the apparent biomass concentration results in more stable cultures, but the concentration of recombinant protein is in the same range as when constant methanol feeding is employed. (c) 2014 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 30:728-735, 2014
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| 6. |
- Bonzom, Cyrielle, 1987, et al.
(författare)
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Enzyme production and immobilization in mesoporous materials
- 2014
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Enzymes display high reactivity and selectivity under normal conditions, but may suffer from denaturation in industrial applications. A strategy to solve this limitation is to immobilize enzymes [1]. Mesoporous silica materials (MPS) have become a common choice as support to immobilized enzymes. MPS offer unique properties such as high enzyme loading and tunable pore size [2].Feruloyl esterase (FAE) is a subclass (EC. 3.1.1.73) of carboxylic ester hydrolases. They catalyze the hydrolysis of ester linkages in plant cell walls materials releasing ferulic acid and other hydroxycinnamic acids [3]. They are also examples of FAEs used for esterification and transesterification [4].From the genomes of Aspergillus glacus and Aspergillus oryzae, some putative FAE were identified. Among them, five were selected for further investigation in order to find a suitable enzyme for catalyzing the reaction of interest. The selected genes were quite distant in an evolutionary tree.The five putative FAEs were cloned into Pichia pastoris and produced by fed-batch fermentation. They were then purified either by IMAC columns or by ion-exchange chromatography. Their activity was assessed against a range of substrate to screen for FAE, tannase and other esterase activities. When the type of the respective enzyme activity was determined, some of them were further characterized. Five new enzymes were recombinantly produced and purified. Their activity type was determined and some of them were immobilized.Enzymes produced in sufficient quantities and having a good free activity were further investigated by immobilization. The selected support for immobilization was mesoporous silica particles (MPS). The conditions of immobilization were investigated and the activity once immobilized was tested and compared to the free one to gain insights on what happens during the immobilization of enzymes. Results were compared to those obtained with a commercially available FAE (E-FAERU, Megazyme).References.[1] Hudson S.; Cooney J.; Magner E., Angew. Chem. Int. Ed. 2008, 47, 8582-8594. [2] Carlsson N.; Gustafsson H.; Thörn C.; Olsson L.; Holmberg K.; Åkerman B. Advances in Colloid and Interface Science 2014, 204, 339-360.[3] Topakas E.; Vafiadi C.; Christakopoulos P. Process Biochemistry 2007, 42, 497-509.[4] Thörn C.; Gustafsson H.; Olsson L. Journal of molecular Catalysis B: Enzymatic 2011, 72, 57-64
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| 8. |
- Peciulyte, Ausra, 1986, et al.
(författare)
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Morphology and enzyme production of Trichoderma reesei Rut C-30 are affected by the physical and structural characteristics of cellulosic substrates
- 2014
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Ingår i: Fungal Genetics and Biology. - : Elsevier BV. - 1087-1845 .- 1096-0937. ; 72, s. 64-72
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Tidskriftsartikel (refereegranskat)abstract
- The industrial production of cellulolytic enzymes is dominated by the filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina). In order to develop optimal enzymatic cocktail, it is of importance to understand the natural regulation of the enzyme profile as response to the growth substrate. The influence of the complexity of cellulose on enzyme production by the microorganisms is not understood. In the present study we attempted to understand how different physical and structural properties of cellulose-rich substrates affected the levels and profiles of extracellular enzymes produced by T. reesei. Enzyme production by T. reesei Rut C-30 was studied in submerged cultures on five different cellulose-rich substrates, namely, commercial cellulose Avicel (R) and industrial-like cellulosic pulp substrates which consist mainly of cellulose, but also contain residual hemicellulose and lignin. In order to evaluate the hydrolysis of the substrates by the fungal enzymes, the spatial polymer distributions were characterised by cross-polarisation magic angle spinning carbon-13 nuclear magnetic resonance (CP/MAS C-13-NMR) in combination with spectral fitting. Proteins in culture supernatants at early and late stages of enzyme production were labeled by Tandem Mass Tags (TMT) and protein profiles were analysed by liquid chromatography-tandem mass spectrometry. The data have been deposited to the ProteomeXchange with identifier PXD001304. In total 124 proteins were identified and quantified in the culture supernatants, including cellulases, hemicellulases, other glycoside hydrolases, lignin-degrading enzymes, auxiliary activity 9 (AA9) family (formerly GH61), supporting activities of proteins and enzymes acting on cellulose, proteases, intracellular proteins and several hypothetical proteins. Surprisingly, substantial differences in the enzyme profiles were found even though there were minor differences in the chemical composition between the cellulose-rich substrates.
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