Cytosolic NADPH balancing in Penicillium chrysogenum cultivated on mixtures of glucose and ethanol

The in vivo flux through the oxidative branch of the pentose phosphate pathway (oxPPP) in Penicillium chrysogenum was determined during growth in glucose/ethanol carbon-limited chemostat cultures, at the same growth rate. Non-stationary 13C flux analysis was used to measure the oxPPP flux. A nearly constant oxPPP flux was found for all glucose/ethanol ratios studied. This indicates that the cytosolic NADPH supply is independent of the amount of assimilated ethanol. The cofactor assignment in the model of van Gulik et al. (Biotechnol Bioeng 68(6):602–618, 2000) was supported using the published genome annotation of P. chrysogenum. Metabolic flux analysis showed that NADPH requirements in the cytosol remain nearly the same in these experiments due to constant biomass growth. Based on the cytosolic NADPH balance, it is known that the cytosolic aldehyde dehydrogenase in P. chrysogenum is NAD +  dependent. Metabolic modeling shows that changing the NAD + -aldehyde dehydrogenase to NADP + -aldehyde dehydrogenase can increase the penicillin yield on substrate

13C labeling experiments at metabolic nonstationary conditions: An exploratory study

<p>Abstract</p> <p>Background</p> <p>Stimulus Response Experiments to unravel the regulatory properties of metabolic networks are becoming more and more popular. However, their ability to determine enzyme kinetic parameters has proven to be limited with the presently available data. In metabolic flux analysis, the use of ¹³C labeled substrates together with isotopomer modeling solved the problem of underdetermined networks and increased the accuracy of flux estimations significantly.</p> <p>Results</p> <p>In this contribution, the idea of increasing the information content of the dynamic experiment by adding ¹³C labeling is analyzed. For this purpose a small example network is studied by simulation and statistical methods. Different scenarios regarding available measurements are analyzed and compared to a non-labeled reference experiment. Sensitivity analysis revealed a specific influence of the kinetic parameters on the labeling measurements. Statistical methods based on parameter sensitivities and different measurement models are applied to assess the information gain of the labeled stimulus response experiment.</p> <p>Conclusion</p> <p>It was found that the use of a (specifically) labeled substrate will significantly increase the parameter estimation accuracy. An overall information gain of about a factor of six is observed for the example network. The information gain is achieved from the specific influence of the kinetic parameters towards the labeling measurements. This also leads to a significant decrease in correlation of the kinetic parameters compared to an experiment without ¹³C-labeled substrate.</p

Impact on bird fauna of a non-native oyster expanding into blue mussel beds in the Dutch Wadden Sea

Intertidal mussel beds are important for intertidal ecosystems, because they feature a high taxonomic diversity and abundance of benthic organisms and are important foraging grounds for many avian species. After the introduction of the Pacific oyster (Crassostrea gigas) into the European Wadden Sea, many mussel beds developed into oyster dominated bivalve beds. Despite the fact that oysters have been colonizing many European intertidal areas for about two decades, their impact on the ecosystem is still poorly understood. Here, we investigated the impact of oysters on the condition of mussels and on the spatial distribution of birds on 18 bivalve beds with different grades of oyster occurrence throughout the Dutch Wadden Sea. Moreover, in comparing bird densities on bivalve beds with densities expected on the total intertidal area, we could detect which species exhibit a preference for the structured habitat. Overall, 50 different bird species were observed on the beds, of which about half regularly frequent intertidal flats. Most of these species showed a preference for bivalve beds. The condition of mussels decreased with the oyster dominance, whereas the majority of bird species was not affected by the oyster occurrence. However, three of the four species that were negatively affected depend on intertidal mussels as food source. Even though the Pacific oyster is a nonnative species, attempts to fight it may do more harm to avian biodiversity than good

Correction of MS data for naturally occurring isotopes in isotope labelling experiments

Mass spectrometry (MS) in combination with isotope labelling experiments is widely used for investigations of metabolism and other biological processes. Quantitative applications-e.g., (13)C metabolic flux analysis-require correction of raw MS data (isotopic clusters) for the contribution of all naturally abundant isotopes. This chapter describes how to perform such correction using the software IsoCor. This flexible, user-friendly software can be used to exploit any isotopic tracer, from well-known ((13)C, (15)N, (18)O, etc.) to unusual ((57)Fe, (77)Se, etc.) isotopes. It also provides options-e.g., correction for the isotopic purity of the tracer-to improve the accuracy of quantitative isotopic studies, and allows automated correction of large datasets that can be collected with modern MS methods