BackgroundSeveral studies have shown that the utilization of mixed carbon feeds instead of methanol as sole carbon source is beneficial for protein production with the methylotrophic yeast Pichia pastoris. In particular, growth under mixed feed conditions appears to alleviate the metabolic burden related to stress responses triggered by protein overproduction and secretion. Yet, detailed analysis of the metabolome and fluxome under mixed carbon source metabolizing conditions are missing. To obtain a detailed flux distribution of central carbon metabolism, including the pentose phosphate pathway under methanol-glucose conditions, we have applied metabolomics and instationary 13C flux analysis in chemostat cultivations.ResultsInstationary 13C-based metabolic flux analysis using GC-MS and LC-MS measurements in time allowed for an accurate mapping of metabolic fluxes of glycolysis, pentose phosphate and methanol assimilation pathways. Compared to previous results from NMR-derived stationary state labelling data (proteinogenic amino acids, METAFoR) more fluxes could be determined with higher accuracy. Furthermore, using a thermodynamic metabolic network analysis the metabolite measurements and metabolic flux directions were validated. Notably, the concentration of several metabolites of the upper glycolysis and pentose phosphate pathway increased under glucose-methanol feeding compared to the reference glucose conditions, indicating a shift in the thermodynamic driving forces. Conversely, the extracellular concentrations of all measured metabolites were lower compared with the corresponding exometabolome of glucose-grown P. pastoris cells.The instationary 13C flux analysis resulted in fluxes comparable to previously obtained from NMR datasets of proteinogenic amino acids, but allowed several additional insights. Specifically, i) in vivo metabolic flux estimations were expanded to a larger metabolic network e.g. by including trehalose recycling, which accounted for about 1.5% of the glucose uptake rate; ii) the reversibility of glycolytic/gluconeogenesis, TCA cycle and pentose phosphate pathways reactions was estimated, revealing a significant gluconeogenic flux from the dihydroxyacetone phosphate/glyceraldehydes phosphate pool to glucose-6P. The origin of this finding could be carbon recycling from the methanol assimilatory pathway to the pentose phosphate pool. Additionally, high exchange fluxes of oxaloacetate with aspartate as well as malate indicated amino acid pool buffering and the activity of the malate/Asp shuttle; iii) the ratio of methanol oxidation vs utilization appeared to be lower (54 vs 79% assimilated methanol directly oxidized to CO2).ConclusionsIn summary, the application of instationary 13C-based metabolic flux analysis to P. pastoris provides an experimental framework with improved capabilities to explore the regulation of the carbon and energy metabolism of this yeast, particularly for the case of methanol and multicarbon source metabolism.

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Nowadays, there is a great interest in the industry for outstanding magnetic materials with low-loss properties. Moreover, with the introduction of novel Wide Bandgap devices, higher frequency operation has been demanded from these novel magnetic materials. Thereby, magnetic characterization for obtaining the iron loss behavior is essential. However, when a magnetic material is experimentally characterized, it is important to select a suitable measurement device, especially when a semiconductor inverter is powering the magnetic material and it is driven at a high switching frequency. Usually measurement devices have a good response in magnitude, but there is not a clear understanding about how their phase response at high frequencies measurements affects the characterization. Consequently, if the phase characteristics of a measurement device is not well-know, it is likely that the iron loss characterization will not be accurate. This paper studies the measurement issues when an iron loss characterization procedure is conducted. This study is carried out with the comparison of using clamp type current probes and high precision shunt resistors at high switching frequency excitations with a GANFET inverter. In addition, an experimental evaluation of iron losses at several switching frequencies is presented. As a result, the phase characteristics of several sensors was obtained, and it was possible to identify the inaccuracies that they can generate at a high frequency measurement.