Application of blind source separation to real-time dissolution dynamic nuclear polarization.

The use of a blind source separation (BSS) algorithm is demonstrated for the analysis of time series of nuclear magnetic resonance (NMR) spectra. This type of data is obtained commonly from experiments, where analytes are hyperpolarized using dissolution dynamic nuclear polarization (D-DNP), both in in vivo and in vitro contexts. High signal gains in D-DNP enable rapid measurement of data sets characterizing the time evolution of chemical or metabolic processes. BSS is based on an algorithm that can be applied to separate the different components contributing to the NMR signal and determine the time dependence of the signals from these components. This algorithm requires minimal prior knowledge of the data, notably, no reference spectra need to be provided, and can therefore be applied rapidly. In a time-resolved measurement of the enzymatic conversion of hyperpolarized oxaloacetate to malate, the two signal components are separated into computed source spectra that closely resemble the spectra of the individual compounds. An improvement in the signal-to-noise ratio of the computed source spectra is found compared to the original spectra, presumably resulting from the presence of each signal more than once in the time series. The reconstruction of the original spectra yields the time evolution of the contributions from the two sources, which also corresponds closely to the time evolution of integrated signal intensities from the original spectra. BSS may therefore be an approach for the efficient identification of components and estimation of kinetics in D-DNP experiments, which can be applied at a high level of automation.

Metabolic changes in a pyruvate kinase gene deletion mutant of Corynebacterium glutamicum ATCC 13032.

To investigate primary effects of a pyruvate kinase (PYK) defect on glucose metabolism in Corynebacterium glutamicum, a pyk-deleted mutant was derived from wild-type C. glutamicum ATCC13032 using the double-crossover chromosome replacement technique. The mutant was then evaluated under glutamic acid-producing conditions induced by biotin limitation. The mutant showed an increased specific rate of glucose consumption, decreased growth, higher glutamic acid production, and aspartic acid formation during the glutamic acid production phase. A significant increase in phosphoenolpyruvate (PEP) carboxylase activity and a significant decrease in PEP carboxykinase activity occurred in the mutant, which suggested an enhanced overall flux of the anaplerotic pathway from PEP to oxaloacetic acid in the mutant. The enhanced anaplerotic flux may explain both the increased rate of glucose consumption and the higher productivity of glutamic acid in the mutant. Since the pyk-complemented strain had similar metabolic profiles to the wild-type strain, the observed changes represented intrinsic effects of pyk deletion on the physiology of C. glutamicum.

Metabolic characterization of Escherichia coli strains adapted to growth on lactate.

In comparison with intensive studies of genetic mechanisms related to biological evolutionary systems, much less analysis has been conducted on metabolic network responses to adaptive evolution that are directly associated with evolved metabolic phenotypes. Metabolic mechanisms involved in laboratory evolution of Escherichia coli on gluconeogenic carbon sources, such as lactate, were studied based on intracellular flux states determined from 13C tracer experiments and 13C-constrained flux analysis. At the end point of laboratory evolution, strains exhibited a more than doubling of the average growth rate and a 50% increase in the average biomass yield. Despite different evolutionary trajectories among parallel evolved populations, most improvements were obtained within the first 250 generations of evolution and were generally characterized by a significant increase in pathway capacity. Partitioning between gluconeogenic and pyruvate catabolic flux at the pyruvate node remained almost unchanged, while flux distributions around the key metabolites phosphoenolpyruvate, oxaloacetate, and acetyl-coenzyme A were relatively flexible over the course of evolution on lactate to meet energetic and anabolic demands during rapid growth on this gluconeogenic carbon substrate. There were no clear qualitative correlations between most transcriptional expression and metabolic flux changes, suggesting complex regulatory mechanisms at multiple levels of genetics and molecular biology. Moreover, higher fitness gains for cell growth on both evolutionary and alternative carbon sources were found for strains that adaptively evolved on gluconeogenic carbon sources compared to those that evolved on glucose. These results provide a novel systematic view of the mechanisms underlying microbial adaptation to growth on a gluconeogenic substrate.

Taenia crassiceps organic acids detected in cysticerci.

Taenia crassiceps cysticerci is used as an experimental model to cysticercosis studies; however there are subcutaneous cases of cysticercosis caused by these cysticerci. It remains unclear in the literature the energetic and fatty acid metabolism in cestodes. Its metabolic study may provide knowledge of pathways that may serve as potential anti-helminthic drugs sites of action. In this work we studied the citric acid cycle organic acids and the fatty acid oxidation in cysticerci removed from mice with 21 and 42 days of infection in two different evolutive stages: growing and final. The organic acids were extracted using perchloric acid and analyzed by HPLC methodology. We found significant statistically differences in oxalate, malate, lactate, and beta-hydroxybutirate concentrations between cysticerci. These results indicate the aerobic metabolism in vivo in spite of the low oxygen concentration of its habitat, and also indicate the presence of fatty acid oxidation as an alternative energetic source.

Metabolic consequences of altered phosphoenolpyruvate carboxykinase activity in Corynebacterium glutamicum reveal anaplerotic regulation mechanisms in vivo.

Corynebacterium glutamicum possesses high in vivo activity of the gluconeogenic phosphoenolpyruvate carboxykinase (PEPCk) during growth on glucose, resulting together with anaplerotic carboxylation reactions in a PEP/pyruvate/oxaloacetate substrate cycle. The present study investigated the changes in intracellular fluxes and metabolite concentrations that are caused by altered PEPCk activity in L-lysine-producing C. glutamicum MH20-22B, applying a recently developed (13)C labeling-based strategy for anaplerotic flux resolution and quantification. Abolition of PEPCk activity by deletion of the respective pck gene resulted in increased intracellular concentrations of oxaloacetate L-aspartate, alpha-ketoglutarate, pyruvate, and L-lysine and in a 60% enhanced flux toward L-lysine biosynthesis, whereas increasing the PEPCk activity by pck overexpression had opposite effects. The results of the combined measurements of enzyme activities, in vivo fluxes, and metabolite concentrations were exploited to elucidate the in vivo regulation of anaplerotic reactions in C. glutamicum, and implications for the metabolic engineering of amino-acid-producing strains are discussed.