Including metabolite concentrations into flux balance analysis: thermodynamic realizability as a constraint on flux distributions in metabolic networks

Background  

In recent years, constrained optimization – usually referred to as flux balance analysis (FBA) – has become a widely applied method for the computation of stationary fluxes in large-scale metabolic networks. The striking advantage of FBA as compared to kinetic modeling is that it basically requires only knowledge of the stoichiometry of the network. On the other hand, results of FBA are to a large degree hypothetical because the method relies on plausible but hardly provable optimality principles that are thought to govern metabolic flux distributions.     

Experimental determination of flux control distribution in biochemical systems: in vitro model to analyze transient metabolite concentrations

Determination of the control coefficients allows the identification of rate-controlling steps in a reaction system. However, the measurement of the flux control coefficients in a biochemical system is not a trivial task, except for some special cases. We have developed a theoretical basis for the direct determination of these coefficients from dynamic responses. In order to show the validity of this methodology experimentally, the dynamic approach is applied to an in vitro reconstituted partial glycolytic pathway to determine the flux control coefficients of hexokinase and phosphofructokinase. It is shown that the dynamic approach gives consistent results, which agree well with values obtained by the direct enzyme titration method. The detailed procedure and potential applications to other systems, such as immobilized enzyme or cell reactors, are discussed. (c) 1993 Wiley & Sons, Inc.     

Metabolic control analysis of anaerobic glycolysis in human hibernating myocardium replaces traditional concepts of flux control

Myocardial hibernation represents an adaptation to sustained ischemia to maintain tissue vitality during severe supply-demand imbalance which is characterized by an increased glucose uptake. To elucidate this adaptive protective mechanism, the regulation of anaerobic glycolysis was investigated using human biopsies. In hibernating myocardium showing an increase in anaerobic glycolytic flux metabolizing exogenous glucose, the adjustment of flux through this pathway was analyzed by flux:metabolite co-responses. By this means, a previously unknown pattern of regulation using multisite modulation was found which largely differs from traditional concepts of metabolic control of the Embden-Meyerhof pathway in normal and diseased myocardium.     

An elementary metabolite unit (EMU) based method of isotopically nonstationary flux analysis

Nonstationary metabolic flux analysis (NMFA) is at present a very computationally intensive exercise, especially for large reaction networks. We applied elementary metabolite unit (EMU) theory to NMFA, dramatically reducing computational difficulty. We also introduced block decoupling, a new method that systematically and comprehensively divides EMU systems of equations into smaller subproblems to further reduce computational difficulty. These improvements led to a 5000-fold reduction in simulation times, enabling an entirely new and more complicated set of problems to be analyzed with NMFA. We simulated a series of nonstationary and stationary GC/MS measurements for a large E. coli network that was then used to estimate parameters and their associated confidence intervals. We found that fluxes could be successfully estimated using only nonstationary labeling data and external flux measurements. Addition of near-stationary and stationary time points increased the precision of most parameters. Contrary to prior reports, the precision of nonstationary estimates proved to be comparable to the precision of estimates based solely on stationary data. Finally, we applied EMU-based NMFA to experimental nonstationary measurements taken from brown adipocytes and successfully estimated fluxes and some metabolite concentrations. By using NFMA instead of traditional MFA, the experiment required only 6 h instead of 50 (the time necessary for most metabolite labeling to reach 99% of isotopic steady state).     

Methanethiol degradation in anaerobic bioreactors at elevated pH (8): reactor performance and microbial community analysis

The degradation of methanethiol (MT) at 30 degrees C under saline-alkaline (pH 8-10, 0.5M Na(+)) conditions was studied in a lab-scale Upflow Anaerobic Sludge Blanket (UASB) reactor inoculated with estuarine sediment from the Wadden Sea (The Netherlands). At a sodium concentration of 0.5M and a pH between 8 and 9 complete MT degradation to sulfide, methane and carbon dioxide was possible at a maximum loading rate of 22mmolMTL(-1)day(-1) and a hydraulic retention time of 6h. The presence of yeast extract (100mg/L) in the medium was essential for complete MT degradation. 16S rRNA based DGGE and sequence analysis revealed that species related to the genera Methanolobus and Methanosarcina dominated the archaeal community in the reactor sludge. Their relative abundance fluctuated in time, possibly as a result of the changing operational conditions in the reactor. The most dominant MT-degrading archaeon was enriched from the reactor and obtained in pure culture. This strain WR1, which was most closely related to Methanolobus taylorii, degraded MT, dimethyl sulfide (DMS), methanol and trimethylamine. Its optimal growth conditions were 0.2M NaCl, 30 degrees C and pH 8.4. In batch and reactor experiments operated at pH 10, MT was not degraded.     

Intracellular flux analysis in hybridomas using mass balances and in vitro (13)C nmr

Intracellular fluxes are important in defining cellular physiology and its changes in response to environmental variations. Stoichiometric balances combined with extra cellular metabolite measurements were applied to the estimation of intracellular fluxes and the study of energy metabolism in the hybridoma cell line ATCC CRL 1606. Redundant measurements allowed the evaluation of the consistency of the stoichiometry, measurements, and pseudo-steady-state assumption leading to refinement of the assumed biochemistry and identification of measurement errors. To validate the flux estimates, two batch experiments were performed with glucose labeled in the 1 position with (13)C. The distribution of (13)C in secreted lactate was measured via nuclear magnetic resonance spectroscopy (NMR) and compared to that predicted from the estimated intracellular fluxes. There was good agreement between the measured and estimated isotope distributions, demonstrating the validity of the flux estimates obtained from stoichiometric balances. (c) 1995 John Wiley & Sons, Inc.     

Thermal degradation processes of end-capped poly(L-lactide)s in the presence and absence of residual zinc catalyst

Thermal degradation processes of end-capped poly(L-lactide)s (PLLAs) were investigated by means of several thermoanalytical techniques under both isothermal and nonisothermal conditions. Based on the thermogravimetric analysis, it was found that two distinct processes at temperatures below and above 330 degrees C were existed in the nonisothermal degradation for PLLA samples depending on the amounts of residual zinc compounds from synthesis process. Isothermal degradation experiments at different temperature regions showed that the sample weight of PLLA decreased linearly with time in both cases, whereas the changes in molecular weight revealed different tendency for the temperature. On the basis of characterization of the residual PLLA molecules after isothermal degradation at 330 degrees C, it was confirmed that the omega chain end of the residual molecules was an acrylic ester unit. Majority of volatile products during thermal degradation of PLLA was the lactide regardless of the degradation temperature. From these results, it is concluded that, during the thermal degradation of PLLA samples in the presence of large amounts of residual zinc compounds, the zinc compounds catalyze both the intermolecular transesterification generating PLLA with low molecular weights and the selective unzipping depolymerization of PLLA with low molecular weights at temperatures below 330 degrees C. In contrast, the primary reaction of thermal degradation for PLLA in the absence of residual zinc compounds above 330 degrees C is a competition between the random chain scission via a cis-elimination reaction and the cyclic rupture via intramolecular transesterification of PLLA molecules. In addition, it was evidenced that the racemization of lactic acid units in the main chain of PLLA molecules occurred at temperatures above 330 degrees C.     

Ecological skills: mind the gap(s)

Anecdotal evidence indicated that there is a shortage of ecological skills. A three year project has been established by the profession, the Institute of Ecology and Environmental Management (IEEM), which runs from May 2008 until April 2011, to examine the issue and to make recommendations. Botany and plant sciences are used as a case study to provide an illustration of what is happening in part of the sector.     

On-line monitoring and control of substrate concentrations in biological processes by flow injection analysis systems

Concentrations of substrates, glucose, and ammionia in biological processes have been on-line monitored by using glucose-flow injection (FIA) and ammonia-FIA systems. Based on the on-line monitored data the concentrations of substrates have been controlled by an on-off controller, a PID controller, and a neural network (NN) based controller. A simulation program has been developed to test the control quality of each controller and to estimate the control parameters. The on-off controller often produced high oscillations at the set point due to its low robustness. The control quality of a PID controller could have been improved by a high analysis frequency and by a short residence time of sample in a FIA system. A NN-based controller with 3 layers has been developed, and a 3(input)-2(hidden)-1(output) network structure has been found to be optimal for the NN-based controller. The performance of the three controllers has been tested in a simulated process as well as in a cultivation process ofSaccharomyces cerevisiae, and the performance has also been compared to simulation results. The NN-based controller with the 3-2-1 network structure was robust and stable against some disturbances, such as a sudden injection of distilled water into a biological process.     

Selection of tracers for 13C-metabolic flux analysis using elementary metabolite units (EMU) basis vector methodology

Metabolic flux analysis (MFA) is a powerful technique for elucidating in vivo fluxes in microbial and mammalian systems. A key step in (13)C-MFA is the selection of an appropriate isotopic tracer to observe fluxes in a proposed network model. Despite the importance of MFA in metabolic engineering and beyond, current approaches for tracer experiment design are still largely based on trial-and-error. The lack of a rational methodology for selecting isotopic tracers prevents MFA from achieving its full potential. Here, we introduce a new technique for tracer experiment design based on the concept of elementary metabolite unit (EMU) basis vectors. We demonstrate that any metabolite in a network model can be expressed as a linear combination of so-called EMU basis vectors, where the corresponding coefficients indicate the fractional contribution of the EMU basis vector to the product metabolite. The strength of this approach is the decoupling of substrate labeling, i.e. the EMU basis vectors, from the dependence on free fluxes, i.e. the coefficients. In this work, we demonstrate that flux observability inherently depends on the number of independent EMU basis vectors and the sensitivities of coefficients with respect to free fluxes. Specifically, the number of independent EMU basis vectors places hard limits on how many free fluxes can be determined in a model. This constraint is used as a guide for selecting feasible substrate labeling. In three example models, we demonstrate that by maximizing the number of independent EMU basis vectors the observability of a system is improved. Inspection of sensitivities of coefficients with respect to free fluxes provides additional constraints for proper selection of tracers. The present contribution provides a fresh perspective on an important topic in metabolic engineering, and gives practical guidelines and design principles for a priori selection of isotopic tracers for (13)C-MFA studies.     

Cyclin degradation: don't mes(s) with meiosis

Cyclin degradation is required for exit from mitosis and enables a new round of DNA replication in the subsequent S phase. A recent study in fission yeast shows that, during exit from meiosis I, the Mes1 protein partially inhibits cyclin degradation and thereby allows entry into meiosis II without an intervening S phase.     

Peroxidase-catalyzed oxidation of eugenol: formation of a cytotoxic metabolite(s)

The oxidation of eugenol (4-allyl-2-methoxyphenol) by horseradish peroxidase was studied. Following the initiation of the reaction with hydrogen peroxide, eugenol was oxidized via a one-electron pathway to a phenoxyl radical which subsequently formed a transient, yellow-colored intermediate which was identified as a quinone methide. The eugenol phenoxyl radical was detected using fast-flow electron spin resonance. The radicals and/or quinone methide further reacted to form an insoluble complex polymeric material. The stoichiometry of the disappearance of eugenol versus hydrogen peroxide was approximately 2:1. The addition of glutathione or ascorbate prevented the appearance of the quinone methide and also prevented the disappearance of the parent compound. In the presence of glutathione, a thiyl radical was detected, and increases in oxygen consumption and in the formation of oxidized glutathione were also observed. These results suggested that glutathione reacted with the eugenol phenoxyl radical and reduced it back to the parent compound. Glutathione also reacted directly with the quinone methide resulting in the formation of a eugenol-glutathione conjugate(s). Using 3H-labeled eugenol, extensive covalent binding to protein was observed. Finally, the oxidation products of eugenol/peroxidase were observed to be highly cytotoxic using isolated rat hepatocytes as target cells.     

土壤和沉积物中多氯代有机化合物厌氧降解研究进展

多氯代有机化合物(PCOCs)是土壤和沉积物中的典型污染物,厌氧条件下PCOCs能够发生脱氯发应,从而使其毒性大大降低,脱氯后形成的低氯代化合物可以进一步好氧降解,直至完全矿化。从PCOCs的降解过程出发,重点综述了几种典型PCOCs的厌氧脱氯机理以及几种重要影响因素;阐明了脱氯反应是PCOCs厌氧降解的关键步骤,反应的发生必须有还原剂提供电子,微生物的参与尤为重要;同时展望了同位素示踪法在研究PCOCs降解机制上的应用,以及开发高效降解PCOCs微生物的必要性等。     

Proton-transfer-reaction mass spectrometry (PTR-MS) for online monitoring of glucose depletion and cell concentrations in HEK 293 gene therapy processes

Biotechnology Letters - The applicability of proton-transfer-reaction mass spectrometry (PTR-MS) as a versatile online monitoring tool to increase consistency and robustness for recombinant...     

Methanogenic degradation of (amino)aromatic compounds by anaerobic microbial communities

degradation of a range of aromatic substrates by anaerobic microbial communities was studied. Active methanogenic microbial communities decomposing aminoaromatic acids and azo dyes into CH₄ and CO₂ were isolated. Products of primary conversion were found to be 2-hydroxybenzyl and benzyl alcohols gradually transforming into benzoate. It was shown that isolated microbial communities are capable of converting the initial substrates—benzyl alcohol, benzoate, salicylic acid, and azo dye Acid Orange 6—into biogas without a lag-phase but with different velocities. Aromatic and linear intermediates of biodegradation of aromatic amines by obtained enrichment cultures were determined for the first time. Selective effect of aromatic substrates on a microbial community that was expressed in decrease in diversity and gradual change of dominant morphotypes was revealed.     

New tools for mass isotopomer data evaluation in (13)C flux analysis: mass isotope correction, data consistency checking, and precursor relationships

13C metabolic flux analysis (MFA) is based on carbon-labeling experiments where a specifically (13)C labeled substrate is fed. The labeled carbon atoms distribute over the metabolic network and the label enrichment of certain metabolic pools is measured by using different methods. Recently, MS methods have been dramatically improved-large and precise datasets are now available. MS data has to be preprocessed and corrected for natural stable mass isotopes. In this article we present (1). a new elegant method to correct MS measurement data for natural stable mass isotopes by infinite dimensional matrix calculus and (2). we statistically analyze and discuss a reconstruction of labeling pattern in metabolic precursors from biosynthesis molecules. Moreover, we establish a new method for consistency checking of MS spectra that can be applied for automatic error recognition in high-throughput flux analysis procedures. Preprocessing the measurement data changes their statistical properties which have to be considered in the subsequent parameter fitting process for (13)C MFA. We show that correcting for stable mass isotopes leads to rather small correlations. On the other hand, a direct reconstruction of a precursor labeling pattern from an aromatic amino acid measurement turns out to be critical. Reasonable results are only obtained if additional, independent information about the labeling of at least one precursor is available. A versatile MatLab tool for the rapid correction and consistency checking of MS spectra is presented. Practical examples for the described methods are also given.     

Production and degradation of geosmin in a stratified lake with anaerobic hypolimnion (Schleinsee)

Abstract Depth profiles of geosmin concentrations were determined over the year in a stratified lake with anaerobic hypolimnion. Two independent sources of geosmin were observed. Most geosmin was produced under anaerobic conditions in the hypolimnion in the autumn. A much smaller source of geosmin production was actinomycetes in the epilimnion. With the onset of the autumnal circulation of the water body, rapid aerobic degradation of geosmin was observed.     

13C tracer experiments and metabolite balancing for metabolic flux analysis: comparing two approaches

Conventional metabolic flux analysis uses the information gained from determination of measurable fluxes and a steady-state assumption for intracellular metabolites to calculate the metabolic fluxes in a given metabolic network. The determination of intracellular fluxes depends heavily on the correctness of the assumed stoichiometry including the presence of all reactions with a noticeable impact on the model metabolite balances. Determination of fluxes in complex metabolic networks often requires the inclusion of NADH and NADPH balances, which are subject to controversial debate. Transhydrogenation reactions that transfer reduction equivalents from NADH to NADPH or vice versa can usually not be included in the stoichiometric model, because they result in singularities in the stoichiometric matrix. However, it is the NADPH balance that, to a large extent, determines the calculated flux through the pentose phosphate pathway. Hence, wrong assumptions on the presence or activity of transhydrogenation reactions will result in wrong estimations of the intracellular flux distribution. Using 13C tracer experiments and NMR analysis, flux analysis can be performed on the basis of only well established stoichiometric equations and measurements of the labeling state of intracellular metabolites. Neither NADH/NADPH balancing nor assumptions on energy yields need to be included to determine the intracellular fluxes. Because metabolite balancing methods and the use of 13C labeling measurements are two different approaches to the determination of intracellular fluxes, both methods can be used to verify each other or to discuss the origin and significance of deviations in the results. Flux analysis based entirely on metabolite balancing and flux analysis, including labeling information, have been performed independently for a wild-type strain of Aspergillus oryzae producing alpha-amylase. Two different nitrogen sources, NH4+ and NO3-, have been used to investigate the influence of the NADPH requirements on the intracellular flux distribution. The two different approaches to the calculation of fluxes are compared and deviations in the results are discussed. Copyright 1998 John Wiley & Sons, Inc.     

Metabolite Imager: customized spatial analysis of metabolite distributions in mass spectrometry imaging

Mass spectrometry (MS) is currently the most utilized analytical instrument for evaluating the metabolite composition of a biological sample at both the qualitative and quantitative level. The exponential growth of raw data generated through increasingly versatile mass spectrometers requires sophisticated algorithms to process and visualize the raw data to address biological questions. The structural and quantitative diversity of a single species’ metabolome (e.g. all metabolite species) under different experimental conditions itself forms a very large and complex dataset to analyze. We have developed a free, Java-based metabolomics application “Metabolite Imager” (www.metaboliteimager.com) that enables customized analysis and visualization of the metabolite distributions in tissues acquired through MS-based imaging approaches. Metabolite Imager algorithms perform customized targeted searching of metabolites through user-defined and publicly-available databases enabling the analysis of spatial distributions of large metabolite numbers in tissue sections. Metabolite Imager’s automated, two-dimensional image generator has several customizable features for producing high-resolution images. Additional Metabolite Imager algorithms support identifying targeted and unknown detected metabolites in selected tissue regions using spatially-based enrichment analysis that could impact metabolic engineering strategies. Co-localization algorithms of metabolites and selected ions by m/z enable analysis of precursor-product relationships in situ that will be important for expanding the biological context of metabolic pathways.