Phylophenetic properties of metabolic pathway topologies as revealed by global analysis

Background  

As phenotypic features derived from heritable characters, the topologies of metabolic pathways contain both phylogenetic and phenetic components. In the post-genomic era, it is possible to measure the "phylophenetic" contents of different pathways topologies from a global perspective.     

Parallel labeling experiments for pathway elucidation and 13C metabolic flux analysis

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13C metabolic flux analysis.

Metabolic flux analysis using 13C-labeled substrates has become an important tool in metabolic engineering. It allows the detailed quantification of all intracellular fluxes in the central metabolism of a microorganism. The method has strongly evolved in recent years by the introduction of new experimental procedures, measurement techniques, and mathematical data evaluation methods. Many of these improvements require advanced skills in the application of nuclear magnetic resonance and mass spectrometry techniques on the one hand and computational and statistical experience on the other hand. This minireview summarizes these recent developments and sketches the major practical problems. An outlook to possible future developments concludes the text.     

Potential metabolic limitations in lysine production by Corynebacterium glutamicum as revealed by metabolic network analysis

Analysis of the metabolic network of lysine-producing Corynebacterium glutamicum showed that lysine yields are limited by the excess energy production in lysine biosynthesis. The most probable maximum yield is 0.47 mol/mol on glucose, when phosphoenolpyruvate carboxylase functions in an anaplerotic rection. When this function is fulfilled by the glyoxylate pathway, a maximum yield of 0.38 mol/mol is obtained.     

13C metabolic flux analysis of recombinant expression hosts

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The biosynthetic pathway of curcuminoid in turmeric (Curcuma longa) as revealed by 13C-labeled precursors

In order to investigate the biosynthesis of curcuminoid in rhizomes of turmeric (Curcuma longa), we established an in vitro culture system of turmeric plants for feeding (13)C-labeled precursors. Analyses of labeled desmethoxycurcumin (DMC), an unsymmetrical curcuminoid, by (13)C-NMR, revealed that one molecule of acetic acid or malonic acid and two molecules of phenylalanine or phenylpropanoids, but not tyrosine, were incorporated into DMC. The incorporation efficiencies of the same precursors into DMC and curcumin were similar, and were in the order malonic acid > acetic acid, and cinnamic acid > p-coumaric acid > ferulic acid. These results suggest the possibility that the pathway to curcuminoids utilized two cinnamoyl CoAs and one malonyl CoA, and that hydroxy- and methoxy-functional groups on the aromatic rings were introduced after the formation of the curcuminoid skeleton.     

Amino acid biosynthesis and sodium-dependent transport in Methanococcus voltae, as revealed by 13C NMR

Of several methanogenic bacteria examined only Methanococcus voltae readily incorporated exogenous amino acids into cell protein. This was easily shown, since growth in the presence of exogenous amino acids resulted in a loss of signal intensities from those carbon atoms normally labelled by [13C]acetate during biosynthesis. From 80% to 95% of the Ser, Lys, Pro or Val incorporated into protein could be supplied directly from the growth medium. In contrast, Asp and Glu, if supplied to the medium, accounted for only a small percentage of the total acidic amino acid used in protein synthesis. Constitutive transport systems took up a wide range of amino acids at rates of 0.1-4.1 nmol min-1 mg-1. The transport systems required Na+, with the possible exception of the basic amino acid lysine, and were inhibited by N-ethylmaleimide or 3,3',4',5-tetrachlorosalicylanilide. No interconversion of Ile to other amino acids was detected when cells were given [13C]Ile during growth, whereas the expected labelling of the Asp and Glu families of amino acids resulted when [13C]Asp was provided to the culture. Mc. voltae synthesized its amino acids from acetate via routes fully consistent with those found in Methanospirillum hungatei [Ekiel, I., Smith, I.C.P. & Sprott, G.D. (1983) J. Bacteriol. 156, 316-326]. Propionate could substitute for an auxotrophic requirement for Ile, resulting in the synthesis of Ile with the beta-carbon originating from the carboxyl of acetate and the alpha-carbon from the carboxyl of propionate. No labelling of Ile from [13C]acetate could occur without the fatty acid. These results provide strong evidence for the carboxylation of propionate to form 2-oxobutyrate as intermediate in Ile biosynthesis, and show that the metabolic defect in Ile biosynthesis occurs prior to 2-oxobutyrate synthesis. The presence of constitutive amino acid transport systems and multiple routes for ile biosynthesis make Methanococcus voltae an attractive methanogen for genetic studies.     

Compartment-specific labeling information in 13C metabolic flux analysis of plants

Metabolic engineering of plants has great potential for the low cost production of chemical feedstocks and novel compounds, but to take full advantage of this potential a better understanding of plant central carbon metabolism is needed. Flux studies define the cellular phenotype of living systems and can facilitate rational metabolic engineering. However the measurements usually made in these analyses are often not sufficient to reliably determine many fluxes that are distributed between different subcellular compartments of eukaryotic cells. We have begun to address this shortcoming by increasing the number and quality of measurements that provide (13)C labeling information from specific compartments within the plant cell. The analysis of fatty acid groups, cell wall components, protein glycans, and starch, using both gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy are presented here. Fatty acid labeling determinations are sometimes highly convoluted. Derivatization to butyl amides reduces the errors in isotopomer resolution and quantification, resulting in better determination of fluxes into seed lipid reserves, including both plastidic and cytosolic reactions. While cell walls can account for a third or more of biomass in many seeds, no quantitative cell wall labeling measurements have been reported for plant flux analysis. Hydrolyzing cell wall and derivatizing sugars to the alditol acetates, provides novel labeling information and thereby can improve identification of flux through upper glycolytic intermediates of the cytosol. These strategies improve the quantification of key carbon fluxes in the compartmentalized flux network of plant cells.     

A universal framework for 13C metabolic flux analysis.

A general methodology is presented for the modeling, simulation, design, evaluation, and statistical analysis of (13)C-labeling experiments for metabolic flux analysis. The universal software framework 13C-FLUX was implemented to support all steps of this process. Guided by the example of anaplerotic flux determination in Corynebacterium glutamicum, the technical details of the model setup, experimental design, and data evaluation are discussed. It is shown how the network structure, the input substrate composition, the assumptions about fluxes, and the measurement configuration are specified within 13C-FLUX. Based on the network model, different experimental designs are computed depending on the goal of the investigations. Finally, a specific experiment is evaluated and the various statistical methods used to analyze the results are briefly explained. The appendix gives some details about the software implementation and availability.     

Autotrophic and mixotrophic metabolism of an anammox bacterium revealed by in vivo 13C and 2H metabolic network mapping

Anaerobic ammonium-oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and use of different carbon and energy substrates beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacterium Candidatus ‘Kuenenia stuttgartiensis’ using time-series ¹³C and ²H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis. Our findings confirm predicted metabolic pathways used for CO₂ fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis, and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of the oxidative branch of an incomplete tricarboxylic acid cycle for alpha-ketoglutarate biosynthesis, despite the genome not having an annotated citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood–Ljungdahl pathway instead of oxidizing it completely to CO₂ followed by reassimilation. In contrast, our data suggest that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and that acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactor’s side population. Together, these findings provide a foundation for understanding the carbon metabolism of anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.Subject terms: Environmental microbiology, Metabolism     

On‐line adaptive metabolic flux analysis: Application to PHB production by mixed microbial cultures

In this work, an algorithm for on‐line adaptive metabolic flux analysis (MFA) is proposed and applied to polyhydroxybutyrate (PHB) production by mixed microbial cultures (MMC). In this process, population dynamics constitutes an important source of perturbation to MFA calculations because some stoichiometric and energetic parameters of the underlying metabolic network are continuously changing over time. The proposed algorithm is based on the application of the observer‐based estimator (OBE) to the central MFA equation, whereby the role of the OBE is to force the accumulation of intracellular metabolites to converge to zero by adjusting the values of unknown network parameters. The algorithm was implemented in a reactor equipped with on‐line analyses of dissolved oxygen and carbon dioxide through respirometric and titrimetric measurements. The oxygen and carbon dioxide fluxes were measured directly, whereas acetate, PHB, and sludge production fluxes were estimated indirectly using a projection of latent structures model calibrated a priori with off‐line measurements. The algorithm was implemented in a way that the network parameters associated with biosynthesis were adjusted on‐line. The algorithm proofed to converge exponentially with the steady state error always below 1 mmol/L. The estimated fluxes passed the consistency index test for experimental error variances as low as 1%. The comparison of measured and estimated respiratory coefficient and of the theoretical and estimated yield of sludge on acetate further confirmed the metabolic consistency of the parameters that were estimated on‐line. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Metabolism of the fast-growing bacterium Vibrio natriegens elucidated by 13C metabolic flux analysis

Vibrio natriegens is a fast-growing, non-pathogenic bacterium that is being considered as the next-generation workhorse for the biotechnology industry. However, little is known about the metabolism of this organism which is limiting our ability to apply rational metabolic engineering strategies. To address this critical gap in current knowledge, here we have performed a comprehensive analysis of V. natriegens metabolism. We constructed a detailed model of V. natriegens core metabolism, measured the biomass composition, and performed high-resolution ¹³C metabolic flux analysis (¹³C-MFA) to estimate intracellular fluxes using parallel labeling experiments with the optimal tracers [1,2−¹³C]glucose and [1,6−¹³C]glucose. During exponential growth in glucose minimal medium, V. natriegens had a growth rate of 1.70 1/h (doubling time of 24 min) and a glucose uptake rate of 3.90 g/g/h, which is more than two 2-fold faster than E. coli, although slower than the fast-growing thermophile Geobacillus LC300. ¹³C-MFA revealed that the core metabolism of V. natriegens is similar to that of E. coli, with the main difference being a 33% lower normalized flux through the oxidative pentose phosphate pathway. Quantitative analysis of co-factor balances provided additional insights into the energy and redox metabolism of V. natriegens. Taken together, the results presented in this study provide valuable new information about the physiology of V. natriegens and establish a solid foundation for future metabolic engineering efforts with this promising microorganism.     

Gas chromatography-mass spectrometry analysis of 13C labeling in sugars for metabolic flux analysis

Metabolic flux analysis, using 13C labeled substrates, has become a powerful methodology for quantifying intracellular fluxes. Most often, analysis is restricted to nuclear magnetic resonance or mass spectrometry measurement of 13C label incorporation into protein amino acids. However, amino acid isotopomer distribution insufficiently covers the entire network of central metabolism, especially in plant cells with highly compartmented metabolism, and analysis of other metabolites is required. Analysis of label in saccharides provides complementary data to better define fluxes around hexose, pentose, and triose phosphate pools. Here, we propose a gas chromatography-mass spectrometry (GC-MS) method to analyze 13C labeling in glucose and fructose moieties of sucrose, free glucose, fructose, maltose, inositol, and starch. Our results show that saccharide labeling for isotopomer quantification is better analyzed by chemical ionization than by electron ionization. The structure of the generated fragments was simulated and validated using labeled standards. The method is illustrated by analysis of saccharides extracted from developing rapeseed (Brassica napus L.) embryos. It is shown that glucose 6-phosphate isomerase and plastidial glucose 6-phosphate transport reactions are not at equilibrium, and light is shed on the pathways leading to fructose, maltose, and inositol synthesis.     

Parallel labeling experiments validate Clostridium acetobutylicum metabolic network model for 13C metabolic flux analysis

In this work, we provide new insights into the metabolism of Clostridium acetobutylicum ATCC 824 obtained using a systematic approach for quantifying fluxes based on parallel labeling experiments and ¹³C-metabolic flux analysis (¹³C-MFA). Here, cells were grown in parallel cultures with [1-¹³C]glucose and [U-¹³C]glucose as tracers and ¹³C-MFA was used to quantify intracellular metabolic fluxes. Several metabolic network models were compared: an initial model based on current knowledge, and extended network models that included additional reactions that improved the fits of experimental data. While the initial network model did not produce a statistically acceptable fit of ¹³C-labeling data, an extended network model with five additional reactions was able to fit all data with 292 redundant measurements. The model was subsequently trimmed to produce a minimal network model of C. acetobutylicum for ¹³C-MFA, which could still reproduce all of the experimental data. The flux results provided valuable new insights into the metabolism of C. acetobutylicum. First, we found that TCA cycle was effectively incomplete, as there was no measurable flux between α-ketoglutarate and succinyl-CoA, succinate and fumarate, and malate and oxaloacetate. Second, an active pathway was identified from pyruvate to fumarate via aspartate. Third, we found that isoleucine was produced exclusively through the citramalate synthase pathway in C. acetobutylicum and that CAC3174 was likely responsible for citramalate synthase activity. These model predictions were confirmed in several follow-up tracer experiments. The validated metabolic network model established in this study can be used in future investigations for unbiased ¹³C-flux measurements in C. acetobutylicum.     

Unique and redundant roles for HOG MAPK pathway components as revealed by whole-genome expression analysis

The Saccharomyces cerevisiae high osmolarity glycerol (HOG) mitogen-activated protein kinase pathway is required for osmoadaptation and contains two branches that activate a mitogen-activated protein kinase (Hog1) via a mitogen-activated protein kinase kinase (Pbs2). We have characterized the roles of common pathway components (Hog1 and Pbs2) and components in the two upstream branches (Ste11, Sho1, and Ssk1) in response to elevated osmolarity by using whole-genome expression profiling. Several new features of the HOG pathway were revealed. First, Hog1 functions during gene induction and repression, cross talk inhibition, and in governing the regulatory period. Second, the phenotypes of pbs2 and hog1 mutants are identical, indicating that the sole role of Pbs2 is to activate Hog1. Third, the existence of genes whose induction is dependent on Hog1 and Pbs2 but not on Ste11 and Ssk1 suggests that there are additional inputs into Pbs2 under our inducing conditions. Fourth, the two upstream pathway branches are not redundant: the Sln1-Ssk1 branch has a much more prominent role than the Sho1-Ste11 branch for activation of Pbs2 by modest osmolarity. Finally, the general stress response pathway and both branches of the HOG pathway all function at high osmolarity. These studies demonstrate that cells respond to increased osmolarity by using different signal transduction machinery under different conditions.     

Intracellular metabolic compartmentation assessed by 13C magnetic resonance spectroscopy

Our understanding of the brain has developed from the theory that it is one continuous cell to the knowledge that there are many brain cells originally termed neurons and, furthermore to the discovery of glial cells and their multiple functions. Thus, an increasing complexity was unraveled and we have not reached a complete understanding of the phenomenon which comprises the compartmentation of metabolic pathways and metabolites. This is an important principle needed to fully understand the metabolic processes of the brain. At the cellular level this concept is well established whereas intracellular compartmentation has yet to be explored. Using magnetic resonance spectroscopy (MRS) for analysis of isotopomer composition combined with quantification of amino acid contents it is possible to construct models that describe intracellular compartmentation. Results of studies of cultures of astrocytes and neurons incubated in media containing [U- 13C]glutamate in the presence or absence of thiopental may be used to propose an intracellular three compartment model of mitochondrial function. Due to the experimental paradigm only certain aspects of metabolism can be described. The present model consists of compartments assigned to CO(2) production, glutamate synthesis from ketoglutarate and finally synthesis of a four-carbon metabolite which is shuttled between compartments. It is likely that metabolism may be far more complex than this and we are only beginning to glimpse some aspects of compartmentation at the cellular level.     

Aspects of the chemical structure of soil organic materials as revealed by solid-state13C NMR spectroscopy

Solid-state cross-polarisation/magic-angle-spinning³C nuclear magnetic resonance (CP/MAS¹³C NMR) spectroscopy was used to characterise semi-quantitatively the organic materials contained in particle size and density fractions isolated from five different mineral soils: two Mollisols, two Oxisols and an Andosol. The acquired spectra were analysed to determine the relative proportion of carboxyl, aromatic, O-alkyl and alkyl carbon contained in each fraction. Although similar types of carbon were present in all of the fractions analysed, an influence of both soil type and particle size was evident.The chemical structure of the organic materials contained in the particle size fractions isolated from the Andosol was similar; however, for the Mollisols and Oxisols, the content of O-alkyl, aromatic and alkyl carbon was greatest in the coarse, intermediate and fine fractions, respectively. The compositional differences noted in progressing from the coarser to finer particle size fractions in the Mollisols and Oxisols were consistent with the changes noted in other studies where CP/MAS¹³C NMR was used to monitor the decomposition of natural organic materials. Changes in the C:N ratio of the particle size fractions supported the proposal that the extent of decomposition of the organic materials contained in the fine fractions was greater than that contained in the coarse fractions. The increased content of aromatic and alkyl carbon in the intermediate size fractions could be explained completely by a selective preservation mechanism; however, the further accumulation of alkyl carbon in the clay fractions appeared to result from both a selective preservation and anin situ synthesis.The largest compositional differences noted for the entire organic fraction of the five soils were observed between soil orders. The differences within orders were smaller. The Mollisols and the Andosol were both dominated by O-alkyl carbon but the Andosol had a lower alkyl carbon content. The Oxisols were dominated by both O-alkyl and alkyl carbon.A model describing the oxidative decomposition of plant materials in mineral soils is proposed and used to explain the influence of soil order and particle size on the chemical composition of soil organic matter in terms of its extent of decomposition and bioavailability.     

Isotopically nonstationary 13C metabolic flux analysis in resting and activated human platelets

Platelet metabolism is linked to platelet hyper- and hypoactivity in numerous human diseases. Developing a detailed understanding of the link between metabolic shifts and platelet activation state is integral to improving human health. Here, we show the first application of isotopically nonstationary ¹³C metabolic flux analysis to quantitatively measure carbon fluxes in both resting and thrombin activated platelets. Metabolic flux analysis results show that resting platelets primarily metabolize glucose to lactate via glycolysis, while acetate is oxidized to fuel the tricarboxylic acid cycle. Upon activation with thrombin, a potent platelet agonist, platelets increase their uptake of glucose 3-fold. This results in an absolute increase in flux throughout central metabolism, but when compared to resting platelets they redistribute carbon dramatically. Activated platelets decrease relative flux to the oxidative pentose phosphate pathway and TCA cycle from glucose and increase relative flux to lactate. These results provide the first report of reaction-level carbon fluxes in platelets and allow us to distinguish metabolic fluxes with much higher resolution than previous studies.     

Optimization of 13C isotopic tracers for metabolic flux analysis in mammalian cells

Mammalian cells consume and metabolize various substrates from their surroundings for energy generation and biomass synthesis. Glucose and glutamine, in particular, are the primary carbon sources for proliferating cancer cells. While this combination of substrates generates static labeling patterns for use in (13)C metabolic flux analysis (MFA), the inability of single tracers to effectively label all pathways poses an obstacle for comprehensive flux determination within a given experiment. To address this issue we applied a genetic algorithm to optimize mixtures of (13)C-labeled glucose and glutamine for use in MFA. We identified tracer combinations that minimized confidence intervals in an experimentally determined flux network describing central carbon metabolism in tumor cells. Additional simulations were used to determine the robustness of the [1,2-(13)C(2)]glucose/[U-(13)C(5)]glutamine tracer combination with respect to perturbations in the network. Finally, we experimentally validated the improved performance of this tracer set relative to glucose tracers alone in a cancer cell line. This versatile method allows researchers to determine the optimal tracer combination to use for a specific metabolic network, and our findings applied to cancer cells significantly enhance the ability of MFA experiments to precisely quantify fluxes in higher organisms.