Academic Staff

David’s group formed nearly thirty years ago with initial interests in computer simulation of metabolism and the theory of metabolic control. To these it has since added interests in modelling signal transduction, in various different approaches to network analysis of metabolism, and in reconstructing metabolic networks from genomic data. In the course of this research, he has addressed problems in microbial, plant and mammalian metabolism, often in conjunction with collaborators who have contributed experimental results.

His work forms part of the emerging field of Systems Biology, in that we are concerned with understanding how biological function arises from the interactions between many components, and with building predictive models. Potential applications of our work include the design of changes in cellular metabolism to improve the output of product such as antibiotics, detecting vulnerable sites in cellular networks that could be targets for drugs to control disease-causing organisms, and improved understanding of how organisms manage to adjust their metabolism in response to environmental changes and other signals.

Research group membership

• Cell Systems Modelling Group

Research projects

Research Supervisions

• Computer modelling of metabolic networks
• Designs for metabolic engineering

Further information

• http://mudshark.brookes.ac.uk/
• http://sysbio.brookes.ac.uk/
• http://mitoscop.brookes.ac.uk/
• http://frim.brookes.ac.uk/

Journal articles

• Woodfield H, Fenyk S, Wallington E, Bates R, Brown A, Guschina I, Marillia E, Taylor D, Fell D, Harwood J, Fawcett T, 'Increase in lysophosphatidate acyltransferase activity in oilseed rape (Brassica napus L.) increases seed triacylglycerol content despite its low intrinsic flux control coefficient'
  New Phytologist (2019)
  ISSN: 0028-646X
  Abstract

  Lysophosphatidate acyltransferase (LPAAT) catalyses the second step of the Kennedy pathway for triacylglycerol (TAG) synthesis. Here we express Trapaeolum majus LPAAT in Brassica napus (B. napus) cv 12075 to evaluate the effects on lipid synthesis and estimate the flux control coefficient for LPAAT. • We estimate the flux control coefficient of LPAAT in a whole plant context by deriving a relationship between it and overall lipid accumulation, given that this is an exponential process. • Increasing LPAAT activity resulted in greater TAG accumulation in seeds of between 25 and 29 %; altered fatty acid distributions in seed lipids (particularly those of the Kennedy pathway); and a redistribution of label from 14C-glycerol between phosphoglycerides. • Greater LPAAT activity in seeds led to an increase in TAG content despite its low intrinsic flux control coefficient on account of the exponential nature of lipid accumulation that amplifies the effect of the small flux increment achieved by increasing its activity. We have also developed a novel application of metabolic control analysis likely to have broad application as it determines the in planta flux control that a single component has upon accumulation of storage products. 
  

  Website 
• Norman ROJ, Millat T, Schatsschneider S, Henstra AM, Breitkopf R, Oander B, Annan FJ, Piatek P, Hartman HB, Poolman MG, Fell DA, Winzer K, Minton NP, Hodgman C., 'A genome-scale model of Clostridium autoethanogenum reveals optimal bioprocess conditions for high-value chemical production from carbon monoxide.'
  IET Engineering Biology 3 (2) (2019) pp.32-40
  ISSN: 2398-6182
  Abstract

  Clostridium autoethanogenum is an industrial microbe used for the commercial-scale production of ethanol from carbon monoxide. While significant progress has been made in the attempted diversification of this bioprocess, further improvements are desirable, particularly in the formation of the high-value platform chemicals, such as 2,3-butanediol. A new, experimentally parameterised genome scale model of C. autoethanogenum predicts dramatically increased 2,3-butanediol production under non-carbon-limited conditions when thermodynamic constraints on hydrogen production are considered.

  
  
  Website 
• Huma B, Kundu S, Poolman MG; Kruger N, Fell DA, 'Stoichiometric analysis of the energetics and metabolic impact of photorespiration in C3 plants'
  Plant Journal 96 (6) (2018) pp.1228-1241
  ISSN: 0960-7412 eISSN: 1365-313X
  Abstract

  Analysis of the impact of photorespiration on plant metabolism is usually based on manual inspection of small network diagrams. Here we create a structural metabolic model that contains the reactions that participate in photorespiration in the plastid, peroxisome, mitochondrion and cytosol and the metabolite exchanges between them. This model was subjected to elementary flux modes analysis, a technique that enumerates all the component, minimal pathways of a network. Any feasible photorespiratory metabolism in the plant will be some combination of the elementary flux modes (EFMs) that contain the Rubisco oxygenase reaction. Amongst the EFMs we obtained was the classic photorespiratory cycle, but there were also modes that involve photorespiration coupled with mitochondrial metabolism and ATP production, the glutathione‐ascorbate (GSH‐ASC) cycle and nitrate reduction to ammonia. The modes analysis demonstrated the underlying basis of the metabolic linkages with photorespiration that have been inferred experimentally. The set of reactions common to all the elementary modes showed good agreement with the gene products of mutants that have been reported to have a defective phenotype in photorespiratory conditions. Finally, the set of modes provided a formal demonstration that photorespiration itself does not impact on the CO2:O2 ratio (assimilation quotient, AQ), except in those modes associated with concomitant nitrate reduction.
  

  Website 
• Fatma Z, Hartman H, Poolman MG, Fell DA, Srivastava S, Shakeela T, ⁠Yazdani SS, 'Model-assisted metabolic engineering of Escherichia coli for long chain alkane and alcohol production'
  Metabolic Engineering 48 (2018) pp.1-12
  ISSN: 1096-7176 eISSN: 1096-7184
  Abstract Biologically-derived hydrocarbons are considered to have great potential as next-generation biofuels owing to the similarity of their chemical properties to contemporary diesel and jet fuels. However, the low yield of these hydrocarbons in biotechnological production is a major obstacle for commercialization. Several genetic and process engineering approaches have been adopted to increase the yield of hydrocarbon, but a model driven approach has not been implemented so far. Here, we applied a constraint-based metabolic modeling approach in which a variable demand for alkane biosynthesis was imposed, and co-varying reactions were considered as potential targets for further engineering of an E. coli strain already expressing cyanobacterial enzymes towards higher chain alkane production. The reactions that co-varied with the imposed alkane production were found to be mainly associated with the pentose phosphate pathway (PPP) and the lower half of glycolysis. An optimal modeling solution was achieved by imposing increased flux through the reaction catalyzed by glucose-6-phosphate dehydrogenase (zwf) and iteratively removing 7 reactions from the network, leading to an alkane yield of 94.2% of the theoretical maximum conversion determined by in silico analysis at a given biomass rate. To validate the in silico findings, we first performed pathway optimization of the cyanobacterial enzymes in E. coli via different dosages of genes, promoting substrate channelling through protein fusion and inducing substantial equivalent protein expression, which led to a 36-fold increase in alka(e)ne production from 2.8 mg/L to 102 mg/L. Further, engineering of E. coli based on in silico findings, including biomass constraint, led to an increase in the alka(e)ne titer to 425 mg/L (major components being 249 mg/L pentadecane and 160 mg/L heptadecene), a 148.6-fold improvement over the initial strain, respectively; with a yield of 34.2% of the theoretical maximum. The impact of model-assisted engineering was also tested for the production of long chain fatty alcohol, another commercially important molecule sharing the same pathway while differing only at the terminal reaction, and a titer of 1506 mg/L was achieved with a yield of 86.4% of the theoretical maximum. Moreover, the model assisted engineered strains had produced 2.54 g/L and 12.5 g/L of long chain alkane and fatty alcohol, respectively, in the bioreactor under fed-batch cultivation condition. Our study demonstrated successful implementation of a combined in silico modeling approach along with the pathway and process optimization in achieving the highest reported titers of long chain hydrocarbons in E. coli.Website 
• Pentjuss A, Stalidzans E, Liepins J, Kokina A, Martynova J, Zikmanis P, Mozga I, Scherbaka R, Hartman H, Poolman M, Fell D, Vigants A, 'Model based biotechnological potential analysis of Kluyveromyces marxianus central metabolism'
  Journal of Industrial Microbiology and Biotechnology 44 (8) (2017) pp.1177-1190
  ISSN: 1367-5435 eISSN: 1476-5535
  Abstract The non-conventional yeast Kluyveromyces marxianus is an emerging industrial producer for many biotechnological processes. Here we show the application of a biomass-linked stoichiometric model of central metabolism that is experimentally validated, and mass and charge balanced for assessing the carbon conversion efficiency of wild type and modified K. marxianus. Pairs of substrates (lactose, glucose, inulin, xylose) and products (ethanol, acetate, lactate, glycerol, ethyl acetate, succinate, glutamate, phenylethanol and phenylalanine) are examined by various modeling and optimisation methods. Our model reveals the organism's potential for industrial application and metabolic engineering. Modeling results imply that the aeration regime can be used as a tool to optimise product yield and flux distribution in K. marxianus. Also rebalancing NADH and NADPH utilisation can be used to improve the efficiency of substrate conversion. Xylose is identified as a biotechnologically promising substrate for K. marxianus.Website 
• Ahmad A, Hartman H , Krishnakumar S, Fell D, Poolman M, Srivastava S, 'A genome scale model of Geobacillus thermoglucosidasius (C56-YS93) reveals its biotechnological potential on rice straw hydrolysate'
  Journal of Biotechnology 251 (2017) pp.30-37
  ISSN: 0168-1656
  Abstract Rice straw is a major crop residue which is burnt in many countries, creating signi�cant air pollution. Thus, alternative routes for disposal of rice straw are needed. Biotechnological treatment of rice straw hydrolysate has potential to convert this agriculture waste into valuable biofuel(s) and platform chemicals. Geobacillus thermoglucosidasius is a thermophile with properties specially suited for use as a biocatalyst in lignocellulosic bioprocesses, such as high optimal temperature and tolerance to high levels of ethanol. However, the capabilities of Geobacillus thermoglucosidasius to utilize sugars in rice straw hydrolysate for making bioethanol and other platform chemicals have not been fully explored. In this work, we have created a genome scale metabolic model (denoted iGT736) of the organism containing 736 gene products, 1159 reactions and 1163 metabolites. The model was validated both by purely theoretical approaches and by comparing the behaviour of the model to previously published experimental results. The model was then used to determine the yields of a variety of platform chemicals from glucose and xylose - two primary sugars in rice straw hydrolysate. A comparison with results from a model of Escherichia coli shows that G. thermoglucosidasius is capable of producing a wider range of products, and that for the products also produced by E. coli , the yields are comparable. We also discuss strategies to utilise arabinose, a minor component of rice straw hydrolysate, and propose additional reactions to lead to the synthesis of xylitol, not currently produced by G. thermoglucosidasius. Our results provide additional motivation for the current exploration of the industrial potential of G. thermoglucosidasius and we make our model publicly available to aid the development of metabolic engineering strategies for this organism.Website 
• Singh D, Carlson R, Fell D, Poolman M, 'Modelling metabolism of the diatom Phaeodactylum tricornutum'
  Biochemical Society Transactions 43 (6) (2015) pp.1182-1186
  ISSN: 0300-5127
  Abstract Marine diatoms have potential as a biotechnological production platform, especially for lipid-derived products, including biofuels. Here we introduce some features of diatom metabolism, particularly with respect to photosynthesis, photorespiration and lipid synthesis and their differences relative to other photosynthetic eukaryotes. Since structural metabolic modelling of other photosynthetic organisms has been shown to be capable of representing their metabolic capabilities realistically, we briefly review the main approaches to this type of modelling. We then propose that genome-scale modelling of the diatom Phaeodactylum tricornutum, in response to varying light intensity, could uncover the novel aspects of the metabolic potential of this organism.Website 
• Poolman MG, Kundu S, Shaw R, Fell DA, 'Metabolic trade-offs between biomass synthesis and photosynthate export at different light intensities in a genome-scale metabolic model of rice'
  Frontiers in Plant Science 5 (2014)
  ISSN: 1664-462X eISSN: 1664-462X
  Abstract

  Previously we have used a genome scale model of rice metabolism to describe how metabolism reconfigures at different light intensities in an expanding leaf of rice. Although this established that the metabolism of the leaf was adequately represented, in the model, the scenario was not that of the typical function of the leaf—to provide material for the rest of the plant. Here we extend our analysis to explore the transition to a source leaf as export of photosynthate increases at the expense of making leaf biomass precursors, again as a function of light intensity. In particular we investigate whether, when the leaf is making a smaller range of compounds for export to the phloem, the same changes occur in the interactions between mitochondrial and chloroplast metabolism as seen in biomass synthesis for growth when light intensity increases. Our results show that the same changes occur qualitatively, though there are slight quantitative differences reflecting differences in the energy and redox requirements for the different metabolic outputs.

  Website 
• Kalnenieks U, Pentjuss A, Rutkis R, Stalidzans E, Fell DA, 'Modeling of Zymomonas mobilis central metabolism for novel metabolic engineering strategies'
  Frontiers in Microbiology 5 (42) (2014)
  ISSN: 1664-302X
  Abstract
  Mathematical modeling of metabolism is essential for rational metabolic engineering. The present work focuses on several types of modeling approach to quantitative understanding of central metabolic network and energetics in the bioethanol-producing bacterium Zymomonas mobilis. Combined use of Flux Balance, Elementary Flux Mode, and thermodynamic analysis of its central metabolism, together with dynamic modeling of the core catabolic pathways, can help to design novel substrate and product pathways by systematically analyzing the solution space for metabolic engineering, and yields insights into the function of metabolic network, hardly achievable without applying modeling tools.
   
  Website 
• Wootton-Beard PC, Brandt K, Fell DA, Warner S, Ryan L, 'Effects of a beetroot juice with high neobetanin content on the early-phase insulin response in healthy volunteers'
  Journal of Nutritional Science 3 (2014)
  ISSN: 2048-6790 eISSN: 2048-6790
  Abstract

  Produce rich in phytochemicals may alter postprandial glucose and insulin responses by interacting with the pathways that regulate glucose uptake and insulin secretion in humans. The aims of the present study were to assess the phytochemical constituents of red beetroot juice and to measure the postprandial glucose and insulin responses elicited by either 225 ml beetroot juice (BEET), a control beverage matched for macronutrient content (MCON) or a glucose beverage in healthy adults. Beetroot juice was a particularly rich source of betalain degradation compounds. The orange/yellow pigment neobetanin was measured in particularly high quantities (providing 1·3 g in the 225 ml). A total of sixteen healthy individuals were recruited, and consumed the test meals in a controlled single-blind cross-over design. Results revealed a significant lowering of the postprandial insulin response in the early phase (0–60 min) (P < 0·05) and a significantly lower glucose response in the 0–30 min phase (P < 0·05) in the BEET treatment compared with MCON. Betalains, polyphenols and dietary nitrate found in the beetroot juice may each contribute to the observed differences in the postprandial insulin concentration.

  Website 
• Hartman HB, Fell DA, Rossell S, Jensen PR, Woodward MJ, Thorndahl L, Jelsbak L, Olsen JE, Raghunathan A, Daefler S, Poolman MG, 'Identification of potential drug targets in Salmonella enterica sv. Typhimurium using metabolic modelling and experimental validation'
  MICROBIOLOGY-SGM 160 (2014) pp.1252-1266
  ISSN: 1350-0872 eISSN: 1465-2080
  Abstract Salmonella enterica sv. Typhimurium is an established model organism for Gram-negative, intracellular pathogens. Owing to the rapid spread of resistance to antibiotics among this group of pathogens, new approaches to identify suitable target proteins are required. Based on the genome sequence of S. Typhimurium and associated databases, a genome-scale metabolic model was constructed. Output was based on an experimental determination of the biomass of Salmonella when growing in glucose minimal medium. Linear programming was used to simulate variations in the energy demand while growing in glucose minimal medium. By grouping reactions with similar flux responses, a subnetwork of 34 reactions responding to this variation was identified (the catabolic core). This network was used to identify sets of one and two reactions that when removed from the genome-scale model interfered with energy and biomass generation. Eleven such sets were found to be essential for the production of biomass precursors. Experimental investigation of seven of these showed that knockouts of the associated genes resulted in attenuated growth for four pairs of reactions, whilst three single reactions were shown to be essential for growth.Website 
• Hartman HB, Fell DA, Rossell S, Jensen PR, Woodward MJ, Thorndahl L, Jelsbak L, Olsen JE, Raghunathan A, Daefler S, Poolman MG, 'Identification of potential drug targets in Salmonella enterica sv. Typhimurium using metabolic modelling and experimental validation'
  MICROBIOLOGY-SGM 160 (2014) pp.1252-1266
  ISSN: 1350-0872 eISSN: 1465-2080
  Abstract Salmonella enterica sv. Typhimurium is an established model organism for Gram-negative, intracellular pathogens. Owing to the rapid spread of resistance to antibiotics among this group of pathogens, new approaches to identify suitable target proteins are required. Based on the genome sequence of S. Typhimurium and associated databases, a genome-scale metabolic model was constructed. Output was based on an experimental determination of the biomass of Salmonella when growing in glucose minimal medium. Linear programming was used to simulate variations in the energy demand while growing in glucose minimal medium. By grouping reactions with similar flux responses, a subnetwork of 34 reactions responding to this variation was identified (the catabolic core). This network was used to identify sets of one and two reactions that when removed from the genome-scale model interfered with energy and biomass generation. Eleven such sets were found to be essential for the production of biomass precursors. Experimental investigation of seven of these showed that knockouts of the associated genes resulted in attenuated growth for four pairs of reactions, whilst three single reactions were shown to be essential for growth.Website 
• Fell D, Poolman M G, Kundu S, Shaw R, 'Responses to Light Intensity in a Genome-Scale Model of Rice Metabolism'
  Plant Physiology 162 (2) (2013) pp.1060-1072
  ISSN: 0032-0889 eISSN: 1532-2548
  Abstract

  We describe the construction and analysis of a genome-scale metabolic model representing a developing leaf cell of rice (Oryza sativa) primarily derived from the annotations in the RiceCyc database. We used flux balance analysis to determine that the model represents a network capable of producing biomass precursors (amino acids, nucleotides, lipid, starch, cellulose, and lignin) in experimentally reported proportions, using carbon dioxide as the sole carbon source. We then repeated the analysis over a range of photon flux values to examine responses in the solutions. The resulting flux distributions show that (1) redox shuttles between the chloroplast, cytosol, and mitochondrion may play a significant role at low light levels, (2) photorespiration can act to dissipate excess energy at high light levels, and (3) the role of mitochondrial metabolism is likely to vary considerably according to the balance between energy demand and availability. It is notable that these organelle interactions, consistent with many experimental observations, arise solely as a result of the need for mass and energy balancing without any explicit assumptions concerning kinetic or other regulatory mechanisms.

  Website 
• Cheung CYM, Williams TCR, Poolman MG, Fell DA, Ratcliffe RG, Sweetlove LJ, 'A method for accounting for maintenance costs in flux balance analysis improves the prediction of plant cell metabolic phenotypes under stress conditions'
  Plant Journal 75 (6) (2013) pp.1050-1061
  ISSN: 0960-7412
  Abstract

  Flux balance models of metabolism generally utilize synthesis of biomass as the main determinant of intracellular fluxes. However, the biomass constraint alone is not sufficient to predict realistic fluxes in central heterotrophic metabolism of plant cells because of the major demand on the energy budget due to transport costs and cell maintenance. This major limitation can be addressed by incorporating transport steps into the metabolic model and by implementing a procedure that uses Pareto optimality analysis to explore the trade-off between ATP and NADPH production for maintenance. This leads to a method for predicting cell maintenance costs on the basis of the measured flux ratio between the oxidative steps of the oxidative pentose phosphate pathway and glycolysis. We show that accounting for transport and maintenance costs substantially improves the accuracy of fluxes predicted from a flux balance model of heterotrophic Arabidopsis cells in culture, irrespective of the objective function used in the analysis. Moreover, when the new method was applied to cells under control, elevated temperature and hyper-osmotic conditions, only elevated temperature led to a substantial increase in cell maintenance costs. It is concluded that the hyper-osmotic conditions tested did not impose a metabolic stress, in as much as the metabolic network is not forced to devote more resources to cell maintenance.

  Website 
• Poolman MG, Kundu S, Shaw R, Fell DA, 'Responses to Light Intensity in a Genome–Scale Model of Rice Metabolism'
  Plant Physiology 162 (2) (2013) pp.1060-1072
  ISSN: 0032-0889 eISSN: 1532-2548
  Abstract

  We describe the construction and analysis of a genome-scale metabolic model representing a developing leaf cell of rice (Oryza sativa) primarily derived from the annotations in the RiceCyc database. We used flux balance analysis to determine that the model represents a network capable of producing biomass precursors (amino acids, nucleotides, lipid, starch, cellulose, and lignin) in experimentally reported proportions, using carbon dioxide as the sole carbon source. We then repeated the analysis over a range of photon flux values to examine responses in the solutions. The resulting flux distributions show that (1) redox shuttles between the chloroplast, cytosol, and mitochondrion may play a significant role at low light levels, (2) photorespiration can act to dissipate excess energy at high light levels, and (3) the role of mitochondrial metabolism is likely to vary considerably according to the balance between energy demand and availability. It is notable that these organelle interactions, consistent with many experimental observations, arise solely as a result of the need for mass and energy balancing without any explicit assumptions concerning kinetic or other regulatory mechanisms.

  Website 
• Pentjuss A, Odzina I, Kostromins A, Fell DA, Stalidzans E, Kalnenieks U, 'Biotechnological potential of respiring Zymomonas mobilis: A stoichiometric analysis of its central metabolism'
  Journal of Biotechnology 165 (2013) pp.1-10
  ISSN: 0168-1656 eISSN: 1873-4863
  Abstract

  The active, yet energetically inefficient electron transport chain of the ethanologenic bacterium Zymomonas mobilis could be used in metabolic engineering for redox-balancing purposes during synthesis of certain products. Although several reconstructions of Z. mobilis metabolism have been published, important aspects of redox balance and aerobic catabolism have not previously been considered. Here, annotated genome sequences and metabolic reconstructions have been combined with existing biochemical evidence to yield a medium-scale model of Z. mobilis central metabolism in the form of COBRA Toolbox model files for flux balance analysis (FBA). The stoichiometric analysis presented here suggests the feasibility of several metabolic engineering strategies for obtaining high-value products, such as glycerate, succinate, and glutamate that would use the electron transport chain to oxidize the excess NAD(P)H, generated during synthesis of these metabolites. Oxidation of the excess NAD(P)H would also be needed for synthesis of ethanol from glycerol. Maximum product yields and the byproduct spectra have been estimated for each product, with glucose, xylose, or glycerol as the carbon substrates. These novel pathways represent targets for future metabolic engineering, as they would exploit both the rapid Entner–Doudoroff glycolysis, and the energetically uncoupled electron transport of Z. mobilis.

  Website 
• Rutkis R, Kalnenieks U, Stalidzans E, Fell DA, 'Kinetic modelling of the Zymomonas mobilis Entner-Doudoroff pathway: insights into control and functionality'
  MICROBIOLOGY-SGM 159 (12) (2013) pp.2674-2689
  ISSN: 1350-0872 eISSN: 1465-2080
  Abstract Zymomonas mobilis, an ethanol-producing bacterium, possesses the Entner-Doudoroff (E-D) pathway, pyruvate decarboxylase and two alcohol dehydrogenase isoenzymes for the fermentative production of ethanol and carbon dioxide from glucose. Using available kinetic parameters, we have developed a kinetic model that incorporates the enzymic reactions of the E-D pathway, both alcohol dehydrogenases, transport reactions and reactions related to ATP metabolism. After optimizing the reaction parameters within likely physiological limits, the resulting kinetic model was capable of simulating glycolysis in vivo and in cell-free extracts with good agreement with the fluxes and steady-state intermediate concentrations reported in previous experimental studies. In addition, the model is shown to be consistent with experimental results for the coupled response of ATP concentration and glycolytic flux to ATPase inhibition. Metabolic control analysis of the model revealed that the majority of flux control resides not inside, but outside the E-D pathway itself, predominantly in ATP consumption, demonstrating why past attempts to increase the glycolytic flux through overexpression of glycolytic enzymes have been unsuccessful. Co-response analysis indicates how homeostasis of ATP concentrations starts to deteriorate markedly at the highest glycolytic rates. This kinetic model has potential for application in Z mobilis metabolic engineering and, since there are currently no E-D pathway models available in public databases, it can serve as a basis for the development of models for other micro-organisms possessing this type of glycolytic pathway.Website 
• Rutkis R, Kalnenieks U, Stalidzans E, Fell DA, 'Kinetic modelling of the Zymomonas mobilis Entner-Doudoroff pathway: insights into control and functionality'
  MICROBIOLOGY-SGM 159 (12) (2013) pp.2674-2689
  ISSN: 1350-0872 eISSN: 1465-2080
  Abstract Zymomonas mobilis, an ethanol-producing bacterium, possesses the Entner-Doudoroff (E-D) pathway, pyruvate decarboxylase and two alcohol dehydrogenase isoenzymes for the fermentative production of ethanol and carbon dioxide from glucose. Using available kinetic parameters, we have developed a kinetic model that incorporates the enzymic reactions of the E-D pathway, both alcohol dehydrogenases, transport reactions and reactions related to ATP metabolism. After optimizing the reaction parameters within likely physiological limits, the resulting kinetic model was capable of simulating glycolysis in vivo and in cell-free extracts with good agreement with the fluxes and steady-state intermediate concentrations reported in previous experimental studies. In addition, the model is shown to be consistent with experimental results for the coupled response of ATP concentration and glycolytic flux to ATPase inhibition. Metabolic control analysis of the model revealed that the majority of flux control resides not inside, but outside the E-D pathway itself, predominantly in ATP consumption, demonstrating why past attempts to increase the glycolytic flux through overexpression of glycolytic enzymes have been unsuccessful. Co-response analysis indicates how homeostasis of ATP concentrations starts to deteriorate markedly at the highest glycolytic rates. This kinetic model has potential for application in Z mobilis metabolic engineering and, since there are currently no E-D pathway models available in public databases, it can serve as a basis for the development of models for other micro-organisms possessing this type of glycolytic pathway.Website 
• Williams TCR, Poolman MG, Howden AJM, Schwarzlander M, Fell DA, Ratcliffe RG, Sweetlove LJ, 'A genome-scale metabolic model accurately predicts fluxes in central carbon metabolism under stress conditions.'
  Plant Physiology 154 (1) (2010) pp.311-323
  ISSN: 0032-0889 eISSN: 1532-2548
  Abstract

  Flux is a key measure of the metabolic phenotype. Recently, complete (genome-scale) metabolic network models have been established for Arabidopsis (Arabidopsis thaliana), and flux distributions have been predicted using constraints-based modeling and optimization algorithms such as linear programming. While these models are useful for investigating possible flux states under different metabolic scenarios, it is not clear how close the predicted flux distributions are to those occurring in vivo. To address this, fluxes were predicted for heterotrophic Arabidopsis cells and compared with fluxes estimated in parallel by 13C-metabolic flux analysis (MFA). Reactions of the central carbon metabolic network (glycolysis, the oxidative pentose phosphate pathway, and the tricarboxylic acid [TCA] cycle) were independently analyzed by the two approaches. Net fluxes in glycolysis and the TCA cycle were predicted accurately from the genome-scale model, whereas the oxidative pentose phosphate pathway was poorly predicted. MFA showed that increased temperature and hyperosmotic stress, which altered cell growth, also affected the intracellular flux distribution. Under both conditions, the genome-scale model was able to predict both the direction and magnitude of the changes in flux: namely, increased TCA cycle and decreased phosphoenolpyruvate carboxylase flux at high temperature and a general decrease in fluxes under hyperosmotic stress. MFA also revealed a 3-fold reduction in carbon-use efficiency at the higher temperature. It is concluded that constraints-based genome-scale modeling can be used to predict flux changes in central carbon metabolism under stress conditions.

  Website 
• Fell DA, 'Evolution of Central Carbon Metabolism'
  Molecular Cell 39 (5) (2010) pp.663-664
  ISSN: 1097-2765 eISSN: 1097-4164
  Abstract

  Organisms share a common core to their metabolic networks. But what determined this: chance, chemical necessity, or evolutionary optimization? In this issue of Molecular Cell, Noor et al. (2010) provide new evidence for selection of a network with optimal features from a broader set of possibilities.

  Website 
• Fell D, Poolman M, Gevorgyan A, 'Building and analysing genome-scale metabolic models'
  Biochemical Society Transactions 38 (5) (2010) pp.1197-1201
  ISSN: 0300-5127 eISSN: 1470-8752
  Abstract

  Reconstructing a model of the metabolic network of an organism from its annotated genome sequence would seem, at first sight, to be one of the most straightforward tasks in functional genomics, even if the various data sources required were never designed with this application in mind. The number of genome-scale metabolic models is, however, lagging far behind the number of sequenced genomes and is likely to continue to do so unless the model-building process can be accelerated. Two aspects that could usefully be improved are the ability to find the sources of error in a nascent model rapidly, and the generation of tenable hypotheses concerning solutions that would improve a model. We will illustrate these issues with approaches we have developed in the course of building metabolic models of Streptococcus agalactiae and Arabidopsis thaliana.

  Website 
• Williams TCR, Poolman MG, Howden AJM, Schwarzlander M, Fell DA, Ratcliffe RG, Sweetlove LJ, 'A genome-scale metabolic model accurately predicts fluxes in central carbon metabolism under stress conditions.'
  Plant Physiology 154 (1) (2010) pp.311-323
  ISSN: 0032-0889 eISSN: 1532-2548
  Abstract

  Flux is a key measure of the metabolic phenotype. Recently, complete (genome-scale) metabolic network models have been established for Arabidopsis (Arabidopsis thaliana), and flux distributions have been predicted using constraints-based modeling and optimization algorithms such as linear programming. While these models are useful for investigating possible flux states under different metabolic scenarios, it is not clear how close the predicted flux distributions are to those occurring in vivo. To address this, fluxes were predicted for heterotrophic Arabidopsis cells and compared with fluxes estimated in parallel by 13C-metabolic flux analysis (MFA). Reactions of the central carbon metabolic network (glycolysis, the oxidative pentose phosphate pathway, and the tricarboxylic acid [TCA] cycle) were independently analyzed by the two approaches. Net fluxes in glycolysis and the TCA cycle were predicted accurately from the genome-scale model, whereas the oxidative pentose phosphate pathway was poorly predicted. MFA showed that increased temperature and hyperosmotic stress, which altered cell growth, also affected the intracellular flux distribution. Under both conditions, the genome-scale model was able to predict both the direction and magnitude of the changes in flux: namely, increased TCA cycle and decreased phosphoenolpyruvate carboxylase flux at high temperature and a general decrease in fluxes under hyperosmotic stress. MFA also revealed a 3-fold reduction in carbon-use efficiency at the higher temperature. It is concluded that constraints-based genome-scale modeling can be used to predict flux changes in central carbon metabolism under stress conditions.

  Website 
• de Figueiredo L, Schuster S, Kaleta C, Fell D, 'Can sugars be produced from fatty acids? A test case for pathway analysis tools'
  Bioinformatics 24 (22) (2009) pp.2615-2621
  ISSN: 1367-4803
  Abstract

  Motivation: In recent years, several methods have been proposed for determining metabolic pathways in an automated way based on network topology. The aim of this work is to analyse these methods by tackling a concrete example relevant in biochemistry. It concerns the question whether even-chain fatty acids, being the most important constituents of lipids, can be converted into sugars at steady state. It was proved five decades ago that this conversion using the Krebs cycle is impossible unless the enzymes of the glyoxylate shunt (or alternative bypasses) are present in the system. Using this example, we can compare the various methods in pathway analysis. Results: Elementary modes analysis (EMA) of a set of enzymes corresponding to the Krebs cycle, glycolysis and gluconeogenesis supports the scientific evidence showing that there is no pathway capable of converting acetyl-CoA to glucose at steady state. This conversion is possible after the addition of isocitrate lyase and malate synthase (forming the glyoxylate shunt) to the system. Dealing with the same example, we compare EMA with two tools based on graph theory available online, PathFinding and Pathway Hunter Tool. These automated network generating tools do not succeed in predicting the conversions known from experiment. They sometimes generate unbalanced paths and reveal problems identifying side metabolites that are not responsible for the carbon net flux. This shows that, for metabolic pathway analysis, it is important to consider the topology (including bimolecular reactions) and stoichiometry of metabolic systems, as is done in EMA.

  Website 
• de Figueiredo LF, Schuster S, Kaleta C, Fell D, 'Can sugars be produced from fatty acids? A test case for pathway analysis tools .(Correction to Bioinformatics 24(22))'
  Bioinformatics 25 (1) (2009) pp.152-158
  ISSN: 1367-4803
  Abstract

  Motivation: In recent years, several methods have been proposed for determining metabolic pathways in an automated way based on network topology. The aim of this work is to analyse these methods by tackling a concrete example relevant in biochemistry. It concerns the question whether even-chain fatty acids, being the most important constituents of lipids, can be converted into sugars at steady state. It was proved five decades ago that this conversion using the Krebs cycle is impossible unless the enzymes of the glyoxylate shunt (or alternative bypasses) are present in the system. Using this example, we can compare the various methods in pathway analysis. Results: Elementary modes analysis (EMA) of a set of enzymes corresponding to the Krebs cycle, glycolysis and gluconeogenesis supports the scientific evidence showing that there is no pathway capable of converting acetyl-CoA to glucose at steady state. This conversion is possible after the addition of isocitrate lyase and malate synthase (forming the glyoxylate shunt) to the system. Dealing with the same example, we compare EMA with two tools based on graph theory available online, PathFinding and Pathway Hunter Tool. These automated network generating tools do not succeed in predicting the conversions known from experiment. They sometimes generate unbalanced paths and reveal problems identifying side metabolites that are not responsible for the carbon net flux. This shows that, for metabolic pathway analysis, it is important to consider the topology (including bimolecular reactions) and stoichiometry of metabolic systems, as is done in EMA.

  Website 
• Poolman MG, Miguet L, Sweetlove LJ, Fell DA, 'A Genome-Scale Metabolic Model of Arabidopsis and Some of its Properties.'
  Plant Physiology 151 (3) (2009) pp.1570-1581
  ISSN: 0032-0889 eISSN: 1532-2548
  Abstract

  We describe the construction and analysis of a genome-scale metabolic model of Arabidopsis (Arabidopsis thaliana) primarily derived from the annotations in the Aracyc database. We used techniques based on linear programming to demonstrate the following: (1) that the model is capable of producing biomass components (amino acids, nucleotides, lipid, starch, and cellulose) in the proportions observed experimentally in a heterotrophic suspension culture; (2) that approximately only 15% of the available reactions are needed for this purpose and that the size of this network is comparable to estimates of minimal network size for other organisms; (3) that reactions may be grouped according to the changes in flux resulting from a hypothetical stimulus (in this case demand for ATP) and that this allows the identification of potential metabolic modules; and (4) that total ATP demand for growth and maintenance can be inferred and that this is consistent with previous estimates in prokaryotes and yeast.

  Website 
• Chase JR, Poolman MG, Fell DA, 'Contribution of NADH Increases to Ethanol's Inhibition of Retinol Oxidation by Human ADH Isoforms'
  Alcoholism: Clinical and Experimental Research 33 (4) (2009) pp.571-580
  ISSN: 0145-6008 eISSN: 1530-0277
  Abstract

  A decrease in retinoic acid levels due to alcohol consumption has been proposed as a contributor to such conditions as fetal alcohol spectrum diseases and ethanol-induced cancers. One molecular mechanism, competitive inhibition by ethanol of the catalytic activity of human alcohol dehydrogenase (EC 1.1.1.1) (ADH) on all-trans-retinol oxidation has been shown for the ADH7 isoform. Ethanol metabolism also causes an increase in the free reduced nicotinamide adenine dinucleotide (NADH) in cells, which might reasonably be expected to decrease the retinol oxidation rate by product inhibition of ADH isoforms. To understand the relative importance of these two mechanisms by which ethanol decreases the retinol oxidation in vivo we need to assess them quantitatively. We have built a model system of 4 reactions: (1) ADH oxidation of ethanol and NAD(+), (2) ADH oxidation of retinol and NAD(+), (3) oxidation of ethanol by a generalized Ethanol(oxidase) that uses NAD(+), (4) NADH(oxidase) which carries out NADH turnover. Using the metabolic modeling package ScrumPy, we have shown that the ethanol-induced increase in NADH contributes from 0% to 90% of the inhibition by ethanol, depending on (ethanol) and ADH isoform. Furthermore, while the majority of flux control of retinaldehyde production is exerted by ADH, Ethanol(oxidase) and the NADH(oxidase) contribute as well. Our results show that the ethanol-induced increase in NADH makes a contribution of comparable importance to the ethanol competitive inhibition throughout the range of conditions likely to occur in vivo, and must be considered in the assessment of the in vivo mechanism of ethanol interference with fetal development and other diseases.

  Website 
• Poolman MG, Miguet L, Sweetlove LJ, Fell DA, 'A Genome-Scale Metabolic Model of Arabidopsis and Some of its Properties.'
  Plant Physiology 151 (3) (2009) pp.1570-1581
  ISSN: 0032-0889 eISSN: 1532-2548
  Abstract

  We describe the construction and analysis of a genome-scale metabolic model of Arabidopsis (Arabidopsis thaliana) primarily derived from the annotations in the Aracyc database. We used techniques based on linear programming to demonstrate the following: (1) that the model is capable of producing biomass components (amino acids, nucleotides, lipid, starch, and cellulose) in the proportions observed experimentally in a heterotrophic suspension culture; (2) that approximately only 15% of the available reactions are needed for this purpose and that the size of this network is comparable to estimates of minimal network size for other organisms; (3) that reactions may be grouped according to the changes in flux resulting from a hypothetical stimulus (in this case demand for ATP) and that this allows the identification of potential metabolic modules; and (4) that total ATP demand for growth and maintenance can be inferred and that this is consistent with previous estimates in prokaryotes and yeast.

  Website 
• Wolkenhauer O, Fell D, De Meyts P, Bluthgen N, Herzel H, Le Novere N, Hofer T, Schurrle K, van Leeuwen I, 'SysBiomed Report: Advancing Systems Biology for Medical Applications'
  Iet Systems Biology 3 (2009) pp.131-136
  ISSN: 1751-8849 eISSN: 1751-8857
  Abstract

  The following report selects and summarises some of the conclusions and recommendations generated throughout a series of workshops and discussions that have lead to the publication of the Science Policy Briefing (SPB) Nr. 35, published by the European Science Foundation. (Large parts of the present text are directly based on the ESF SPB. Detailed recommendations with regard to specific application areas are not given here but can be found in the SPB. Issues related to mathematical modelling, including training and the need for an infrastructure supporting modelling are discussed in greater detail in the present text.)The numerous reports and publications about the advances within the rapidly growing field of systems biology have led to a plethora of alternative definitions for key concepts. Here, with ‘mathematical modelling’ the authors refer to the modelling and simulation of subcellular, cellular and macro-scale phenomena, using primarily methods from dynamical systems theory. The aim of such models is encoding and testing hypotheses about mechanisms underlying the functioning of cells. Typical examples are models for molecular networks, where the behaviour of cells is expressed in terms of quantitative changes in the levels of transcripts and gene products. Bioinformatics provides essential complementary tools, including procedures for pattern recognition, machine learning, statistical modelling (testing for differences, searching for associations and correlations) and secondary data extracted from databases.Dynamical systems theory is the natural language to investigate complex biological systems demonstrating nonlinear spatio-temporal behaviour. However, the generation of experimental data suitable to parameterise, calibrate and validate such models is often time consuming and expensive or not even possible with the technology available today. In our report, we use the term ‘computational model’ when mathematical models are complemented with information generated from bioinformatics resources. Hence, ‘the model’ is, in reality, an integrated collection of data and models from various (possibly heterogeneous) sources. The present report focuses on a selection of topics, which were identified as appropriate case studies for medical systems biology, and adopts a particular perspective which the authors consider important. We strongly believe that mathematical modelling represents a natural language with which to integrate data at various levels and, in doing so, to provide insight into complex diseases: 1. Modelling necessitates the statement of explicit hypotheses, a process which often enhances comprehension of the biological system and can uncover critical points where understanding is lacking. 2. Simulations can reveal hidden patterns and/or counter-intuitive mechanisms in complex systems. 3. Theoretical thinking and mathematical modelling constitute powerful tools to integrate and make sense of the biological and clinical information being generated and, more importantly, to generate new hypotheses that can then be tested in the laboratory.Medical Systems Biology projects carried out recently across Europe have revealed a need for action: 4. While the need for mathematical modelling and interdisciplinary collaborations is becoming widely recognised in the biological sciences, with substantial implications for the training and research funding mechanisms within this area, the medical sciences have yet to follow this lead. 5. To achieve major breakthroughs in Medical Systems Biology, existing academic funding schemes for large-scale projects need to be reconsidered. 6. The hesitant stance of the pharmaceutical industry towards major investment in systems biology research has to be addressed. 7. Leading medical journals should be encouraged to promote mathematical modelling.

  Website 
• de Figueiredo LF, Schuster S, Kaleta C, Fell DA, 'Response to comment on 'Can sugars be produced from fatty acids ? A test case for pathway analysis tools''
  BIOINFORMATICS 25 (24) (2009) pp.3330-3331
  ISSN: 1367-4803
  Abstract LetterWebsite 
• Schuster S, Pfeiffer T, Fell DA, 'Is maximization of molar yield in metabolic networks favoured by evolution?'
  Journal of Theoretical Biology 252 (3) (2008) pp.497-504
  ISSN: 0022-5193
  Abstract

  Stoichiometric analysis of metabolic networks allows the calculation of possible metabolic flux distributions in the absence of kinetic data. In order to predict which of the possible fluxes are present under certain conditions, additional constraints and optimization principles can be applied. One approach of calculating unknown fluxes (frequently called flux balance analysis) is based on the optimality principle of maximizing the molar yield of biotransformations. Here, the relevance and applicability of that approach are examined, and it is compared with the principle of maximizing pathway flux. We discuss diverse experimental evidence showing that, often, those biochemical pathways are operative that allow fast but low-yield synthesis of important products, such as fermentation in Saccharomyces cerevisiae and several other yeast species. Together with arguments based on evolutionary game theory, this leads us to the conclusion that maximization of molar yield is by no means a universal principle.

  Website 
• Gevorgyan A, Poolman MG, Fell D, 'Detection of stoichiometric inconsistencies in biomolecular models'
  Bioinformatics 24 (19) (2008) pp.2245-2251
  ISSN: 1367-4803
  Abstract

  Motivation: Metabolic modelling provides a mathematically rigorous basis for system-level analysis of biochemical networks. However, the growing sizes of metabolic models can lead to serious problems in their construction and validation. In this work, we describe a relatively poorly investigated type of modelling error, called stoichiometric inconsistencies. These errors are caused by incorrect definitions of reaction stoichiometries and result in conflicts between two fundamental physical constraints to be satisfied by any valid metabolic model: positivity of molecular masses of all metabolites and mass conservation in all interconversions. Results: We introduce formal definitions of stoichiometric inconsistencies, inconsistent net stoichiometries, elementary leakage modes and other important fundamental properties of incorrectly defined biomolecular networks. Algorithms are described for the verification of stoichiometric consistency of a model, detection of unconserved metabolites and inconsistent minimal net stoichiometries. The usefulness of these algorithms for effective resolving of inconsistencies and for detection of input errors is demonstrated on a published genome-scale metabolic model of Saccharomyces cerevisiae and one of Streptococcus agalactiae constructed using the KEGG database.

  Website 
• de Figueiredo LF, Schuster S, Kaleta C, Fell DA, 'Can Sugars be Produced from Fatty Acids? A Test Case for Pathway Analysis Tools.'
  Bioinformatics 24 (1) (2008) pp.2615-2621
  ISSN: 1367-4803
  Abstract

  Motivation: In recent years, several methods have been proposed for determining metabolic pathways in an automated way based on network topology. The aim of this work is to analyse these methods by tackling a concrete example relevant in biochemistry. It concerns the question whether even-chain fatty acids, being the most important constituents of lipids, can be converted into sugars at steady state. It was proved five decades ago that this conversion using the Krebs cycle is impossible unless the enzymes of the glyoxylate shunt (or alternative bypasses) are present in the system. Using this example, we can compare the various methods in pathway analysis.

• Sweetlove LJ, Fell D, Fernie AR, 'Getting to Grips With the Plant Metabolic Network'
  Biochemical Journal 409 (1) (2008) pp.27-41
  Abstract

  Of all the molecular-interaction networks in plants, metabolism is by far and away the most completely described both in terms of network topology and in terms of the properties of its molecular components. Moreover, experimental tools exist that allow systematic measurement of its behaviour and there is now a wealth of data describing metabolic systems at the molecular level. Nevertheless, we remain some way off being able to exploit this knowledge to rationally engineer metabolism and we are unable to predict the nature of metabolic change under different conditions or in response to genetic intervention. One of the great problems is the persistence of the metabolic pathway paradigm when in reality, metabolism is a highly interconnected network and fixed pathways do not exist. To generate a more realistic view of metabolism, one must encapsulate information about metabolic behaviour into a model of the network as a whole. The construction of a mathematical model that describes the metabolic network of plants at ‘genome scale’ is now a plausible goal (especially in the case of model plants such as Arabidopsis). We have begun the construction of such a model and in my talk I will outline our current progress.

  Website 
• Fell D, Poolman M, Sebu C, Pidcock M, 'Modular decomposition of metabolic systems via null space analysis'
  Journal of Theoretical Biology 249 (4) (2007) pp.691-705
  ISSN: 0022-5193
  Abstract

  We describe a method by which the reactions in a metabolic system may be grouped hierarchically into sets of modules to form a metabolic reaction tree. In contrast to previous approaches, the method described here takes into account the fact that, in a viable network, reactions must be capable of sustaining a steady-state flux. In order to achieve this decomposition we introduce a new concept-”the reaction correlation coefficient, φ, and show that this is a logical extension of the concept of enzyme (or reaction) subsets. In addition to their application to modular decomposition, reaction correlation coefficients have a number of other interesting properties, including a convenient means for identifying disconnected subnetworks in a system and potential applications to metabolic engineering. The method computes reaction correlation coefficients from an orthonormal basis of the null-space of the stoichiometry matrix. We show that reaction correlation coefficients are uniquely defined, even though the basis of the null-space is not. Once a complete set of reaction correlation coefficients is calculated, a metabolic reaction tree can be determined through the application of standard programming techniques. Computation of the reaction correlation coefficients, and the subsequent construction of the metabolic reaction tree is readily achievable for genome-scale models using a commodity desk-top PC.

  Website 
• Poolman MG, Sebu C, Pidcock MK, Fell DA, 'Modular decomposition of metabolic systems via null-space analysis'
  JOURNAL OF THEORETICAL BIOLOGY 249 (4) (2007) pp.691-705
  ISSN: 0022-5193
  Abstract Once a complete set of reaction correlation coefficients is calculated, a metabolic reaction tree can be determined through the application of standard programming techniques. Computation of the reaction correlation coefficients, and the Subsequent construction of the metabolic reaction tree is readily achievable for genorne-scale models using a commodity desk-top PC. (c) 2007 Elsevier Ltd. All rights reserved.Website 
• Visser AE, Fell DA, 'Systems biology meets chromatin function - Workshop on nuclear organization'
  EMBO REPORTS 8 (5) (2007) pp.446-450
  ISSN: 1469-221X eISSN: 1469-3178
  Abstract Meeting report at the EMBO Workshop on Nuclear Organization. Fourth ELMAU Conference on Nuclear Organization, 12-15 October 2006Website 
• Poolman MG, Bonde BK, Gevorgyan A, Patel HH, Fell DA, 'Challenges to be faced in the reconstruction of metabolic networks from public databases'
  IEE Proceedings - Systems Biology 153 (5) (2006) pp.379-384
  ISSN: 1741-2471 eISSN: 1741-248X
  Abstract

  In the post-genomic era, the biochemical information for individual compounds, enzymes, reactions to be found within named organisms has become readily available. The well-known KEGG and BioCyc databases provide a comprehensive catalogue for this information and have thereby substantially aided the scientific community. Using these databases, the complement of enzymes present in a given organism can be determined and, in principle, used to reconstruct the metabolic network. However, such reconstructed networks contain numerous properties contradicting biological expectation. The metabolic networks for a number of organisms are reconstructed from KEGG and BioCyc databases, and features of these networks are related to properties of their originating database.

  Website 
• Vogt AM, Elsasser A, Pott-Beckert A, Ackermann C, Vetter SY, Yildiz M, Schoels W, Fell DA, Katus HA, Kubler W, 'Myocardial energy metabolism in ischemic preconditioning and cardioplegia: A metabolic control analysis'
  MOLECULAR AND CELLULAR BIOCHEMISTRY 278 (1/2) (2005) pp.223-232
  ISSN: 0300-8177 eISSN: 1573-4919
  Abstract For both, cardioplegia (CP) and ischemic preconditioning (IP), increased ischemic tolerance with reduction in infarct size is well documented. These cardioprotective effects are related to a limitation of high energy phosphate (HEP) depletion. As CP and IP have to be assumed to act by different mechanisms, their effects on myocardial HEP metabolism cannot be assumed to be identical. Therefore, a systematic analysis of myocardial HEP metabolism for both procedures and their combination was performed, addressing the question whether there are different effects on myocardial HEP metabolism by IP and CP. In this study, metabolic control analysis was used to analyze the regulation of HEP metabolism. In open chest pigs subjected to 45 min LAD occlusion ( index ischemia), CP and IP preserved myocardial ATP ( control ( C) 0.14 +/- 0.05 mu mol/ g wwt; CP: 0.95 +/- 0.14, IP: 0.61 +/- 0.12; p < 0.05 C vs. CP and IP) and reduced myocardial necrosis ( infarct size IA/RA: C: 90.0 +/- 3.0%; CP: 0.0 +/- 0.0% but patchy necroses; IP: 5.05 +/- 2.1%; p < 0.05 C vs. CP and IP). The effects on HEP metabolism, however, were different: CP acted predominantly by slowing down the breakdown of phosphocreatine (PCr) during early phases of ischemia ( C: Delta PCr 0 - 2 min: 5.24 +/- 0.32 mu mol/ g wwt; CP: Delta PCr 0 - 2 min: 3.38 +/- 0.23 eta mol/ g wwt, p < 0.05 vs. C), leaving ATP breakdown during later stages unaffected ( C: Delta ATP 5 - 45 min: 1.77 +/- 0.11 mu mol/ g wwt CP: Delta ATP 5 - 45 min: 1.59 +/- 0.28 mu mol/ g wwt, n. s. vs. C). In contrast to CP, in IP PCr breakdown was even increased ( IP: Delta PCr 0 - 2 min: 7.06 +/- 0.34 mu mol/ g wwt, p < 0.05 vs. C), but ATP depletion greatly attenuated ( IP: Delta ATP 5 - 45 min: 0.48 +/- 0.10 mu mol/ g wwt, p < 0.05 vs. C and CP). Combining IP and CP yielded an additive effect with slowing down the breakdown of both PCr ( IP+ CP: Delta PCr 0 - 2 min: 5.09 +/- 0.35 mu mol/ g wwt, p < 0.05 vs. C and IP) and ATP ( IP+ CP: Delta ATP 5 - 45 min: 0.56 +/- 0.48 mu mol/ g wwt, p < 0.05 vs. C and CP), resulting in a higher ATP content at the end of index ischemia (1.86 +/- 0.46 mu mol/ g wwt, p < 0.05 vs. C, CP and IP). Compared to IP, combining IP+ CP achieved also a further reduction in infarct size (IA/RA: 0.0 +/- 0.0%, p < 0.05 vs IP) and - compared to CP - a disappearance of the patchy necroses. The concept of major differences in myocardial HEP metabolism during CP and IP is further supported at a molecular level by metabolic control analysis. CP but not IP slowed down the CK reaction velocity at high PCr levels. In contrast to CP exerting a continuous decline in vATPase for any given ATP level, in IP myocardium ATPase reaction velocity was even increased at higher ATP contents, whereas a marked decrease in ATPase reaction velocity was found if ATP levels decreased. The equilibrium of the CK-reaction remained unchanged following CP, whereas IP induced a changing CK equilibrium, which was the more shifted towards PCr the more myocardial HEP content decreased. The data demonstrate different effects of CP and IP on myocardial HEP metabolism, i.e. PCr and ATP breakdown as well as the apparent equilibrium of the creatine kinase (CK)-reaction. For these reasons the combination of the two protective interventions has an additive effect.Website 
• Schafer JRA, Fell DA, Rothman D, Shulman RG, 'Protein Phosphorylation Can Regulate Metabolite Concentrations Rather Than Control Flux: The Example of Glycogen Synthase'
  Proceedings of the National Academy of Sciences 101 (2004) pp.1485-1490
  ISSN: 0027-8424 eISSN: 1091-6490
  Abstract

  Despite dramatic increases in glucose influx during the transition from fasting to fed states, plasma glucose concentration remains tightly controlled. This constancy is in large part due to the capacity of skeletal muscle to absorb excess glucose and store it as glycogen. The magnitude of this capacity is controlled by insulin by way of regulated insertion of glucose transporters into the muscle cell membrane. Here, we examine the mechanism by which muscle cells are able to tolerate large flux increases across their transporters without significantly changing their own metabolite pools. MCA was used to probe data sets that measured the effects of changing plasma glucose and/or insulin concentrations on the rates of glycogen synthesis and the concentrations of metabolites, particularly glucose-6-phosphate. We find that homeostasis is achieved by insulin-dependent phosphorylation changes in GSase sensitivity to the upstream metabolite glucose-6-phosphate. The centrality of GSase to homeostasis resolves the paradox of its sensitivity to allosteric and covalent regulation despite its minimal role in flux control. The importance of this role for enzymatic phosphorylation to diabetes pathology is discussed, and its general applicability is suggested.

  Website 
• Poolman MG, Venkatesh KV, Pidcock MK, Fell DA, 'A Method for the Determination of Flux in Elementary Modes, and its Application to Lactobacillus rhamnosus'
  Biotechnology and Bioengineering 88 (2004) pp.601-612
  ISSN: 0006-3592
  Website 
• Poolman MG, Assmus HE, Fell DA, 'Applications of Metabolic Modelling to Plant Metabolism'
  Journal of Experimental Botany 55 (2004) pp.1177-1186
  ISSN: 0022-0957 eISSN: 1460-2431
  Abstract

  In this paper some of the general concepts underpinning the computer modelling of metabolic systems are introduced. The difference between kinetic and structural modelling is emphasized, and the more important techniques from both, along with the physiological implications, are described. These approaches are then illustrated by descriptions of other work, in which they have been applied to models of the Calvin cycle, sucrose metabolism in sugar cane, and starch metabolism in potatoes.

  Website 
• Papin JA, Price ND, Wiback SJ, Fell DA, Palsson BO, 'Metabolic pathways in the post-genome era'
  TRENDS IN BIOCHEMICAL SCIENCES 28 (5) (2003) pp.250-258
  ISSN: 0968-0004 eISSN: 0167-7640
  Abstract Metabolic pathways are a central paradigm in biology. Historically, they have been defined on the basis of their step-by-step discovery. However, the genome-scale metabolic networks now being reconstructed from annotation of genome sequences demand new network-based definitions of pathways to facilitate analysis of their capabilities and functions, such as metabolic versatility and robustness, and optimal growth rates. This demand has led to the development of a new mathematically based analysis of complex, metabolic networks that enumerates all their unique pathways that take into account all requirements for cofactors and byproducts. Applications include the design of engineered biological systems, the generation of testable hypotheses regarding network structure and function, and the elucidation of properties that can not be described by simple descriptions of individual components (such as product yield, network robustness, correlated reactions and predictions of minimal media). Recently, these properties have also been studied in genome-scale networks. Thus, network-based pathways are emerging as an important paradigm for analysis of biological systems.Website 
• Chassagnole C, Quentin E, Fell DA, de Atauri P, Mazat JP, 'Dynamic simulation of pollutant effects on the threonine pathway in Escherichia coli'
  COMPTES RENDUS BIOLOGIES 326 (5) (2003) pp.501-508
  ISSN: 1631-0691
  Abstract The enzymatic activities of threonine pathway in Escherichia coli are sensitive to pollutants such as cadmium, copper and mercury. which. even at low concentration, can substantially decrease or even block the pathway at several steps. Our aim was to investigate the complex effects on a metabolic pathway of such general enzyme inhibitors with several sites of action, using a previously developed computer simulation of the pathway. For this purpose, the inhibition parameters were experimentally determined and incorporated in the model. The calculation of the flux control coefficient distribution between the five steps of the threonine pathway showed that control remains shared between the three first steps under most inhibition conditions. Response coefficient analysis shows that the inhibition of aspartate semialdehyde dehydrogenase is quantitatively dominant in most circumstances. (C) 2003 Academie des sciences. Published by editions scientifiques et medicales Elsevier SAS. All rights reserved.Website 
• Poolman MG, Fell DA, Raines CA, 'Elementary modes analysis of photosynthate metabolism in the chloroplast stroma'
  EUROPEAN JOURNAL OF BIOCHEMISTRY 270 (2003) pp.430-439
  ISSN: 0014-2956 eISSN: 1432-1033
  Abstract We briefly review the metabolism of the chloroplast stroma, and describe the structural modelling technique of elementary modes analysis. The technique is applied to a model of chloroplast metabolism to investigate viable pathways in the light, in the dark, and in the dark with the addition of sedoheptulose-1,7-bisphosphatase (normally inactive in the dark). The results of the analysis show that it is possible for starch degradation to enhance photosynthetic triose phosphate export in the light, but the reactions of the Calvin cycle alone are not capable of providing a sustainable flux from starch to triose phosphate in the dark. When reactions of the oxidative pentose phosphate pathway are taken into consideration, triose phosphate export in the dark becomes possible by the operation of a cyclic pathway not previously described. The effect of introducing sedoheptulose-1,7-bisphosphatase to the system are relatively minor and, we predict, innocuous in vivo. We conclude that, in contrast with the traditional view of the Calvin cycle and oxidative pentose phosphate pathway as separate systems, they are in fact, in the context of the chloroplast, complementary and overlapping components of the same system.Website 
• Vogt AM, Nef H, Schaper J, Poolman M, Fell DA, Kubler W, Elsasser A, 'Metabolic Control Analysis of Anaerobic Glycolysis in Human Hibernating Myocardium Replaces Traditional Concepts of Flux Control'
  Febs Letters 517 (2002) pp.245-250
  ISSN: 0014-5793 eISSN: 1873-3468
  Abstract

  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.

  Website 
• Vogt AM, Poolman M, Ackermann C, Yildiz M, Schoels W, Fell DA, Kubler W, 'Regulation of Glycolytic Flux in Ischemic Preconditioning - a Study Employing Metabolic Control Analysis'
  Journal of Biological Chemistry 277 (2002) pp.24411-24419
  ISSN: 0021-9258
  Abstract

  Exact adjustment of the Embden-Meyerhof pathway (EMP) is an important issue in ischemic preconditioning (IP) because an attenuated ischemic lactate accumulation contributes to myocardial protection. However, precise mechanisms of glycolytic flux and its regulation in IP remain to be elucidated. In open chest pigs, IP was achieved by two cycles of 10-min coronary artery occlusion and 30-min reperfusion prior to a 45-min index ischemia and 120-min reperfusion. Myocardial contents in glycolytic intermediates were assessed by high performance liquid chromatographic analysis of serial myocardial biopsies under control conditions and IP. Detailed time courses of metabolite contents allow an in-depth description of EMP regulation during index ischemia using metabolic control analysis. IP reduced myocardial infarct size (control, 90.0 ± 3.1 versus 5.05 ± 2.1%;p < 0.001) and attenuated myocardial lactate accumulation (end-ischemic contents, 31.9 ± 4.47versus 10.3 ± 1.26 μmol/wet weight,p < 0.0001), whereby a decrease in anaerobic glycolytic flux by at least 70% could constantly be observed throughout index ischemia. By calculation of flux:metabolite co-responses, the mechanisms of glycolytic regulation were investigated. The continuous deceleration of EMP flux in control myocadium could neither be explained on the basis of substrate availability nor be attributed to regulatory “key enzymes,” as multisite regulation was employed for flux adjustment. In myocardium subjected to IP, an even pronounced deceleration of EMP flux during index ischemia was observed. Again, the adjustment of EMP flux was because of multisite modulation without any evidence for flux limitation by substrate availability or a key enzyme. However, IP changed the regulatory properties of most EMP enzymes, and some of these patterns could not be explained on the basis of substrate kinetics. Instead, other regulatory mechanisms, which have previously not yet been described for EMP enzymes, must be considered. These altered biochemical properties of the EMP enzymes have not yet been described.

  Website 
• Vauclare P, Kopriva S, Fell D, Suter M, Sticher L, von Ballmoos P, Krahenbuhl U, den Camp RO, Brunold C, 'Flux Control of Sulphate Assimilation in Arabidopsis Thaliana: Adenosine 5 -phosphosulphate Reductase Is More Susceptible Than Atp Sulphurylase to Negative Control By Thiols'
  Plant Journal 31 (6) (2002) pp.729-740
  ISSN: 0960-7412
  Abstract

  The effect of externally applied L-cysteine and glutathione (GSH) on ATP sulphurylase and adenosine 5'-phosphosulphate reductase (APR), two key enzymes of assimilatory sulphate reduction, was examined in Arabidopsis thaliana root cultures. Addition of increasing L-cysteine to the nutrient solution increased internal cysteine, gamma-glutamylcysteine and GSH concentrations, and decreased APR mRNA, protein and extractable activity. An effect on APR could already be detected at 0.2 mm L-cysteine, whereas ATP sulphurylase was significantly affected only at 2 mm L-cysteine. APR mRNA, protein and activity were also decreased by GSH at 0.2 mm and higher concentrations. In the presence of L-buthionine-S, R-sulphoximine (BSO), an inhibitor of GSH synthesis, 0.2 mm L-cysteine had no effect on APR activity, indicating that GSH formed from cysteine was the regulating substance. Simultaneous addition of BSO and 0.5 mm GSH to the culture medium decreased APR mRNA, enzyme protein and activity. ATP sulphurylase activity was not affected by this treatment. Tracer experiments using (35)SO(4)(2-) in the presence of 0.5 mm L-cysteine or GSH showed that both thiols decreased sulphate uptake, APR activity and the flux of label into cysteine, GSH and protein, but had no effect on the activity of all other enzymes of assimilatory sulphate reduction and serine acetyltransferase. These results are consistent with the hypothesis that thiols regulate the flux through sulphate assimilation at the uptake and the APR step. Analysis of radioactive labelling indicates that the flux control coefficient of APR is more than 0.5 for the intracellular pathway of sulphate assimilation. This analysis also shows that the uptake of external sulphate is inhibited by GSH to a greater extent than the flux through the pathway, and that the flux control coefficient of APR for the pathway, including the transport step, is proportionately less, with a significant share of the control exerted by the transport step.

   

   

• Carlson R, Fell D, Srienc F, 'Metabolic Pathway Analysis of a Recombinant Yeast for Rational Strain Development'
  Biotechnology and Bioengineering 79 (2002) pp.121-134
  ISSN: 978-94-010-6021-9
  Abstract

  Elementary mode analysis has been used to study a metabolic pathway model of a recombinant Saccharomyces cerevisiae system that was genetically engineered to produce the bacterial storage compound poly-β-hydroxybutyrate (PHB). The model includes biochemical reactions from the intermediary metabolism and takes into account cellular compartmentalization as well as the reversibility/irreversibility of the reactions. The reaction network connects the production and/or consumption of eight external metabolites including glucose, acetate, glycerol, ethanol, PHB, CO2, succinate, and adenosine triphosphate (ATP). Elementary mode analysis of the wild-type S. cerevisiae system reveals 241 unique reaction combinations that balance the eight external metabolites. When the recombinant PHB pathway is included, and when the reaction model is altered to simulate the experimental conditions when PHB accumulates, the analysis reveals 20 unique elementary modes. Of these 20 modes, 7 produce PHB with the optimal mode having a theoretical PHB carbon yield of 0.67. Elementary mode analysis was also used to analyze the possible effects of biochemical network modifications and altered culturing conditions. When the natively absent ATP citrate-lyase activity is added to the recombinant reaction network, the number of unique modes increases from 20 to 496, with 314 of these modes producing PHB. With this topological modification, the maximum theoretical PHB carbon yield increases from 0.67 to 0.83. Adding a transhydrogenase reaction to the model also improves the theoretical conversion of substrate into PHB. The recombinant system with the transhydrogenase reaction but without the ATP citrate-lyase reaction has an increase in PHB carbon yield from 0.67 to 0.71. When the model includes both the ATP citrate-lyase reaction and the transhydrogenase reaction, the maximum theoretical carbon yield increases to 0.84. The reaction model was also used to explore the possibility of producing PHB under anaerobic conditions. In the absence of oxygen, the recombinant reaction network possesses two elementary modes capable of producing PHB. Interestingly, both modes also produce ethanol. Elementary mode analysis provides a means of deconstructing complex metabolic networks into their basic functional units. This information can be used for analyzing existing pathways and for the rational design of further modifications that could improve the system's conversion of substrate into product. 

  Website 
• Schuster S, Hilgetag C, Woods JH, Fell DA, 'Reaction routes in biochemical reaction systems: Algebraic properties, validated calculation procedure and example from nucleotide metabolism'
  JOURNAL OF MATHEMATICAL BIOLOGY 45 (2) (2002) pp.153-181
  ISSN: 0303-6812 eISSN: 1432-1416
  Abstract Elementary flux modes (direct reaction routes) are minimal sets of enzymes that can operate at steady state, with all irreversible reactions used in the appropriate direction. They can be interpreted as component,pathways of a (bio)chemical reaction network. Here, two different definitions of elementary modes are given and their equivalence is proved. Several algebraic properties of elementary modes are then presented and proved. This concerns, amongst other features, the minimal number of enzymes of the network not used in an elementary mode and the situations where irreversible reactions are replaced by reversible ones. Based on these properties, a refined algorithm is presented, and it is formally proved that this algorithm will exclusively generate all the elementary flux modes of an arbitrary network containing reversible or irreversible reactions or both. The algorithm is illustrated by a biochemical example relevant in nucleotide metabolism. The computer implementation in two different programming languages is discussed.Website 
• de Atauri P, Fell DA, Chassagnole C, Magret D, Mazat JP, Cascante M, 'Dependence of control coefficient distribution on the boundaries of a metabolic system: A generalized analysis of the effects of additional input and output reactions to a linear pathway'
  JOURNAL OF THEORETICAL BIOLOGY 215 (2) (2002) pp.239-251
  ISSN: 0022-5193
  Abstract Both experimental and theoretical studies of metabolism are likely to relate to a segment that has been isolated for analytical purposes. In practice, it will be embedded in the whole of cellular metabolism. Thus, it is necessary to consider how conclusions about the control of an isolated pathway may be modified in this wider context where the input and output metabolites are considered as variables of cellular metabolism. Here, we analyse the effect of expanding a linear metabolic pathway by adding an extra input or an extra output. In particular, we analyse the effect of the elasticities of the extra steps on control coefficients. We derive matrix algebraic relationships for obtaining flux and concentration control coefficients from expressions depending on these extra elasticities and on parameters (elasticities and control coefficients) of the original pathway. These equations can be shown in certain cases to be generalized versions of earlier rescaling relationships and to be related to top-down analysis, but also apply where the new variable metabolite of the expanded pathway is an effector of more than one step of the original pathway. We use our relationships to analyse the dependence or independence of control coefficients upon these extra elasticities for the published analyses of the pathway of mammalian serine biosynthesis (Fell & Snell, 1988) and Escherischia coli threonine biosynthesis (Chassagnole et al., 2001). The same analysis can be applied to determine whether the transport reactions of substrates and products of a pathway in and out of a cell need to be included in estimations of the control coefficients of the enzymes. (C) 2002 Elsevier Science Ltd. All rights reserved.Website 
• Chassagnole C, Rais B, Quentin E, Fell DA, Mazat JP, 'An integrated study of threonine-pathway enzyme kinetics in Escherichia coli'
  Biochemical Journal 356 (2001) pp.415-423
  ISSN: 0264-6021
  Abstract

  We have determined the kinetic parameters of the individual steps of the threonine pathway from aspartate inEscherichia coli under a single set of experimental conditions chosen to be physiologically relevant. Our aim was to summarize the kinetic behaviour of each enzyme in a single tractable equation that takes into account the effect of the products as competitive inhibitors of the substrates in the forward reaction and also, when appropriate (e.g. near-equilibrium reactions), as substrates of the reverse reactions. Co-operative feedback inhibition by threonine and lysine was also included as necessary. We derived the simplest rate equations that describe the salient features of the enzymes in the physiological range of metabolite concentrations in order to incorporate them ultimately into a complete model of the threonine pathway, able to predict quantitatively the behaviour of the pathway under natural or engineered conditions.

  Website 
• Chassagnole C, Fell DA, Rais B, Kudla B, Mazat JP, 'Control of the threonine-synthesis pathway in Escherichia coli: A theoretical and experimental approach'
  Biochemical Journal 356 (2001) pp.433-444
  ISSN: 0264-6021
  Abstract

  A computer simulation of the threonine-synthesis pathway in Escherichia coli Tir-8 has been developed based on our previous measurements of the kinetics of the pathway enzymes under near-physiological conditions. The model successfully simulates the main features of the time courses of threonine synthesis previously observed in a cell-free extract without alteration of the experimentally determined parameters, although improved quantitative fits can be obtained with small parameter adjustments. At the concentrations of enzymes, precursors and products present in cells, the model predicts a threonine-synthesis flux close to that required to support cell growth. Furthermore, the first two enzymes operate close to equilibrium, providing an example of a near-equilibrium feedback-inhibited enzyme. The predicted flux control coefficients of the pathway enzymes under physiological conditions show that the control of flux is shared between the first three enzymes: aspartate kinase, aspartate semialdehyde dehydrogenase and homoserine dehydrogenase, with no single activity dominating the control. The response of the model to the external metabolites shows that the sharing of control between the three enzymes holds across a wide range of conditions, but that the pathway flux is sensitive to the aspartate concentration. When the model was embedded in a larger model to simulate the variable demands for threonine at different growth rates, it showed the accumulation of free threonine that is typical of the Tir-8 strain at low growth rates. At low growth rates, the control of threonine flux remains largely with the pathway enzymes. As an example of the predictive power of the model, we studied the consequences of over-expressing different enzymes in the pathway.

   
  Website 
• Wagner A, Fell DA, 'The small world inside large metabolic networks'
  PROCEEDINGS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES 268 (2001) pp.1803-1810
  ISSN: 0962-8452
  Abstract The metabolic network of the catabolic, energy and biosynthetic metabolism of Escherichia colt is a paradigmatic case for the large genetic and metabolic networks that functional genomics efforts are beginning to elucidate. To analyse the structure of previously unknown networks involving hundreds or thousands of components by simple visual inspection is impossible, and quantitative approaches are needed to analyse them. We have undertaken a graph theoretical analysis of the E. coli metabolic network and find that this network is a small-world graph, a type of graph distinct from both regular and random networks and observed in a variety of seemingly unrelated areas, such as friendship networks in sociology, the structure of electrical power grids, and the nervous system of Caenorhabditis elegans. Moreover, the connectivity of the metabolites follows a power law, another unusual but by no means rare statistical distribution. This provides an objective criterion for the centrality of the tricarboxylic acid cycle to metabolism. The small-world architecture may serve to minimize transition times between metabolic states, and contains evidence about the evolutionary history of metabolism.
• Rais B, Chassagnole C, Letellier T, Fell DA, Mazat JP, 'Threonine synthesis from aspartate in Escherichia coli cell-free extracts: pathway dynamics'
  BIOCHEMICAL JOURNAL 356 (2001) pp.425-432
  ISSN: 0264-6021
  Abstract We have developed an experimental model of the whole threonine pathway that allows us to study the production of threonine from aspartate under different conditions. The model consisted of a desalted crude extract of Escherichia coli to which we added the substrates and necessary cofactors of the pathway: aspartate, ATP and NADPH. In this experimental model we measured not only the production of threonine, but also the time dependence of all the intermediate metabolites and of the initial substrates, aspartate, ATP and NADPH. A stoichiometric conversion of precursors into threonine was observed. We have derived conditions in which a quasi steady state can be transiently observed and used to simulate physiological conditions of functioning of the pathway in the cell. The dependence of threonine synthesis and of the aspartate and NADPH consumption on the initial aspartate and threonine concentrations exhibits greater sensitivity to the aspartate concentration than to the threonine concentration in these non-steady-state conditions. A response to threonine is only observed in a narrow concentration range from 0.23 to 2 mM.
• Poolman MG, Olcer H, Lloyd JC, Raines CA, Fell DA, 'Computer modelling and experimental evidence for two steady states in the photosynthetic Calvin cycle'
  EUROPEAN JOURNAL OF BIOCHEMISTRY 268 (2001) pp.2810-2816
  ISSN: 0014-2956 eISSN: 1432-1033
  Abstract We present observations of photosynthetic carbon dioxide assimilation, and leaf starch content from genetically modified tobacco (Nicotiana tabacum) plants in which the activity of the Calvin cycle enzyme, sedoheptulose-1,7-bisphosphatase, is reduced by an antisense construct. The measurements were made on leaves of varying ages and used to calculate the flux control coefficients of sedoheptulose-1,7-bisphosphatase over photosynthetic assimilation and starch synthesis. These calculations suggest that control coefficients for both are negative in young leaves, and positive in mature leaves. This behaviour is compared to control coefficients obtained from a detailed computer model of the Calvin cycle. The comparison demonstrates that the experimental observations are consistent with bistable behaviour exhibited by the model, and provides the first experimental evidence that such behaviour in the Calvin cycle occurs in vivo as well as in silico.
• Poolman MG, Fell DA, Thomas S, 'Modelling Photosynthesis and Its Control'
  Journal of Experimental Botany 51 (1) (2000) pp.319-328
  ISSN: 0022-0957 eISSN: 1460-2431
  Abstract

  The dynamic and steady‐state behaviour of a computer simulation of the Calvin cycle reactions of the chloroplast, including starch synthesis and degradation, and triose phosphate export have been investigated. A major difference compared with previous models is that none of the reversible reactions are assumed to be at equilibrium. The model can exhibit alternate steady states of low or high carbon assimilation flux, with hysteresis in the transitions between the steady states induced by environmental factors such as phosphate and light intensity. The enzymes which have the greatest influence on the flux have been investigated by calculation of their flux control coefficients. Different patterns of control are exhibited over the assimilation flux, the flux to starch and the flux to cytosolic triose phosphate. The assimilation flux is mostly sensitive to sedoheptulose bisphosphatase and Rubisco, with the exact distribution depending on their relative activities. Other enzymes, particularly the triose phosphate translocator, become more influential when other fluxes are considered. These results are shown to be broadly consistent with observations on transgenic plants.

  Website 
• Brightman FA, Fell DA, 'Differential Feedback Regulation of the MAPK Cascade Underlies the Quantitative Differences in EGF and NGF Signalling in PC12 Cells'
  Febs Letters 482 (2000) pp.169-174
  ISSN: 0014-5793 eISSN: 1873-3468
  Abstract

  Although epidermal growth factor (EGF) induces transient activation of Ras and the mitogen-activated protein kinase (MAPK) cascade in PC12 cells, whereas nerve growth factor (NGF) stimulates sustained activation, the basis for these contrasting responses is not known. We have developed a computer simulation of EGF-induced MAPK cascade activation, which provides quantitative evidence that feedback inhibition of the MAPK cascade is the most important factor in determining the duration of cascade activation. Hence, we propose that the observed quantitative differences in EGF and NGF signalling can be accounted for by differential feedback regulation of the MAPK cascade.

   

   

  Website 
• Fell D, 'Tonic Blues'
  New Scientist 165 (2000) pp.U1-U1
  ISSN: 0262-4079
  
• Schuster S, Fell DA, Dandekar T, 'A general definition of metabolic pathways useful for systematic organization and analysis of complex metabolic networks'
  NATURE BIOTECHNOLOGY 18 (2000) pp.326-332
  ISSN: 1087-0156 eISSN: 1546-1696
  Abstract A set of linear pathways often does not capture the full range of behaviors of a metabolic network. The concept of 'elementary flux modes' provides a mathematical tool to define and comprehensively describe all metabolic routes that are both stoichiometrically and thermodynamically feasible for a group of enzymes. We have used this concept to analyze the interplay between the pentose phosphate pathway (PPP) and glycolysis. The set of elementary modes for this system involves conventional glycolysis, a futile cycle, all the modes of PPP function described in biochemistry textbooks, and additional modes that are a priori equally entitled to pathway status. Applications include maximizing product yield in amino acid and antibiotic synthesis, reconstruction and consistency checks of metabolism from genome data, analysis of enzyme deficiencies, and drug target identification in metabolic networks.
• Thomas S, Fell DA, 'A Control Analysis Exploration of the Role of ATP Utilisation in Glycolytic-flux Control and Glycolytic-metabolite-concentration Regulation'
  European Journal of Biochemistry 258 (1999) pp.956-967
  ISSN: 0014-2956 eISSN: 1432-1033
  Abstract

  A theoretical metabolic-control-analysis approach has been used to study aspects of glycolytic-flux control and carbon-metabolite regulation, particularly the role of ATP demand (ATPase), in order to determine what general features of the regulation of energy metabolism would be consistent with good carbon-metabolite homeostasis in the face of large changes in carbon flux. On the basis of a semi-quantitative control-analysis model, incorporating estimates of substrate, product and effector actions on the enzymes, the experimentally observed characteristics of glycolytic-flux changes prove to impose constraints on the feasible ranges of these estimates. This leads to the identification of several features of energy metabolism, each of which is necessary but not sufficient to explain the observations; although most of these have been advocated previously (such as AMP activation of phosphofructokinase (PFK), ADP inhibition of ATPase and the role of energy charge or ATP/ADP ratio), our analysis allows their relative importance to be assessed. In the model, the distribution of flux control depends primarily on ADP inhibition of ATPase, and on the activation of PFK by AMP; increase in ADP inhibition of ATPase increases the control on PFK; increase in AMP activation of PFK increases control on ATPase. PFK exerts greater flux control than does ATPase over approximately 50 % of the ranges (parameter space) studied, but its control is sufficiently high to achieve sizeable flux increases over less than 20 % of the space. Furthermore, control by alteration in PFK activity is shown to result in poor glycolytic metabolite homeostasis over the entire parameter space studied. However, over a large proportion of the parameter space, control by activation of ATPase can lead to large flux changes, i.e. high flux control, coupled with excellent glycolytic-metabolite homeostasis, similar to that observed in working muscle. As well as altering the relative degrees of flux control invested in PFK and ATPase, ADP inhibition of ATPase and AMP activation of PFK have pronounced effects on the homeostatic properties of the system. Stronger ADP inhibition of ATPase results in improved homeostasis of glycolytic metabolites, ATP and ADP in response to PFK activation, whereas stronger activation of PFK by AMP improves the homeostasis of these three quantities in response to ATPase activation. The results are further evidence of the potential for physiological ATP demand to exert control over glycolytic flux, but additionally show that the known effector interactions, in addition to their previously known role in ATP regulation, could contribute to the remarkable homeostasis of glycolytic-metabolite levels observed in vivo. They further indicate that quantitative characterisation of likely domains of behaviour of metabolic systems can be achieved by an algebraic analysis that is not highly dependent on a full and precise knowledge of the molecular details of the kinetic/regulatory properties of the enzymes, but that still allows an assessment of whether hypotheses regarding the system are feasible and sufficient to account for the observations.

  Website 
• Schuster S, Dandekar T, Fell DA, 'Detection of elementary flux modes in biochemical networks: a promising tool for pathway analysis and metabolic engineering'
  TRENDS IN BIOTECHNOLOGY 17 (1999) pp.53-60
  ISSN: 0167-7799 eISSN: 1879-3096
  Abstract Rational metabolic engineering requires powerful theoretical methods such as pathway analysis, in which the topology of metabolic networks is considered. All metabolic capabilities in steady states are composed of elementary flux modes, which are minimal sets of enzymes that can each generate valid steady states. The modes of the fructose-2,6-bisphosphate cycle, the combined tricarboxylic-acjd-cycle-glyoxylate-shunt system and tryptophan synthesis are used here for illustration. This approach can be used for many biotechnological applications such as increasing the yield of a product, channelling a product into desired pathways and in functional reconstruction from genomic data.
• Thomas S, Fell DA, 'The Role of Multiple Enzyme Activation in Metabolic Flux Control'
  Advances in Enzyme Regulation 38 (1) (1998) pp.65-85
  ISSN: 0065-2571
  Abstract

  Over recent years, the concept that flux through a metabolic pathway is controlled by a single rate-limiting enzyme has been challenged from a number of quarters. We have presented three lines of evidence that the control of pathway flux by activation of multiple several steps, including steps responsible for consumption of pathway products, is an important feature of physiological flux control in response to external stimmuli. Theoretical tools exist that can be used to analyze the distribution of flux control between the steps involved in signal transduction and enzyme activation.

  Website 
• Fell DA, 'Increasing the flux in metabolic pathways: A metabolic control analysis perspective'
  BIOTECHNOLOGY AND BIOENGINEERING 58 (1998) pp.121-124
  ISSN: 0006-3592
  Abstract The problems of engineering increased flux in metabolic pathways are analyzed in terms of the understanding provided by metabolic control analysis. Overexpression of a single enzyme is unlikely to be effective unless it is known to have a high flux control coefficient, which can be used as an approximate predictive tool. This is likely to rule out enzymes subject to feedback inhibition, because it transfers control downstream from the inhibited enzyme to the enzymes utilizing the feedback metabolite. Although abolishing feedback inhibition can restore flux control to an enzyme, it is also likely to cause large increases in the concentrations of metabolic intermediates. Simultaneous and coordinated overexpression of most of the enzymes in a pathway can, in principle, produce substantial flux increases without changes in metabolite levels, though technically it may be difficult to achieve. It is, however, closer to the method used by cells to change flux levels, where coordinated changes in the level of activity of pathway enzymes are the norm. Another option is to increase the demand for the pathway product, perhaps by increasing its rate of excretion or removal. (C) 1998 John Wiley & Sons, Inc.
• Thomas S, Mooney PJ, Burrell MM, Fell DA, 'Metabolic Control Analysis of Glycolysis in Tuber Tissue of Potato (solanum Tuberosum): Explanation for the Low Control Coefficient of Phosphofructokinase Over Respiratory Flux'
  Biochemical Journal 322 (1) (1997) pp.119-127
  ISSN: 0264-6021
  Abstract

  We have applied Metabolic Control Analysis (MCA) in an attempt to determine the distribution of glycolytic flux control between the steps of glycolysis in aged disks of potato tuber under aerobic conditions, using concentrations of glycolytic metabolites in tuber tissue from a range of transgenic potato plants and published enzyme kinetic data. We modelled the substrate and effector kinetics of potato tuber phosphofructokinase (PFK) by reanalysing published results. Despite the scarcity of reliable kinetic data, our results are in agreement with experimental findings namely that, under the conditions described, PFK has little control over glycolytic flux. Furthermore our analysis predicts that under these conditions far more control lies in the dephosphorylation of phosphoenolpyruvate and/or in the steps beyond. We have validated the results of our analysis in two ways. First, predictions based on calculated concentration control coefficients from the analysis show generally good agreement with observed metabolite deviation indices discussed in the preceding paper [Thomas, Mooney, Burrell, and Fell (1997) Biochem. J. 322, 111-117]. Second, sensitivity analysis of our results shows that the calculated control coefficients are robust to errors in the elasticities used in the analysis, of which relatively few need to be known accurately. Experimental and control analysis results agree with previous predictions of MCA that strong co-operative feedback inhibition of enzymes serves to move flux control downstream of the inhibiting metabolite. We conclude that MCA can successfully model the outcome of experiments in the genetic manipulation of enzyme amounts.

• Thomas S, Mooney PJF, Burrell MM, Fell DA, 'Finite Change Analysis of Glycolytic Intermediates in Tuber Tissue of Lines of Transgenic Potato (solanum Tuberosum) Overexpressing Phosphofructokinase'
  Biochemical Journal 322 (1997) pp.111-117
  ISSN: 0264-6021
  Abstract

  Genetically engineered organisms overexpressing phosphofructokinase (PFK), a supposed 'regulatory' step of glycolysis, often show little or no measurable change in glycolytic or respiratory flux, although the concentrations of glycolytic intermediates may change. We have used the finite change theory of Metabolic Control Analysis (MCA) to analyse the concentrations of glycolytic metabolites in aged disks of tuber tissue from four lines of transgenic potatoes expressing different amounts of PFK that, under aerobic conditions, showed statistically indistinguishable rates of respiration. The constancy of the metabolites' concentration deviation indices for different increases in PFK expression indicated that the metabolite changes from a graded series, excluding the possibility of anomalous behaviour that might be observed in a single transgenic line. Consequently we were able to use the finite change method to validate the results of an MCA model of tuber glycolysis [Thomas, Mooney, Burrell and Fell (1997) Biochem. J. 322, 119-127]. Furthermore the metabolite changes with PFK activity are evidence that near-equilibrium steps do not transmit increased substrate concentrations down the pathway without attenuation. Our results support the view that flux increase by activation of a single enzyme early in the pathway will, contrary to expectations, be of limited effectiveness in achieving flux increases.

   

• Thomas S, Fell DA, 'Design of Metabolic Control for Large Flux Changes'
  Journal of Theoretical Biology 182 (1996) pp.285-298
  ISSN: 0022-5193
  Abstract

  Metabolic Control Analysis has invalidated many traditional biochemical concepts of control, in particular the rate-limiting step. However, it has not been used to question the mechanisms by which pathway flux is thought to be controlled, such as the action of allosteric effectors or of covalent modification mechanisms. Here we use Control Analysis and computer simulation to examine the response of pathway segments to change in flux imposed by action on an enzyme outside the segment. Whether these segments contain near-equilibrium enzyme-catalysed reactions, cooperative enzymes, feedforward activation loops or feedback inhibition loops, their responses are significantly different from those observedin vivo. In particular, they do not exhibit the remarkable degrees of metabolite homoeostasis during large flux changes that have frequently been observed experimentally. On the other hand, near-constant levels of metabolites in spite of large changes of flux are consistent with our recent proposal thatmulti-site modulation—simultaneous activation of many pathway steps—is the normal method by which metabolism is controlled.

  Website 
• Fell D, 'Desert Naivety'
  New Scientist 149 (1996) pp.49-49
  ISSN: 0262-4079
  Abstract

  Your article describes a proposal by Japanese researchers to improve the desert tolerance of plants by giving them the gene for a more efficient form of the CO2-fixing enzyme RuBisCo (Technology, 20 January). This gives credence to a naive proposal that is very unlikely to work.

   

  Despite the support given in many biochemistry textbooks to the supposed role of RuBisCo in limiting the rate of photosynthesis, it is unlikely that the enzyme would pose a serious limitation in desert conditions. Mark Stitt and his colleagues, then at the University of Bayreuth, Germany, quantified the effect of varying the activity of RuBisCo in tobacco plants under a variety of environmental conditions. They found that RuBisCo alone does not limit the rate, except when photosynthesis is being driven at high rates by above normal light intensity, carbon dioxide levels and humidity. Other factors are at least as important under conditions of reduced ...

• Fell D, 'Dr Henrik Kacser (1918-1995)'
  Journal of Theoretical Biology 182 (3) (1996) pp.193-194
  ISSN: 0022-5193
  Abstract

  No abstract: reproduced with minor modifications from the obituary published in the The Biochemist 17 (3) 26-27

  Website 
• Veech RL, Fell DA, 'Distribution control of metabolic flux'
  CELL BIOCHEMISTRY AND FUNCTION 14 (4) (1996) pp.229-236
  ISSN: 0263-6484 eISSN: 1099-0844
  Website 
• Thomas S, Fell DA, 'Error and Bias in Control Coefficients Calculated From Elasticities'
  Biochemical Society Transactions 23 (1995) pp.S294-S294
  ISSN: 0300-5127 eISSN: 1470-8752
  
• Thomas S, Moniz-Barrito JP, Fell DA, Woods JH, Poolman MG, 'Flux Control in Ecosystems'
  Trends in Ecology and Evolution 10 (1995) pp.245-245
  ISSN: 0169-5347
  Abstract

  Letter

• Fell DA, Thomas S, 'Physiological Control of Metabolic Flux - the Requirement for Multisite Modulation'
  Biochemical Journal 311 (1995) pp.35-39
  ISSN: 0264-6021
  Abstract

  Since Blackman [1] proposed the concept of the 'rate-limiting

  step' in 1905, it has dominated the approach to understanding

  the control of metabolic pathways. For example, it was endorsed

  in Krebs' concept of 'pacemaker' enzymes [2], which he saw as

  the target sites for hormone and drug action on metabolism.

  Even though the theory of Metabolic Control Analysis [3,4] has

  since shown that control can be distributed over many steps in a

  pathway, and that the degree of control of any given step can be

  quantified by its flux control coefficient, qualitative explanations

  of how a pathway can be controlled have not been greatly

  affected. Indeed, although Metabolic Control Analysis has been

  increasingly adopted in metabolic biochemistry, and experiments

  have confirmed both that control is generally distributed [5] and

  that genuinely rate-limiting enzymes are rare, it has also

  legitimized the concept that an enzyme that responds to some

  external controlling factor can be an agent of metabolic control

  provided the enzyme has a finite flux control coefficient. However,

  we shall cite arguments that such mechanisms cannot be responsible

  for large changes in metabolic flux. On the other hand,

  recent theoretical developments arising from Metabolic Control

  Analysis do allow us to characterize how large changes in

  metabolic flux could be implemented; they can only be achieved

  with minimal disturbance ofmetabolite concentrations and fluxes

  in other pathways by co-ordinated changes in the activities of

  many of the enzymes in the pathway, and this can be shown to

  be a common mechanism of control.

  Related experimental and theoretical evidence also contradicts

  the view that regulatory enzymes exhibiting allosteric properties

  are effective agents for control of metabolic flux. Our conclusion

  is that their more significant role is in homoeostasis. In consequence,

  different approaches are needed to both the study and

  explanation of metabolic control.

• Thomas S, Fell DA, 'Error and Bias in Control Coefficients Calculated From Elasticities'
  Biochemical Society Transactions 23 (1995) pp.S294-S294
  ISSN: 0300-5127 eISSN: 1470-8752
  
• Kashiwaya Y, Sato K, Tsuchiya N, Thomas S, Fell DA, Veech RL, Passoneau JV, 'Control of Glucose-utilization in Working Perfused Rat-heart'
  Journal of Biological Chemistry 269 (1994) pp.25502-25514
  ISSN: 0021-9258
  Abstract

  Metabolic control analyses of glucose utilization were performed for four groups of working rat hearts perfused with Krebs-Henseleit buffer containing 10 mM glucose only, or with the addition of 4 mM D-beta-hydroxybutyrate/1 mM acetoacetate, 100 nM insulin (0.05 unit/ml), or both. Net glycogen breakdown occurred in the glucose group only and was converted to net glycogen synthesis in the presence of all additions. The flux of [2-3H]glucose through P-glucoisomerase (EC 5.3.1.9) was reduced with ketones, elevated with insulin, and unchanged with the combination. Net glycolytic flux was reduced in the presence of ketones and the combination. The flux control coefficients were determined for the portion of the pathway involving glucose transport to the branches of glycogen synthesis and glycolysis. Major control was divided between the glucose transporter and hexokinase (EC 2.7.1.1) in the glucose group. The distribution of the control was slightly shifted to hexokinase with ketones, and control at the glucose transport step was abolished in the presence of insulin. Analysis of the pathway from 3-P-glycerate to pyruvate determined that the major control was shared by enolase (EC 4.2.1.1) and pyruvate kinase (EC 2.7.1.40) in the glucose group. Addition of ketones, insulin, or the combination shifted the control to P-glycerate mutase (EC 5.4.2.1) and pyruvate kinase. These results illustrate that the control of the metabolic flux in glucose metabolism of rat heart is not exerted by a single enzyme but variably distributed among enzymes depending upon substrate availability, hormonal stimulation, or other changes of conditions.

• Thomas S, Fell DA, 'Metabolic Control Analysis - Sensitivity of Control Coefficients to Experimentally Determined Variables'
  Journal of Theoretical Biology 167 (1994) pp.175-200
  ISSN: 0022-5193
  Abstract

  Metabolic Control Analysis has provided tools for understanding and quantifying the regulation of biochemical systems, but the values of control coefficients in non-trivial systems can depend on the values of a large number of quantities: metabolite concentrations, fluxes and elasticity coefficients. This poses two related questions. One concerns the accuracy and precision of the values obtained for the control coefficients, depending as they do on a large number of experimentally determined values. The second concerns the importance of the variables in determining the values of the control coefficients: as these variables represent components of the biochemical system under consideration, this corresponds to the question of how do the components determine the regulatory properties of the system? This paper describes an extension to an earlier sensitivity analysis of the control coefficients that can be used to answer both of these questions. The analysis has been incorporated into a computer program (MetaCon), so enabling rapid and automatic determination of the sensitivities of each of the control coefficients to each of the experimental quantities. The program has been used to analyse the control coefficients of two experimental systems taken from the literature. The results of the analysis are presented. Two important points arise: control coefficients in a given biochemical system can show wide variations in their s