What Process Is Glycolytic Stoichiometry Optimal For?

It has been proposed that the glycolytic stoichiometry of 2 ATP per glucose is the result of an optimization that maximizes the rate of ATP production. However, using a nonequilibrium thermodynamic approach, we show here that glycolysis operates under optimal output power and not at optimal flow of ATP production. Furthermore, it can be proved that the same maximal output power can be achieved with different stoichiometries. However, changes in the glycolytic stoichiometry would dramatically affect the efficiency of all those cellular processes powered by ATP. Our results suggest that the stoichiometric coefficient, as found in most contemporary cells, may be the outcome of an evolutionary process leading to yield an operative quantum energy for the hydrolysis of ATP. [Reviewing Editor: Dr. Antony Dean]     

Estimation of the energetic biomass yield and efficiency of oxidative phosphorylation in cell-recycle cultures of Schizosaccharomyces pombe

The energetics of growth of the fission yeast Schizosaccharomyces pombe was studied in continuous high-cell concentration cultures using a cell-recycle fermentor. Under non-O₂-limited conditions, steady-states were obtained at various specific growth rates (partial cell-recycle) with purely oxidative (glucose limitation) or respiro-fermentative (glucose excess) metabolic behaviour. The stoichiometry of biomass synthesis was established from the elemental composition of the cells and measurements of all the specific metabolic rates, i.e. consumption of glucose and O₂ and production of CO₂, ethanol and other products. The theoretical yield factor for biomass on glucose was Y_G,X = 0.85 C-mol·C-mol^–1 and maintenance requirements were negligible. Assuming a constant coupling between energy generation and biomass formation for both respirative and respiro-fermentative breakdown of glucose, the biomass yield from ATP (Y_ATP) and the efficiency of oxidative phosphorylation (P/O ratio) could be determined as 9.8 g biomass·mol ATP and 1.28 mol ATP·atom of O₂, respectively. Correspondence to: A. Pareilleux     

A structured metabolic model for anaerobic and aerobic stoichiometry and kinetics of the biological phosphorus removal process

A structured metabolic model is developed that describes the stoichiometry and kinetics of the biological P removal process. In this approach all relevant metabolic reactions underlying the metabolism, considering also components like adenosine triphosphate (ATP) and nic-otinamide-adenine dinucleotide (NADH(2)) are describedbased on biochemical pathways. As a consequence of the relations between the stoichiometry of the metabolic reactions and the reaction rates of components, the required number of kinetic relations to describe the process is reduced. The model describes the dynamics of the storage compounds which are considered separately from the active biomass. The model was validated in experiments at a constant sludge retention time of 8 days, over the anaerobic and aerobic phases in which the external oncentrations as well as the internal fractions of the relevant components involved in the P-removal process were monitored. These measurements include dissolved acetate, phosphate, and ammonium; oxygen consumption; poly-beta-hydroxybutyrate (PHB); glycogen; and active biomass. The model satisfactorily describes the dynamic behavior of all components during the anaerobicand aerobic phases.(c) 1995 John Wiley & Sons, Inc.     

Biochemical production capabilities of Escherichia coli

Microbial metabolism provides at mechanism for the conversion of substrates into useful biochemicals. Utilization of microbes in industrial processes requires a modification of their natural metabolism in order to increase the efficiency of the desired conversion. Redirection of metabolic fluxes forms the basis of the newly defined field of metabolic engineering. In this study we use a flux balance based approach to study the biosynthesis of the 20 amino acids and 4 nucleotides as biochemical products. These amino acids and nucleotides are primary products of biosynthesis as well as important industrial products and precursors for the production of other biochemicals. The biosynthetic reactions of the bacterium Escherichia coli have been formulated into a metabolic network, and growth has been defined as a balanced drain on the metabolite pools corresponding to the cellular composition. Theoretical limits on the conversion of glucose, glycerol, and acetate substrates to biomass as well as the biochemical products have been computed. The substrate that results in the maximal carbon conversion to a particular product is identified. Criteria have been developed to identify metabolic constraints in the optimal solutions. The constraints of stoichiometry, energy, and redox have been determined in the conversions of glucose, glycerol, and acetate substrates into the biochemicals. Flux distributions corresponding to the maximal production of the biochemicals are presented. The goals of metabolic engineering are the optimal redirection of fluxes from generating biomass toward producing the desired biochemical. Optimal biomass generation is shown to decrease in a piecewise linear manner with increasing product formation. In some cases, synergy is observed between biochemical production and growth, leading to an increased overall carbon conversion. Balanced growth and product formation are important in a bioprocess, particularly for nonsecreted products. (c) 1993 John Wiley & Sons, Inc.     

Mass-energy balance of the growth of phototropic purple bacteria

The principles of the theory of mass-energy balance of the growth of cellular populations are described. Based on this theory, the effect of biochemical parameters of cell metabolism on the efficiency of phototrophic growth of bacterial culture was studied. The metabolism of phototrophic bacteria was subdivided into constructive and energetic partial metabolisms. The stoichiometric coefficients describing the energetics of the two metabolism types were calculated. An equation system of the mass-energy balance for the flows of reductivity, high-energy bonds, and protons--carriers of the transmembrane electrochemical potential was derived. Equations for biomass quantum yield were obtained. The calculated yield values are in agreement with experimental data.     

Flux-balance analysis of mitochondrial energy metabolism: consequences of systemic stoichiometric constraints

Mitochondrial metabolism is a critical component in the functioning and maintenance of cellular organs. The stoichiometry of biochemical reaction networks imposes constraints on mitochondrial function. A modeling framework, flux-balance analysis (FBA), was used to characterize the optimal flux distributions for maximal ATP production in the mitochondrion. The model predicted the expected ATP yields for glucose, lactate, and palmitate. Genetic defects that affect mitochondrial functions have been implicated in several human diseases. FBA can characterize the metabolic behavior due to genetic deletions at the metabolic level, and the effect of mutations in the tricarboxylic acid (TCA) cycle on mitochondrial ATP production was simulated. The mitochondrial ATP production is severely affected by TCA-cycle mutations. In addition, the model predicts the secretion of TCA-cycle intermediates, which is observed in clinical studies of mitochondriopathies such as those associated with fumarase deficiency. The model provides a systemic perspective to characterize the effect of stoichiometric constraints and specific metabolic fluxes on mitochondrial function.     

Genome‐derived minimal metabolic models for Escherichia coli MG1655 with estimated in vivo respiratory ATP stoichiometry

Metabolic network models describing growth of Escherichia coli on glucose, glycerol and acetate were derived from a genome scale model of E. coli. One of the uncertainties in the metabolic networks is the exact stoichiometry of energy generating and consuming processes. Accurate estimation of biomass and product yields requires correct information on the ATP stoichiometry. The unknown ATP stoichiometry parameters of the constructed E. coli network were estimated from experimental data of eight different aerobic chemostat experiments carried out with E. coli MG1655, grown at different dilution rates (0.025, 0.05, 0.1, and 0.3 h^?1) and on different carbon substrates (glucose, glycerol, and acetate). Proper estimation of the ATP stoichiometry requires proper information on the biomass composition of the organism as well as accurate assessment of net conversion rates under well‐defined conditions. For this purpose a growth rate dependent biomass composition was derived, based on measurements and literature data. After incorporation of the growth rate dependent biomass composition in a metabolic network model, an effective P/O ratio of 1.49 ± 0.26 mol of ATP/mol of O, K_X (growth dependent maintenance) of 0.46 ± 0.27 mol of ATP/C‐mol of biomass and m_ATP (growth independent maintenance) of 0.075 ± 0.015 mol of ATP/C‐mol of biomass/h were estimated using a newly developed Comprehensive Data Reconciliation (CDR) method, assuming that the three energetic parameters were independent of the growth rate and the used substrate. The resulting metabolic network model only requires the specific rate of growth, µ, as an input in order to accurately predict all other fluxes and yields. Biotechnol. Bioeng. 2010;107: 369–381. © 2010 Wiley Periodicals, Inc.     

Energy coupling in Saccharomyces cerevisiae: selected opportunities for metabolic engineering

Free-energy (ATP) conservation during product formation is crucial for the maximum product yield that can be obtained, but often overlooked in metabolic engineering strategies. Product pathways that do not yield ATP or even demand input of free energy (ATP) require an additional pathway to supply the ATP needed for product formation, cellular maintenance, and/or growth. On the other hand, product pathways with a high ATP yield may result in excess biomass formation at the expense of the product yield. This mini-review discusses the importance of the ATP yield for product formation and presents several opportunities for engineering free-energy (ATP) conservation, with a focus on sugar-based product formation by Saccharomyces cerevisiae. These engineering opportunities are not limited to the metabolic flexibility within S.?cerevisiae itself, but also expression of heterologous reactions will be taken into account. As such, the diversity in microbial sugar uptake and phosphorylation mechanisms, carboxylation reactions, product export, and the flexibility of oxidative phosphorylation via the respiratory chain and H(+) -ATP synthase can be used to increase or decrease free-energy (ATP) conservation. For product pathways with a negative, zero or too high ATP yield, analysis and metabolic engineering of the ATP yield of product formation will provide a promising strategy to increase the product yield and simplify process conditions.     

Revisiting the Thermodynamic Theory of Optimal ATP Stoichiometries by Analysis of Various ATP-Producing Metabolic Pathways

The stoichiometry of ATP-producing metabolic pathways had been analysed theoretically by several authors by using evolutionary arguments and optimality principles. Waddell et al. (Biochem Educ 27:12–13, 1999) analysed (lactate-producing) glycolysis and used linear irreversible thermodynamics. The result was that half of the free-energy difference should be converted into free-energy of ATP and the remaining half should be used to drive the pathway. The calculated stoichiometry is in agreement with the observed yield of two moles of ATP per mole of glucose. Using the same approach, we here analyse eight other metabolic pathways. Although the deviation is not very large, the calculated values do not fit as nicely as for glycolysis as leading to lactate. For example, for O₂ respiration, the theoretical ATP yield equals 27.9. The real value varies among organisms between 26 and 38. For mixed-acid fermentation in Escherichia coli, the theoretical and experimental values are 2.24 and 2, respectively. For arginine degradation in M. pneumoniae, the calculated value is 2.43 mol of ATP, while in vivo only one mole is produced. During evolution, some pathways may not have reached their optimal ATP net production because energy yield is not their only function. Moreover, it should be acknowledged that the approach by linear irreversible thermodynamics is a rough approximation.     

Metal plasmon-coupled fluorescence imaging and label free coenzyme detection in cells

Flavin adenine dinucleotide (FAD) is a key metabolite in cellular energy conversion. Flavin can also bind with some enzymes in the metabolic pathway and the binding sites may be changed due to the disease progression. Thus, there is interest on studying its expression level, distribution, and redox state within the cells. FAD is naturally fluorescent, but it has a modest extinction coefficient and quantum yield. Hence the intrinsic emission from FAD is generally too weak to be isolated distinctly from the cellular backgrounds in fluorescence cell imaging. In this article, the metal nanostructures on the glass coverslips were used as substrates to measure FAD in cells. Particulate silver films were fabricated with an optical resonance near the absorption and the emission wavelengths of FAD which can lead to efficient coupling interactions. As a result, the emission intensity and quantum yield by FAD were greatly increased and the lifetime was dramatically shortened resulting in less interference from the longer lived cellular background. This feature may overcome the technical limits that hinder the direct observation of intrinsically fluorescent coenzymes in the cells by fluorescence microscopy. Fluorescence cell imaging on the metallic particle substrates may provide a non-invasive strategy for collecting the information of coenzymes in cells.     

Serial propagation in water-in-oil emulsions selects for Saccharomyces cerevisiae strains with a reduced cell size or an increased biomass yield on glucose

In S. cerevisiae and many other micro-organisms an increase in metabolic efficiency (i.e. ATP yield on carbon) is accompanied by a decrease in growth rate. From a fundamental point of view, studying these yield-rate trade-offs provides insight in for example microbial evolution and cellular regulation. From a biotechnological point of view, increasing the ATP yield on carbon might increase the yield of anabolic products. We here aimed to select S. cerevisiae mutants with an increased biomass yield. Serial propagation of individual cells in water-in-oil emulsions previously enabled the selection of lactococci with increased biomass yields, and adapting this protocol for yeast allowed us to enrich an engineered Crabtree-negative S. cerevisiae strain with a high biomass yield on glucose. When we started the selection with an S. cerevisiae deletion collection, serial propagation in emulsion enriched hxk2Δ and reg1Δ strains with an increased biomass yield on glucose. Surprisingly, a tps1Δ strain was highly abundant in both emulsion- and suspension-propagated populations. In a separate experiment we propagated a chemically mutagenized S. cerevisiae population in emulsion, which resulted in mutants with a higher cell number yield on glucose, but no significantly changed biomass yield. Genome analyses indicate that genes involved in glucose repression and cell cycle processes play a role in the selected phenotypes. The repeated identification of mutations in genes involved in glucose-repression indicates that serial propagation in emulsion is a valuable tool to study metabolic efficiency in S. cerevisiae.     

A structured approach to the study of metabolic control principles in intact and impaired mitochondria

We devised an approach to extract control principles of cellular bioenergetics for intact and impaired mitochondria from ODE-based models and applied it to a recently established bioenergetic model of cancer cells. The approach used two methods for varying ODE model parameters to determine those model components that, either alone or in combination with other components, most decisively regulated bioenergetic state variables. We found that, while polarisation of the mitochondrial membrane potential (ΔΨ(m)) and, therefore, the protomotive force were critically determined by respiratory complex I activity in healthy mitochondria, complex III activity was dominant for ΔΨ(m) during conditions of cytochrome-c deficiency. As a further important result, cellular bioenergetics in healthy, ATP-producing mitochondria was regulated by three parameter clusters that describe (1) mitochondrial respiration, (2) ATP production and consumption and (3) coupling of ATP-production and respiration. These parameter clusters resembled metabolic blocks and their intermediaries from top-down control analyses. However, parameter clusters changed significantly when cells changed from low to high ATP levels or when mitochondria were considered to be impaired by loss of cytochrome-c. This change suggests that the assumption of static metabolic blocks by conventional top-down control analyses is not valid under these conditions. Our approach is complementary to both ODE and top-down control analysis approaches and allows a better insight into cellular bioenergetics and its pathological alterations.     

Growth rate–stoichiometry couplings in diverse biota

Biological stoichiometry provides a mechanistic theory linking cellular and biochemical features of co‐evolving biota with constraints imposed by ecosystem energy and nutrient inputs. Thus, understanding variation in biomass carbon : nitrogen : phosphorus (C : N : P) stoichiometry is a major priority for integrative biology. Among various factors affecting organism stoichiometry, differences in C : P and N : P stoichiometry have been hypothesized to reflect organismal P‐content because of altered allocation to P‐rich ribosomal RNA at different growth rates (the growth rate hypothesis, GRH). We tested the GRH using data for microbes, insects, and crustaceans and we show here that growth, RNA content, and biomass P content are tightly coupled across species, during ontogeny, and under physiological P limitation. We also show, however, that this coupling is relaxed when P is not limiting for growth. The close relationship between P and RNA contents indicates that ribosomes themselves represent a biogeochemically significant repository of P in ecosystems and that allocation of P to ribosome generation is a central process in biological production in ecological systems.     

Metabolic profiling of hypoxic cells revealed a catabolic signature required for cell survival

Hypoxia is one of the features of poorly vascularised areas of solid tumours but cancer cells can survive in these areas despite the low oxygen tension. The adaptation to hypoxia requires both biochemical and genetic responses that culminate in a metabolic rearrangement to counter-balance the decrease in energy supply from mitochondrial respiration. The understanding of metabolic adaptations under hypoxia could reveal novel pathways that, if targeted, would lead to specific death of hypoxic regions. In this study, we developed biochemical and metabolomic analyses to assess the effects of hypoxia on cellular metabolism of HCT116 cancer cell line. We utilized an oxygen fluorescent probe in anaerobic cuvettes to study oxygen consumption rates under hypoxic conditions without the need to re-oxygenate the cells and demonstrated that hypoxic cells can maintain active, though diminished, oxidative phosphorylation even at 1% oxygen. These results were further supported by in situ microscopy analysis of mitochondrial NADH oxidation under hypoxia. We then used metabolomic methodologies, utilizing liquid chromatography-mass spectrometry (LC-MS), to determine the metabolic profile of hypoxic cells. This approach revealed the importance of synchronized and regulated catabolism as a mechanism of adaptation to bioenergetic stress. We then confirmed the presence of autophagy under hypoxic conditions and demonstrated that the inhibition of this catabolic process dramatically reduced the ATP levels in hypoxic cells and stimulated hypoxia-induced cell death. These results suggest that under hypoxia, autophagy is required to support ATP production, in addition to glycolysis, and that the inhibition of autophagy might be used to selectively target hypoxic regions of tumours, the most notoriously resistant areas of solid tumours.     

Cellular mechanisms of brain energy metabolism and their relevance to functional brain imaging.

Despite striking advances in functional brain imaging, the cellular and molecular mechanisms that underlie the signals detected by these techniques are still largely unknown. The basic physiological principle of functional imaging is represented by the tight coupling existing between neuronal activity and the associated local increase in both blood flow and energy metabolism. Positron emission tomography (PET) signals detect blood flow, oxygen consumption and glucose use associated with neuronal activity; the degree of blood oxygenation is currently thought to contribute to the signal detected with functional magnetic resonance imaging, while magnetic resonance spectroscopy (MRS) identifies the spatio-temporal pattern of the activity-dependent appearance of certain metabolic intermediates such as glucose or lactate. Recent studies, including those of neurotransmitter-regulated metabolic fluxes in purified preparations and analyses of the cellular localization of enzymes and transporters involved in energy metabolism, as well as in vivo microdialysis and MRS approaches have identified the neurotransmitter glutamate and astrocytes, a specific type of glial cell, as pivotal elements in the coupling of synaptic activity with energy metabolism. Astrocytes are ideally positioned to sense increases in synaptic activity and to couple them with energy metabolism. Indeed they possess specialized processes that cover the surface of intraparenchymal capillaries, suggesting that astrocytes may be a likely site of prevalent glucose uptake. Other astrocyte processes are wrapped around synaptic contacts which possess receptors and reuptake sites for neurotransmitters. Glutamate stimulates glucose uptake into astrocytes. This effect is mediated by specific glutamate transporters present on these cells. The activity of these transporters, which is tightly coupled to the synaptic release of glutamate and operates the clearance of glutamate from the extracellular space, is driven by the electrochemical gradient of Na+. This Na(+)-dependent uptake of glutamate into astrocytes triggers a cascade of molecular events involving the Na+/K(+)-ATPase leading to the glycolytic processing of glucose and the release of lactate by astrocytes. The stoichiometry of this process is such that for one glutamate molecule taken up with three Na+ ions, one glucose molecule enters an astrocyte, two ATP molecules are produced through aerobic glycolysis and two lactate molecules are released. Within the astrocyte, one ATP molecule fuels one 'turn of the pump' while the other provides the energy needed to convert glutamate to glutamine by glutamine synthase. Evidence has been accumulated from structural as well as functional studies indicating that, under aerobic conditions, lactate may be the preferred energy substrate of activated neurons. Indeed, in the presence of oxygen, lactate is converted to pyruvate, which can be processed through the tricarboxylic acid cycle and the associated oxidative phosphorylation, to yield 17 ATP molecules per lactate molecule. These data suggest that during activation the brain may transiently resort to aerobic glycolysis occurring in astrocytes, followed by the oxidation of lactate by neurons. The proposed model provides a direct mechanism to couple synaptic activity with glucose use and is consistent with the notion that the signals detected during physiological activation with 18F-deoxyglucose (DG)-PET may reflect predominantly uptake of the tracer into astrocytes. This conclusion does not question the validity of the 2-DG-based techniques, rather it provides a cellular and molecular basis for these functional brain imaging techniques.     

The effect of nucleotides on the rate of spontaneous quantum bumps in Limulus ventral photoreceptors

The effect of intracellular nucleotides on the rate of spontaneous quantum bumps in Limulus ventral photoreceptors has been examined. Internal dialysis of photoreceptors with solutions lacking nucleotide leads to an elevation of the quantum bump rate that can be reversed by introduction of nucleotide. Similarly, elevation occurs after treating intact cells with the metabolic inhibitor 2-deoxyglucose. This effect can be reversed by intracellular injection of ATP. The rate of spontaneous quantum bumps in unpoisoned cells can be reduced to below normal levels by injection of ATP. These results support the hypothesis that high-energy nucleotides suppress the rate of spontaneous quantum bumps.     

The role of ATP and free ADP in metabolic coupling during fuel-stimulated insulin release from islet beta-cells in the isolated perfused rat pancreas.

The isolated perfused rat pancreas was used to test the hypothesis that total cellular ATP or the ratio of ATP/free ADP plays the primary role in coupling intermediary metabolism to the biophysical events that are the basis of glucose-stimulated insulin release. The pancreas was preperfused for 20 min with 4.0 mM of a physiological mixture of 20 amino acids plus 4.2 mM glucose, and insulin release was then stimulated for 150 s by suddenly increasing the glucose to 8.3 mM. The pancreas was sampled at 24, 48, 72, and 150 s after the switch. The content of total ATP, ADP, AMP, Pi, phosphocreatine, and creatine were measured in beta-cell enriched cores of pancreatic islets microdissected from freeze-dried pancreas cryostat sections. Metabolites were measured by quantitative histochemical enzymatic cycling techniques. Modeling studies were carried out to assess the impact of biochemical analytical results on the membrane potential of the beta-cells. The level of free ADP was calculated using the creatine kinase equilibrium reaction and an intracellular pH of 7.2. First phase insulin release was stimulated at least 10-fold with the maximum reached 45 s after adding high glucose. The biochemical analytical data demonstrate that the total cellular level of the putative coupling factor ATP and of the ratios ATP/free ADP and ATP/free ADP x Pi are not significantly influenced by a glucose level change that causes a more than 10-fold surge of insulin release. The strength and limitations of the present experimental strategy and the implications of the results for our understanding of metabolic coupling in glucose-stimulated insulin release are discussed.     

Looking inside the box: using Raman microspectroscopy to deconstruct microbial biomass stoichiometry one cell at a time

Stoichiometry of microbial biomass is a key determinant of nutrient recycling in a wide variety of ecosystems. However, little is known about the underlying causes of variance in microbial biomass stoichiometry. This is primarily because of technological constraints limiting the analysis of macromolecular composition to large quantities of microbial biomass. Here, we use Raman microspectroscopy (MS), to analyze the macromolecular composition of single cells of two species of bacteria grown on minimal media over a wide range of resource stoichiometry. We show that macromolecular composition, determined from a subset of identified peaks within the Raman spectra, was consistent with macromolecular composition determined using traditional analytical methods. In addition, macromolecular composition determined by Raman MS correlated with total biomass stoichiometry, indicating that analysis with Raman MS included a large proportion of a cell''s total macromolecular composition. Growth phase (logarithmic or stationary), resource stoichiometry and species identity each influenced each organism''s macromolecular composition and thus biomass stoichiometry. Interestingly, the least variable peaks in the Raman spectra were those responsible for differentiation between species, suggesting a phylogenetically specific cellular architecture. As Raman MS has been previously shown to be applicable to cells sampled directly from complex environments, our results suggest Raman MS is an extremely useful application for evaluating the biomass stoichiometry of environmental microorganisms. This includes the ability to partition microbial biomass into its constituent macromolecules and increase our understanding of how microorganisms in the environment respond to resource heterogeneity.