On the stability of metabolic cycles

We investigate the stability properties of two different classes of metabolic cycles using a combination of analytical and computational methods. Using principles from structural kinetic modeling (SKM), we show that the stability of metabolic networks with certain structural regularities can be studied using a combination of analytical and computational techniques. We then apply these techniques to a class of single input, single output metabolic cycles, and find that the cycles are stable under all conditions tested. Next, we extend our analysis to a small autocatalytic cycle, and determine parameter regimes within which the cycle is very likely to be stable. We demonstrate that analytical methods can be used to understand the relationship between kinetic parameters and stability, and that results from these analytical methods can be confirmed with computational experiments. In addition, our results suggest that elevated metabolite concentrations and certain crucial saturation parameters can strongly affect the stability of the entire metabolic cycle. We discuss our results in light of the possibility that evolutionary forces may select for metabolic network topologies with a high intrinsic probability of being stable. Furthermore, our conclusions support the hypothesis that certain types of metabolic cycles may have played a role in the development of primitive metabolism despite the absence of regulatory mechanisms.     

Proteomics approaches to study genetic and metabolic disorders

Several proteomics approaches to study different aspects of genetic and metabolic diseases are presented. The choice of technique is strongly dependent on the biological question to be addressed and the availability and amount of sample. In general, there are three approaches that may be used to study genetic and metabolic diseases: protein profiling of complex biological samples, identification of affected proteins, or a functional proteomics approach to study protein interactions and function.     

Application of the metabolic control theory to the study of the dynamics of substrate cycles

Substrate cycles are ubiquitous structures of the cellular metabolism (e.g. Krebs cycle, fatty acids -oxydation cycles, etc... ). Moiety-conserved cycles (e.g. adenine nucleotides and NADH/NAD, etc...) are also important.The role played by such cycles in the metabolism and its regulation is not clearly understood so far. However, it was shown that these cycles can generate multistationarity (bistability), irreversible transitions, enhancement of sensitivity, temporal oscillations and chaotic motions (Hervagault & Canu, 1987; Hervagault & Cimino, 1989; Reich & Sel'kov, 1981; Ricard & Soulié, 1982). Fig. 1: Scheme of the open binary substrate cycle under study. The substrate S is converted into P with a net rate v₂. Substrate P is converted in turn into S with a net rate v₃. Step v₂ is inhibited by excess of the substrate, S. In addition, the cycle operates under open conditions, that is zero-order input of S at rates \ga₀(v₁) and first order outputs of S and P at rates \gaS and \gaP(v₄), respectively.The metabolic control theory (see also Fell, 1990), which shows how a metabolic network reacts to small perturbations in the vicinity of a steady state, and is formulated with the so-called control coefficients, was applied to such a cycle in order to get a better knowledge on the importance of each step at the regulatory point of view.The behaviour of a binary substrate cycle (fig. 1) in which one of the enzymes may be subjected to inhibition by excess of its substrate (v₂) was studied theoretically. The flux and concentration control coefficients were calculated for various steady states of the system. The evolution of the different control coefficients is compared to the evolution of the steady states. We mainly focused our study on situations for which the steady states are stable.     

On the analysis of substrate cycles in large metabolic systems

The simultaneous operation of paired, opposing reactions (substrate cycles) or parallel reactions (dual pathways) with seeming wastage of ATP is widespread in cellular metabolism. Analysis of such “futile” pathways has hitherto been limited to loci with only two or three interconnecting fluxes. We introduce here a method that allows straightforward analysis of more complex systems. The method involves the linear superposition of “fundamental” modes, one or more of which may be energetically wasteful. Decomposition of a flux pattern into such modes allows computation of the amount of free energy “wasted” at any locus. Appropriate normalizations of energy wastage yield a number of indices useful for assessing the energetic impact of futile pathways on the cell and for comparing the degree of regulation of substrate cycles or dual pathways under different metabolic conditions. This approach is applied to steady-state flux data obtained in the protozoanTetrahymena pyriformis and in isolated rat hepatocytes under a variety of conditions.     

High-resolution analysis of metabolic cycles in the intertidal mussel Mytilus californianus

Inhabitants of the marine rocky intertidal live in an environment that alternates between aquatic and terrestrial due to the rise and fall of the tide. The tide creates a cyclical availability of oxygen with animals having access to oxygenated water during episodes of submergence, while access to oxygen is restricted during aerial emergence. Here we performed liquid chromatography and gas chromatography-mass spectrometry enabled metabolomic profiling of gill samples isolated from the California ribbed mussel, Mytilus californianus, to investigate how metabolism is orchestrated in this variable environment. We created a simulated intertidal environment in which mussels were acclimated to alternating high and low tides of 6 h duration, and samples were taken every 2 h for 72 h to capture reproducible changes in metabolite levels over six high and six low tides. We quantified 169 named metabolites of which 24 metabolites cycled significantly with a 12-h period that was linked to the tidal cycle. These data confirmed the presence of alternating phases of fermentation and aerobic metabolism and highlight a role for carnitine-conjugated metabolites during the anaerobic phase of this cycle. Mussels at low tide accumulated eight carnitine-conjugated metabolites, arising from the degradation of fatty acids, branched-chain amino acids, and mitochondrial β-oxidation end products. The data also implicate sphingosine as a potential signaling molecule during aerial emergence. These findings identify new levels of metabolic control whose role in intertidal adaptation remains to be elucidated.     

Systems metabolic engineering for chemicals and materials

Metabolic engineering has contributed significantly to the enhanced production of various value-added and commodity chemicals and materials from renewable resources in the past two decades. Recently, metabolic engineering has been upgraded to the systems level (thus, systems metabolic engineering) by the integrated use of global technologies of systems biology, fine design capabilities of synthetic biology, and rational-random mutagenesis through evolutionary engineering. By systems metabolic engineering, production of natural and unnatural chemicals and materials can be better optimized in a multiplexed way on a genome scale, with reduced time and effort. Here, we review the recent trends in systems metabolic engineering for the production of chemicals and materials by presenting general strategies and showcasing representative examples.     

Epistasis for growth rate and total metabolic flux in yeast

Studies of interactions between gene deletions repeatedly show that the effect of epistasis on the growth of yeast cells is roughly null or barely positive. These observations relate generally to the pace of growth, its costs in terms of required metabolites and energy are unknown. We measured the maximum rate at which yeast cultures grow and amounts of glucose they consume per synthesized biomass for strains with none, single, or double gene deletions. Because all strains were maintained under a fermentative mode of growth and thus shared a common pattern of metabolic processes, we used the rate of glucose uptake as a proxy for the total flux of metabolites and energy. In the tested sample, the double deletions showed null or slightly positive epistasis both for the mean growth and mean flux. This concordance is explained by the fact that average efficiency of converting glucose into biomass was nearly constant, that is, it did not change with the strength of growth effect. Individual changes in the efficiency caused by gene deletions did have a genetic basis as they were consistent over several environments and transmitted between single and double deletion strains indicating that the efficiency of growth, although independent of its rate, was appreciably heritable. Together, our results suggest that data on the rate of growth can be used as a proxy for the rate of total metabolism when the goal is to find strong individual interactions or estimate the mean epistatic effect. However, it may be necessary to assay both growth and flux in order to detect smaller individual effects of epistasis.     

Optimizing pentose utilization in yeast: the need for novel tools and approaches

Background

The two-step dilute acid hydrolysis (DAH) of softwood is costly in energy demands and capital costs. However, it has the advantage that hydrolysis and subsequent removal of hemicellulose-derived sugars can be carried out under conditions of low severity, resulting in a reduction in the level of sugar degradation products during the more severe subsequent steps of cellulose hydrolysis. In this paper, we discuss a single-step DAH method that incorporates a temperature profile at two levels. This profile should simulate the two-step process while removing its major disadvantage, that is, the washing step between the runs, which leads to increased energy demand.

Results

The experiments were conducted in a reactor with a controlled temperature profile. The total dry matter content of the hydrolysate was up to 21.1% w/w, corresponding to a content of 15.5% w/w of water insoluble solids. The highest measured glucose yield, (18.3 g glucose per 100 g dry raw material), was obtained after DAH cycles of 3 min at 209°C and 6 min at 211°C with 1% H₂SO₄, which resulted in a total of 26.3 g solubilized C6 sugars per 100 g dry raw material. To estimate the remaining sugar potential, enzymatic hydrolysis (EH) of the solid fraction was also performed. EH of the solid residue increased the total level of solubilized C6 sugars to a maximum of 35.5 g per 100 g dry raw material when DAH was performed as described above (3 min at 210°C and 2 min at 211°C with 1% H₂SO₄).

Conclusion

The dual-temperature DAH method did not yield decisively better results than the single-temperature, one-step DAH. When we compared the results with those of earlier studies, the hydrolysis performance was better than with the one-step DAH but not as well as that of the two-step, single-temperature DAH. Additional enzymatic hydrolysis resulted in lower levels of solubilized sugars compared with other studies on one-step DAH and two-step DAH followed by enzymatic hydrolysis. A two-step steam pretreatment with EH gave rise to a considerably higher sugar yield in this study.     

On the potential of mass spectrometry-based metabolite profiling approaches to the study of biochemical adaptation in psychrophilic yeast

To move beyond targeted approaches to the biochemical characterization of psychrophilic yeast and provide a more holistic understanding of the chemistry of physiological adaptation of psychrophiles at the molecular level, ultraperformance liquid chromatography combined with simultaneous acquisition of low- and high-collision energy mass spectra (UPLC/MS^e) was employed for a preliminary comparative analysis of cell extracts of psychrophilic Antarctic yeasts Cryptococcus vishniacii CBS 10616 and Dioszegia cryoxerica CBS 10919 versus the mesophile Saccharomyces cerevisiae ‘cry havoc’. A detailed workflow for providing high-confidence preliminary identifications of psychrophilic yeast-specific molecular features is presented. Preliminary identifications of psychrophile-specific features in C. vishniacii and D. cryoxerica determined with the described method include the glycerophospholipids lysophosphatidylcholine 18:2, lysophosphatidylcholine 18:3, lysophosphatidylethanolamine 18:3, and lysophosphatidylethanolamine 18:2. In addition, levels of guanosine diphosphate appear significantly elevated in cell extracts of the psychrophilic yeasts as compared to Saccharomyces cerevisiae. Finally, five psychrophilic yeast-specific peptides have been discovered. All of these are demonstrated to be glycine- and/or proline-rich, a known structural characteristic of many naturally occurring bioactive peptides. The potential of this untargeted metabolite profiling approach as a tool for knowledge discovery and hypothesis generation in the study of biodiversity and microbial adaptation is highlighted.     

Functional essentiality from topology features in metabolic networks: a case study in yeast

The relation between the position of mutations in Saccharomyces cerevisiae metabolic network and their lethality is the subject of this work. We represent the topology of the network by a directed graph: nodes are metabolites and arcs represent the reactions; a mutation corresponds to the removal of all the arcs referring to the deleted enzyme. Using publicly available knock-out data, we show that lethality corresponds to the lack of alternative paths in the perturbed network linking the nodes affected by the enzyme deletion. Such feature is at the basis of the recently recognized importance of 'marginal' arcs of metabolic networks.     

Recombinant DNA approaches for the development of metabolic systems used in in vitro toxicology.

In the past few years there has been considerable progress in the development of mammalian cell systems for use in genetic toxicology by the stable transfer of genes/cDNAs coding for drug metabolizing enzymes directly into the target cell. Alternative approaches have also been developed in which mammalian cells are transiently transfected with cDNAs coding for drug-metabolizing enzymes and S9 preparations expressing a single metabolizing enzyme isolated and used for metabolic activation. Progress in these areas is reviewed here and the relative merits of the different approaches are discussed. Work to date has focused primarily on the cytochrome P450 family of enzymes, although other enzyme systems involved in xenobiotic metabolism have been used. The central theme of this review is the transfer of genetic information to improve the metabolic capability of cell systems used in genetic toxicology. However, a basic philosophy of the review is that genetic manipulation of cultured mammalian cells has the potential for developing systems to be used to better understand chemically induced toxicological effects.     

Turnover of yeast fructose-bisphosphatase in different metabolic conditions

Earlier work demonstrated that addition of glucose to yeast growing on noncarbohydrate carbon sources sharply reduces the levels of fructose bisphosphatase. This report indicates that the decrease in the levels of fructose bisphosphatase is accompanied by a parallel decrease of cross-reacting material to specific antibody to fructose bisphosphatase. Use of the specific antibody shows that the loss of activity is irreversible and that its reapperance requires synthesis of protein de novo. The protein is highly stable during growth in ethanol (half life about 90 h). Addition of glucose increases the rate of degradation abut 200-fold. It is shown that the values of the rates of synthesis and degradation of fructose bisphosphatase vary with the metabolic situation of the yeast.