Metabolomic and 13C-metabolic flux analysis of a xylose-consuming Saccharomyces cerevisiae strain expressing xylose isomerase

Over the past two decades, significant progress has been made in the engineering of xylose-consuming Saccharomyces cerevisiae strains for production of lignocellulosic biofuels. However, the ethanol productivities achieved on xylose are still significantly lower than those observed on glucose for reasons that are not well understood. We have undertaken an analysis of central carbon metabolite pool sizes and metabolic fluxes on glucose and on xylose under aerobic and anaerobic conditions in a strain capable of rapid xylose assimilation via xylose isomerase in order to investigate factors that may limit the rate of xylose fermentation. We find that during xylose utilization the flux through the non-oxidative Pentose Phosphate Pathway (PPP) is high but the flux through the oxidative PPP is low, highlighting an advantage of the strain employed in this study. Furthermore, xylose fails to elicit the full carbon catabolite repression response that is characteristic of glucose fermentation in S. cerevisiae. We present indirect evidence that the incomplete activation of the fermentation program on xylose results in a bottleneck in lower glycolysis, leading to inefficient re-oxidation of NADH produced in glycolysis.Shell Oil CompanyNational Institute of General Medical Sciences (U.S.) Biotechnology Training Progra

Benefit incidence analysis in developing countries

As interesting and difficult as it is to allocate tax burdens to individuals, the profession knows even less about allocating benefits. The authors survey the literature on benefit incidence since DeWulf's (1975) review, focusing on the methodology and results of benefit incidence analysis in developing countries. Research in this area faces all the general-equilibrium difficulties faced by tax incidence analysis as well as the difficult task of measuring benefits from publicly provided goods and services. Despite the inherent pitfalls of this methodology, the authors believe that benefit incidence analysis can provide an important perspective on the budget by combining data on household use with data on project costs. In particular, benefit incidence analyses can help illuminate the distributional impacts of proposed reallocations of government resources among projects. The value of such research is especially high considering the scarcity of recent research in this area. The authors review the existing methodology, survey the available results, and point out areas in which further research might have large payoffs. They also make specific methodological suggestions that might help ensure that future research is as useful for policymakers as possible. For example: Aggregate results based on the zero-government counterfactual rely on strong assumptions about fixed relative prices and incomes, government efficiency, and the relationship between marginal and total benefits. And those studies are often not designed to identify which types of public services benefit the poor. Researchers should focus more on providing benefit incidence studies on specific government functions or programs that can help policymakers reach conclusions about proposed reallocations of resources among government programs. Benefit incidence should be assigned to households based on household survey information on usage rather than on ad hoc assumptions that assign benefits based on income or the number of members in the household. Improved annual cost measures for services need to be developed, particulary for capital inputs. Researchers should group households by deciles and whenever possible should consider other groupings based on household income adjusted for household composition, age, location, and other relevant socioeconomic variables. Careful attention to life-cycle benefits, benefit shifting, rent-seeking, out-of-pocket costs, displacement of private sector efforts, average versus marginal incidence, and several other issues can significantly increase the value of benefit incidence analysis to policymakers.Economic Theory&Research,Environmental Economics&Policies,Poverty Assessment,Health Economics&Finance,Banks&Banking Reform

Benefit incidence analysis in developing countries

As interesting and difficult as it is to allocate tax burdens to individuals, the profession knows even less about allocating benefits. The authors survey the literature on benefit incidence since DeWulf\u27s (1975) review, focusing on the methodology and results of benefit incidence analysis in developing countries. Research in this area faces all the general-equilibrium difficulties faced by tax incidence analysis as well as the difficult task of measuring benefits from publicly provided goods and services. Despite the inherent pitfalls of this methodology, the authors believe that benefit incidence analysis can provide an important perspective on the budget by combining data on household use with data on project costs. In particular, benefit incidence analyses can help illuminate the distributional impacts of proposed reallocations of government resources among projects. The value of such research is especially high considering the scarcity of recent research in this area. The authors review the existing methodology, survey the available results, and point out areas in which further research might have large payoffs. They also make specific methodological suggestions that might help ensure that future research is as useful for policymakers as possible. For example: Aggregate results based on the zero-government counterfactual rely on strong assumptions about fixed relative prices and incomes, government efficiency, and the relationship between marginal and total benefits. And those studies are often not designed to identify which types of public services benefit the poor. Researchers should focus more on providing benefit incidence studies on specific government functions or programs that can help policymakers reach conclusions about proposed reallocations of resources among government programs. Benefit incidence should be assigned to households based on household survey information on usage rather than on ad hoc assumptions that assign benefits based on income or the number of members in the household. Improved annual cost measures for services need to be developed, particulary for capital inputs. Researchers should group households by deciles and whenever possible should consider other groupings based on household income adjusted for household composition, age, location, and other relevant socioeconomic variables. Careful attention to life-cycle benefits, benefit shifting, rent-seeking, out-of-pocket costs, displacement of private sector efforts, average versus marginal incidence, and several other issues can significantly increase the value of benefit incidence analysis to policymakers

Metabolic requirements for cancer cell proliferation

Background: The study of cancer metabolism has been largely dedicated to exploring the hypothesis that oncogenic transformation rewires cellular metabolism to sustain elevated rates of growth and division. Intense examination of tumors and cancer cell lines has confirmed that many cancer-associated metabolic phenotypes allow robust growth and survival; however, little attention has been given to explicitly identifying the biochemical requirements for cell proliferation in a rigorous manner in the context of cancer metabolism. Results: Using a well-studied hybridoma line as a model, we comprehensively and quantitatively enumerate the metabolic requirements for generating new biomass in mammalian cells; this indicated a large biosynthetic requirement for ATP, NADPH, NAD+, acetyl-CoA, and amino acids. Extension of this approach to serine/glycine and glutamine metabolic pathways suggested lower limits on serine and glycine catabolism to supply one-carbon unit synthesis and significant availability of glutamine-derived carbon for biosynthesis resulting from nitrogen demands alone, respectively. We integrated our biomass composition results into a flux balance analysis model, placing upper bounds on mitochondrial NADH oxidation to simulate metformin treatment; these simulations reproduced several empirically observed metabolic phenotypes, including increased reductive isocitrate dehydrogenase flux. Conclusions: Our analysis clarifies the differential needs for central carbon metabolism precursors, glutamine-derived nitrogen, and cofactors such as ATP, NADPH, and NAD+, while also providing justification for various extracellular nutrient uptake behaviors observed in tumors. Collectively, these results demonstrate how stoichiometric considerations alone can successfully predict empirically observed phenotypes and provide insight into biochemical dynamics that underlie responses to metabolic perturbations.National Institutes of Health (U.S.) (Grant 1R01DK075850-01)National Institutes of Health (U.S.) (Grant 1R01CA160458-01A1

Reductive glutamine metabolism is a function of the α-ketoglutarate to citrate ratio in cells

Reductively metabolized glutamine is a major cellular carbon source for fatty acid synthesis during hypoxia or when mitochondrial respiration is impaired. Yet, a mechanistic understanding of what determines reductive metabolism is missing. Here we identify several cellular conditions where the α-ketoglutarate/citrate ratio is changed due to an altered acetyl-CoA to citrate conversion, and demonstrate that reductive glutamine metabolism is initiated in response to perturbations that result in an increase in the α-ketoglutarate/citrate ratio. Thus, targeting reductive glutamine conversion for a therapeutic benefit might require distinct modulations of metabolite concentrations rather than targeting the upstream signalling, which only indirectly affects the process.German Science Foundation (Grant FE1185)National Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award Postdoctoral Fellowship F32 CA132358)National Institutes of Health (U.S.) (Grant 5-P30-CA14051-39)Damon Runyon Cancer Research FoundationBurroughs Wellcome FundSmith Family FoundationNational Institutes of Health (U.S.) (Grant 1R01CA160458-01A1

Environment Impacts the Metabolic Dependencies of Ras-Driven Non-Small Cell Lung Cancer

Cultured cells convert glucose to lactate, and glutamine is the major source of tricarboxylic acid (TCA)-cycle carbon, but whether the same metabolic phenotype is found in tumors is less studied. We infused mice with lung cancers with isotope-labeled glucose or glutamine and compared the fate of these nutrients in tumor and normal tissue. As expected, lung tumors exhibit increased lactate production from glucose. However, glutamine utilization by both lung tumors and normal lung was minimal, with lung tumors showing increased glucose contribution to the TCA cycle relative to normal lung tissue. Deletion of enzymes involved in glucose oxidation demonstrates that glucose carbon contribution to the TCA cycle is required for tumor formation. These data suggest that understanding nutrient utilization by tumors can predict metabolic dependencies of cancers in vivo. Furthermore, these data argue that the in vivo environment is an important determinant of the metabolic phenotype of cancer cells.National Science Foundation (U.S.) (Grant T32GM007287

¹³C-metabolic flux analysis of recombinant yeasts for biofuels applications

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2015.Cataloged from PDF version of thesis.Includes bibliographical references.Fossil fuels have powered the transportation industry since the Industrial Revolution. However, rising transportation energy demand and new knowledge about the environmental impact of burning fossil fuels have motivated the development of technologies for sustainable production of renewable, carbon-neutral liquid fuels. To that end, biological systems may be leveraged to fix carbon dioxide and to catalyze the conversion of renewable feed stocks to fuel molecules. Today, the gasoline additive ethanol and biodiesel are produced by yeast fermentation of sugars derived from cornstarch and sucrose and transesterification of vegetable oils, respectively. However ethanol has many drawbacks as a fuel additive, and both biofuels are currently produced from edible feed stocks. For biofuels to contribute significantly to meeting total transportation energy demand, processes for production of fuel molecules from non-food feed stocks must be engineered. Two promising solutions are fermentation of sugars derived from "woody," lignocellulosic biomass and production of fuels from volatile fatty acids (VFAs) such as acetate, which can be produced by fermentation of organics in municipal solid waste and sewage or syngas. The production of biofuels from lignocellulosic material or VFAs will require metabolic engineering of biocatalysts to improve yields, productivities, and final titers. These metabolic engineering efforts can be facilitated by ¹³C-Metabolic Flux Analysis (MFA), a method for elucidating the otherwise unobservable intracellular metabolic fluxes in biological systems. We first developed protocols for extraction and LC-MS/MS analysis of intracellular metabolites, which provides data that may be used for metabolic flux estimation. We then performed an analysis of both the measurement and modeling errors associated with using these data for flux determination. Finally, we applied ¹³C-MFA to two industrially relevant systems: 1) Fermentation of xylose, a sugar present in lignocellulosic biomass, to ethanol in Saccharomyces cerevisiae, and 2) overproduction of fatty acids that may be transesterified to biodiesel from either glucose or acetate in the oleaginous yeast Yarrowia lipolytica. These experiments identified a potential bottleneck in xylose fermentation in S. cerevisiae and the primary source of NADPH for fatty acid biosynthesis in Y. lipolytica, and also suggested potential strategies for improving lipid yields in Y. lipolytica.by Thomas M. Wasylenko.Ph. D

Kinetic isotope effects significantly influence intracellular metabolite [superscript 13]C labeling patterns and flux determination

Rigorous mathematical modeling of carbon-labeling experiments allows estimation of fluxes through the pathways of central carbon metabolism, yielding powerful information for basic scientific studies as well as for a wide range of applications. However, the mathematical models that have been developed for flux determination from [superscript 13]C labeling data have commonly neglected the influence of kinetic isotope effects on the distribution of [superscript 13]C label in intracellular metabolites, as these effects have often been assumed to be inconsequential. We have used measurements of the [superscript 13]C isotope effects on the pyruvate dehydrogenase enzyme from the literature to model isotopic fractionation at the pyruvate node and quantify the modeling errors expected to result from the assumption that isotope effects are negligible. We show that under some conditions kinetic isotope effects have a significant impact on the [superscript 13]C labeling patterns of intracellular metabolites, and the errors associated with neglecting isotope effects in [superscript 13]C-metabolic flux analysis models can be comparable in size to measurement errors associated with GC–MS. Thus, kinetic isotope effects must be considered in any rigorous assessment of errors in [superscript 13]C labeling data, goodness-of-fit between model and data, confidence intervals of estimated metabolic fluxes, and statistical significance of differences between estimated metabolic flux distributions.United States. Dept. of Energy (Grant DE-SC0008744)MIT-National Institute of General Medical Sciences (U.S.) (Biotechnology Training Program

Bifurcations of lurching waves in a thalamic neuronal network

We consider a two-layer, one-dimensional lattice of neurons; one layer consists of excitatory thalamocortical neurons, while the other is comprised of inhibitory reticular thalamic neurons. Such networks are known to support “lurching” waves, for which propagation does not appear smooth, but rather progresses in a saltatory fashion; these waves can be characterized by different spatial widths (different numbers of neurons active at the same time). We show that these lurching waves are fixed points of appropriately defined Poincaré maps, and follow these fixed points as parameters are varied. In this way, we are able to explain observed transitions in behavior, and, in particular, to show how branches with different spatial widths are linked with each other. Our computer-assisted analysis is quite general and could be applied to other spatially extended systems which exhibit this non-trivial form of wave propagation.Universidad de los Andes (Bogotá, Colombia) (Grant (Project FAI ING-002-09))United States. Dept. of EnergyUnited States. Air Force Office of Scientific Researc