Lipid production in Yarrowia lipolytica is maximized by engineering cytosolic redox metabolism.

Microbial factories have been engineered to produce lipids from carbohydrate feedstocks for production of biofuels and oleochemicals. However, even the best yields obtained to date are insufficient for commercial lipid production. To maximize the capture of electrons generated from substrate catabolism and thus increase substrate-to-product yields, we engineered 13 strains of Yarrowia lipolytica with synthetic pathways converting glycolytic NADH into the lipid biosynthetic precursors NADPH or acetyl-CoA. A quantitative model was established and identified the yield of the lipid pathway as a crucial determinant of overall process yield. The best engineered strain achieved a productivity of 1.2 g/L/h and a process yield of 0.27 g-fatty acid methyl esters/g-glucose, which constitutes a 25% improvement over previously engineered yeast strains. Oxygen requirements of our highest producer were reduced owing to decreased NADH oxidization by aerobic respiration. We show that redox engineering could enable commercialization of microbial carbohydrate-based lipid production.

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.

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.

The oxidative pentose phosphate pathway is the primary source of NADPH for lipid overproduction from glucose in Yarrowia lipolytica.

Oleaginous microbes represent an attractive means of converting a diverse range of feedstocks into oils that can be transesterified to biodiesel. However, the mechanism of lipid overproduction in these organisms is incompletely understood, hindering the development of strategies for engineering superior biocatalysts for "single-cell oil" production. In particular, it is unclear which pathways are used to generate the large quantities of NADPH required for overproduction of the highly reduced fatty acid species. While early studies implicated malic enzyme as having a key role in production of lipogenic NADPH in oleaginous fungi, several recent reports have cast doubts as to whether malic enzyme may contribute to production of lipogenic NADPH in the model oleaginous yeast Yarrowia lipolytica. To address this problem we have used (13)C-Metabolic Flux Analysis to estimate the metabolic flux distributions during lipid accumulation in two Y. lipolytica strains; a control strain and a previously published engineered strain capable of producing lipids at roughly twice the yield. We observe a dramatic rearrangement of the metabolic flux distribution in the engineered strain which supports lipid overproduction. The NADPH-producing flux through the oxidative Pentose Phosphate Pathway is approximately doubled in the engineered strain in response to the roughly two-fold increase in fatty acid biosynthesis, while the flux through malic enzyme does not differ significantly between the two strains. Moreover, the estimated rate of NADPH production in the oxidative Pentose Phosphate Pathway is in good agreement with the estimated rate of NADPH consumption in fatty acid biosynthesis in both strains. These results suggest the oxidative Pentose Phosphate Pathway is the primary source of lipogenic NADPH in Y. lipolytica.

Metabolomic and (13)C-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.

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.

Kinetic isotope effects significantly influence intracellular metabolite (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 (13) C labeling data have commonly neglected the influence of kinetic isotope effects on the distribution of (13) C label in intracellular metabolites, as these effects have often been assumed to be inconsequential. We have used measurements of the (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 (13) C labeling patterns of intracellular metabolites, and the errors associated with neglecting isotope effects in (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 (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.

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.