Large-Scale 13C Flux Profiling Reveals Conservation of the Entner-Doudoroff Pathway as a Glycolytic Strategy among Marine Bacteria That Use Glucose

Marine bacteria form one of the largest living surfaces on Earth, and their metabolic activity is of fundamental importance for global nutrient cycling. Here, we explored the largely unknown intracellular pathways in 25 microbes representing different classes of marine bacteria that use glucose: Alphaproteobacteria, Gammaproteobacteria, and Flavobacteriia of the Bacteriodetes phylum. We used ¹³C isotope experiments to infer metabolic fluxes through their carbon core pathways. Notably, 90% of all strains studied use the Entner-Doudoroff (ED) pathway for glucose catabolism, whereas only 10% rely on the Embden-Meyerhof-Parnas (EMP) pathway. This result differed dramatically from the terrestrial model strains studied, which preferentially used the EMP pathway yielding high levels of ATP. Strains using the ED pathway exhibited a more robust resistance against the oxidative stress typically found in this environment. An important feature contributing to the preferential use of the ED pathway in the oceans could therefore be enhanced supply of NADPH through this pathway. The marine bacteria studied did not specifically rely on a distinct anaplerotic route, but the carboxylation of phosphoenolpyruvate (PEP) or pyruvate for fueling of the tricarboxylic acid (TCA) cycle was evenly distributed. The marine isolates studied belong to clades that dominate the uptake of glucose, a major carbon source for bacteria in seawater. Therefore, the ED pathway may play a significant role in the cycling of mono- and polysaccharides by bacterial communities in marine ecosystems.     

Carbon flux distribution in antibiotic-producing chemostat cultures of Streptomyces lividans

The carbon metabolism of derivatives of Streptomyces lividans growing under phosphate limitation in chemostat cultures and producing the antibiotics actinorhodin and undecylprodigiosin was investigated. By applying metabolic flux analysis to a stoichiometric model, the relationship between antibiotic production, biomass accumulation, and carbon flux through the major carbon metabolic pathways (the Embden Meyerhoff Parnas and pentose-phosphate pathways) was analyzed. Distribution of carbon flux through the catabolic pathways was shown to be dependent on growth rate, as well as on the carbon and energy source (glucose or gluconate) used. Increasing growth rates promoted an increase in the flux of carbon through glycolysis and the pentose-phosphate pathway. The synthesis of both actinorhodin and undecylprodigiosin was found to be inversely related to flux through the pentose-phosphate pathway.     

Metabolic-flux profiling of the yeasts Saccharomyces cerevisiae and Pichia stipitis

The so far largely uncharacterized central carbon metabolism of the yeast Pichia stipitis was explored in batch and glucose-limited chemostat cultures using metabolic-flux ratio analysis by nuclear magnetic resonance. The concomitantly characterized network of active metabolic pathways was compared to those identified in Saccharomyces cerevisiae, which led to the following conclusions. (i) There is a remarkably low use of the non-oxidative pentose phosphate (PP) pathway for glucose catabolism in S. cerevisiae when compared to P. stipitis batch cultures. (ii) Metabolism of P. stipitis batch cultures is fully respirative, which contrasts with the predominantly respiro-fermentative metabolic state of S. cerevisiae. (iii) Glucose catabolism in chemostat cultures of both yeasts is primarily oxidative. (iv) In both yeasts there is significant in vivo malic enzyme activity during growth on glucose. (v) The amino acid biosynthesis pathways are identical in both yeasts. The present investigation thus demonstrates the power of metabolic-flux ratio analysis for comparative profiling of central carbon metabolism in lower eukaryotes. Although not used for glucose catabolism in batch culture, we demonstrate that the PP pathway in S. cerevisiae has a generally high catabolic capacity by overexpressing the Escherichia coli transhydrogenase UdhA in phosphoglucose isomerase-deficient S. cerevisiae.     

Application of metabolic flux analysis for the identification of metabolic bottlenecks in the biosynthesis of penicillin-G

A detailed stoichiometric model was developed for growth and penicillin-G production in Penicillium chrysogenum. From an a priori metabolic flux analysis using this model it appeared that penicillin production requires significant changes in fluxes through the primary metabolic pathways. This is brought about by the biosynthesis of carbon precursors for the beta-lactan nucleus and an increased demand for NADPH, mainly for sulfate reduction. As a result, significant changes in flux partitioning occur around four principal nodes in primary metabolism. These are located at: (1) glucose-6-phosphate; (2) 3-phosphoglycerate; (3) mitochondrial pyruvate; and (4) mitochondrial isocitrate. These nodes should be regarded as potential bottlenecks for increased productivity. The flexibility of these principal nodes was investigated by experimental manipulation of the fluxes through the central metabolic pathways using a high-producing strain of P. chrysogenum. Metabolic fluxes were manipulated through growth of the cells on different substrates in carbon-limited chemostat culture. Metabolic flux analysis, based on measured input and output fluxes, was used to calculate the fluxes around the principal nodes. It was found that, for growth on glucose, ethanol, and acetate, the flux partitioning around these nodes differed significantly. However, this had hardly any effect on penicillin productivity, showing that primary carbon metabolism is not likely to contain potential bottlenecks. Further experiments were performed to manipulate the total metabolic demand for the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). NADPH demand was increased stepwise by cultivating the cells on glucose or xylose as the carbon source combined with either ammonia or nitrate as the nitrogen source, which resulted in a stepwise decrease of penicillin production. This clearly shows that, in penicillin fermentation, possible limitations in primary metabolism reside in the supply/regeneration of cofactors (NADPH) rather than in the supply of carbon precursors.     

Redox rebalance against genetic perturbations and modulation of central carbon metabolism by the oxidative stress regulation

The micro-aerophilic organisms and aerobes as well as yeast and higher organisms have evolved to gain energy through respiration (via oxidative phosphorylation), thereby enabling them to grow much faster than anaerobes. However, during respiration, reactive oxygen species (ROSs) are inherently (inevitably) generated, and threaten the cell’s survival. Therefore, living organisms (or cells) must furnish the potent defense systems to keep such ROSs at harmless level, where the cofactor balance plays crucial roles. Namely, NADH is the source of energy generation (catabolism) in the respiratory chain reactions, through which ROSs are generated, while NADPH plays important roles not only for the cell synthesis (anabolism) but also for detoxifying ROSs. Therefore, the cell must rebalance the redox ratio by modulating the fluxes of the central carbon metabolism (CCM) by regulating the multi-level regulation machinery upon genetic perturbations and the change in the growth conditions.Here, we discuss about how aerobes accomplish such cofactor homeostasis against redox perturbations. In particular, we consider how single-gene mutants (including pgi, pfk, zwf, gnd and pyk mutants) modulate their metabolisms in relation to cofactor rebalance (and also by adaptive laboratory evolution). We also discuss about how the overproduction of NADPH (by the pathway gene mutation) can be utilized for the efficient production of useful value-added chemicals such as medicinal compounds, polyhydroxyalkanoates, and amino acids, all of which require NADPH in their synthetic pathways. We then discuss about the metabolic responses against oxidative stress, where αketoacids play important roles not only for the coordination between catabolism and anabolism, but also for detoxifying ROSs by non-enzymatic reactions, as well as for reducing the production of ROSs by repressing the activities of the TCA cycle and respiration (via carbon catabolite repression). Thus, we discuss about the mechanisms (basic strategies) that modulate the metabolism from respiration to respiro-fermentative metabolism causing overflow, based on the role of Pyk activity, affecting the NADPH production at the oxidative pentose phosphate (PP) pathway, and the roles of αketoacids for the change in the source of energy generation from the oxidative phosphorylation to the substrate level phosphorylation.     

Carbon Metabolism in Spores of the Arbuscular Mycorrhizal Fungus Glomus intraradices as Revealed by Nuclear Magnetic Resonance Spectroscopy

Arbuscular mycorrhizal (AM) fungi are obligate symbionts that colonize the roots of over 80% of plants in all terrestrial environments. Understanding why AM fungi do not complete their life cycle under free-living conditions has significant implications for the management of one of the world's most important symbioses. We used (13)C-labeled substrates and nuclear magnetic resonance spectroscopy to study carbon fluxes during spore germination and the metabolic pathways by which these fluxes occur in the AM fungus Glomus intraradices. Our results indicate that during asymbiotic growth: (a) sugars are made from stored lipids; (b) trehalose (but not lipid) is synthesized as well as degraded; (c) glucose and fructose, but not mannitol, can be taken up and utilized; (d) dark fixation of CO(2) is substantial; and (e) arginine and other amino acids are synthesized. The labeling patterns are consistent with significant carbon fluxes through gluconeogenesis, the glyoxylate cycle, the tricarboxylic acid cycle, glycolysis, non-photosynthetic one-carbon metabolism, the pentose phosphate pathway, and most or all of the urea cycle. We also report the presence of an unidentified betaine-like compound. Carbon metabolism during asymbiotic growth has features in between those presented by intraradical and extraradical hyphae in the symbiotic state.     

Identification of the Entner-Doudoroff pathway in an antibiotic-producing actinomycete species

The metabolic network of the central carbon metabolism represents the backbone of cellular metabolism and provides the precursors and cofactors required for synthesis of secondary metabolites. It is therefore pivotal to map the operating metabolic network in the central carbon metabolism in order to design metabolic engineering strategies towards construction of more efficient producers of specific metabolites. In this context, methods that allow rapid and reliable mapping of the central carbon metabolism are valuable. In the present study, a (13)C labelling-based method was used to identify the primary metabolic pathways of the poorly characterized antibiotic-producing actinomycete Nonomuraea sp. ATCC 39727. Surprisingly, it was found that Nonomuraea sp. ATCC 39272 predominantly metabolizes glucose via the Entner-Doudoroff (ED) pathway. This represents the first time that the ED pathway has been recognized as the main catabolic pathway in an actinomycete. The Nonomuraea genes encoding the key enzymes of the ED pathway were subsequently identified, sequenced and functionally described.     

Parameter estimation and dynamic control analysis of central carbon metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The central carbon metabolic pathway is the most important among metabolic pathways in all microorganisms since it produces energy and precursors for biosynthesis. In this study, a dynamic model for central carbon metabolism in Escherichia coli (E. coli) consisting of the phosphotransferase (PTS) system, glycolysis, pentose-phosphate pathway (PPP), and storage materials was obtained by ameliorating the model proposed by Chassagnole et al. (2002). In order to improve the performance of the model, principal parameters were estimated through the experimental measurements of intracellular concentrations of metabolites under transient conditions. Through dynamic metabolic control analysis (MCA), the tendencies of the metabolic fluxes at branch points were investigated, and the key parameters and enzyme kinetics that most dominantly affected the productivity of the desired metabolites were determined.     

Dynamic flux cartography of hairy roots primary metabolism

A dynamic model for plant cell and hairy root primary metabolism is presented. The model includes nutrient uptake (Pi, sugars, nitrogen sources), the glycolysis and pentose phosphate pathways, the TCA cycle, amino acid biosynthesis, respiratory chain, biosynthesis of cell building blocks (structural hexoses, organic acids, lipids, and organic phosphated molecules). The energy shuttles (ATP, ADP) and cofactors (NAD/H, NADP/H) are also included. The model describes the kinetics of 44 biochemical reactions (fluxes) of the primary metabolism of plant cells and includes 41 biochemical species (metabolites, nutrients, biomass components). Multiple Michaelis-Menten type kinetics are used to describe biochemical reaction rates. Known regulatory phenomena on metabolic pathways are included using sigmoid switch functions. A visualization framework showing fluxes and metabolite concentrations over time is presented. The visualization of fluxes and metabolites is used to analyze simulation results from Catharanthus roseus hairy root 50 d batch cultures. The visualization of the metabolic system allows analyzing split ratios between pathways and flux time-variations. For carbon metabolism, the cells were observed to have relatively high and stable fluxes for the central carbon metabolism and low and variable fluxes for anabolic pathways. For phosphate metabolism, a very high free intracellular Pi turnover rate was observed with higher flux variations than for the carbon metabolism. Nitrogen metabolism also exhibited large flux variations. The potential uses of the model are also discussed.     

Resolving the TCA cycle and pentose-phosphate pathway of Clostridium acetobutylicum ATCC 824: Isotopomer analysis, in vitro activities and expression analysis

Solventogenic clostridia are an important class of microorganisms that can produce various biofuels. One of the bottlenecks in engineering clostridia stems from the fact that central metabolic pathways remain poorly understood. Here, we utilized the power of (13) C-based isotopomer analysis to re-examine central metabolic pathways of Clostridium acetobutylicum ATCC 824. We demonstrate using [1,2-(13) C]glucose, MS analysis of intracellular metabolites, and enzymatic assays that C. acetobutylicum has a split TCA cycle where only Re-citrate synthase (CS) contributes to the production of α-ketoglutarate via citrate. Furthermore, we show that there is no carbon exchange between α-ketoglutarate and fumarate and that the oxidative pentose-phosphate pathway (oxPPP) is inactive. Dynamic gene expression analysis of the putative Re-CS gene (CAC0970), its operon, and all glycolysis, pentose-phosphate pathway, and TCA cycle genes identify genes and their degree of involvement in these core pathways that support the powerful primary metabolism of this industrial organism.     

Elucidating the role of copper in CHO cell energy metabolism using 13C metabolic flux analysis

¹³C‐metabolic flux analysis was used to understand copper deficiency‐related restructuring of energy metabolism, which leads to excessive lactate production in recombinant protein‐producing CHO cells. Stationary‐phase labeling experiments with U‐¹³C glucose were conducted on CHO cells grown under high and limiting copper in 3 L fed‐batch bioreactors. The resultant labeling patterns of soluble metabolites were measured by GC‐MS and used to estimate metabolic fluxes in the central carbon metabolism pathways using OpenFlux. Fluxes were evaluated 300 times from stoichiometrically feasible random guess values and their confidence intervals calculated by Monte Carlo simulations. Results from metabolic flux analysis exhibited significant carbon redistribution throughout the metabolic network in cells under Cu deficiency. Specifically, glycolytic fluxes increased (25%–79% relative to glucose uptake) whereas fluxes through the TCA and pentose phosphate pathway (PPP) were lower (15%–23% and 74%, respectively) compared with the Cu‐containing condition. Furthermore, under Cu deficiency, 33% of the flux entering TCA via the pyruvate node was redirected to lactate and malate production. Based on these results, we hypothesize that Cu deficiency disrupts the electron transport chain causing ATP deficiency, redox imbalance, and oxidative stress, which in turn drive copper‐deficient CHO cells to produce energy via aerobic glycolysis, which is associated with excessive lactate production, rather than the more efficient route of oxidative phosphorylation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1179–1186, 2015     

Hierarchy in Pentose Sugar Metabolism in Clostridium acetobutylicum

Bacterial metabolism of polysaccharides from plant detritus into acids and solvents is an essential component of the terrestrial carbon cycle. Understanding the underlying metabolic pathways can also contribute to improved production of biofuels. Using a metabolomics approach involving liquid chromatography-mass spectrometry, we investigated the metabolism of mixtures of the cellulosic hexose sugar (glucose) and hemicellulosic pentose sugars (xylose and arabinose) in the anaerobic soil bacterium Clostridium acetobutylicum. Simultaneous feeding of stable isotope-labeled glucose and unlabeled xylose or arabinose revealed that, as expected, glucose was preferentially used as the carbon source. Assimilated pentose sugars accumulated in pentose phosphate pathway (PPP) intermediates with minimal flux into glycolysis. Simultaneous feeding of xylose and arabinose revealed an unexpected hierarchy among the pentose sugars, with arabinose utilized preferentially over xylose. The phosphoketolase pathway (PKP) provides an alternative route of pentose catabolism in C. acetobutylicum that directly converts xylulose-5-phosphate into acetyl-phosphate and glyceraldehyde-3-phosphate, bypassing most of the PPP. When feeding the mixture of pentose sugars, the labeling patterns of lower glycolytic intermediates indicated more flux through the PKP than through the PPP and upper glycolysis, and this was confirmed by quantitative flux modeling. Consistent with direct acetyl-phosphate production from the PKP, growth on the pentose mixture resulted in enhanced acetate excretion. Taken collectively, these findings reveal two hierarchies in clostridial pentose metabolism: xylose is subordinate to arabinose, and the PPP is used less than the PKP.     

Quantitative analysis of energy metabolic pathways in MCF-7 breast cancer cells by selected reaction monitoring assay

To investigate the quantitative response of energy metabolic pathways in human MCF-7 breast cancer cells to hypoxia, glucose deprivation, and estradiol stimulation, we developed a targeted proteomics assay for accurate quantification of protein expression in glycolysis/gluconeogenesis, TCA cycle, and pentose phosphate pathways. Cell growth conditions were selected to roughly mimic the exposure of cells in the cancer tissue to the intermittent hypoxia, glucose deprivation, and hormonal stimulation. Targeted proteomics assay allowed for reproducible quantification of 76 proteins in four different growth conditions after 24 and 48 h of perturbation. Differential expression of a number of control and metabolic pathway proteins in response to the change of growth conditions was found. Elevated expression of the majority of glycolytic enzymes was observed in hypoxia. Cancer cells, as opposed to near-normal MCF-10A cells, exhibited significantly increased expression of key energy metabolic pathway enzymes (FBP1, IDH2, and G6PD) that are known to redirect cellular metabolism and increase carbon flux through the pentose phosphate pathway. Our quantitative proteomic protocol is based on a mass spectrometry-compatible acid-labile detergent and is described in detail. Optimized parameters of a multiplex selected reaction monitoring (SRM) assay for 76 proteins, 134 proteotypic peptides, and 401 transitions are included and can be downloaded and used with any SRM-compatible mass spectrometer. The presented workflow is an integrated tool for hypothesis-driven studies of mammalian cells as well as functional studies of proteins, and can greatly complement experimental methods in systems biology, metabolic engineering, and metabolic transformation of cancer cells.     

Comparative study on central metabolic fluxes of <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strains in continuous culture using <Superscript>13</Superscript>C labelled substrates

Fluxes of central carbon metabolism [glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA cycle), biomass formation] were determined for several Bacillus megaterium strains (DSM319, WH320, WH323, MS941) in C- and N-limited chemostat cultures by ¹³C labelling experiments. The labelling patterns of proteinogenic amino acids were analysed by GC/MS and therefrom flux ratios at important nodes within the metabolic network could be calculated. On the basis of a stoichiometric metabolic model flux distributions were estimated for the different B. megaterium strains used at various cultivation conditions. Generally all strains exhibited similar metabolic flux distributions, however, several significant changes were found in (1) the glucose flux entering the PPP via the oxidative branch, (2) the reversibilities within the PPP, (3) the relative fluxes of pyruvate and acetyl-CoA fed to the TCA cycle, (4) the fluxes around the pyruvate node involving a futile cycle.