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Corynebacterium glutamicum is known for its ability to produce glutamic acid and has been utilized for the fermentative production of various amino acids. Glutamic acid production in C. glutamicum is induced by penicillin. In this study, the transcriptome and metabolome of C. glutamicum is analyzed to understand the mechanism of penicillin-induced glutamic acid production. Transcriptomic analysis with DNA microarray revealed that expression of some glycolysis- and TCA cycle-related genes, which include those encoding the enzymes involved in conversion of glucose to 2-oxoglutaric acid, is upregulated after penicillin addition. Meanwhile, expression of some TCA cycle-related genes, encoding the enzymes for conversion of 2-oxoglutaric acid to oxaloacetic acid, and the anaplerotic reactions decreased. In addition, expression of NCgl1221 and odhI, encoding proteins involved in glutamic acid excretion and inhibition of the 2-oxoglutarate dehydrogenase, respectively, is upregulated. Functional category enrichment analysis of genes upregulated and downregulated after penicillin addition revealed that genes for signal transduction systems are enriched among upregulated genes, whereas those for energy production and carbohydrate and amino acid metabolisms are enriched among the downregulated genes. As for the metabolomic analysis using capillary electrophoresis time-of-flight mass spectrometry, the intracellular content of most metabolites of the glycolysis and the TCA cycle decreased dramatically after penicillin addition. Overall, these results indicate that the cellular metabolism and glutamic acid excretion are mainly optimized at the transcription level during penicillin-induced glutamic acid production by C. glutamicum.

Covering two-thirds of our planet, the global deep ocean plays a central role in supporting life on Earth. Among other processes, this biggest ecosystem buffers the rise of atmospheric CO2. Despite carbon sequestration in the deep ocean has been known for a long time, microbial activity in the meso- and bathypelagic realm via the “assimilation of bicarbonate in the dark” (ABD) has only recently been described in more details. Based on recent findings, this process seems primarily the result of chemosynthetic and anaplerotic reactions driven by different groups of deep-sea prokaryoplankton. We quantified bicarbonate assimilation in relation to total prokaryotic abundance, prokaryotic heterotrophic production and respiration in the meso- and bathypelagic Mediterranean Sea. The measured ABD values, ranging from 133 to 370 μg C m−3 d−1, were among the highest ones reported worldwide for similar depths, likely due to the elevated temperature of the deep Mediterranean Sea (13–14°C also at abyssal depths). Integrated over the dark water column (≥200 m depth), bicarbonate assimilation in the deep-sea ranged from 396 to 873 mg C m−2 d−1. This quantity of produced de novo organic carbon amounts to about 85–424% of the phytoplankton primary production and covers up to 62% of deep-sea prokaryotic total carbon demand. Hence, the ABD process in the meso- and bathypelagic Mediterranean Sea might substantially contribute to the inorganic and organic pool and significantly sustain the deep-sea microbial food web. To elucidate the ABD key-players, we established three actively nitrifying and CO2-fixing prokaryotic enrichments. Consortia were characterized by the co-occurrence of chemolithoautotrophic Thaumarchaeota and chemoheterotrophic proteobacteria. One of the enrichments, originated from Ionian bathypelagic waters (3,000 m depth) and supplemented with low concentrations of ammonia, was dominated by the Thaumarchaeota “low-ammonia-concentration” deep-sea ecotype, an enigmatic and ecologically important group of organisms, uncultured until this study.

Bacillus licheniformis which can produce cellulase including endo glucanase and glucosidase is an important industrial microbe for cellulose degradation. The purpose of this research was to assess the effect of endo glucanase gene bglC and glucosidase gene bglH on the central metabolic flux in B. licheniformis. bglC and bglH were knocked out using homologous recombination method, respectively, and the corresponding knockout strains were obtained for 13C metabolic flux analysis. A significant change was observed in metabolic fluxes after 13C metabolic flux ratio analysis. In both of the knockout strains, the increased fluxes of the pentose phosphate pathway and malic enzyme reaction enabled an elevated supply of NADPH which provided enough reducing power for the in vivo synthesis reactions. The fluxes through tricarboxylic acid cycle and anaplerotic reactions increased fast in the two knockout strains, which meant more energy generated. The changed fluxes in central carbon metabolism provided a holistic view of the physiological status in B. licheniformis and possible targets for further strain engineering. This article is protected by copyright. All rights reserved.

In contrast to stoichiometric-based models, the development of large-scale kinetic models of metabolism has been hindered by the challenge of identifying kinetic parameter values and kinetic rate laws applicable to a wide range of environmental and/or genetic perturbations. The recently introduced ensemble modeling (EM) procedure provides a promising remedy to address these challenges by decomposing metabolic reactions into elementary reaction steps and incorporating all phenotypic observations, upon perturbation, in its model parameterization scheme. Here, we present a kinetic model of Escherichia coli core metabolism that satisfies the fluxomic data for wild-type and seven mutant strains by making use of the EM concepts. This model encompasses 138 reactions, 93 metabolites and 60 substrate-level regulatory interactions accounting for glycolysis/gluconeogenesis, pentose phosphate pathway, TCA cycle, major pyruvate metabolism, anaplerotic reactions and a number of reactions in other parts of the metabolism. Parameterization is performed using a formal optimization approach that minimizes the discrepancies between model predictions and flux measurements. The predicted fluxes by the model are within the uncertainty range of experimental flux data for 78% of the reactions (with measured fluxes) for both the wild-type and seven mutant strains. The remaining flux predictions are mostly within three standard deviations of reported ranges. Converting the EM-based parameters into a Michaelis-Menten equivalent formalism revealed that 35% of Km and 77% of kcat parameters are within uncertainty range of the literature-reported values. The predicted metabolite concentrations by the model are also within uncertainty ranges of metabolomic data for 68% of the metabolites. A leave-one-out cross-validation test to evaluate the flux prediction performance of the model showed that metabolic fluxes for the mutants located in the proximity of mutations used for training the model can be predicted more accurately. The constructed model and the parameterization procedure presented in this study pave the way for the construction of larger-scale kinetic models with more narrowly distributed parameter values as new metabolomic/fluxomic data sets are becoming available for E. coli and other organisms.

The central process in energy production is the oxidation of acetyl-CoA to CO2 by the tricarboxylic acid (TCA, Krebs, citric acid) cycle. However, this cycle functions also as a biosynthetic pathway from which intermediates leave to be converted primarily to glutamate, GABA, glutamine and aspartate and to a smaller extent to glucose derivatives and fatty acids in the brain. When TCA cycle ketoacids are removed, they must be replaced to permit the continued function of this essential pathway, by a process termed anaplerosis. Since the TCA cycle cannot act as a carbon sink, anaplerosis must be coupled with cataplerosis; the exit of intermediates from the TCA cycle. The role of anaplerotic reactions for cellular metabolism in the brain has been studied extensively. However, the coupling of this process with cataplerosis and the roles that both pathways play in the regulation of amino acid, glucose, and fatty acid homeostasis have not been emphasized. The concept of a linkage between anaplerosis and cataplerosis should be underscored, because the balance between these two processes is essential. The hypothesis that cataplerosis in the brain is achieved by exporting the lactate generated from the TCA cycle intermediates into the blood and perivascular area is presented. This shifts the generally accepted paradigm of lactate generation as simply derived from glycolysis to that of oxidation and might present an alternative explanation for aerobic glycolysis. Intermediates leave the tricarboxylic acid cycle and must be replaced by a process termed anaplerosis that must be coupled to cataplerosis. We hypothesize that cataplerosis is achieved by exporting the lactate generated from the cycle into the blood and perivascular area. This shifts the paradigm of lactate generation as solely derived from glycolysis to that of oxidation and might present an alternative explanation for aerobic glycolysis.

Fed-batch production of the aromatic amino acid L-phenylalanine was studied with recombinant Escherichia coli strains on a 15 L-scale using glycerol as carbon source. Flux Variability Analysis (FVA) was applied for intracellular flux estimation to obtain an insight into intracellular flux distribution during L-phenylalanine production. Variability analysis revealed great flux uncertainties in the central carbon metabolism, especially concerning malate consumption. Due to these results two recombinant strains were genetically engineered differing in the ability of malate degradation and anaplerotic reactions (E. coli FUS4.11 ΔmaeA pF81kan and E. coli FUS4.11 ΔmaeA ΔmaeB pF81kan). Applying these malic enzyme knock-out mutants in the standardized L-phenylalanine production process resulted in almost identical process performances (e.g., L-phenylalanine concentration, production rate and byproduct formation). This clearly highlighted great redundancies in central metabolism in E. coli. Uncertainties of intracellular flux estimations by constraint-based analyses during fed-batch production of L-phenylalanine were drastically reduced by application of the malic enzyme knock-out mutants. Biotechnol. Bioeng. 2014;111: 1406–1416. © 2014 Wiley Periodicals, Inc.

The glyoxylate cycle is a sequence of anaplerotic reactions catalyzed by the key enzymes isocitrate lyase (ICL) and malate synthase, and plays an important role in the pathogenesis of microorganisms during infection. An icl-deletion mutant of Candida albicans exhibited reduced virulence in mice compared with the wild type. Five diketopiperazines, which are small and stable cyclic peptides, isolated from the marine-derived Streptomyces puniceus Act1085, were evaluated for their inhibitory effects on C. albicans ICL. The structures of these compounds were elucidated based on spectroscopic data and comparisons with previously reported data. Cyclo(L-Phe-L-Val) was identified as a potent ICL inhibitor, with a half maximal inhibitory concentration of 27 &mu, g/mL. Based on the growth phenotype of the icl-deletion mutants and icl expression analyses, we demonstrated that cyclo(L-Phe-L-Val) inhibits the gene transcription of ICL in C. albicans under C2-carbon-utilizing conditions.

We investigated the growth kinetics and transcriptional responses of Corynebacterium glutamicum in environments with low (pCO2

Biological soil crusts (BSCs) cover extensive portions of the earth’s deserts. In order to survive desiccation cycles and utilize short periods of activity during infrequent precipitation, crust microorganisms must rely on the unique capabilities of vegetative cells to enter a dormant state and be poised for rapid resuscitation upon wetting. To elucidate the key events involved in the exit from dormancy, we performed a wetting experiment of a BSC and followed the response of the dominant cyanobacterium, Microcoleus vaginatus, in situ using a whole-genome transcriptional time course that included two diel cycles. Immediate, but transient, induction of DNA repair and regulatory genes signaled the hydration event. Recovery of photosynthesis occurred within 1 h, accompanied by upregulation of anabolic pathways. Onset of desiccation was characterized by the induction of genes for oxidative and photo-oxidative stress responses, osmotic stress response and the synthesis of C and N storage polymers. Early expression of genes for the production of exopolysaccharides, additional storage molecules and genes for membrane unsaturation occurred before drying and hints at preparedness for desiccation. We also observed signatures of preparation for future precipitation, notably the expression of genes for anaplerotic reactions in drying crusts, and the stable maintenance of mRNA through dormancy. These data shed light on possible synchronization between this cyanobacterium and its environment, and provides key mechanistic insights into its metabolism in situ that may be used to predict its response to climate, and or, land-use driven perturbations.

Roseobacter clade bacteria are ubiquitous in marine environments and now thought to be significant contributors to carbon and sulfur cycling. However, only a few strains of roseobacters have been isolated from the deep-sea water column and have not been thoroughly investigated. Here, we present the complete genomes of phylogentically closed related Thiobacimonas profunda JLT2016 and Pelagibaca abyssi JLT2014 isolated from deep-sea water of the Southeastern Pacific. The genome sequences showed that the two deep-sea roseobacters carry genes for versatile metabolisms with functional capabilities such as ribulose bisphosphate carboxylase-mediated carbon fixation and inorganic sulfur oxidation. Physiological and biochemical analysis showed that T. profunda JLT2016 was capable of autotrophy, heterotrophy, and mixotrophy accompanied by the production of exopolysaccharide. Heterotrophic carbon fixation via anaplerotic reactions contributed minimally to bacterial biomass. Comparative proteomics experiments showed a significantly up-regulated carbon fixation and inorganic sulfur oxidation associated proteins under chemolithotrophic conditions compared to heterotrophic conditions. Collectively, rosebacters show a high metabolic flexibility, suggesting a considerable capacity for adaptation to the marine environment.

Background Glycerol, a byproduct of biodiesel, has become a readily available and inexpensive carbon source for the production of high-value products. However, the main drawback of glycerol utilization is the low consumption rate and shortage of NADPH formation, which may limit the production of NADPH-requiring products. To overcome these problems, we constructed a carbon catabolite repression-negative ΔptsGglpK* mutant by both blocking a key glucose PTS transporter and enhancing the glycerol conversion. The mutant can recover normal growth by co-utilization of glycerol and glucose after loss of glucose PTS transporter. To reveal the metabolic potential of the ΔptsGglpK* mutant, this study examined the flux distributions and regulation of the co-metabolism of glycerol and glucose in the mutant. Results By labeling experiments using [1,3-13C]glycerol and [1-13C]glucose, 13C metabolic flux analysis was employed to decipher the metabolisms of both the wild-type strain and the ΔptsGglpK* mutant in chemostat cultures. When cells were maintained at a low dilution rate (0.1 h−1), the two strains showed similar fluxome profiles. When the dilution rate was increased, both strains upgraded their pentose phosphate pathway, glycolysis and anaplerotic reactions, while the ΔptsGglpK* mutant was able to catabolize much more glycerol than glucose (more than tenfold higher). Compared with the wild-type strain, the mutant repressed its flux through the TCA cycle, resulting in higher acetate overflow. The regulation of fluxomes was consistent with transcriptional profiling of several key genes relevant to the TCA cycle and transhydrogenase, namely gltA, icdA, sdhA and pntA. In addition, cofactor fluxes and their pool sizes were determined. The ΔptsGglpK* mutant affected the redox NADPH/NADH state and reduced the ATP level. Redox signaling activated the ArcA regulatory system, which was responsible for TCA cycle repression. Conclusions This work employs both 13C-MFA and transcription/metabolite analysis for quantitative investigation of the co-metabolism of glycerol and glucose in the ΔptsGglpK* mutant. The ArcA regulatory system dominates the control of flux redistribution. The ΔptsGglpK* mutant can be used as a platform for microbial cell factories for the production of biofuels and biochemicals, since most of fuel molecule (e.g., alcohols) synthesis requires excess reducing equivalents. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0591-1) contains supplementary material, which is available to authorized users.

Cancer cells have a greater need for energy and a ready supply of the building blocks necessary for the synthesis of macromolecules (nucleotides, protein, lipids) in order to duplicate genome and biomass. The hypothesis can be postulated that those precursors for synthetic processes, which can only be furnished by glycolysis, cannot be sufficiently recruited from external sources (the blood stream) and that glycolysis is necessarily markedly activated. It can also be hypothesized that the Krebs cycle, which also furnishes precursors for macromolecule synthesis to meet the requirements of proliferating cells, is depleted of intermediates. In view of its cyclic nature requiring not only pyruvate but also oxalacetate as the "last" metabolite of the reaction sequence for its sustenance, the Krebs cycle may be partially inactivated. While anaplerotic reactions and other sources (amino acids and fatty acids) could supply the cycle with intermediates, those pathways constitute futile cycles for amino and fatty acids as they would be partially degraded in the cycle and the intermediates thus obtained would be exported into the cytoplasm for synthetic processes with no advantage for the cell. It is also hypothesized that glutamine, an important fuel for cancer cells and playing a critical role in anaplerosis, may not contribute to reinforce the cycle; malate and α-ketoglutarate, two products of glutamine metabolism, might be exported from the mitochondria as precursors of biosynthetic pathways. It is possible then that malate, used for NADPH production required in the biosynthetic pathways, and glycerol-phosphate, too used for biosynthetic purposes (lipid biosynthesis), are unable to sustain the mitochondrial redox shuttles reducing the respiratory capacity of the mitochondria. Low shuttle capacity implies that NADH generated by glycolysis needs to be continuously re-oxidized in the cytoplasm via lactate dehydrogenase to maintain glycolysis fully activated, causing the abnormal lactate production observed in cancer. The paper goes onto discuss the essential role of glucose in cancer cell proliferation also in inducing the Crabtree effect. It is finally hypothesized that respiration inhibition after cancer cells have been supplied with glucose is due to reactivation in a suited medium of biosynthetic pathways with the consequences described above.

This work is a computational study based on a new detailed metabolic network model comprising well-mixed compartments representing separate cytosol and mitochondria of astrocytes, glutamatergic and gamma aminobutyric acid (GABA)ergic neurons, communicating through an extracellular space compartment and fed by arterial blood flow. Our steady-state analysis assumes statistical mass balance of both carbons and amino groups. The study is based on Bayesian flux balance analysis, which uses Markov chain Monte Carlo sampling techniques and provides a quantitative description of steady states when the two exchangers aspartate-glutamate carrier (AGC1) and oxoglutarate carrier (OGC) in the malate-aspartate shuttle in astrocyte are not in equilibrium, as recent studies suggest. It also highlights the importance of anaplerotic reactions, pyruvate carboxylase in astrocyte and malic enzyme in neurons, for neurotransmitter synthesis and recycling. The model is unbiased with respect to the glucose partitioning between cell types, and shows that determining the partitioning cannot be done by stoichiometric constraints alone. Furthermore, the intercellular lactate trafficking is found to depend directly on glucose partitioning, suggesting that a steady state may support different scenarios. At inhibitory steady state, characterized by high rate of GABA release, there is elevated oxidative activity in astrocyte, not in response to specific energetic needs.

Biosynthesis is up-regulated in tumors and thus the demand for anabolic intermediates is increased. The metabolic routes providing the building blocks for macromolecules are thus a very attractive target as they are not normally up-regulated in a normal quiescent cell. Some routes for glycolysis-derived intermediates production have been identified, but these do not constitute the whole pool of biosynthetic molecules in the cell, as many of these derive from mitochondria in the Krebs cycle. Indeed, this metabolic pathway is considered a “biosynthetic hub” from which anabolism is fed. If a metabolite efflux is indeed occurring, anaplerotic reactions must keep a steady supply of substrates. In spite of this obvious relevance of anaplerosis, it has been poorly characterized in the malignant cell context. Glutaminolysis and and pyruvate carboxylation are two pathways that function in an anaplerotic fashion. In spite of the increasing evidence implicating these two processes in cancer metabolism their role as intermediate providers is overlooked. In this review we analyze the implications of an active anaplerosis in cancer and we discuss experimental evidence showing the relevance of these metabolic routes in tumor physiology.

Improved design of metabolic flux estimation using mixed label 13C labeling experiments and identifiability analysis motivated re-examination of metabolic fluxes during anaerobic fermentation in the Escherichia coli. Comprehensive metabolic flux maps were determined by using a mixture of differently labeled glucose and compared to conventional flux maps obtained using extracellular measurements and comprehensive metabolic flux maps obtained using only U-13C glucose as the substrate. As expected, conventional flux analysis performs poorly in comparison to 13C-MFA, especially in the Embden-Meyerhof-Parnas (EMP) and pentose phosphate (PP) pathways. Identifiability analysis indicated and experiments confirmed that a mixture of 10% U-l3C glucose, 25% 1-13C glucose, and 65% naturally labeled glucose significantly improved the statistical quality of all calculated fluxes in the PP pathway, the EMP pathway, the anaplerotic reactions, and the tricarboxylic acid cycle. Modifying the network topology for the presence and absence of the Entner-Doudoroff pathway and the glyoxylate shunt did not affect the value or quality of estimated fluxes significantly. Extracellular measurement of formate production was necessary for the accurate estimation of the fluxes around the formate node.

SUMMARY Plasma cell survival and the consequent duration of immunity vary widely with infection or vaccination. Using fluorescent glucose analog uptake, we defined multiple developmentally independent mouse plasma cell populations with varying lifespans. Long-lived plasma cells imported more fluorescent glucose analog, expressed higher surface levels of the amino acid transporter CD98, and had more autophagosome mass than did short-lived cells. Low amino acid concentrations triggered reductions in both antibody secretion and mitochondrial respiration, especially by short-lived plasma cells. To explain these observations, we found that glutamine was used for both mitochondrial respiration and anaplerotic reactions, yielding glutamate and aspartate for antibody synthesis. Endoplasmic reticulum (ER) stress responses, which link metabolism to transcriptional outcomes, were similar between long- and short-lived subsets. Accordingly, population and single-cell transcriptional comparisons across mouse and human plasma cell subsets revealed few consistent and conserved differences. Thus, plasma cell antibody secretion and lifespan are primarily defined by non-transcriptional metabolic traits., In Brief Plasma cell survival and the consequent duration of immunity vary widely with infection or vaccination. Lam et al. demonstrate that short- and long-lived plasma cells are distinguished by metabolic properties such as nutrient uptake. In contrast, very few conserved transcriptional changes are observed between plasma cells of varying longevity., Graphical Abstract

Aspergillus terreus is successfully used for industrial production of itaconic acid. The acid is formed from cis-aconitate, an intermediate of the tricarboxylic (TCA) cycle, by catalytic action of cis-aconitate decarboxylase. It could be assumed that strong anaplerotic reactions that replenish the pool of the TCA cycle intermediates would enhance the synthesis and excretion rate of itaconic acid. In the phylogenetic close relative Aspergillus niger, upregulated metabolic flux through glycolysis has been described that acted as a strong anaplerotic reaction. Deregulated glycolytic flux was caused by posttranslational modification of 6-phosphofructo-1-kinase (PFK1) that resulted in formation of a highly active, citrate inhibition-resistant shorter form of the enzyme. In order to avoid complex posttranslational modification, the native A. niger pfkA gene has been modified to encode for an active shorter PFK1 fragment. By the insertion of the modified A. niger pfkA genes into the A. terreus strain, increased specific productivities of itaconic acid and final yields were documented by transformants in respect to the parental strain. On the other hand, growth rate of all transformants remained suppressed which is due to the low initial pH value of the medium, one of the prerequisites for the accumulation of itaconic acid by A. terreus mycelium.

Streptococcus thermophilus is a major component of dairy starter cultures used for the manufacture of yoghurt and cheese. In this study, the CO2 metabolism of S. thermophilus DSM 20617T, grown in either a N2 atmosphere or an enriched CO2 atmosphere, was analysed using both genetic and proteomic approaches. Growth experiments performed in a chemically defined medium revealed that CO2 depletion resulted in bacterial arginine, aspartate and uracil auxotrophy. Moreover, CO2 depletion governed a significant change in cell morphology, and a high reduction in biomass production. A comparative proteomic analysis revealed that cells of S. thermophilus showed a different degree of energy status depending on the CO2 availability. In agreement with proteomic data, cells grown under N2 showed a significantly higher milk acidification rate compared with those grown in an enriched CO2 atmosphere. Experiments carried out on S. thermophilus wild-type and its derivative mutant, which was inactivated in the phosphoenolpyruvate carboxylase and carbamoyl-phosphate synthase activities responsible for fixing CO2 to organic molecules, suggested that the anaplerotic reactions governed by these enzymes have a central role in bacterial metabolism. Our results reveal the capnophilic nature of this micro-organism, underlining the essential role of CO2 in S. thermophilus physiology, and suggesting potential applications in dairy fermentation processes.

Nitropropionate effect on the lactate and acetate metabolism of neurons and astrocytes grown in vitro with perinatal concen- trations. Anaplerotic reactions are an essential metabolic mechanism for the postnatal continuity of the brain development, contributing in processes that require substrates synthesized from Krebs cycle intermediates; however, their role during the presuckling period in the neonate is unknown. Objective. To estimate the anaplerotic capacity of neurons and astrocytes grown in vitro under perinatal conditions. Materials and methods. The effect of 3-nitropropionate (3-NPA)(2 mM) an inhibitor of the succinate dehydrogenase (SDH) on the oxidative and lipogenic metabolism of 14 C-derived from acetate and lactate in perinatal concentrations. The results were compared with its respective controls without inhibitor. Results. In spite of the presence of 3-NPA, respiratory activity with lactate was 40% in neurons and 73% in astrocytes, the lipogenesis was 53% in neurons and 52% in astrocytes. With acetate, the oxidation in neurons was 15% and 63% in astrocytes, lipogenesis was maintained in astrocytes but in neurons it increased up to 174% (p

Motivated by the finding that Pseudomonas knackmussii B13 but not Rhodococcus opacus 1CP grows in the absence of externally provided CO(2), we investigated the assimilation of (13)CO(2) into active cells cultivated with non-labelled glucose as sole energy substrate. (13)C found in the bulk biomass indicated a substantial but different CO(2) assimilation by Pseudomonas and Rhodococcus, respectively (3500 per thousand and 2600 per thousand). Cellular fatty acids were labelled from -15 per thousand to 470 per thousand and amino acids from 500 per thousand to 24,000 per thousand demonstrating clear differences between various compound classes. 'You are what you eat plus 1 per thousand' is therefore only valid for the average bulk C without specific isotope signature deviation of the external CO(2) or carbonates. Odd-numbered and 10-methyl fatty acids, which are much more abundant in Rhodococcus or other Gram-positive bacteria, were up to fivefold higher enriched in (13)C relative to the Pseudomonas fatty acids. A high-level growth-phase-independent, labelling of the oxaloacetate-derived amino acids indicated heterotrophic CO(2) fixation by anaplerotic reactions known to replenish the tricarboxylic acid cycle. Although both strains assimilate CO(2) via similar general pathways, Rhodococcus depends to a much higher extent on carboxylations reactions with external CO(2) owing to the formation of odd-numbered fatty acids. As a general consequence, heterotrophic fixation of CO(2) should be taken into account in investigations of degradation experiments using isotope tracer compounds.

Aspergillus niger AB1.13 cultures with glucoamylase production (with D-glucose as substrate) and without glucoamylase production (with D-xylose as substrate) were characterized by metabolic flux analysis. Two comprehensive metabolic models for d-glucose- as well as for D-xylose-consumption were used to quantify and compare the metabolic fluxes through the central pathways of carbon metabolism at different pH-values. The models consist of the most relevant metabolic pathways for A. niger including glycolysis, pentose-phosphate pathway, citrate cycle, energy metabolism and anaplerotic reactions comprising two intracellular compartments, the cytoplasm and mitochondrion. When D-xylose was used as the sole carbon source, the relative flux of the substrate through the oxidative pentose-phosphate pathway (PPP) via G6P-dehydrogenase was unaffected by the pH-value of the culture medium. About 30% of D-xylose consumed was routed through the oxidative PPP. In contrast, the flux of D-glucose (i.e., under glucoamylase-producing conditions) through the oxidative PPP was remarkably higher and, in addition was significantly affected by the pH-value of the culture medium (40% at pH 5.5, 56% at pH 3.7, respectively). Summarizing, the flux through the PPP under glucoamylase producing conditions was 30-90% higher than for non-producing conditions.

Tracking metabolic profiles has the potential to reveal crucial enzymatic steps that could be targeted in the drug discovery process. It is of special importance for various types of cancer known to be associated with substantial rewiring of metabolic networks. Here we introduce an integrated approach for the analysis of metabolome that allows us to simultaneously assess pathway activities (fluxes) and concentrations of a large number of the key components involved in central metabolism of human cells. This is accomplished by in vivo labeling with [U-13C]glucose followed by two-dimensional nuclear magnetic resonance (NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS) analysis. A comprehensive isotopomer model was developed, which enabled us to compare fluxes through the key central metabolic pathways including glycolysis, pentose phosphate pathway, tricarboxylic acid cycle, anaplerotic reactions, and biosynthetic pathways of fatty acids and amino acids. The validity and strength of this approach is illustrated by its application to a number of perturbations to breast cancer cells, including exposure to hypoxia, drug treatment, and tumor progression. We observed significant differences in the activities of specific metabolic pathways resulting from these perturbations and providing new mechanistic insights. Based on these findings we conclude that the developed metabolomic approach constitutes a promising analytical tool for revealing specific metabolic phenotypes in a variety of cell types and pathological conditions.

A scheme of investigating the intracellular metabolic fluxes in central metabolism of Saccharomyces cerevisiae based on isotope model and tracer experiment was developed. The metabolic model applied in this study includes the Embden-Meyerhof-Parnas pathway, the pentose phosphate pathway, the tricarboxylic acid cycle, CO 2 anaplerotic reactions, ethanol and acetate formation, and pathways involved in amino acid synthesis. The approach of hybridized genetic algorithm combined with the sequential simplex technique was used to optimize a quadratic error function without the requirement of the information on the partial derivatives. The impact of some key parameters on the algorithm was studied. This approach was proved to be rapid and numerically stable in the analysis of the central metabolism of S.cerevisiae.

INTRODUCTION: Medulloblastoma is the most common malignant paediatric brain tumour. Current treatments include surgery and radio/chemotherapy, which are associated with significant side effects, such as neurological, intellectual and physical disabilities. Metabolic adaptation has emerged as a hallmark of cancer and as a promising therapeutic target. Rapidly proliferating cancer cells adapt their metabolism by increasing nutrient uptake and by reorganising metabolic flux to support cell growth, promoting macromolecule synthesis and ATP production. Glutamine is a conditionally essential amino acid in cancer cells, being utilized as a carbon and nitrogen source for macromolecule production as well as for anaplerotic reactions fuelling the tricarboxylic acid cycle. METHOD: In this study, we used the DAOY medulloblastoma cell line. Amino acid starvation reduced cell proliferation but only glutamine starvation induced apoptosis. DAOY cells express high levels of ASCT2. Chemical inhibition (GPNA) or shRNA knockdown of ASCT2 in DAOY cells reduced cell growth and migration in the scratch assay. RESULTS: We demonstrate that the glutamine transporter ASCT2 is highly expressed in the DAOY medulloblastoma cell line. We show that chemical or shRNA-mediated inhibition of ASCT2 in vitro significantly inhibits cell growth, induce apoptosis and inhibit cell migration. CONCLUSION: Glutamine contributes to essentially every core metabolic task of proliferating tumour cells. ASCT2-mediated glutamine uptake is essential for multiple pathways regulating cell growth and cell migration in a medulloblastoma cell line, and it could therefore be a putative therapeutic target for medulloblastoma.

The effect of sucA or sucC gene knockout on the metabolism in Escherichia coli was investigated for the aerobic cell growth in batch and continuous cultivations based on gene expressions, enzyme activities, intracellular metabolite concentrations and metabolic flux analysis. In the batch cultivation, the cell growth rate and the glucose uptake rate were lower for sucA mutant as compared with the parent strain, while it was not the case for sucC mutant. A significantly higher amount of acetate was produced, and it was not utilized in sucC mutant, while a little less acetate was produced in sucA mutant as compared with the parent strain. Unlike the parent strain and sucC mutant, sucA mutant excreted a little amount of l -glutamate. Enzyme activity results show that some of the glycolytic enzymes such as Tpi and Pgk were up-regulated, while Pfk, Fba and Pyk activities were down-regulated for sucA mutant as compared with the parent strain. For sucC mutant, the activities of Pfk, Fba, Tpi, GAPDH, Pgk and Pyk activities were down-regulated. As for the TCA cycle enzymes, the activities of CS and ICDH were down-regulated, while those of Icl, MS, Fum and MDH were up-regulated for sucA mutant. The activities of the oxidative pentose phosphate (PP) pathway enzymes such as G6PDH and 6PGDH and the gluconeogenic pathway enzyme such as Mez were up-regulated in sucA mutant. The Ack activity was down-regulated for sucA mutant, but not for sucC mutant. In continuous cultivation, the gene expression results indicate that the global regulatory genes such as fadR and iclR were slightly down-regulated in sucA mutant, which enhanced the expression of aceA gene and caused the up-regulation of the isocitrate lyase activity in sucA mutant, while fadR and iclR of sucC mutant changed little and no isocitrate lyase activation was observed for sucC mutant. Some other global regulatory genes such as arcA and fnr genes were down-regulated in both mutants, which caused some of the TCA cycle genes to be up-regulated. The effect of the sucA gene knockout on the metabolic flux distributions was investigated based on 1H–13C NMR spectra and GC–MS signals obtained from 13C-labeling experiments. Flux analysis results indicate that the knockout of sucA gene caused the activation of PP pathway and the glyoxylate shunt. The fluxes through glycolysis and the TCA cycle were down-regulated in the sucA mutant. On the other hand, the fluxes through PP pathway and the anaplerotic reactions of Ppc-Pck and Mez increased.

In many organisms, metabolite interconversion at the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate node involves a structurally entangled set of reactions that interconnects the major pathways of carbon metabolism and thus, is responsible for the distribution of the carbon flux among catabolism, anabolism and energy supply of the cell. While sugar catabolism proceeds mainly via oxidative or non-oxidative decarboxylation of pyruvate to acetyl-CoA, anaplerosis and the initial steps of gluconeogenesis are accomplished by C3- (PEP- and/or pyruvate-) carboxylation and C4- (oxaloacetate- and/or malate-) decarboxylation, respectively. In contrast to the relatively uniform central metabolic pathways in bacteria, the set of enzymes at the PEP-pyruvate-oxaloacetate node represents a surprising diversity of reactions. Variable combinations are used in different bacteria and the question of the significance of all these reactions for growth and for biotechnological fermentation processes arises. This review summarizes what is known about the enzymes and the metabolic fluxes at the PEP-pyruvate-oxaloacetate node in bacteria, with a particular focus on the C3-carboxylation and C4-decarboxylation reactions in Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum. We discuss the activities of the enzymes, their regulation and their specific contribution to growth under a given condition or to biotechnological metabolite production. The present knowledge unequivocally reveals the PEP-pyruvate-oxaloacetate nodes of bacteria to be a fascinating target of metabolic engineering in order to achieve optimized metabolite production.

Using our recently developed sensor reactor approach, lysine-producing, nongrowing Corynebacterium glutamicum MH20-22B cells were subjected to serial (13)C-labeling experiments for flux analysis during the leucine-limited fed-batch production phase in a 300-L bioreactor. Based on two-dimensional (2D) nuclear magnetic resonance (NMR) measurements of (13)C-labeling patterns of cytoplasmic free metabolites, metabolic flux distributions in the central metabolism were successfully determined. Focusing on the highly concentrated metabolite L-glutamate, the working hypothesis was validated that the equilibration of labeling patterns in intracellular pools was much faster (up to 9.45 min) than the labeling period (3 h) used in the experiments. Analysis of anaplerotic reactions revealed that highly selective lysine production was accompanied by a significant reduction of decarboxylating reactions from 10 mol% to only 2 mol%, whereas PEP/pyruvate-carboxylating fluxes remained constant at about 40 mol% of consumed glucose. These results support the conclusion that an optimized C. glutamicum L-lysine producer should possess increased PEP carboxylase and/or pyruvate carboxylase activity combined with downregulated, decarboxylating fluxes consuming oxaloacetate/malate. The findings also illustrate the usefulness of the sensor reactor approach in the study of industrial fermentations.

Our aim in this commentary is to provide evidence that certain oxoacids formed in anaplerotic reactions control cell proliferation/apoptosis. In tumour cells with impaired Krebs cycle enzymes, some anaplerotic reactions do compensate for the deficit in oxoacids. One of these, oxaloacetate, derived from the transamination of asparagine but not of aspartate, is decarboxylated 4-fold more efficiently in polyoma-virus transformed cells than in their non-transformed counterparts. The deamidation of asparagine, in the cell culture medium, to aspartate by asparaginase decreases asparagine transamination and inhibits concomitantly the growth of asparaginase-sensitive lymphoma cells, suggesting a causal relationship between asparagine transamination and growth. Another oxoacid that can provide ATP when metabolised in mitochondria, but by the branched-chain oxoacid dehydrogenase complex (BCOADC), is 2-oxobutanoate. It has two origins: (a) deamination of threonine, and (b) cleavage of cystathionine, a metabolite derived from methionine. 2-Oxobutanoate in the presence of insulin promotes growth in G1/S arrested cells. But methionine also gives rise to another substrate of BCOADC, 4-methylthio-2-oxobutanoate (MTOB), which is synthesised exclusively from methylthioadenosine (MTA) by the action of MTA phosphorylase. In Met-dependent tumour cells with defective MTA phosphorylase, 2-oxobutanoate production would exceed that of MTOB. Further, BCOADC also has 3-fold greater affinity for 2-oxobutanoate than for MTOB; hence, the deficiency in 3-methylthio propionyl CoA, the final product of MTOB decarboxylation, would be exacerbated. Methional, the transient metabolic precursor in 3-methylthio propionyl CoA biosynthesis, is apoptogenic for both normal and bcl(2)-negative transformed cells in culture. Investigations of other causal relationships between the genes/enzymes mediating the homeostasis of anaplerotic oxoacids and cell growth/death may be worthwhile.

The use of parallel labeling experiments for (13)C metabolic flux analysis ((13)C-MFA) has emerged in recent years as the new gold standard in fluxomics. The methodology has been termed COMPLETE-MFA, short for complementary parallel labeling experiments technique for metabolic flux analysis. In this contribution, we have tested the limits of COMPLETE-MFA by demonstrating integrated analysis of 14 parallel labeling experiments with Escherichia coli. An effort on such a massive scale has never been attempted before. In addition to several widely used isotopic tracers such as [1,2-(13)C]glucose and mixtures of [1-(13)C]glucose and [U-(13)C]glucose, four novel tracers were applied in this study: [2,3-(13)C]glucose, [4,5,6-(13)C]glucose, [2,3,4,5,6-(13)C]glucose and a mixture of [1-(13)C]glucose and [4,5,6-(13)C]glucose. This allowed us for the first time to compare the performance of a large number of isotopic tracers. Overall, there was no single best tracer for the entire E. coli metabolic network model. Tracers that produced well-resolved fluxes in the upper part of metabolism (glycolysis and pentose phosphate pathways) showed poor performance for fluxes in the lower part of metabolism (TCA cycle and anaplerotic reactions), and vice versa. The best tracer for upper metabolism was 80% [1-(13)C]glucose+20% [U-(13)C]glucose, while [4,5,6-(13)C]glucose and [5-(13)C]glucose both produced optimal flux resolution in the lower part of metabolism. COMPLETE-MFA improved both flux precision and flux observability, i.e. more independent fluxes were resolved with smaller confidence intervals, especially exchange fluxes. Overall, this study demonstrates that COMPLETE-MFA is a powerful approach for improving flux measurements and that this methodology should be considered in future studies that require very high flux resolution.

Computational strain-design prediction accuracy has been the focus for many recent efforts through the selective integration of kinetic information into metabolic models. In general, kinetic model prediction quality is determined by the range and scope of genetic and/or environmental perturbations used during parameterization. In this effort, we apply the k-OptForce procedure on a kinetic model of E. coli core metabolism constructed using the Ensemble Modeling (EM) method and parameterized using multiple mutant strains data under aerobic respiration with glucose as the carbon source. Minimal interventions are identified that improve succinate yield under both aerobic and anaerobic conditions to test the fidelity of model predictions under both genetic and environmental perturbations. Under aerobic condition, k-OptForce identifies interventions that match existing experimental strategies while pointing at a number of unexplored flux re-directions such as routing glyoxylate flux through the glycerate metabolism to improve succinate yield. Many of the identified interventions rely on the kinetic descriptions that would not be discoverable by a purely stoichiometric description. In contrast, under fermentative (anaerobic) condition, k-OptForce fails to identify key interventions including up-regulation of anaplerotic reactions and elimination of competitive fermentative products. This is due to the fact that the pathways activated under anaerobic condition were not properly parameterized as only aerobic flux data were used in the model construction. This study shed light on the importance of condition-specific model parameterization and provides insight on how to augment kinetic models so as to correctly respond to multiple environmental perturbations.

The TCA cycle is an amphibolic pathway. Anaplerotic reactions replenish TCA cycle intermediates when they leak away from the cycle. Oxaloacetate leaks away from the TCA cycle to form pyrimidines and glucose. Succinyl-CoA leaks away from the TCA cycle to form the porphyrins (including heme). Several intermediates may leak away from the TCA cycle to form proteins. Citrate may leak away from the TCA cycle to reform acetyl-CoA and OAA in the cytoplasm, with the former entering into lipid biosynthesis. Leaks in the TCA cycle of skeletal muscle fibers are largely replenished through glucose oxidation. Amino acids may be used to replenish leaks in the TCA cycle.

Tuberculosis, the second leading infectious disease killer after HIV, remains a top public health priority. The causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), which can cause both acute and clinically latent infections, reprograms metabolism in response to the host niche. Phosphoenolpyruvate carboxykinase (Pck) is the enzyme at the center of the phosphoenolpyruvate-pyruvate-oxaloacetate node, which is involved in regulating the carbon flow distribution to catabolism, anabolism, or respiration in different states of Mtb infection. Under standard growth conditions, Mtb Pck is associated with gluconeogenesis and catalyzes the metal-dependent formation of phosphoenolpyruvate. In non-replicating Mtb, Pck can catalyze anaplerotic biosynthesis of oxaloacetate. Here, we present insights into the regulation of Mtb Pck activity by divalent cations. Through analysis of the X-ray structure of Pck-GDP and Pck-GDP-Mn2+ complexes, mutational analysis of the GDP binding site, and quantum mechanical (QM)-based analysis, we explored the structural determinants of efficient Mtb Pck catalysis. We demonstrate that Mtb Pck requires presence of Mn2+ and Mg2+ cations for efficient catalysis of gluconeogenic and anaplerotic reactions. The anaplerotic reaction, which preferably functions in reducing conditions that are characteristic for slowed or stopped Mtb replication, is also effectively activated by Fe2+ in the presence of Mn2+ or Mg2+ cations. In contrast, simultaneous presence of Fe2+ and Mn2+ or Mg2+ inhibits the gluconeogenic reaction. These results suggest that inorganic ions can contribute to regulation of central carbon metabolism by influencing the activity of Pck. Furthermore, the X-ray structure determination, biochemical characterization, and QM analysis of Pck mutants confirmed the important role of the Phe triad for proper binding of the GDP-Mn2+ complex in the nucleotide binding site and efficient catalysis of the anaplerotic reaction.

Background: Previous studies have shown an antiasthenic effect of citrulline/malate (CM) but the mechanism of action at the muscular level remains unknown.Objective: To investigate the effects of CM supplementation on muscle energetics.Methods: Eighteen men complaining of fatigue but with no documented disease were included in the study. A rest-exercise (finger flexions)-recovery protocol was performed twice before (D−7 and D0), three times during (D3, D8, D15), and once after (D22) 15 days of oral supplementation with 6 g/day CM. Metabolism of the flexor digitorum superficialis was analysed by31P magnetic resonance spectroscopy at 4.7 T.Results: Metabolic variables measured twice before CM ingestion showed no differences, indicating good reproducibility of measurements and no learning effect from repeating the exercise protocol. CM ingestion resulted in a significant reduction in the sensation of fatigue, a 34% increase in the rate of oxidative ATP production during exercise, and a 20% increase in the rate of phosphocreatine recovery after exercise, indicating a larger contribution of oxidative ATP synthesis to energy production. Considering subjects individually and variables characterising aerobic function, extrema were measured after either eight or 15 days of treatment, indicating chronological heterogeneity of treatment induced changes. One way analysis of variance confirmed improved aerobic function, which may be the result of an enhanced malate supply activating ATP production from the tricarboxylic acid cycle through anaplerotic reactions.Conclusion: The changes in muscle metabolism produced by CM treatment indicate that CM may promote aerobic energy production.

C4 photosynthesis represents a most remarkable case of convergent evolution of a complex trait, which includes the reprogramming of the expression patterns of thousands of genes. Anatomical, physiological, and phylogenetic and analyses as well as computational modeling indicate that the establishment of a photorespiratory carbon pump (termed C2 photosynthesis) is a prerequisite for the evolution of C4. However, a mechanistic model explaining the tight connection between the evolution of C4 and C2 photosynthesis is currently lacking. Here we address this question through comparative transcriptomic and biochemical analyses of closely related C3, C3–C4, and C4 species, combined with Flux Balance Analysis constrained through a mechanistic model of carbon fixation. We show that C2 photosynthesis creates a misbalance in nitrogen metabolism between bundle sheath and mesophyll cells. Rebalancing nitrogen metabolism requires anaplerotic reactions that resemble at least parts of a basic C4 cycle. Our findings thus show how C2 photosynthesis represents a pre-adaptation for the C4 system, where the evolution of the C2 system establishes important C4 components as a side effect. DOI: http://dx.doi.org/10.7554/eLife.02478.001, eLife digest Environmental pressures sometimes cause different organisms to independently evolve the same traits. A dramatic example of this phenomenon, which is called convergent evolution, can be seen in the modes used by plants to convert carbon dioxide from the air into starch during photosynthesis. Early plants existed in an environment with high levels of carbon dioxide in the air. Over time, carbon dioxide levels decreased, so plants evolved more efficient types of photosynthesis to cope. A very efficient type of photosynthesis, called C4 photosynthesis essentially represents a carbon dioxide concentration mechanism. It has evolved at least 62 times independently in 19 different families of flowering plants. Scientists have shown that a less advanced, low-efficiency version of photosynthetic carbon dioxide concentration, called C2 photosynthesis, is a stepping-stone to C4 photosynthesis. It is also known that the evolution of C4 photosynthesis required changes to the expression patterns of thousands of genes, but the exact mechanism that leads from C2 photosynthesis to C4 photosynthesis is not clear. To explore this in greater detail, Mallmann, Heckmann et al. studied plants from the genus Flaveria, which belongs to the same family as sunflowers and asters. Under identical greenhouse conditions, plants that use three different photosynthetic pathways—C3 photosynthesis, C4 photosynthesis, or an intermediate between the two—were grown and their gene expression patterns were compared. Computer simulations were used to model the metabolism of plants that relied on C2 photosynthesis. Based on the modeling, it appears that C2 photosynthesis shifts the balance of nitrogen metabolism between two types of cell that are critical to photosynthesis. To rebalance the nitrogen, several genes are expressed to trigger an ammonia recycling mechanism. The same genes are turned on during C4 photosynthesis, and this recycling mechanism include parts of the C4 process. The findings of Mallmann, Heckmann et al. suggest that the initial steps in C4 photosynthesis evolved to prevent nitrogen imbalance. Over time, this mechanism was co-opted to become part of a more efficient form of photosynthesis, which may explain why so many different plants evolved from C2 to C4 photosynthesis. DOI: http://dx.doi.org/10.7554/eLife.02478.002

The glyoxylate cycle is a sequence of anaplerotic reactions catalyzed by the key enzymes isocitrate lyase (ICL) and malate synthase (MLS). Mutants of Candida albicans lacking ICL are markedly less virulent in mice than the wild-type. Suvanine sesterterpenes (1−9) isolated from a tropical sponge Coscinoderma sp. were evaluated for their inhibitory activities toward recombinant ICL from C. albicans. These studies led to the identification of a potent ICL inhibitor, suvanine salt (2), which possesses a sodium counterion and displays an inhibitory concentration value (IC50) of 6.35 μM. The growth phenotype of ICL deletion mutants and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses indicated that compound 2 inhibits the ICL mRNA expression in C. albicans under C2-carbon-utilizing conditions. The present data highlight the potential for suvanine sesterterpenes treatment of C. albicans infections via inhibition of ICL activity.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the reversible decarboxylation of oxaloacetate to yield phosphoenolpyruvate and CO2. The role of the enzyme in gluconeogenesis and anaplerotic reactions in a range of organisms is discussed, along with the important function in C4 and CAM photosynthesis in higher plants. In addition, new data are presented indicating that PEPCK may play a key role in amino acid metabolism. It is proposed that PEPCK is involved in the conversion of the carbon skeleton of asparagine/aspartate (oxaloacetate) to that of glutamate/glutamine (2-oxoglutarate). This metabolism is particularly important in the transport system, seeds and fruits of higher plants.

De novo biosynthesis of amino acids uses intermediates provided by the TCA cycle that must be replenished by anaplerotic reactions to maintain the respiratory competency of the cell. Genome-wide expression analyses in Saccharomyces cerevisiae reveal that many of the genes involved in these reactions are repressed in the presence of the preferred nitrogen sources glutamine or glutamate. Expression of these genes in media containing urea or ammonia as a sole nitrogen source requires the heterodimeric bZip transcription factors Rtg1 and Rtg3 and correlates with a redistribution of the Rtg1p/Rtg3 complex from a predominantly cytoplasmic to a predominantly nuclear location. Nuclear import of the complex requires the cytoplasmic protein Rtg2, a previously identified upstream regulator of Rtg1 and Rtg3, whereas export requires the importin-β-family member Msn5. Remarkably, nuclear accumulation of Rtg1/Rtg3, as well as expression of their target genes, is induced by addition of rapamycin, a specific inhibitor of the target of rapamycin (TOR) kinases. We demonstrate further that Rtg3 is a phosphoprotein and that its phosphorylation state changes after rapamycin treatment. Taken together, these results demonstrate that target of rapamycin signaling regulates specific anaplerotic reactions by coupling nitrogen quality to the activity and subcellular localization of distinct transcription factors.

A comprehensive metabolic network that considers 147 reaction fluxes and 105 metabolites is used in a mass-flux-balance-based stoichiometric model for Bacillus licheniformis for serine alkaline protease (SAP) overproduction. The theoretical capacity analysis leading to optimized SAP overproduction was carried out by using a linear constrained optimization technique for several specific growth rates and the variation of the fluxes were calculated by fixing the sole carbon source citrate’s uptake rate at 10 mmol/gDW/h. The theoretical data-based capacity analysis was conducted by using the model in combination with the off-line extracellular analyses of the dry cell and the metabolites that were citrate, organic acids, amino acids, and SAP; and the variations in the intracellular fluxes were obtained for the three periods of the batch bioprocess. The flux distribution maps of the analyses showed that the tricarboxylic acid cycle was active and the cells utilized the gluconeogenesis pathway, the pentose phosphate pathway, and the anaplerotic reactions; nevertheless, the glyoxylate shunt and the glycolysis pathway were inactive. The theoretical capacity analysis showed that SAP synthesis flux increased with the decrease in the specific growth rate, and was the highest at μ = 0 h −1 as 0.0260 mmol/gDW/h. Both in the theoretical capacity and the theoretical data-based capacity analyses, among the fluxes towards the amino acid groups, aspartic acid group had the highest value and aromatic acid group had the lowest flux value; the flux distributions are similar. The flux values towards SAP was maximum in Period II, whereas it was minimum in Period I. In Period II of the theoretical data-based capacity analysis, the fluxes of alanine and valine are higher than the other amino acid fluxes; and the pyruvate branch point seems to be the potential metabolic engineering site. The results reveal that SAP production can theoretically be increased 1.09, 16.68, and 7.21 folds, respectively, in Periods I, II, and III. The diversions in the pathways and certain metabolic reactions depending on the bioprocess periods and potential strategies for improving SAP production are also discussed.

A cycle remains a cycle only as long as the spokes of the wheel are not stolen. To keep the citric acid cycle going requires anaplerotic reactions such as the glyoxylate shunt to restore the cycle intermediates that are withdrawn for the biosynthesis of cell constituents, e.g. amino acids and haemin precursors. The article by Erb et al. in this issue of Molecular Microbiology documents an alternative path that replenishes four-carbon intermediates during growth on acetate in the absence of the glyoxylate shunt. The reaction sequence forms malate and succinyl-CoA from three acetyl-CoA, one CO(2) and one HCO(3) in a linear pathway. This new pathway was discovered in phototrophic anoxygenic bacteria and in few aerobic bacteria, but it is probably widespread among many metabolic groups of bacteria.

The effect of prolonged anoxia (96 h under a N2 atmosphere) during either winter (November) or summer (July) was investigated by measuring the maximal activities of 20 metabolic enzymes in gill, mantle, hepatopancreas, and phasic and catch adductor muscles of the oyster, Crassostrea virginica. The enzymes analyzed are involved in carbohydrate and amino acid metabolism, the pentose phosphate shunt, anaplerotic reactions of the TCA cycle, and phosphagen/adenylate metabolism. The data demonstrate that oyster metabolism is influenced by both long-term seasonal change and by shorter-term environmental insult (anoxia). Seasonal changes were concentrated among enzymes involved in glycogen metabolism whereas the prominent response to anoxia was suppression of PK activity. Anoxia exposure induced tissue-specific changes in enzyme activities suggesting a substantial metabolic reorganization involving both coarse controls on enzyme amount and reversible covalent modification. In addition, the effects of anoxia on enzymes of intermediary metabolism were seasonally dependent and more widespread in the winter. These results demonstrate the interaction of two environmental variables (season, anoxia) and suggest the importance of season as a modifying factor in the anoxic response.

A mass flux balance-based stoichiometric model of Bacillus licheniformis for the serine alkaline protease (SAP) fermentation process has been established. The model considers 147 reaction fluxes, and there are 105 metabolites that are assumed to be in pseudo-steady state. Metabolic flux distributions were obtained from the solution of the model based on the minimum SAP accumulation rate assumption in B. licheniformis in combination with the off-line extracellular analyses of the metabolites that were the sole carbon source citrate, dry cell, organic acids, amino acids, and SAP; variations in the intracellular fluxes were demonstrated for the three periods of the batch bioprocess. The flux distribution maps showed that the cells completed the TCA cycle and utilized the gluconeogenesis pathway, pentose phosphate pathway, and anaplerotic reactions throughout the fermentation; however, the glycolysis pathway was inactive in all the periods of the fermentation. The flux values toward SAP increased throughout the bioprocess and slightly decreased in the last period; however, SAP selectivity values were almost the same in Periods II and III and higher than Period I. The diversions in the pathways and certain metabolic reactions depending on the bioprocess periods are also presented and the results indicated that the intracellular amino acid fluxes played an important role in the SAP fermentation process.

The product of the ACR1 gene is essential for growth of Saccharomyces cerevisiae on ethanol or acetate as sole carbon source, and its expression is subject to glucose repression. It was previously shown that Acr1p is a membrane protein which specifically transports succinate and fumarate. Its suggested function is to shuttle cytosolic succinate from the glyoxylate cycle into the mitochondria in exchange for fumarate, an activity that is essential during gluconeogenic growth on C2 compounds. In this study we show that ACR1 is coregulated with the genes coding for the key enzymes of the glyoxylate cycle and gluconeogenesis: ICL1, MLS1 and PCK1, FBP1 respectively. We demonstrate that derepression of ACR1 is strictly dependent on the Zn2Cys6-type transcriptional activator Cat8p. A detailed deletion analysis of the ACR1 promoter revealed that 69% of the derepression of ACR1 is mediated by three cis-acting elements, located between positions -679 and -569 relative to the translational start, which show a high degree of similarity to the UAS/CSRE elements of PCK1, FBP1, ICL1 and MLS1. Our results, in conjunction with previous biochemical data, clearly identify Acr1p as an element which is directly involved in gluconeogenesis, functioning as the mitochondrial carrier which links the anaplerotic reactions of the glyoxylate cycle to the TCA cycle.

A prototrophic pyruvate-carboxylase-negative (Pyc-) mutant was constructed by deleting the PYC1 and PYC2 genes in a CEN.PK strain of Saccharomyces cerevisiae. Its maximum specific growth rate on ethanol was identical to that of the isogenic wild type but it was unable to grow in batch cultures in glucose-ammonia media. Consistent with earlier reports, growth on glucose could be restored by supplying aspartate as a sole nitrogen source. Ethanol could not replace aspartate as a source of oxaloacetate in batch cultures. To investigate whether alleviation of glucose repression allowed expression of alternative pathways for oxaloacetate synthesis, the Pyc- strain and an isogenic wild-type strain were grown in aerobic carbon-limited chemostat cultures at a dilution rate of 0.10 h-1 on mixtures of glucose and ethanol. In such mixed-substrate chemostat cultures of the Pyc- strain, steady-state growth could only be obtained when ethanol contributed 30% or more of the substrate carbon in the feed. Attempts to further decrease the ethanol content of the feed invariably resulted in washout. In Pyc- as well as in wild-type cultures, levels of isocitrate lyase, malate synthase and phospho-enol-pyruvate carboxykinase in cell extracts decreased with a decreasing ethanol content in the feed. Nevertheless, at the lowest ethanol fraction that supported growth of the Pyc- mutant, activities of the glyoxylate cycle enzymes in cell extracts were still sufficient to meet the requirement for C4-compounds in biomass synthesis. This suggests that factors other than glucose repression of alternative routes for oxaloacetate synthesis prevent growth of Pyc-mutants on glucose.

The recent discovery that phosphoenolpyruvate carboxylase (PEPCx) is dispensable for growth and lysine production in Corynebacterium glutamicum implies that this organism possesses (an) alternative anaplerotic enzyme(s). In permeabilized cells of C. glutamicum, we detected pyruvate carboxylase (PCx) activity. This activity was effectively inhibited by low concentrations of ADP, AMP and acetyl-CoA. PCx activity was highest [45 ± 5 nmol min−1 (mg dry wt)−1] in cells grown on lactate or pyruvate, and was about two- to threefold lower when the cells were grown on glucose or acetate, suggesting that formation of PCx is regulated by the carbon source in the growth medium. In cells grown at low concentrations of biotin (< 5 μg I−1), PCx activity was drastically reduced, indicating that the enzyme is a biotin protein. Growth experiments with the wild-type and a defined PEPCx-negative mutant of C. glutamicum on glucose showed that the mutant has a significantly higher demand for biotin than the wild-type, whereas both strains have the same high biotin requirement for growth on lactate and the same low biotin requirement for growth on acetate. These results indicate that (i) PCx is an essential anaplerotic enzyme for growth on glucose in the absence of PEPCx, (ii) PCx is an essential anaplerotic enzyme for growth on lactate even in the presence of PEPCx, and (iii) PCx has no anaplerotic significance for growth on acetate as the carbon source. In support of these conclusions, screening for clones unable to grow on a minimal medium containing lactate, but able to grow on a medium containing glucose or acetate, led to the isolation of PCx-defective mutants of C. glutamicum.

Phosphoenolpyruvate carboxylase (PEPCx) has recently been found to be dispensable as an anaplerotic enzyme for growth and lysine production of Corynebacterium glutamicum. To clarify the role of the glyoxylate cycle as a possible alternative anaplerotic sequence, defined PEPCx- and isocitrate-lyase (ICL)-negative double mutants of C. glutamicum wild-type and of the L-lysine-producing strain MH20-22B were constructed by disruption of the respective genes. Analysis of these mutants revealed that the growth on glucose and the lysine productivity were identical to that of the parental strains. These results show that PEPCx and the glyoxylate cycle are not essential for growth of C. glutamicum on glucose and for lysine production and prove the presence of another anaplerotic reaction in this organism. To study the anaplerotic pathways in C. glutamicum further, (HCO3-)-C-13-labeling experiments were performed with cells of the wild-type and a PEPCx-negative strain growing on glucose. Proton nuclear magnetic resonance analysis of threonine isolated from cell protein of both strains revealed the same labeling pattern: about 37% C-13 enrichment in C-4 and 3.5% C-13 enrichment in C-1. Since the carbon backbone of threonine corresponds to that of oxaloacetate, the label in C-4 of threonine positively identifies the anaplerotic pathway as a C-3-carboxylation reaction that also takes place in the absence of PEPCx.

Mycobacterium tuberculosis (MTb), the causative agent of tuberculosis, can persist in macrophages for decades, maintaining its basic metabolic activities. Phosphoenolpyruvate carboxykinase (Pck; EC 4.1.1.32) is a key player in central carbon metabolism regulation. In replicating MTb, Pck is associated with gluconeogenesis, but in non-replicating MTb, it also catalyzes the reverse anaplerotic reaction. Here, we explored the role of selected cysteine residues in function of MTb Pck under different redox conditions. Using mass spectrometry analysis we confirmed formation of S-S bridge between cysteines C391 and C397 localized in the C-terminal subdomain. Molecular dynamics simulations of C391-C397 bridged model indicated local conformation changes needed for formation of the disulfide. Further, we used circular dichroism and Raman spectroscopy to analyze the influence of C391 and C397 mutations on Pck secondary and tertiary structures, and on enzyme activity and specificity. We demonstrate the regulatory role of C391 and C397 that form the S-S bridge and in the reduced form stabilize Pck tertiary structure and conformation for gluconeogenic and anaplerotic reactions.

Metabolic flux redirection during nitrogen-limited growth was investigated in the Synechocystis sp. PCC 6803 glucose-tolerant (GT) strain under photoautotrophic conditions by isotopically non-stationary metabolic flux analysis (INST-MFA). A ΔnrtABCD mutant of Synechocystis sp. PCC 6803 was constructed to reproduce phenotypes arising during nitrogen starvation. The ΔnrtABCD mutant and the wild-type GT strain were cultured under photoautotrophic conditions by a photobioreactor. Intracellular metabolites were labeled over a time course using NaH13CO3 as a carbon source. Based on these data, the metabolic flux distributions in the wild-type and ΔnrtABCD cells were estimated by INST-MFA. The wild-type GT and ΔnrtABCD strains displayed similar distribution patterns, although the absolute levels of metabolic flux were lower in ΔnrtABCD. Furthermore, the relative flux levels for glycogen metabolism, anaplerotic reactions and the oxidative pentose phosphate pathway were increased in ΔnrtABCD. This was probably due to the increased expression of enzyme genes that respond to nitrogen depletion. Additionally, we found that the ratio of ATP/NADPH demand increased slightly in the ΔnrtABCD mutant. These results indicated that futile ATP consumption increases under nitrogen-limited conditions because the Calvin-Benson cycle and the oxidative pentose phosphate pathway form a metabolic futile cycle that consumes ATP without CO2 fixation and NADPH regeneration.