GC/MS-based 13C metabolic flux analysis resolves the parallel and cyclic photomixotrophic metabolism of Synechocystis sp. PCC 6803 and selected deletion mutants including the Entner-Doudoroff and phosphoketolase pathways

Background Cyanobacteria receive huge interest as green catalysts. While exploiting energy from sunlight, they co-utilize sugar and CO2. This photomixotrophic mode enables fast growth and high cell densities, opening perspectives for sustainable biomanufacturing. The model cyanobacterium Synechocystis sp. PCC 6803 possesses a complex architecture of glycolytic routes for glucose breakdown that are intertwined with the CO2-fixing Calvin-Benson-Bassham (CBB) cycle. To date, the contribution of these pathways to photomixotrophic metabolism has remained unclear. Results Here, we developed a comprehensive approach for 13C metabolic flux analysis of Synechocystis sp. PCC 6803 during steady state photomixotrophic growth. Under these conditions, the Entner-Doudoroff (ED) and phosphoketolase (PK) pathways were found inactive but the microbe used the phosphoglucoisomerase (PGI) (63.1%) and the oxidative pentose phosphate pathway (OPP) shunts (9.3%) to fuel the CBB cycle. Mutants that lacked the ED pathway, the PK pathway, or phosphofructokinases were not affected in growth under metabolic steady-state. An ED pathway-deficient mutant (Δeda) exhibited an enhanced CBB cycle flux and increased glycogen formation, while the OPP shunt was almost inactive (1.3%). Under fluctuating light, ∆eda showed a growth defect, different to wild type and the other deletion strains. Conclusions The developed approach, based on parallel 13C tracer studies with GC–MS analysis of amino acids, sugars, and sugar derivatives, optionally adding NMR data from amino acids, is valuable to study fluxes in photomixotrophic microbes to detail. In photomixotrophic cells, PGI and OPP form glycolytic shunts that merge at switch points and result in synergistic fueling of the CBB cycle for maximized CO2 fixation. However, redirected fluxes in an ED shunt-deficient mutant and the impossibility to delete this shunt in a GAPDH2 knockout mutant, indicate that either minor fluxes (below the resolution limit of 13C flux analysis) might exist that could provide catalytic amounts of regulatory intermediates or alternatively, that EDA possesses additional so far unknown functions. These ideas require further experiments

PLoS Biol

Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, known as "catabolite repression," allowing redirection of cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism named "metabolic contest" for regulating the use of carbon sources without nutrient sensing and signaling. Trypanosoma brucei is a unicellular eukaryote transmitted by tsetse flies and causing human African trypanosomiasis, or sleeping sickness. We showed that, in contrast to most microorganisms, the insect stages of this parasite developed a preference for glycerol over glucose, with glucose consumption beginning after the depletion of glycerol present in the medium. This "metabolic contest" depends on the combination of 3 conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-related organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways both being ATP-dependent (glycerol kinase and hexokinase, respectively); and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being approximately 80-fold higher than the hexokinase activity. As predicted by our model, an approximately 50-fold down-regulation of the GK expression abolished the preference for glycerol over glucose, with glucose and glycerol being metabolized concomitantly. In theory, a metabolic contest could be found in any organism provided that the 3 conditions listed above are met

Procyclic trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in the presence of physiological amounts of proline

Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly where the procyclic forms of the parasite develop in the proline-rich (1–2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and α-ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of thirteen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of α-ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) α-ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an α-ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6 showed a growth defect in the presence of 2 mM proline, which is rescued by α-ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly

Increasing field strength versus advanced isotope labeling for NMR based fluxomics

International audienc

Reaching metabolomic niches through miniaturization of 1 H-NMR experiments.

International audienceNMR spectroscopy is a great source of information for metabolomics studies. However, one major drawback of NMR is its lack of sensitivity which limits its use for small samples. In such cases, the samples have to be diluted and the experiment time lengthened to accumulate enough signal, which is not optimal. Miniaturization of NMR experiments in the framework of the REACT-EU OCSSIGEN project could be the way to overcome these limitations and reach new metabolomic applications

MDH2 is a metabolic switch rewiring the fuelling of respiratory chain and TCA cycle

ABSTRACT The mitochondrial respiratory chain (RC) enables many metabolic processes by regenerating both mitochondrial and cytosolic NAD + and ATP. The oxidation by the RC of the NADH metabolically produced in the cytosol involves redox shuttles as the malate-aspartate shuttle (MAS) and is of paramount importance for cell fate. However, the specific metabolic regulations allowing mitochondrial respiration to prioritize NADH oxidation in response to high NADH/NAD + redox stress have not been elucidated. Our work demonstrates the crucial role played by MDH2 in orchestrating the electron fuelling of the RC. On one hand, MDH2 mediated oxaloacetate (OAA) production allows mitochondria to metabolize pyruvate and glutamate and to feed complex I with NADH, whereas on the other hand, OAA inhibits complex II. This regulatory mechanism synergistically increases RC’s NADH oxidative capacity and rewires MDH2 driven anaplerosis of the TCA favouring the oxidation of malate produced by the MAS

(15)N-NMR-based approach for amino acids-based (13)C-metabolic flux analysis of metabolism

NMR analysis of the isotope incorporation in amino acids can be used to derive information about the topology and operation of cellular metabolism. Although traditionally performed by (1)H and/or (13)C NMR, we present here novel experiments that exploit the (15)N nucleus to derive the same information with increased efficiency. Combined with a novel Hα-(13)CO experiment, we increase the coverage of the isotopic space that can be probed by obtaining the complete distribution of isotopic species for the first two carbons of amino acids in cellular biomass hydrolysates. Our approach was evaluated using as reference material a biologically produced sample containing (15)N-labeled metabolites with fully predictable (13)C-labeling patterns. Results show excellent agreement between measured and expected isotopomer abundances for the different NMR experiments, with an accuracy and precision within 1%. We also demonstrate how these experiments can give detailed information about metabolic fluxes depending on the expression level of a critical enzyme. Hence, exploiting the (15)N labeling of a cellular sample accelerates subsequent analysis of the hydrolyzed biomass and increases the coverage of isotopomers that can be quantified, making it a promising tool to increase the throughput and the resolution of (13)C-fluxomics studies

Development of high throughput miniaturized robotic platforms for metabolism investigations of microorganisms

During the past few years, we have developed a fully integrated solution to analyze the fluxome (i.e. in vivo reaction rate associated with a cellular network of an organism), which combines two robotic cultivation and sampling workstation for 13C-labelling experiments with MS and NMR-based isotopic profiling, and tools for processing and interpreting isotopic data.First of all, we developed in collaboration with TECAN, a platform for automated HT-fluxome profiling of metabolic variants. Controlled by two software, this robotic system prepares, runs, monitors and controls the fermentations, and adjusts the pH, temperature and stirrer speed for 48 micro-scale (10 ml) fermentations in parallel. The robotic system also features fully automated metabolite harvesting and extraction (e.g. cell pellets, supernatants and intracellular metabolites) for downstream analysis.Drastically increasing the quantity of biological samples produced, a second robotic platform has been acquired to fully automatize the preparation of biological samples for analysis by liquid chromatography coupled to mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). The device is able to handle a complete range of sample racks as well as carrying out various types of manipulation such as dilution, aliquoting, addition of buffer and internal standard, filtration, centrifugation, sample pre-treatment (i.e SPE) all while ensuring traceability of all samples.Recently, we focus our development on the handling of low-volume or small-quantity samples in order to analyze matrices for which we have few amounts available or to concentrate classical samples and have access to minority metabolites. We have adapted our robotic platform to pipette a few microliters (2µL) of sample while maintaining excellent precision (CV<10%), and equipped the pipetting arm with capillary needles to fill 1.7mm internal diameter NMR tubes. This opens up new prospects, including the possibility of studying the metabolism of new sample niches such as spheroids or others extracts with very low volumes.We have now a complete high-throughput, miniaturized workflow for microorganisms cultivations, preparing and analyzing miniaturized samples, as well as bioinformatics tools for processing the large datasets generated. This entire workflow is now available to scientists wishing to carry out targeted or non-targeted metabolomics and fluxomic experiments

Isotopic profiling of C-13-labeled biological samples by two-dimensional heteronuclear J-resolved nuclear magnetic resonance spectroscopy

The use of two-dimensional heteronuclear J-resolved (2D H-JRES) nuclear magnetic resonance (NMR) spectroscopy for fast and reliable measurement of isotopic patterns from C-13-enriched compounds resulting from carbon labeling experiments was evaluated. Its use with biological samples of increasing complexity showed that 2D H-JRES spectroscopy is suitable for high-throughput isotopic profiling of any kind of labeled samples. Moreover, the method enabled accurate quantification of C-13 enrichments and, thus, can be used for metabolic flux analysis. The excellent trade-off between reduced experimental time and the number of measurable isotopic data makes 2D H-JRES NMR a promising approach for high-throughput flux analysis of samples of intermediate complexity. (C) 2012 Elsevier Inc. All rights reserved

Ultrafast Quantitative 2D NMR: An Efficient Tool for the Measurement of Specific Isotopic Enrichments in Complex Biological Mixtures

International audienceTwo-dimensional nuclear magnetic resonance (2D NMR) is a promising tool for studying metabolic fluxes by measuring C-13-enrichments in complex mixtures of C-13-labeled metabolites. However, the methods reported so far are hampered by very long acquisition durations limiting the use of 2D NMR as a quantitative tool for fluxomics. In this paper, we propose a new approach for measuring specific C-13-enrichments in a very fast way, by using new experiments based on ultrafast 2D NMR. Two homonudear 2D experiments (ultrafast COSY and zTOCSY) are proposed to measure C-13-enrichments in a single scan. Their advantages and limitations are discussed, and their high analytical potentialities are highlighted. Both methods are characterized by an accuracy of 1-2%, an average precision of 3%, and an excellent linearity. The analytical performance is equivalent or better than any of the conventional methods previously reported. The two ultrafast experiments are applied to the measurement of C-13-enrichments on a biomass hydrolyzate, showing the first known application of ultrafast 2D NMR to a real biological extract. The experiment duration is divided by 200 compared to the conventional methods, while preserving 80% of the quantitative information. This new approach opens new perspectives of application for fluxomics and metabonomics