Search

Background: Cyanobacteria receive huge interest as green catalysts. While exploiting energy from sunlight, they co-utilize sugar and CO 2 . 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 CO 2 -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 13 C 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 13 C 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 CO 2 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 13 C 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., (© 2022. The Author(s).)

The nucleoredoxin gene NXNL2 encodes for two products through alternative splicing, rod-derived cone viability factor-2 (RdCVF2) that mediates neuronal survival and the thioredoxin-related protein (RdCVF2L), an enzyme that regulates the phosphorylation of TAU. To investigate the link between NXNL2 and tauopathies, we studied the Nxnl2 knockout mouse (Nxnl2 -/- ). We established the expression pattern of the Nxnl2 gene in the brain using a Nxnl2 reporter mouse line, and characterized the behavior of the Nxnl2 -/- mouse at 2 months of age. Additionally, long term potentiation and metabolomic from hippocampal specimens were collected at 2 months of age. We studied TAU oligomerization, phosphorylation and aggregation in Nxnl2 -/- brain at 18 months of age. Finally, newborn Nxnl2 -/- mice were treated with adeno-associated viral vectors encoding for RdCVF2, RdCVF2L or both and measured the effect of this therapy on long-term potential, glucose metabolism and late-onset tauopathy. Nxnl2 -/- mice at 2 months of age showed severe behavioral deficiency in fear, pain sensitivity, coordination, learning and memory. The Nxnl2 -/- also showed deficits in long-term potentiation, demonstrating that the Nxnl2 gene is involved in regulating brain functions. Dual delivery of RdCVF2 and RdCVF2L in newborn Nxnl2 -/- mice fully correct long-term potentiation through their synergistic action. The expression pattern of the Nxnl2 gene in the brain shows a predominant expression in circumventricular organs, such as the area postrema. Glucose metabolism of the hippocampus of Nxnl2 -/- mice at 2 months of age was reduced, and was not corrected by gene therapy. At 18-month-old Nxnl2 -/- mice showed brain stigmas of tauopathy, such as oligomerization, phosphorylation and aggregation of TAU. This late-onset tauopathy can be prevented, albeit with modest efficacy, by recombinant AAVs administrated to newborn mice. The Nxnl2 -/- mice have memory dysfunction at 2-months that resembles mild-cognitive impairment and at 18-months exhibit tauopathy, resembling to the progression of Alzheimer's disease. We propose the Nxnl2 -/- mouse is a model to study multistage aged related neurodegenerative diseases. The NXNL2 metabolic and redox signaling is a new area of therapeutic research in neurodegenerative diseases., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Suppressive regulatory T cell (Treg) differentiation is controlled by diverse immunometabolic signaling pathways and intracellular metabolites. Here we show that cell-permeable α-ketoglutarate (αKG) alters the DNA methylation profile of naive CD4 T cells activated under Treg polarizing conditions, markedly attenuating FoxP3+ Treg differentiation and increasing inflammatory cytokines. Adoptive transfer of these T cells into tumor-bearing mice results in enhanced tumor infiltration, decreased FoxP3 expression, and delayed tumor growth. Mechanistically, αKG leads to an energetic state that is reprogrammed toward a mitochondrial metabolism, with increased oxidative phosphorylation and expression of mitochondrial complex enzymes. Furthermore, carbons from ectopic αKG are directly utilized in the generation of fatty acids, associated with lipidome remodeling and increased triacylglyceride stores. Notably, inhibition of either mitochondrial complex II or DGAT2-mediated triacylglyceride synthesis restores Treg differentiation and decreases the αKG-induced inflammatory phenotype. Thus, we identify a crosstalk between αKG, mitochondrial metabolism and triacylglyceride synthesis that controls Treg fate., Competing Interests: Declaration of interests C.M., S.K., V.D., and N.T. are inventors on patents describing the use of ligands for detection of and modulation of metabolite transporters (N.T. gave up her rights), licensed to METAFORA-biosystems., (Copyright © 2021. Published by Elsevier Inc.)

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., Competing Interests: The authors have declared that no competing interests exist.

Stable-isotope labeling experiments are widely used to investigate the topology and functioning of metabolic networks. Label incorporation into metabolites can be quantified using a broad range of mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy methods, but in general, no single approach can completely cover isotopic space, even for small metabolites. The number of quantifiable isotopic species could be increased and the coverage of isotopic space improved by integrating measurements obtained by different methods; however, this approach has remained largely unexplored because no framework able to deal with partial, heterogeneous isotopic measurements has yet been developed. Here, we present a generic computational framework based on symbolic calculus that can integrate any isotopic data set by connecting measurements to the chemical structure of the molecules. As a test case, we apply this framework to isotopic analyses of amino acids, which are ubiquitous to life, central to many biological questions, and can be analyzed by a broad range of MS and NMR methods. We demonstrate how this integrative framework helps to (i) clarify and improve the coverage of isotopic space, (ii) evaluate the complementarity and redundancy of different techniques, (iii) consolidate isotopic data sets, (iv) design experiments, and (v) guide future analytical developments. This framework, which can be applied to any labeled element, isotopic tracer, metabolite, and analytical platform, has been implemented in IsoSolve (available at https://github.com/MetaSys-LISBP/IsoSolve and https://pypi.org/project/IsoSolve), an open-source software that can be readily integrated into data analysis pipelines.

Lactate, a metabolite produced when the glycolytic flux exceeds mitochondrial oxidative capacities, is now viewed as a critical regulator of metabolism by acting as both a carbon and electron carrier and a signaling molecule between cells and tissues. In recent years, increasing evidence report its key role in white, beige, and brown adipose tissue biology, and highlights new mechanisms by which lactate participates in the maintenance of whole-body energy homeostasis. Lactate displays a wide range of biological effects in adipose cells not only through its binding to the membrane receptor but also through its transport and the subsequent effect on intracellular metabolism notably on redox balance. This study explores how lactate regulates adipocyte metabolism and plasticity by balancing intracellular redox state and by regulating specific signaling pathways. We also emphasized the contribution of adipose tissues to the regulation of systemic lactate metabolism, their roles in redox homeostasis, and related putative physiopathological repercussions associated with their decline in metabolic diseases and aging., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lagarde, Jeanson, Portais, Galinier, Ader, Casteilla and Carrière.)

Glycosomes are peroxisome-related organelles of trypanosomatid parasites containing metabolic pathways, such as glycolysis and biosynthesis of sugar nucleotides, usually present in the cytosol of other eukaryotes. UDP-glucose pyrophosphorylase (UGP), the enzyme responsible for the synthesis of the sugar nucleotide UDP-glucose, is localized in the cytosol and glycosomes of the bloodstream and procyclic trypanosomes, despite the absence of any known peroxisome-targeting signal (PTS1 and PTS2). The questions that we address here are (i) is the unusual glycosomal biosynthetic pathway of sugar nucleotides functional and (ii) how is the PTS-free UGP imported into glycosomes? We showed that UGP is imported into glycosomes by piggybacking on the glycosomal PTS1-containing phosphoenolpyruvate carboxykinase (PEPCK) and identified the domains involved in the UGP/PEPCK interaction. Proximity ligation assays revealed that this interaction occurs in 3 to 10% of glycosomes, suggesting that these correspond to organelles competent for protein import. We also showed that UGP is essential for the growth of trypanosomes and that both the glycosomal and cytosolic metabolic pathways involving UGP are functional, since the lethality of the knockdown UGP mutant cell line ( RNAi UGP, where RNAi indicates RNA interference) was rescued by expressing a recoded UGP (rUGP) in the organelle ( RNAi UGP/ EXP rUGP-GPDH, where GPDH is glycerol-3-phosphate dehydrogenase). Our conclusion was supported by targeted metabolomic analyses (ion chromatography-high-resolution mass spectrometry [IC-HRMS]) showing that UDP-glucose is no longer detectable in the RNAi UGP mutant, while it is still produced in cells expressing UGP exclusively in the cytosol (PEPCK null mutant) or glycosomes ( RNAi UGP/ EXP rUGP-GPDH). Trypanosomatids are the only known organisms to have selected functional peroxisomal (glycosomal) sugar nucleotide biosynthetic pathways in addition to the canonical cytosolic ones. IMPORTANCE Unusual compartmentalization of metabolic pathways within organelles is one of the most enigmatic features of trypanosomatids. These unicellular eukaryotes are the only organisms that sequestered glycolysis inside peroxisomes (glycosomes), although the selective advantage of this compartmentalization is still not clear. Trypanosomatids are also unique for the glycosomal localization of enzymes of the sugar nucleotide biosynthetic pathways, which are also present in the cytosol. Here, we showed that the cytosolic and glycosomal pathways are functional. As in all other eukaryotes, the cytosolic pathways feed glycosylation reactions; however, the role of the duplicated glycosomal pathways is currently unknown. We also showed that one of these enzymes (UGP) is imported into glycosomes by piggybacking on another glycosomal enzyme (PEPCK); they are not functionally related. The UGP/PEPCK association is unique since all piggybacking examples reported to date involve functionally related interacting partners, which broadens the possible combinations of carrier-cargo proteins being imported as hetero-oligomers.

Mutations in IDH induce epigenetic and transcriptional reprogramming, differentiation bias, and susceptibility to mitochondrial inhibitors in cancer cells. Here, we first show that cell lines, PDXs, and patients with acute myeloid leukemia (AML) harboring an IDH mutation displayed an enhanced mitochondrial oxidative metabolism. Along with an increase in TCA cycle intermediates, this AML-specific metabolic behavior mechanistically occurred through the increase in electron transport chain complex I activity, mitochondrial respiration, and methylation-driven CEBPα-induced fatty acid β-oxidation of IDH1 mutant cells. While IDH1 mutant inhibitor reduced 2-HG oncometabolite and CEBPα methylation, it failed to reverse FAO and OxPHOS. These mitochondrial activities were maintained through the inhibition of Akt and enhanced activation of peroxisome proliferator-activated receptor-γ coactivator-1 PGC1α upon IDH1 mutant inhibitor. Accordingly, OxPHOS inhibitors improved anti-AML efficacy of IDH mutant inhibitors in vivo. This work provides a scientific rationale for combinatory mitochondrial-targeted therapies to treat IDH mutant AML patients, especially those unresponsive to or relapsing from IDH mutant inhibitors., Competing Interests: Disclosures: B. Nicolay reported "other" from Agios Pharmaceuticals outside the submitted work and is an employee and shareholder of Agios Pharmaceuticals. J.R. Marszalek reported a patent to IACS-010759 issued. K. Takahashi reported personal fees from Celgene during the conduct of the study; and personal fees from Symbio Pharmaceuticals, GSK, and Novartis outside the submitted work. C.D. DiNardo reported personal fees from Agios Pharmaceuticals, Celgene, and AbbVie outside the submitted work. M. Konopleva reported "other" from Amgen, Kisoji, and Reata Pharmaceutical; and grants from AbbVie, Genentech, and Stemline Therapeutics, F. Hoffman La-Roche, Forty Seven, Eli Lilly, Cellectis, Calithera, Ablynx, Agios, Ascentage, Astra Zeneca, Rafael Pharmaceutical, and Sanofi outside the submitted work. In addition, M. Konopleva had a patent to Novartis pending (62/993,166), a patent to Eli Lilly issued, and a patent to Reata Pharmaceutical issued (7,795,305 B2 CDDO). C. Récher reported grants from Celgene, Amgen, Novartis, Jazz, AbbVie, Astellas, MaatPharma, Agios, Daiichi-Sankyo, and Roche; personal fees from Incyte, Macrogenics, Otsuka, Janssen, Pfizer, and Takeda; and non-financial support from Sanofi and Gilead outside the submitted work. No other disclosures were reported., (© 2021 Stuani et al.)

We have developed a robust workflow to measure high-resolution fluxotypes (metabolic flux phenotypes) for large strain libraries under fully controlled growth conditions. This was achieved by optimizing and automating the whole high-throughput fluxomics process and integrating all relevant software tools. This workflow allowed us to obtain highly detailed maps of carbon fluxes in the central carbon metabolism in a fully automated manner. It was applied to investigate the glucose fluxotypes of 180 Escherichia coli strains deleted for y-genes. Since the products of these y-genes potentially play a role in a variety of metabolic processes, the experiments were designed to be agnostic as to their potential metabolic impact. The obtained data highlight the robustness of E. coli 's central metabolism to y-gene deletion. For two y-genes, deletion resulted in significant changes in carbon and energy fluxes, demonstrating the involvement of the corresponding y-gene products in metabolic function or regulation. This work also introduces novel metrics to measure the actual scope and quality of high-throughput fluxomics investigations.

Overflow metabolism refers to the production of seemingly wasteful by-products by cells during growth on glucose even when oxygen is abundant. Two theories have been proposed to explain acetate overflow in Escherichia coli - global control of the central metabolism and local control of the acetate pathway - but neither accounts for all observations. Here, we develop a kinetic model of E. coli metabolism that quantitatively accounts for observed behaviours and successfully predicts the response of E. coli to new perturbations. We reconcile these theories and clarify the origin, control, and regulation of the acetate flux. We also find that, in turns, acetate regulates glucose metabolism by coordinating the expression of glycolytic and TCA genes. Acetate should not be considered a wasteful end-product since it is also a co-substrate and a global regulator of glucose metabolism in E. coli . This has broad implications for our understanding of overflow metabolism., Competing Interests: PM, BE, SU, JP, FL No competing interests declared, (© 2021, Millard et al.)

Relapses driven by chemoresistant leukemic cell populations are the main cause of mortality for patients with acute myeloid leukemia (AML). Here, we show that the ectonucleotidase CD39 (ENTPD1) is upregulated in cytarabine-resistant leukemic cells from both AML cell lines and patient samples in vivo and in vitro . CD39 cell-surface expression and activity is increased in patients with AML upon chemotherapy compared with diagnosis, and enrichment in CD39-expressing blasts is a marker of adverse prognosis in the clinics. High CD39 activity promotes cytarabine resistance by enhancing mitochondrial activity and biogenesis through activation of a cAMP-mediated adaptive mitochondrial stress response. Finally, genetic and pharmacologic inhibition of CD39 ecto-ATPase activity blocks the mitochondrial reprogramming triggered by cytarabine treatment and markedly enhances its cytotoxicity in AML cells in vitro and in vivo . Together, these results reveal CD39 as a new residual disease marker and a promising therapeutic target to improve chemotherapy response in AML. SIGNIFICANCE: Extracellular ATP and CD39-P2RY13-cAMP-OxPHOS axis are key regulators of cytarabine resistance, offering a new promising therapeutic strategy in AML. This article is highlighted in the In This Issue feature, p. 1426 ., (©2020 American Association for Cancer Research.)

Well-differentiated and dedifferentiated liposarcomas (LPSs) are characterized by a systematic amplification of the MDM2 oncogene, which encodes a key negative regulator of the p53 pathway. The molecular mechanisms underlying MDM2 overexpression while sparing wild-type p53 in LPS remain poorly understood. Here, we show that the p53-independent metabolic functions of chromatin-bound MDM2 are exacerbated in LPS and mediate an addiction to serine metabolism that sustains nucleotide synthesis and tumor growth. Treatment of LPS cells with Nutlin-3A, a pharmacological inhibitor of the MDM2-p53 interaction, stabilized p53 but unexpectedly enhanced MDM2-mediated control of serine metabolism by increasing its recruitment to chromatin, likely explaining the poor clinical efficacy of this class of MDM2 inhibitors. In contrast, genetic or pharmacological inhibition of chromatin-bound MDM2 by SP141, a distinct MDM2 inhibitor triggering its degradation, or interfering with de novo serine synthesis, impaired LPS growth both in vitro and in clinically relevant patient-derived xenograft models. Our data indicate that targeting MDM2 functions in serine metabolism represents a potential therapeutic strategy for LPS., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Thermogenic (brown and beige) adipose tissues improve glucose and lipid homeostasis and therefore represent putative targets to cure obesity and related metabolic diseases including type II diabetes. Beside decades of research and the very well-described role of noradrenergic signaling, mechanisms underlying adipocytes plasticity and activation of thermogenic adipose tissues remain incompletely understood. Recent studies show that metabolites such as lactate control the oxidative capacity of thermogenic adipose tissues. Long time viewed as a metabolic waste product, lactate is now considered as an important metabolic substrate largely feeding the oxidative metabolism of many tissues, acting as a signaling molecule and as an inter-cellular and inter-tissular redox carrier. In this review, we provide an overview of the recent findings highlighting the importance of lactate in adipose tissues, from its production to its role as a browning inducer and its metabolic links with brown adipose tissue. We also discuss additional function(s) than thermogenesis ensured by brown and beige adipose tissues, i.e., their ability to dissipate high redox pressure and oxidative stress thanks to the activity of the uncoupling protein-1, helping to maintain tissue and whole organism redox homeostasis and integrity.

Studies of the topology, functioning, and regulation of metabolic systems are based on two main types of information that can be measured by mass spectrometry: the (absolute or relative) concentration of metabolites and their isotope incorporation in 13 C-labeling experiments. These data are currently obtained from two independent experiments because the 13 C-labeled internal standard (IS) used to determine the concentration of a given metabolite overlaps the 13 C-mass fractions from which its 13 C-isotopologue distribution (CID) is quantified. Here, we developed a generic method with a dedicated processing workflow to obtain these two sets of information simultaneously in a unique sample collected from a single cultivation, thereby reducing by a factor of 2 both the number of cultivations to perform and the number of samples to collect, prepare, and analyze. The proposed approach is based on an IS labeled with other isotope(s) that can be resolved from the 13 C-mass fractions of interest. As proof-of-principle, we analyzed amino acids using a doubly labeled 15 N 13 C-cell extract as IS. Extensive evaluation of the proposed approach shows a similar accuracy and precision compared to state-of-the-art approaches. We demonstrate the value of this approach by investigating the dynamic response of amino acids metabolism in mammalian cells upon activation of the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), a key component of the unfolded protein response. Integration of metabolite concentrations and isotopic profiles reveals a reduced de novo biosynthesis of amino acids upon PERK activation. The proposed approach is generic and can be applied to other (micro)organisms, analytical platforms, isotopic tracers, or classes of metabolites.

Background: The fungus Ashbya gossypii is an important industrial producer of the vitamin riboflavin. Using this microbe, riboflavin is manufactured in a two-stage process based on a rich medium with vegetable oil, yeast extract and different precursors: an initial growth and a subsequent riboflavin production phase. So far, our knowledge on the intracellular metabolic fluxes of the fungus in this complex process is limited, but appears highly relevant to better understand and rationally engineer the underlying metabolism. To quantify intracellular fluxes of growing and riboflavin producing A. gossypii, studies with different 13 C tracers were embedded into a framework of experimental design, isotopic labeling analysis by MS and NMR techniques, and model-based data processing. The studies included the use 13 C of yeast extract, a key component used in the process., Results: During growth, the TCA cycle was found highly active, whereas the cells exhibited a low flux through gluconeogenesis as well as pentose phosphate pathway. Yeast extract was the main carbon donor for anabolism,  while vegetable oil selectively contributed to the proteinogenic amino acids glutamate, aspartate, and alanine. During the subsequent riboflavin biosynthetic phase, the carbon flux through the TCA cycle remained high. Regarding riboflavin formation, most of the vitamin's carbon originated from rapeseed oil (81 ± 1%), however extracellular glycine and yeast extract also contributed with 9 ± 0% and 8 ± 0%, respectively. In addition, advanced yeast extract-based building blocks such as guanine and GTP were directly incorporated into the vitamin., Conclusion: Intracellular carbon fluxes for growth and riboflavin production on vegetable oil provide the first flux insight into a  fungus on complex industrial medium. The knowledge gained therefrom is valuable for further strain and process improvement. Yeast extract, while being the main carbon source during growth, contributes valuable building blocks to the synthesis of vitamin B 2 . This highlights the importance of careful selection of the right yeast extract for a process based on its unique composition.

Metabolism of an organism is composed of hundreds to thousands of interconnected biochemical reactions responding to environmental or genetic constraints. This metabolic network provides a rich knowledge to contextualize omics data and to elaborate hypotheses on metabolic modulations. Nevertheless, performing this kind of integrative analysis is challenging for end users with not sufficiently advanced computer skills since it requires the use of various tools and web servers. MetExplore offers an all-in-one online solution composed of interactive tools for metabolic network curation, network exploration and omics data analysis. In particular, it is possible to curate and annotate metabolic networks in a collaborative environment. The network exploration is also facilitated in MetExplore by a system of interactive tables connected to a powerful network visualization module. Finally, the contextualization of metabolic elements in the network and the calculation of over-representation statistics make it possible to interpret any kind of omics data. MetExplore is a sustainable project maintained since 2010 freely available at https://metexplore.toulouse.inra.fr/metexplore2/.

Metabolic engineering strategies applied over the last two decades to produce shikimate (SA) in Escherichia coli have resulted in a battery of strains bearing many expression systems. However, the effects that these systems have on the host physiology and how they impact the production of SA are still not well understood. In this work we utilized an engineered E. coli strain to determine the consequences of carrying a vector that promotes SA production from glucose with a high-yield but that is also expected to impose a significant cellular burden. Kinetic comparisons in fermentors showed that instead of exerting a negative effect, the sole presence of the plasmid increased glucose consumption without diminishing the growth rate. By constitutively expressing a biosynthetic operon from this vector, the more active glycolytic metabolism was exploited to redirect intermediates toward the production of SA, which further increased the glucose consumption rate and avoided excess acetate production. Fluxomics and metabolomics experiments revealed a global remodeling of the carbon and energy metabolism in the production strain, where the increased SA production reduced the carbon available for oxidative and fermentative pathways. Moreover, the results showed that the production of SA relies on a specific setup of the pentose phosphate pathway, where both its oxidative and non-oxidative branches are strongly activated to supply erythrose-4-phosphate and balance the NADPH requirements. This work improves our understanding of the metabolic reorganization observed in E. coli in response to the plasmid-based expression of the SA biosynthetic pathway. Biotechnol. Bioeng. 2017;114: 1319-1330. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

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.

The mouse double minute 2 (MDM2) oncoprotein is recognized as a major negative regulator of the p53 tumor suppressor, but growing evidence indicates that its oncogenic activities extend beyond p53. Here, we show that MDM2 is recruited to chromatin independently of p53 to regulate a transcriptional program implicated in amino acid metabolism and redox homeostasis. Identification of MDM2 target genes at the whole-genome level highlights an important role for ATF3/4 transcription factors in tethering MDM2 to chromatin. MDM2 recruitment to chromatin is a tightly regulated process that occurs during oxidative stress and serine/glycine deprivation and is modulated by the pyruvate kinase M2 (PKM2) metabolic enzyme. Depletion of endogenous MDM2 in p53-deficient cells impairs serine/glycine metabolism, the NAD(+)/NADH ratio, and glutathione (GSH) recycling, impacting their redox state and tumorigenic potential. Collectively, our data illustrate a previously unsuspected function of chromatin-bound MDM2 in cancer cell metabolism., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Summary: Several methods and computational tools have been developed to design novel metabolic pathways. A major challenge is evaluating the metabolic efficiency of the designed pathways in the host organism. Here we present FindPath, a unified system to predict and rank possible pathways according to their metabolic efficiency in the cellular system. This tool uses a chemical reaction database to generate possible metabolic pathways and exploits constraint-based models (CBMs) to identify the most efficient synthetic pathway to achieve the desired metabolic function in a given host microorganism. FindPath can be used with common tools for CBM manipulation and uses the standard SBML format for both input and output files., Availability and Implementation: http://metasys.insa-toulouse.fr/software/findpath/., Contact: heux@insa-toulouse.fr, Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The growing demand for (13) C-metabolic flux analysis ((13) C-MFA) in the field of metabolic engineering and systems biology is driving the need to rationalize expensive and time-consuming (13) C-labeling experiments. Experimental design is a key step in improving both the number of fluxes that can be calculated from a set of isotopic data and the precision of flux values. We present IsoDesign, a software that enables these parameters to be maximized by optimizing the isotopic composition of the label input. It can be applied to (13) C-MFA investigations using a broad panel of analytical tools (MS, MS/MS, (1) H NMR, (13) C NMR, etc.) individually or in combination. It includes a visualization module to intuitively select the optimal label input depending on the biological question to be addressed. Applications of IsoDesign are described, with an example of the entire (13) C-MFA workflow from the experimental design to the flux map including important practical considerations. IsoDesign makes the experimental design of (13) C-MFA experiments more accessible to a wider biological community. IsoDesign is distributed under an open source license at http://metasys.insa-toulouse.fr/software/isodes/, (© 2013 Wiley Periodicals, Inc.)

Mass spectrometry (MS) in combination with isotope labelling experiments is widely used for investigations of metabolism and other biological processes. Quantitative applications-e.g., (13)C metabolic flux analysis-require correction of raw MS data (isotopic clusters) for the contribution of all naturally abundant isotopes. This chapter describes how to perform such correction using the software IsoCor. This flexible, user-friendly software can be used to exploit any isotopic tracer, from well-known ((13)C, (15)N, (18)O, etc.) to unusual ((57)Fe, (77)Se, etc.) isotopes. It also provides options-e.g., correction for the isotopic purity of the tracer-to improve the accuracy of quantitative isotopic studies, and allows automated correction of large datasets that can be collected with modern MS methods.

Adaptive metabolic behavior of photoautotrophic microorganisms toward genetic and environmental perturbations can be interpreted in a quantitative depiction of carbon flow through a biochemical reaction network using isotopic non-stationary (13) C-metabolic flux analysis (INST (13) C-MFA). To evaluate (13) C-metabolic flux maps for Chlamydomonas reinhardtii, an original experimental framework was designed allowing rapid, reliable collection of high-quality isotopomer data against time. It involved (i) a short-time (13) C labeling injection device based on mixing control in a torus-shaped photobioreactor with plug-flow hydrodynamics allowing a sudden step-change in the (13) C proportion in the substrate feed and (ii) a rapid sampling procedure using an automatic fast filtration method coupled to a manual rapid liquid nitrogen quenching step. (13) C-substrate labeling enrichment was controlled through the total dissolved inorganic carbon concentration in the pulsed solution. First results were obtained from steady-state continuous culture measurements allowing the characterization of the kinetics of label incorporation into light-limited growing cells cultivated in a photobioreactor operating at the maximal biomass productivity for an incident photon flux density of 200 µmol m(-2) s(-1). (13)C label incorporation was measured for 21 intracellular metabolites using IC-MS/MS in 58 samples collected across a labeling experiment duration of 7 min. The fastest labeling rate was observed for 2/3-phosphoglycerate with an apparent isotopic stationary state reached after 300 s. The labeling rate was consistent with the optimized mixing time of about 4.9 s inside the reactor and the shortest reliable sampling period assessed at 5 s., (Copyright © 2012 Wiley Periodicals, Inc.)

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 ¹³C-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 ¹³C 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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Motivation: The problem of stationary metabolic flux analysis based on isotope labelling experiments first appeared in the early 1950s and was basically solved in early 2000s. Several algorithms and software packages are available for this problem. However, the generic stochastic algorithms (simulated annealing or evolution algorithms) currently used in these software require a lot of time to achieve acceptable precision. For deterministic algorithms, a common drawback is the lack of convergence stability for ill-conditioned systems or when started from a random point., Results: In this article, we present a new deterministic algorithm with significantly increased numerical stability and accuracy of flux estimation compared with commonly used algorithms. It requires relatively short CPU time (from several seconds to several minutes with a standard PC architecture) to estimate fluxes in the central carbon metabolism network of Escherichia coli., Availability: The software package influx_s implementing this algorithm is distributed under an OpenSource licence at http://metasys.insa-toulouse.fr/software/influx/., Supplementary Information: Supplementary data are available at Bioinformatics online.

Trypanosoma brucei is a parasitic protist that undergoes a complex life cycle during transmission from its mammalian host (bloodstream forms) to the midgut of its insect vector (procyclic form). In both parasitic forms, most glycolytic steps take place within specialized peroxisomes, called glycosomes. Here, we studied metabolic adaptations in procyclic trypanosome mutants affected in their maintenance of the glycosomal redox balance. T. brucei can theoretically use three strategies to maintain the glycosomal NAD(+)/NADH balance as follows: (i) the glycosomal succinic fermentation branch; (ii) the glycerol 3-phosphate (Gly-3-P)/dihydroxyacetone phosphate (DHAP) shuttle that transfers reducing equivalents to the mitochondrion; and (iii) the glycosomal glycerol production pathway. We showed a hierarchy in the use of these glycosomal NADH-consuming pathways by determining metabolic perturbations and adaptations in single and double mutant cell lines using a combination of NMR, ion chromatography-MS/MS, and HPLC approaches. Although functional, the Gly-3-P/DHAP shuttle is primarily used when the preferred succinate fermentation pathway is abolished in the Δpepck knock-out mutant cell line. In the absence of these two pathways (Δpepck/(RNAi)FAD-GPDH.i mutant), glycerol production is used but with a 16-fold reduced glycolytic flux. In addition, the Δpepck mutant cell line shows a 3.3-fold reduced glycolytic flux compensated by an increase of proline metabolism. The inability of the Δpepck mutant to maintain a high glycolytic flux demonstrates that the Gly-3-P/DHAP shuttle is not adapted to the procyclic trypanosome context. In contrast, this shuttle was shown earlier to be the only way used by the bloodstream forms of T. brucei to sustain their high glycolytic flux.

Functional metabolomics of skeletal muscle involves the simultaneous identification and quantification of a large number of metabolites. For this purpose, the extraction of metabolites from animal tissues is a crucial technical step that needs to be optimized. In this work, five extraction methods for skeletal muscle metabolome analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS) were tested. Bird skeletal muscles sampled postmortem and quenched in liquid nitrogen were used. Three replicates of the same sample were extracted using the following solvent systems of varying polarity: boiling water (BW, +100 degrees C), cold pure methanol (CPM, -80 degrees C), methanol/chloroform/water (MCW, -20 degrees C), boiling ethanol (BE, +80 degrees C), and perchloric acid (PCA, -20 degrees C). Three injections by extraction were performed. The BW extraction showed the highest recovery of metabolites with the lowest variability (<10%) except for creatine-phosphate (creatine-P). Considering yield (area of the peaks), reproducibility, and ease, the current experiment drew a scale for the muscle metabolome extraction starting from the best to the least convenient: BW>MCW>CPM>PCABE. In addition, the semiquantification of metabolites in two muscles showing different metabolic and contractile properties was carried out after BW extraction and showed expected differences in metabolite contents, thereby validating the technique for biological investigations. In conclusion, the BW extraction is recommended for analysis of skeletal muscle metabolome except for creatine-P, which was poorly recovered with this technique., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

The assimilation of one-carbon (C1) compounds, such as methanol, by serine cycle methylotrophs requires the continuous regeneration of glyoxylate. Instead of the glyoxylate cycle, this process is achieved by a not yet established pathway where CoA thioesters are known to play a key role. We applied state-of-the-art metabolomics and (13)C metabolomics strategies to demonstrate how glyoxylate is generated during methylotrophic growth in the isocitrate lyase-negative methylotroph Methylobacterium extorquens AM1. High-resolution mass spectrometry showed the presence of CoA thioesters specific to the recently proposed ethylmalonyl-CoA pathway. The operation of this pathway was demonstrated by short-term (13)C-labeling experiments, which allowed determination of the sequence of reactions from the order of label incorporation into the different CoA derivatives. Analysis of (13)C positional enrichment in glycine by NMR was consistent with the predicted labeling pattern as a result of the operation of the ethylmalonyl-CoA pathway and the unique operation of the latter for glyoxylate generation during growth on methanol. The results also revealed that 2 molecules of glyoxylate were regenerated in this process. This work provides a complete pathway for methanol assimilation in the model methylotroph M. extorquens AM1 and represents an important step toward the determination of the overall topology of its metabolic network. The operation of the ethylmalonyl-CoA pathway in M. extorquens AM1 has major implications for the physiology of these methylotrophs and their role in nature, and it also provides a common ground for C1 and C2 compound assimilation in isocitrate lyase-negative bacteria.

In this report, we introduce a liquid chromatography single-mass spectrometry method for metabolome quantification, using the LTQ Orbitrap high-resolution mass spectrometer. Analytes were separated with hydrophilic interaction liquid chromatography. At a working resolution of 30,000 (at m/z 400), the limit of detection varied from 50 fmol to 5 pmol for 25 metabolites tested. In terms of metabolite concentration, the linearity was about 2 to 3 orders of magnitude for most compounds (R(2)>0.99). To determine the accuracy of the system in complex sample matrices, the isotope dilution method was evaluated from mixtures of pure compounds and uniformly 13C-labeled cell extracts. With the application of this method, quantification was possible within single runs even when the pool sizes of individual metabolites varied from 0.13 to 55.6 microM. As a case study, intracellular concentrations of central metabolites were determined for Methylobacterium extorquens AM1 during growth on two different carbon sources, methanol and succinate. Reproducible results from technical and biological repetitions were obtained that revealed significant variations of intracellular metabolite pool sizes, depending on the carbon source. The LTQ Obitrap offers new perspectives and strategies for metabolome quantification.

A 2D-NMR method based on zero-quantum filtered (ZQF-) TOtal Correlation SpectroscopY (TOCSY) was applied to measure 13C-enrichments in complex mixtures of 13C-labeled metabolites generated in carbon-labeling experiments. Using ZQF-TOCSY, more than 30 13C-enrichments could be potentially measured from the analysis of a biomass hydrolyzate prepared from Escherichia coli cells grown on a mixture of 20% [U-13C]-glucose and 80% [1-13C]-glucose, without need for separation of metabolites. The method is applicable to biomass hydrolyzates, cell extracts, and other complex biological samples. It is also applicable to any combination of labeled substrates and provides a basis for examining non-steady-state conditions.

With the increasing use of metabolomics as a means to study a large number of different biological research questions, there is a need for a minimal set of reporting standards that allow the scientific community to evaluate, understand, repeat, compare and re-investigate metabolomics studies. Here we propose, a first draft of minimal requirements to effectively describe the biological context of metabolomics studies that involve microbial or in vitro biological subjects. This recommendation has been produced by the microbiology and in vitro biology working subgroup of the Metabolomics Standards Initiative in collaboration with the yeast systems biology network as part of a wider standardization initiative led by the Metabolomics Society. Microbial and in vitro biology metabolomics is defined by this sub-working group as studies with any cell or organism that require a defined external medium to facilitate growth and propagation. Both a minimal set and a best practice set of reporting standards for metabolomics experiments have been defined. The minimal set of reporting standards for microbial or in vitro biology metabolomics experiments includes those factors that are specific for metabolomics experiments and that critically determine the outcome of the experiments. The best practice set of reporting standards contains both the factors that are specific for metabolomics experiments and general aspects that critically determine the outcome of any microbial or in vitro biological experiment.

Formate dehydrogenase has traditionally been assumed to play an essential role in energy generation during growth on C(1) compounds. However, this assumption has not yet been experimentally tested in methylotrophic bacteria. In this study, a whole-genome analysis approach was used to identify three different formate dehydrogenase systems in the facultative methylotroph Methylobacterium extorquens AM1 whose expression is affected by either molybdenum or tungsten. A complete set of single, double, and triple mutants was generated, and their phenotypes were analyzed. The growth phenotypes of the mutants suggest that any one of the three formate dehydrogenases is sufficient to sustain growth of M. extorquens AM1 on formate, while surprisingly, none is required for growth on methanol or methylamine. Nuclear magnetic resonance analysis of the fate of [(13)C]methanol revealed that while cells of wild-type M. extorquens AM1 as well as cells of all the single and the double mutants continuously produced [(13)C]bicarbonate and (13)CO(2), cells of the triple mutant accumulated [(13)C]formate instead. Further studies of the triple mutant showed that formate was not produced quantitatively and was consumed later in growth. These results demonstrated that all three formate dehydrogenase systems must be inactivated in order to disrupt the formate-oxidizing capacity of the organism but that an alternative formate-consuming capacity exists in the triple mutant.

The kinetics of tropane alkaloid biosynthesis, particularly the isomerization of littorine into hyoscyamine, were studied by analyzing the kinetics of carbon-13 ((13)C) in metabolites of Datura innoxia hairy root cultures fed with labeled tropoyl moiety precursors. Both littorine and hyoscyamine were the major alkaloids accumulated, while scopolamine was never detected. Feeding root cultures with (RS)-phenyl[1,3-(13)C(2)]lactic acid led to (13)C spin-spin coupling detected on C-1' and C-2' of the hyoscyamine skeleton, which validated the intramolecular rearrangement of littorine into hyoscyamine. Label from phenyl[1-(13)C]alanine or (RS)-phenyl[1,3-(13)C(2)]lactic acid was incorporated at higher levels in littorine than in hyoscyamine. Initially, the apparent hyoscyamine biosynthesized rate (v(app)()hyo = 0.9 micromol (13)C.flask(-1).d(-1)) was lower than littorine formation (v(app)()litto = 1.8 micromol (13)C.flask(-1).d(-1)), suggesting that the isomerization reaction could be rate limiting. The results obtained for the kinetics of littorine biosynthesis were in agreement with the role of this compound as a direct precursor of hyoscyamine biosynthesis.