Glycerol suppresses glucose consumption in trypanosomes through metabolic contest.

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.

Chromatin-Bound MDM2 Regulates Serine Metabolism and Redox Homeostasis Independently of p53.

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.

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.

Lactate fluxes mediated by the monocarboxylate transporter-1 are key determinants of the metabolic activity of beige adipocytes.

Activation of energy-dissipating brown/beige adipocytes represents an attractive therapeutic strategy against metabolic disorders. While lactate is known to induce beiging through the regulation of Ucp1 gene expression, the role of lactate transporters on beige adipocytes' ongoing metabolic activity remains poorly understood. To explore the function of the lactate-transporting monocarboxylate transporters (MCTs), we used a combination of primary cell culture studies, 13C isotopic tracing, laser microdissection experiments, and in situ immunofluorescence of murine adipose fat pads. Dissecting white adipose tissue heterogeneity revealed that the MCT1 is expressed in inducible beige adipocytes as the emergence of uncoupling protein 1 after cold exposure was restricted to a subpopulation of MCT1-expressing adipocytes suggesting MCT1 as a marker of inducible beige adipocytes. We also observed that MCT1 mediates bidirectional and simultaneous inward and outward lactate fluxes, which were required for efficient utilization of glucose by beige adipocytes activated by the canonical ß3-adrenergic signaling pathway. Finally, we demonstrated that significant lactate import through MCT1 occurs even when glucose is not limiting, which feeds the oxidative metabolism of beige adipocytes. These data highlight the key role of lactate fluxes in finely tuning the metabolic activity of beige adipocytes according to extracellular metabolic conditions and reinforce the emerging role of lactate metabolism in the control of energy homeostasis.

An optimization method for untargeted MS-based isotopic tracing investigations of metabolism.

INTRODUCTION: Stable isotope tracer studies are increasingly applied to explore metabolism from the detailed analysis of tracer incorporation into metabolites. Untargeted LC/MS approaches have recently emerged and provide potent methods for expanding the dimension and complexity of the metabolic networks that can be investigated. A number of software tools have been developed to process the highly complex MS data collected in such studies; however, a method to optimize the extraction of valuable isotopic data is lacking. OBJECTIVES: To develop and validate a method to optimize automated data processing for untargeted MS-based isotopic tracing investigations of metabolism. METHODS: The method is based on the application of a suitable reference material to rationally perform parameter optimization throughout the complete data processing workflow. It was applied in the context of 13C-labelling experiments and with two different software, namely geoRge and X13CMS. It was illustrated with the study of a E. coli mutant impaired for central metabolism. RESULTS: The optimization methodology provided significant gain in the number and quality of extracted isotopic data, independently of the software considered. Pascal triangle samples are well suited for such purpose since they allow both the identification of analytical issues and optimization of data processing at the same time. CONCLUSION: The proposed method maximizes the biological value of untargeted MS-based isotopic tracing investigations by revealing the full metabolic information that is encoded in the labelling patterns of metabolites.

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.

IsoSolve: An Integrative Framework to Improve Isotopic Coverage and Consolidate Isotopic Measurements by Mass Spectrometry and/or Nuclear Magnetic Resonance.

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.

ScalaFlux: A scalable approach to quantify fluxes in metabolic subnetworks.

13C-metabolic flux analysis (13C-MFA) allows metabolic fluxes to be quantified in living organisms and is a major tool in biotechnology and systems biology. Current 13C-MFA approaches model label propagation starting from the extracellular 13C-labeled nutrient(s), which limits their applicability to the analysis of pathways close to this metabolic entry point. Here, we propose a new approach to quantify fluxes through any metabolic subnetwork of interest by modeling label propagation directly from the metabolic precursor(s) of this subnetwork. The flux calculations are thus purely based on information from within the subnetwork of interest, and no additional knowledge about the surrounding network (such as atom transitions in upstream reactions or the labeling of the extracellular nutrient) is required. This approach, termed ScalaFlux for SCALAble metabolic FLUX analysis, can be scaled up from individual reactions to pathways to sets of pathways. ScalaFlux has several benefits compared with current 13C-MFA approaches: greater network coverage, lower data requirements, independence from cell physiology, robustness to gaps in data and network information, better computational efficiency, applicability to rich media, and enhanced flux identifiability. We validated ScalaFlux using a theoretical network and simulated data. We also used the approach to quantify fluxes through the prenyl pyrophosphate pathway of Saccharomyces cerevisiae mutants engineered to produce phytoene, using a dataset for which fluxes could not be calculated using existing approaches. A broad range of metabolic systems can be targeted with minimal cost and effort, making ScalaFlux a valuable tool for the analysis of metabolic fluxes.

Simultaneous Measurement of Metabolite Concentration and Isotope Incorporation by Mass Spectrometry.

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 13C-labeling experiments. These data are currently obtained from two independent experiments because the 13C-labeled internal standard (IS) used to determine the concentration of a given metabolite overlaps the 13C-mass fractions from which its 13C-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 13C-mass fractions of interest. As proof-of-principle, we analyzed amino acids using a doubly labeled 15N13C-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.

Glycerol supports growth of the Trypanosoma brucei bloodstream forms in the absence of glucose: Analysis of metabolic adaptations on glycerol-rich conditions.

The bloodstream forms of Trypanosoma brucei (BSF), the parasite protist causing sleeping sickness, primarily proliferate in the blood of their mammalian hosts. The skin and adipose tissues were recently identified as additional major sites for parasite development. Glucose was the only carbon source known to be used by bloodstream trypanosomes to feed their central carbon metabolism, however, the metabolic behaviour of extravascular tissue-adapted parasites has not been addressed yet. Since the production of glycerol is an important primary function of adipocytes, we have adapted BSF trypanosomes to a glucose-depleted but glycerol-rich culture medium (CMM_Glyc/GlcNAc) and compared their metabolism and proteome to those of parasites grown in standard glucose-rich conditions (CMM_Glc). BSF were shown to consume 2-folds more oxygen per consumed carbon unit in CMM_Glyc/GlcNAc and were 11.5-times more sensitive to SHAM, a specific inhibitor of the plant-like alternative oxidase (TAO), which is the only mitochondrial terminal oxidase expressed in BSF. This is consistent with (i) the absolute requirement of the mitochondrial respiratory activity to convert glycerol into dihydroxyacetone phosphate, as deduced from the updated metabolic scheme and (ii) with the 1.8-fold increase of the TAO expression level compared to the presence of glucose. Proton NMR analysis of excreted end products from glycerol and glucose metabolism showed that these two carbon sources are metabolised through the same pathways, although the contributions of the acetate and succinate branches are more important in the presence of glycerol than glucose (10.2% versus 3.4% of the excreted end products, respectively). In addition, metabolomic analyses by mass spectrometry showed that, in the absence of glucose, 13C-labelled glycerol was incorporated into hexose phosphates through gluconeogenesis. As expected, RNAi-mediated down-regulation of glycerol kinase expression abolished glycerol metabolism and was lethal for BSF grown in CMM_Glyc/GlcNAc. Interestingly, BSF have adapted their metabolism to grow in CMM_Glyc/GlcNAc by concomitantly increasing their rate of glycerol consumption and decreasing that of glucose. However, the glycerol kinase activity was 7.8-fold lower in CMM_Glyc/GlcNAc, as confirmed by both western blotting and proteomic analyses. This suggests that the huge excess in glycerol kinase that is not absolutely required for glycerol metabolism, might be used for another yet undetermined non-essential function in glucose rich-conditions. Altogether, these data demonstrate that BSF trypanosomes are well-adapted to glycerol-rich conditions that could be encountered by the parasite in extravascular niches, such as the skin and adipose tissues.

Gluconeogenesis is essential for trypanosome development in the tsetse fly vector.

In the glucose-free environment that is the midgut of the tsetse fly vector, the procyclic form of Trypanosoma brucei primarily uses proline to feed its central carbon and energy metabolism. In these conditions, the parasite needs to produce glucose 6-phosphate (G6P) through gluconeogenesis from metabolism of non-glycolytic carbon source(s). We showed here that two phosphoenolpyruvate-producing enzymes, PEP carboxykinase (PEPCK) and pyruvate phosphate dikinase (PPDK) have a redundant function for the essential gluconeogenesis from proline. Indeed, incorporation of 13C-enriched proline into G6P was abolished in the PEPCK/PPDK null double mutant (Δppdk/Δpepck), but not in the single Δppdk and Δpepck mutant cell lines. The procyclic trypanosome also uses the glycerol conversion pathway to feed gluconeogenesis, since the death of the Δppdk/Δpepck double null mutant in glucose-free conditions is only observed after RNAi-mediated down-regulation of the expression of the glycerol kinase, the first enzyme of the glycerol conversion pathways. Deletion of the gene encoding fructose-1,6-bisphosphatase (Δfbpase), a key gluconeogenic enzyme irreversibly producing fructose 6-phosphate from fructose 1,6-bisphosphate, considerably reduced, but not abolished, incorporation of 13C-enriched proline into G6P. In addition, the Δfbpase cell line is viable in glucose-free conditions, suggesting that an alternative pathway can be used for G6P production in vitro. However, FBPase is essential in vivo, as shown by the incapacity of the Δfbpase null mutant to colonise the fly vector salivary glands, while the parental phenotype is restored in the Δfbpase rescued cell line re-expressing FBPase. The essential role of FBPase for the development of T. brucei in the tsetse was confirmed by taking advantage of an in vitro differentiation assay based on the RNA-binding protein 6 over-expression, in which the procyclic forms differentiate into epimastigote forms but not into mammalian-infective metacyclic parasites. In total, morphology, immunofluorescence and cytometry analyses showed that the differentiation of the epimastigote stages into the metacyclic forms is abolished in the Δfbpase mutant.

MetExplore: collaborative edition and exploration of metabolic networks.

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/.

Biofilm microenvironment induces a widespread adaptive amino-acid fermentation pathway conferring strong fitness advantage in Escherichia coli.

Bacterial metabolism has been studied primarily in liquid cultures, and exploration of other natural growth conditions may reveal new aspects of bacterial biology. Here, we investigate metabolic changes occurring when Escherichia coli grows as surface-attached biofilms, a common but still poorly characterized bacterial lifestyle. We show that E. coli adapts to hypoxic conditions prevailing within biofilms by reducing the amino acid threonine into 1-propanol, an important industrial commodity not known to be naturally produced by Enterobacteriaceae. We demonstrate that threonine degradation corresponds to a fermentation process maintaining cellular redox balance, which confers a strong fitness advantage during anaerobic and biofilm growth but not in aerobic conditions. Whereas our study identifies a fermentation pathway known in Clostridia but previously undocumented in Enterobacteriaceae, it also provides novel insight into how growth in anaerobic biofilm microenvironments can trigger adaptive metabolic pathways edging out competition with in mixed bacterial communities.

Functional analysis of isoprenoid precursors biosynthesis by quantitative metabolomics and isotopologue profiling.

INTRODUCTION: Isoprenoids are amongst the most abundant and diverse biological molecules and are involved in a broad range of biological functions. Functional understanding of their biosynthesis is thus key in many fundamental and applicative fields, including systems biology, medicine and biotechnology. However, available methods do not yet allow accurate quantification and tracing of stable isotopes incorporation for all the isoprenoids precursors. OBJECTIVES: We developed and validated a complete methodology for quantitative metabolomics and isotopologue profiling of isoprenoid precursors in the yeast Saccharomyces cerevisiae. METHODS: This workflow covers all the experimental and computational steps from sample collection and preparation to data acquisition and processing. It also includes a novel quantification method based on liquid chromatography coupled to high-resolution mass spectrometry. Method validation followed the Metabolomics Standards Initiative guidelines. RESULTS: This workflow ensures accurate absolute quantification (RSD < 20%) of all mevalonate and prenyl pyrophosphates intermediates with a high sensitivity over a large linear range (from 0.1 to 50 pmol). In addition, we demonstrate that this workflow brings crucial information to design more efficient phytoene producers. Results indicate stable turnover rates of prenyl pyrophosphate intermediates in the constructed strains and provide quantitative information on the change of the biosynthetic flux of phytoene precursors. CONCLUSION: This methodology fills one of the last technical gaps for functional studies of isoprenoids biosynthesis and should be applicable to other eukaryotic and prokaryotic (micro)organisms after adaptation of some organism-dependent steps. This methodology also opens the way to 13C-metabolic flux analysis of isoprenoid biosynthesis.

Combined transcriptomics-metabolomics profiling of the heat shock response in the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus is a remarkable archaeon able to grow at temperatures around 100 °C. To gain insight into how this model hyperthermophile copes with heat stress, we compared transcriptomic and metabolomic data of cells subjected to a temperature shift from 90 °C to 97 °C. In this study, we used RNA-sequencing to characterize the global variation in gene expression levels, while nuclear magnetic resonance (NMR) and targeted ion exchange liquid chromatography-mass spectrometry (LC-MS) were used to determine changes in metabolite levels. Of the 552 differentially expressed genes in response to heat shock conditions, 257 were upregulated and 295 were downregulated. In particular, there was a significant downregulation of genes for synthesis and transport of amino acids. At the metabolite level, 37 compounds were quantified. The level of di-myo-inositol phosphate, a canonical heat stress solute among marine hyperthermophiles, increased considerably (5.4-fold) at elevated temperature. Also, the levels of mannosylglycerate, UDP-N-acetylglucosamine (UDPGlcNac) and UDP-N-acetylgalactosamine were enhanced. The increase in the pool of UDPGlcNac was concurrent with an increase in the transcript levels of the respective biosynthetic genes. This work provides the first metabolomic analysis of the heat shock response of a hyperthermophile.

E4F1 controls a transcriptional program essential for pyruvate dehydrogenase activity.

The mitochondrial pyruvate dehydrogenase (PDH) complex (PDC) acts as a central metabolic node that mediates pyruvate oxidation and fuels the tricarboxylic acid cycle to meet energy demand. Here, we reveal another level of regulation of the pyruvate oxidation pathway in mammals implicating the E4 transcription factor 1 (E4F1). E4F1 controls a set of four genes [dihydrolipoamide acetlytransferase (Dlat), dihydrolipoyl dehydrogenase (Dld), mitochondrial pyruvate carrier 1 (Mpc1), and solute carrier family 25 member 19 (Slc25a19)] involved in pyruvate oxidation and reported to be individually mutated in human metabolic syndromes. E4F1 dysfunction results in 80% decrease of PDH activity and alterations of pyruvate metabolism. Genetic inactivation of murine E4f1 in striated muscles results in viable animals that show low muscle PDH activity, severe endurance defects, and chronic lactic acidemia, recapitulating some clinical symptoms described in PDC-deficient patients. These phenotypes were attenuated by pharmacological stimulation of PDH or by a ketogenic diet, two treatments used for PDH deficiencies. Taken together, these data identify E4F1 as a master regulator of the PDC.

Improved Isotopic Profiling by Pure Shift Heteronuclear 2D J-Resolved NMR Spectroscopy.

Quantitative information on the carbon isotope content of metabolites is essential for flux analysis. Whereas this information is in principle present in proton NMR spectra through both direct and long-range heteronuclear coupling constants, spectral overlap and homonuclear coupling constants both hinder its extraction. We demonstrate here how pure shift 2D J-resolved NMR spectroscopy can simultaneously remove the homonuclear couplings and separate the chemical shift information from the heteronuclear coupling patterns. We demonstrate the power of this method on cell lysates from different bacterial cultures and investigate in detail the branched chain amino acid biosynthesis.

Methodology for the Validation of Isotopic Analyses by Mass Spectrometry in Stable-Isotope Labeling Experiments.

Stable-isotope labeling experiments (ILEs) are widely used to investigate the topology and operation of metabolic networks. The quality of isotopic data collected in ILEs is of utmost importance to ensure reliable biological interpretations, but current evaluation approaches are limited due to a lack of suitable reference material and relevant evaluation criteria. In this work, we present a complete methodology to evaluate mass spectrometry (MS) methods used for quantitative isotopic studies of metabolic systems. This methodology, based on a biological sample containing metabolites with controlled labeling patterns, exploits different quality metrics specific to isotopic analyses (accuracy and precision of isotopologue masses, abundances, and mass shifts and isotopic working range). We applied this methodology to evaluate a novel LC-MS method for the analysis of amino acids, which was tested on high resolution (Orbitrap operating in full scan mode) and low resolution (triple quadrupole operating in multiple reaction monitoring mode) mass spectrometers. Results show excellent accuracy and precision over a large working range and revealed matrix-specific as well as mode-specific characteristics. The proposed methodology can identify reliable (and unreliable) isotopic data in an easy and straightforward way and efficiently supports the identification of sources of systematic biases as well as of the main factors that influence the overall accuracy and precision of measurements. This approach is generic and can be used to validate isotopic analyses on different matrices, analytical platforms, labeled elements, or classes of metabolites. It is expected to strengthen the reliability of isotopic measurements and thereby the biological value of ILEs.

Lysine production from the sugar alcohol mannitol: Design of the cell factory Corynebacterium glutamicum SEA-3 through integrated analysis and engineering of metabolic pathway fluxes.

The amino acid lysine is among the world's most important biotechnological products, and enabling its manufacture from the most attractive new materials is an ever-present challenge. In this study, we describe a cell factory of Corynebacterium glutamicum, which produces lysine from mannitol. A preliminary mutant C. glutamicum SEA-1 obtained by the deletion of the mannitol repressor MtlR in the glucose-based, lysine-producing strain C. glutamicum LYS-12 produced only small amounts of lysine. This limitation was due to a significant accumulation of fructose and a limited NADPH supply, which caused a low flux of only 6% into the oxidative pentose phosphate (PP) pathway. Subsequent expression of fructokinase slightly increased production but failed to substantially redirect the flux from the Emden-Meyerhof-Parnas (EMP) pathway to the PP pathway. This suggested the design of C. glutamicum SEA-3, which overexpressed the NADP-dependent glyceraldehyde 3-phosphate dehydrogenase GapN from Streptococcus mutans and coupled the EMP pathway flux to NADPH formation. When grown on mannitol, the SEA-3 strain had a lysine yield of 0.24 mol mol-1 and a specific productivity of 1.3 mmol g-1 h-1, approximately 60% and 75% higher, respectively, than those of the basic producer SEA-1. A computational pathway analysis revealed that this design would potentially enable a lysine yield of 0.9 mol mol-1, providing room for further development. Our findings open new avenues for lysine production from marine macroalgae, which is farmed globally as an attractive third-generation renewable resource. Mannitol is a major constituent of these algae (up to 30% and higher) and can be easily extracted from their biomass with hot water.

Improved riboflavin production with Ashbya gossypii from vegetable oil based on 13C metabolic network analysis with combined labeling analysis by GC/MS, LC/MS, 1D, and 2D NMR.

The fungus Ashbya gossypii is an important industrial producer of riboflavin, i.e. vitamin B2. In order to meet the constantly increasing demands for improved production processes, it appears essential to better understand the underlying metabolic pathways of the vitamin. Here, we used a highly sophisticated set-up of parallel 13C tracer studies with labeling analysis by GC/MS, LC/MS, 1D, and 2D NMR to resolve carbon fluxes in the overproducing strain A. gossypii B2 during growth and subsequent riboflavin production from vegetable oil as carbon source, yeast extract, and supplemented glycine. The studies provided a detailed picture of the underlying metabolism. Glycine was exclusively used as carbon-two donor of the vitamin's pyrimidine ring, which is part of its isoalloxazine ring structure, but did not contribute to the carbon-one metabolism due to the proven absence of a functional glycine cleavage system. The pools of serine and glycine were closely connected due to a highly reversible serine hydroxymethyltransferase. Transmembrane formate flux simulations revealed that the one-carbon metabolism displayed a severe bottleneck during initial riboflavin production, which was overcome in later phases of the cultivation by intrinsic formate accumulation. The transiently limiting carbon-one pool was successfully replenished by time-resolved feeding of small amounts of formate and serine, respectively. This increased the intracellular availability of glycine, serine, and formate and resulted in a final riboflavin titer increase of 45%.