NMR metabolite quantification of a synthetic urine sample: an inter-laboratory comparison of processing workflows.

INTRODUCTION: Absolute quantification of individual metabolites in complex biological samples is crucial in targeted metabolomic profiling. OBJECTIVES: An inter-laboratory test was performed to evaluate the impact of the NMR software, peak-area determination method (integration vs. deconvolution) and operator on quantification trueness and precision. METHODS: A synthetic urine containing 32 compounds was prepared. One site prepared the urine and calibration samples, and performed NMR acquisition. NMR spectra were acquired with two pulse sequences including water suppression used in routine analyses. The pre-processed spectra were sent to the other sites where each operator quantified the metabolites using internal referencing or external calibration, and his/her favourite in-house, open-access or commercial NMR tool. RESULTS: For 1D NMR measurements with solvent presaturation during the recovery delay (zgpr), 20 metabolites were successfully quantified by all processing strategies. Some metabolites could not be quantified by some methods. For internal referencing with TSP, only one half of the metabolites were quantified with a trueness below 5%. With peak integration and external calibration, about 90% of the metabolites were quantified with a trueness below 5%. The NMRProcFlow integration module allowed the quantification of several additional metabolites. The number of quantified metabolites and quantification trueness improved for some metabolites with deconvolution tools. Trueness and precision were not significantly different between zgpr- and NOESYpr-based spectra for about 70% of the variables. CONCLUSION: External calibration performed better than TSP internal referencing. Inter-laboratory tests are useful when choosing to better rationalize the choice of quantification tools for NMR-based metabolomic profiling and confirm the value of spectra deconvolution tools.

PeakForest: a multi-platform digital infrastructure for interoperable metabolite spectral data and metadata management.

INTRODUCTION: Accuracy of feature annotation and metabolite identification in biological samples is a key element in metabolomics research. However, the annotation process is often hampered by the lack of spectral reference data in experimental conditions, as well as logistical difficulties in the spectral data management and exchange of annotations between laboratories. OBJECTIVES: To design an open-source infrastructure allowing hosting both nuclear magnetic resonance (NMR) and mass spectra (MS), with an ergonomic Web interface and Web services to support metabolite annotation and laboratory data management. METHODS: We developed the PeakForest infrastructure, an open-source Java tool with automatic programming interfaces that can be deployed locally to organize spectral data for metabolome annotation in laboratories. Standardized operating procedures and formats were included to ensure data quality and interoperability, in line with international recommendations and FAIR principles. RESULTS: PeakForest is able to capture and store experimental spectral MS and NMR metadata as well as collect and display signal annotations. This modular system provides a structured database with inbuilt tools to curate information, browse and reuse spectral information in data treatment. PeakForest offers data formalization and centralization at the laboratory level, facilitating shared spectral data across laboratories and integration into public databases. CONCLUSION: PeakForest is a comprehensive resource which addresses a technical bottleneck, namely large-scale spectral data annotation and metabolite identification for metabolomics laboratories with multiple instruments. PeakForest databases can be used in conjunction with bespoke data analysis pipelines in the Galaxy environment, offering the opportunity to meet the evolving needs of metabolomics research. Developed and tested by the French metabolomics community, PeakForest is freely-available at https://github.com/peakforest .

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.

Exploring the Glucose Fluxotype of the E. coli y-ome Using High-Resolution Fluxomics.

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.

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.

Chemical and Metabolic Controls on Dihydroxyacetone Metabolism Lead to Suboptimal Growth of Escherichia coli.

In this work, we shed light on the metabolism of dihydroxyacetone (DHA), a versatile, ubiquitous, and important intermediate for various chemicals in industry, by analyzing its metabolism at the system level in Escherichia coli Using constraint-based modeling, we show that the growth of E. coli on DHA is suboptimal and identify the potential causes. Nuclear magnetic resonance analysis shows that DHA is degraded nonenzymatically into substrates known to be unfavorable to high growth rates. Transcriptomic analysis reveals that DHA promotes genes involved in biofilm formation, which may reduce the bacterial growth rate. Functional analysis of the genes involved in DHA metabolism proves that under the aerobic conditions used in this study, DHA is mainly assimilated via the dihydroxyacetone kinase pathway. In addition, these results show that the alternative routes of DHA assimilation (i.e., the glycerol and fructose-6-phosphate aldolase pathways) are not fully activated under our conditions because of anaerobically mediated hierarchical control. These pathways are therefore certainly unable to sustain fluxes as high as the ones predicted in silico for optimal aerobic growth on DHA. Overexpressing some of the genes in these pathways releases these constraints and restores the predicted optimal growth on DHA.IMPORTANCE DHA is an attractive triose molecule with a wide range of applications, notably in cosmetics and the food and pharmaceutical industries. DHA is found in many species, from microorganisms to humans, and can be used by Escherichia coli as a growth substrate. However, knowledge about the mechanisms and regulation of this process is currently lacking, motivating our investigation of DHA metabolism in E. coli We show that under aerobic conditions, E. coli growth on DHA is far from optimal and is hindered by chemical, hierarchical, and possibly allosteric constraints. We show that optimal growth on DHA can be restored by releasing the hierarchical constraint. These results improve our understanding of DHA metabolism and are likely to help unlock biotechnological applications involving DHA as an intermediate, such as the bioconversion of glycerol or C1 substrates into value-added chemicals.

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.

Metabolic flux analysis in Ashbya gossypii using 13C-labeled yeast extract: industrial riboflavin production under complex nutrient conditions.

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 13C 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 13C 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 B2. This highlights the importance of careful selection of the right yeast extract for a process based on its unique composition.

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%.

Metabolic footprint of epiphytic bacteria on Arabidopsis thaliana leaves.

The phyllosphere, which is defined as the parts of terrestrial plants above the ground, is a large habitat for different microorganisms that show a high extent of adaption to their environment. A number of hypotheses were generated by culture-independent functional genomics studies to explain the competitiveness of specialized bacteria in the phyllosphere. In contrast, in situ data at the metabolome level as a function of bacterial colonization are lacking. Here, we aimed to obtain new insights into the metabolic interplay between host and epiphytes upon colonization of Arabidopsis thaliana leaves in a controlled laboratory setting using environmental metabolomics approaches. Quantitative nuclear magnetic resonance (NMR) and imaging high-resolution mass spectrometry (IMS) methods were used to identify Arabidopsis leaf surface compounds and their possible involvement in the epiphytic lifestyle by relative changes in compound pools. The dominant carbohydrates on the leaf surfaces were sucrose, fructose and glucose. These sugars were significantly and specifically altered after epiphytic leaf colonization by the organoheterotroph Sphingomonas melonis or the phytopathogen Pseudomonas syringae pv. tomato, but only to a minor extent by the methylotroph Methylobacterium extorquens. In addition to carbohydrates, IMS revealed surprising alterations in arginine metabolism and phytoalexin biosynthesis that were dependent on the presence of bacteria, which might reflect the consequences of bacterial activity and the recognition of not only pathogens but also commensals by the plant. These results highlight the power of environmental metabolomics to aid in elucidating the molecular basis underlying plant-epiphyte interactions in situ.

Gluconeogenesis, an essential metabolic pathway for pathogenic Francisella.

Intracellular multiplication and dissemination of the infectious bacterial pathogen Francisella tularensis implies the utilization of multiple host-derived nutrients. Here, we demonstrate that gluconeogenesis constitutes an essential metabolic pathway in Francisella pathogenesis. Indeed, inactivation of gene glpX, encoding the unique fructose 1,6-bisphosphatase of Francisella, severely impaired bacterial intracellular multiplication when cells were supplemented by gluconeogenic substrates such as glycerol or pyruvate. The ΔglpX mutant also showed a severe virulence defect in the mouse model, confirming the importance of this pathway during the in vivo life cycle of the pathogen. Isotopic profiling revealed the major role of the Embden-Meyerhof (glycolysis) pathway in glucose catabolism in Francisella and confirmed the importance of glpX in gluconeogenesis. Altogether, the data presented suggest that gluconeogenesis allows Francisella to cope with the limiting glucose availability it encounters during its infectious cycle by relying on host amino acids. Hence, targeting the gluconeogenic pathway might constitute an interesting therapeutic approach against this pathogen.

Developmental stage dependent metabolic regulation during meiotic differentiation in budding yeast.

BACKGROUND: The meiotic developmental pathway in yeast enables both differentiation of vegetative cells into haploid spores that ensure long-term survival, and recombination of the parental DNA to create genetic diversity. Despite the importance of proper metabolic regulation for the supply of building blocks and energy, little is known about the reprogramming of central metabolic pathways in meiotically differentiating cells during passage through successive developmental stages. RESULTS: Metabolic regulation during meiotic differentiation in budding yeast was analysed by integrating information on genome-wide transcriptional activity, 26 enzymatic activities in the central metabolism, the dynamics of 67 metabolites, and a metabolic flux analysis at mid-stage meiosis. Analyses of mutants arresting sporulation at defined stages demonstrated that metabolic reprogramming is tightly controlled by the progression through the developmental pathway. The correlation between transcript levels and enzymatic activities in the central metabolism varies significantly in a developmental-stage dependent manner. The complete loss of phosphofructokinase activity at mid-stage meiosis enables a unique setup of the glycolytic pathway which facilitates carbon flux repartitioning into synthesis of spore-wall precursors during the co-assimilation of glycogen and acetate. The need for correct homeostasis of purine nucleotides during the meiotic differentiation was demonstrated by the sporulation defect of the AMP deaminase mutant, amd1, which exhibited hyper-accumulation of ATP accompanied by depletion of guanosine nucleotides. CONCLUSIONS: Our systems-level analysis shows that reprogramming of the central metabolism during the meiotic differentiation is controlled at different hierarchical levels to meet the metabolic and energetic needs at successive developmental stages.

Isotopic profiling of ¹³C-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 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.

Photobioreactor design for isotopic non-stationary 13C-metabolic flux analysis (INST 13C-MFA) under photoautotrophic conditions.

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.