Stéphane Massou

Demonstration of the ethylmalonyl-CoA pathway by using 13C metabolomics

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 13C 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 13C-labeling experiments, which allowed determination of the sequence of reactions from the order of label incorporation into the different CoA derivatives. Analysis of 13C 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.

Genome-scale reconstruction and system level investigation of the metabolic network of Methylobacterium extorquens AM1

BackgroundMethylotrophic microorganisms are playing a key role in biogeochemical processes - especially the global carbon cycle - and have gained interest for biotechnological purposes. Significant progress was made in the recent years in the biochemistry, genetics, genomics, and physiology of methylotrophic bacteria, showing that methylotrophy is much more widespread and versatile than initially assumed. Despite such progress, system-level description of the methylotrophic metabolism is currently lacking, and much remains to understand regarding the network-scale organization and properties of methylotrophy, and how the methylotrophic capacity emerges from this organization, especially in facultative organisms.ResultsIn this work, we report on the integrated, system-level investigation of the metabolic network of the facultative methylotroph Methylobacterium extorquens AM1, a valuable model of methylotrophic bacteria. The genome-scale metabolic network of the bacterium was reconstructed and contains 1139 reactions and 977 metabolites. The sub-network operating upon methylotrophic growth was identified from both in silico and experimental investigations, and 13C-fluxomics was applied to measure the distribution of metabolic fluxes under such conditions. The core metabolism has a highly unusual topology, in which the unique enzymes that catalyse the key steps of C1 assimilation are tightly connected by several, large metabolic cycles (serine cycle, ethylmalonyl-CoA pathway, TCA cycle, anaplerotic processes). The entire set of reactions must operate as a unique process to achieve C1 assimilation, but was shown to be structurally fragile based on network analysis. This observation suggests that in nature a strong pressure of selection must exist to maintain the methylotrophic capability. Nevertheless, substantial substrate cycling could be measured within C2/C3/C4 inter-conversions, indicating that the metabolic network is highly versatile around a flexible backbone of central reactions that allows rapid switching to multi-carbon sources.ConclusionsThis work emphasizes that the metabolism of M. extorquens AM1 is adapted to its lifestyle not only in terms of enzymatic equipment, but also in terms of network-level structure and regulation. It suggests that the metabolism of the bacterium has evolved both structurally and functionally to an efficient but transitory utilization of methanol. Besides, this work provides a basis for metabolic engineering to convert methanol into value-added products.

A new and specific mode of stabilization of metallic nanoparticles

We report in this paper the stabilization of ruthenium nanoparticles using a very simple ligand (4-(3-phenylpropyl)pyridine), through strong pi-coordination of the phenyl moiety.

Response of the central metabolism of Escherichia coli to modified expression of the gene encoding the glucose-6-phosphate dehydrogenase

The deletion of the zwf gene encoding G6PDH activity led to restructuring of the carbon flux through central metabolism in Escherichia coli, though over‐expression of this gene had only minor consequences for overall carbon flux. The modified carbon flux seen in the zwf deletion mutant enabled alternative routes of anabolic precursor formation and an adequate supply of NADPH synthesis via a modified TCA cycle to be generated so as to sustain growth rates comparable to the WT.

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

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

Heterologous expression of a deuterated membrane-integrated receptor and partial deuteration in methylotrophic yeasts.

Methylotrophic yeast has previously been shown to be an excellent system for the cost-effective production of perdeuterated biomass and for the heterologous expression of membrane receptors. A protocol for the expression of 85% deuterated, functional human μ-opiate receptor was established. For partially deuterated biomass, deuteration level and distribution were determined for fatty acids, amino acids and carbohydrates. It was shown that prior to biosynthesis of lipids and amino acids (and of carbohydrates, to a lower extent), exchange occurs between water and methanol hydrogen atoms, so that 80%–90% randomly deuterated biomass and over-expressed proteins may be obtained using only deuterated water.

Sampling of intracellular metabolites for stationary and non-stationary 13C metabolic flux analysis in Escherichia coli

The analysis of metabolic intermediates is a rich source of isotopic information for (13)C metabolic flux analysis ((13)C-MFA) and extends the range of its applications. The sampling of labeled metabolic intermediates is particularly important to obtain reliable isotopic information. The assessment of the different sampling procedures commonly used to generate such data, therefore, is crucial. In this work, we thoroughly evaluated several sampling procedures for stationary and non-stationary (13)C-MFA using Escherichia coli. We first analyzed the efficiency of these procedures for quenching metabolism and found that procedures based on cold or boiling solvents are reliable, in contrast to fast filtration, which is not. We also showed that separating the cells from the broth is not necessary in isotopic stationary state conditions. On the other hand, we demonstrated that the presence of metabolic intermediates outside the cells strongly affects the transient isotopic data monitored during non-stationary (13)C-labeling experiments. Meaningful isotopic data can be obtained by recovering intracellular labeled metabolites from pellets of cells centrifuged in cold solvent. We showed that if the intracellular pools are not separated from the extracellular ones, accurate flux maps can be established provided that the contribution of exogenous compounds is taken into account in the metabolic flux model.

NMR-based structural glycomics for high-throughput screening of carbohydrate-active enzyme specificity.

We report here the development of a straightforward, sensitive, and quantitative NMR-based method for high-throughput characterization of carbohydrate structure and screening of carbohydrate active enzyme (CAZyme) specificity. Automated assays starting from gene library expression to carbohydrate structure determination directly from crude reaction media have been established and successfully used to screen a library of 4032 CAZymes obtained by combinatorial engineering, at a rate of 480 enzyme variants per day. This allowed one to accurately discriminate 303 enzyme variants with altered specificity. The results demonstrate the potential of high-throughput NMR technology in glycomics, to mine artificial and natural enzyme diversity for novel biocatalysts.

Fast Spatially Encoded 3D NMR Strategies for 13C-Based Metabolic Flux Analysis

The measurement of site-specific (13)C enrichments in complex mixtures of (13)C-labeled metabolites is a powerful tool for metabolic flux analysis. One of the main methods to measure such enrichments is homonuclear (1)H 2D NMR. However, the major limitation of this technique is the acquisition time, which can amount to a few hours. This drawback was recently overcome by the design of fast COSY experiments for measuring specific (13)C-enrichments, based on single-scan 2D NMR. However, these experiments are still limited by overlaps because of(1)H-(13)C splittings, thus limiting the metabolic information accessible for complex biological mixtures. To circumvent this limitation, we propose to tilt the (1)H-(13)C coupling into a third dimension via fast-hybrid 3D NMR methods combining the speed of ultrafast 2D NMR with the high resolution of conventional methods. Two strategies are described that allow the acquisition of a complete 3D J-resolved-COSY spectrum in 12 min (for concentrations as low as 10 mM). The analytical potentialities of both methods are evaluated on a series of (13)C-enriched glucose samples and on a biomass hydrolyzate obtained from Escherichia coli cells. Once optimized, the two complementary experiments lead to a trueness and a precision of a few percent and an excellent linearity. The advantages and drawbacks of these approaches are discussed and their potentialities are highlighted.

Hydrogenation Processes at the Surface of Ruthenium Nanoparticles: A NMR Study

AbstractThe reactivity of ruthenium nanoparticles stabilized by 4-(3-phenylpropyl)pyridine in hydrogen transfer and hydrogenation processes was monitored by NMR spectroscopy. Unsaturated substrates such as styrene, 4-vinylpyridine and 4-phenyl-but-3-en-2-one were used as model molecules to investigate the surface properties of nanoparticles by a combination of NMR studies. Interestingly, the hydrides present at the metallic surface after nanoparticles synthesis are selectively transferred to vinylic groups without reducing the aromatic rings, under dihydrogen-free atmosphere. DOSY and NOE NMR experiments permitted to propose a way of interaction of the organic compounds at the metallic surface. In particular, the coordination of the substrate could be evidenced for 4-vinylpyridine and 4-ethylpyridine but not for styrene derivatives.Graphical AbstractCurved double arrows represent magnetization exchanges. Straight arrows represent adsorption/desorption phenomena.