6-Phosphogluconolactonase is critical for the efficient functioning of the pentose phosphate pathway.

The metabolic networks of microorganisms are remarkably robust to genetic and environmental perturbations. This robustness stems from redundancies such as gene duplications, isoenzymes, alternative metabolic pathways, and also from non-enzymatic reactions. In the oxidative branch of the pentose phosphate pathway (oxPPP), 6-phosphogluconolactone hydrolysis into 6-phosphogluconate is catalysed by 6-phosphogluconolactonase (Pgl) but in the absence of the latter, the oxPPP flux is thought to be maintained by spontaneous hydrolysis.

Metabolic impact of heterologous protein production in Pseudomonas putida: Insights into carbon and energy flux control.

For engineered microorganisms, the production of heterologous proteins that are often useless to host cells represents a burden on resources, which have to be shared with normal cellular processes. Within a certain metabolic leeway, this competitive process has no impact on growth. However, once this leeway, or free capacity, is fully utilized, the extra load becomes a metabolic burden that inhibits cellular processes and triggers a broad cellular response, reducing cell growth and often hindering the production of heterologous proteins.

Dynamic Metabolic Response to (p)ppGpp Accumulation in .

The stringent response is a ubiquitous bacterial reaction triggered by nutrient deprivation and mediated by the intracellular concentrations of ppGpp and pppGpp. These alarmones, jointly referred to as (p)ppGpp, control gene transcription, mRNA translation and protein activity to adjust the metabolism and growth rate to environmental changes. While the ability of (p)ppGpp to mediate cell growth slowdown and metabolism adaptation has been demonstrated in , it's role in remains unclear.

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.

Functional Analysis of Deoxyhexose Sugar Utilization in Escherichia coli Reveals Fermentative Metabolism under Aerobic Conditions.

l-Rhamnose and l-fucose are the two main 6-deoxyhexoses Escherichia coli can use as carbon and energy sources. Deoxyhexose metabolism leads to the formation of lactaldehyde, whose fate depends on oxygen availability. Under anaerobic conditions, lactaldehyde is reduced to 1,2-propanediol, whereas under aerobic conditions, it should be oxidized into lactate and then channeled into the central metabolism.

Control and regulation of acetate overflow in .

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 - 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 metabolism that quantitatively accounts for observed behaviours and successfully predicts the response of to new perturbations.

Engineering precursor pools for increasing production of odd-chain fatty acids in .

Microbial production of lipids is one of the promising alternatives to fossil resources with increasing environmental and energy concern. Odd-chain fatty acids (OCFA), a type of unusual lipids, are recently gaining a lot of interest as target compounds in microbial production due to their diverse applications in the medical, pharmaceutical, and chemical industries. In this study, we aimed to enhance the pool of precursors with three-carbon chain (propionyl-CoA) and five-carbon chain (β-ketovaleryl-CoA) for the production of OCFAs in .

Crucial Role of ppGpp in the Resilience of Escherichia coli to Growth Disruption.

Bacteria grow in constantly changing environments that can suddenly become completely depleted of essential nutrients. The stringent response, a rewiring of the cellular metabolism mediated by the alarmone (p)ppGpp, plays a crucial role in adjusting bacterial growth to the severity of the nutritional stress. The ability of (p)ppGpp to trigger a slowdown of cell growth or induce bacterial dormancy has been widely investigated.

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 C-labeling experiments. These data are currently obtained from two independent experiments because the C-labeled internal standard (IS) used to determine the concentration of a given metabolite overlaps the C-mass fractions from which its 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.

Harvesting of Prebiotic Fructooligosaccharides by Nonbeneficial Human Gut Bacteria.

Prebiotic oligosaccharides, such as fructooligosaccharides, are increasingly being used to modulate the composition and activity of the gut microbiota. However, carbohydrate utilization analyses and metagenomic studies recently revealed the ability of deleterious and uncultured human gut bacterial species to metabolize these functional foods. Moreover, because of the difficulties of functionally profiling transmembrane proteins, only a few prebiotic transporters have been biochemically characterized to date, while carbohydrate binding and transport are the first and thus crucial steps in their metabolization.

Increasing field strength versus advanced isotope labeling for NMR-based fluxomics.

Nuclear magnetic resonance (NMR)-based fluxomics seeks to measure the incorporation of isotope labels in selected metabolites to follow kinetically the synthesis of the latter. It can however equally be used to understand the biosynthetic origin of the same metabolites. We investigate here different NMR approaches to optimize such experiments in terms of resolution and time requirement.

IsoCor: isotope correction for high-resolution MS labeling experiments.

Summary: Mass spectrometry (MS) is widely used for isotopic studies of metabolism and other (bio)chemical processes. Quantitative applications in systems and synthetic biology require to correct the raw MS data for the contribution of naturally occurring isotopes. Several tools are available to correct low-resolution MS data, and recent developments made substantial improvements by introducing resolution-dependent correction methods, hence opening the way to the correction of high-resolution MS (HRMS) data.

Absolute Quantification of ppGpp and pppGpp by Double-Spike Isotope Dilution Ion Chromatography-High-Resolution Mass Spectrometry.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) play a central role in the adaptation of bacterial and plant cells to nutritional and environmental stresses and in bacterial resistance to antibiotics. These compounds have historically been detected and quantified by two-dimensional thin-layer chromatography of P-radiolabeled nucleotides. We report a new method to quantify ppGpp and pppGpp in complex biochemical matrix using ion chromatography coupled to high-resolution mass spectrometry.

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.

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.

Systems and synthetic biology perspective of the versatile plant-pathogenic and polysaccharide-producing bacterium Xanthomonas campestris.

Bacteria of the genus Xanthomonas are a major group of plant pathogens. They are hazardous to important crops and closely related to human pathogens. Being collectively a major focus of molecular phytopathology, an increasing number of diverse and intricate mechanisms are emerging by which they communicate, interfere with host signalling and keep competition at bay.

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.

Plasmid-encoded biosynthetic genes alleviate metabolic disadvantages while increasing glucose conversion to shikimate in an engineered Escherichia coli strain.

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.

Acetate fluxes in Escherichia coli are determined by the thermodynamic control of the Pta-AckA pathway.

Escherichia coli excretes acetate upon growth on fermentable sugars, but the regulation of this production remains elusive. Acetate excretion on excess glucose is thought to be an irreversible process. However, dynamic C-metabolic flux analysis revealed a strong bidirectional exchange of acetate between E.

Multilevel interaction of the DnaK/DnaJ(HSP70/HSP40) stress-responsive chaperone machine with the central metabolism.

Networks of molecular chaperones maintain cellular protein homeostasis by acting at nearly every step in the biogenesis of proteins and protein complexes. Herein, we demonstrate that the major chaperone DnaK/HSP70 of the model bacterium Escherichia coli is critical for the proper functioning of the central metabolism and for the cellular response to carbon nutrition changes, either directly or indirectly via the control of the heat-shock response. We identified carbon sources whose utilization was positively or negatively affected by DnaK and isolated several central metabolism genes (among other genes identified in this work) that compensate for the lack of DnaK and/or DnaK/Trigger Factor chaperone functions in vivo.

Recent advances in high-throughput C-fluxomics.

The rise of high throughput (HT) strain engineering tools accompanying the area of synthetic biology is supporting the generation of a large number of microbial cell factories. A current bottleneck in process development is our limited capacity to rapidly analyze the metabolic state of the engineered strains, and in particular their intracellular fluxes. HT C-fluxomics workflows have not yet become commonplace, despite the existence of several HT tools at each of the required stages.

Differential quantitative proteome analysis of Escherichia coli grown on acetate versus glucose.

Relative protein abundances of Escherichia coli MG1655 growing exponentially on minimal medium with acetate or glucose as the sole carbon source were investigated in a quantitative shotgun proteome analysis with TMT6-plex isobaric tags. Peptides were separated by high resolution high/low pH 2D-LC, using an optimized fraction pooling scheme followed by mass spectrometric analysis. Quantitative data were acquired for 2099 proteins covering 49% of the predicted E.

The post-transcriptional regulatory system CSR controls the balance of metabolic pools in upper glycolysis of Escherichia coli.

Metabolic control in Escherichia coli is a complex process involving multilevel regulatory systems but the involvement of post-transcriptional regulation is uncertain. The post-transcriptional factor CsrA is stated as being the only regulator essential for the use of glycolytic substrates. A dozen enzymes in the central carbon metabolism (CCM) have been reported as potentially controlled by CsrA, but its impact on the CCM functioning has not been demonstrated.

Acetate Exposure Determines the Diauxic Behavior of Escherichia coli during the Glucose-Acetate Transition.

Unlabelled: Growth of Escherichia coli on glucose in batch culture is accompanied by the excretion of acetate, which is consumed by the cells when glucose is exhausted. This glucose-acetate transition is classically described as a diauxie (two successive growth stages). Here, we investigated the physiological and metabolic properties of cells after glucose exhaustion through the analysis of growth parameters and gene expression.