Intermediates Of Central Carbon Metabolism Research Articles

Metabolic changes associated with polysaccharide utilization reduce susceptibility to some β-lactams in Bacteroides thetaiotaomicron.

Antibiotic therapy alters bacterial abundance and metabolism in the gut microbiome, leading to dysbiosis and opportunistic infections. Bacteroides thetaiotaomicron (Bth) is both a commensal in the gut and an opportunistic pathogen in other body sites. Past work has shown that Bth responds to β-lactam treatment differently depending on the metabolic environment both in vitro and in vivo. Studies of other bacteria show that an increase in respiratory metabolism independent of growth rate promotes susceptibility to bactericidal antibiotics. We propose that Bth enters a protected state linked to an increase in polysaccharide utilization and a decrease in the use of simple sugars. Here, we apply antibiotic susceptibility testing, transcriptomic analysis, and genetic manipulation to characterize this polysaccharide-mediated tolerance (PM tolerance) phenotype. We found that a variety of mono- and disaccharides increased the susceptibility of Bth to several different β-lactams compared to polysaccharides. Transcriptomics indicated a metabolic shift from reductive to oxidative branches of the tricarboxylic acid cycle on polysaccharides. Accordingly, supplementation with intermediates of central carbon metabolism had varying effects on PM tolerance. Transcriptional analysis also showed a decrease in the expression of the electron transport chain (ETC) protein NQR and an increase in the ETC protein NUO, when given fiber versus glucose. Deletion of NQR increased Bth susceptibility while deletion of NUO and a third ETC protein NDH2 had no effect. This work confirms that carbon source utilization modulates antibiotic susceptibility in Bth and that anaerobic respiratory metabolism and the ETC play an essential role.IMPORTANCEAntibiotics are indispensable medications that revolutionized modern medicine. However, their effectiveness is challenged by a large array of resistance and tolerance mechanisms. Treatment with antibiotics also disrupts the gut microbiome which can adversely affect health. Bacteroides are prevalent in the gut microbiome and yet are frequently involved in anaerobic infections. Thus, understanding how antibiotics affect these bacteria is necessary to implement proper treatment. Recent work has investigated the role of metabolism in antibiotic susceptibility in distantly related bacteria such as Escherichia coli. Using antibiotic susceptibility testing, transcriptomics, and genetic manipulation, we demonstrate that polysaccharides reduce β-lactam susceptibility when compared to monosaccharides. This finding underscores the profound impact of metabolic adaptation on the therapeutic efficacy of antibiotics. In the long term, this work indicates that modulation of metabolism could make Bacteroides more susceptible during infections or protect them in the context of the microbiome.

Bioplastic degradation and assimilation processes by a novel bacterium isolated from the marine plastisphere

Biosourced and biodegradable plastics offer a promising solution to reduce environmental impacts of plastics for specific applications. Here, we report a novel bacterium named Alteromonas plasticoclasticus MED1 isolated from the marine plastisphere that forms biofilms on foils of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Experiments of degradation halo, plastic matrix weight loss, bacterial oxygen consumption and heterotrophic biosynthetic activity showed that the bacterial isolate MED1 is able to degrade PHBV and to use it as carbon and energy source. The likely entire metabolic pathway specifically expressed by this bacterium grown on PHBV matrices was shown by further genomic and transcriptomic analysis. In addition to a gene coding for a probable secreted depolymerase, a gene cluster was located that encodes characteristic enzymes involved in the complete depolymerization of PHBV, the transport of oligomers, and in the conversion of the monomers into intermediates of central carbon metabolism. The transcriptomic experiments showed the activation of the glyoxylate shunt during PHBV degradation, setting the isocitrate dehydrogenase activity as regulated branching point of the carbon flow entering the tricarboxylic acid cycle. Our study also shows the potential of exploring the natural plastisphere to discover new bacteria with promising metabolic capabilities.

Chemical isotope labeling assisted liquid chromatography-mass spectrometry method for simultaneous analysis of central carbon metabolism intermediates

Central carbon metabolism pathway (CCM) is one of the most important metabolic pathways in all living organisms and play crucial function in aspect of organism life. However, the simultaneous detection of CCM intermediates remains challenging. Here, we developed a chemical isotope labeling combined with LC-MS method for simultaneous determination of CCM intermediates with high coverage and accuracy. By chemical derivatization with 2-(diazo-methyl)-N-methyl-N-phenyl-benzamide (2-DMBA) and d5-2-DMBA, all CCM intermediates obtain better separation and accurate quantification at a single LC-MS run. The obtained limits of detection of CCM intermediates ranged from 5 to 36 pg/mL. Using this method, we achieved simultaneous and accurate quantification of 22 CCM intermediates in different biological samples. Take account of the high detection sensitivity of the developed method, this method was further applied to the quantification of CCM intermediates at single-cell level. Finally, 21 CCM intermediates were detected in 1000 HEK-293T cells and 9 CCM intermediates were detected in mouse kidney glomeruli optical slice samples (10∼100 cells).

Improvement of acetyl-CoA supply and glucose utilization increases l-leucine production in Corynebacterium glutamicum.

l-Leucine is an important essential amino acid with multiple industrial applications, whose market requirements cannot be met because of the low productivity. In this study, a strain of Corynebacterium glutamicum with high l-leucine yield was constructed to enhance its acetyl-CoA supply and glucose utilization. One copy of leuA under the control of a strong promoter was incorporated into the C. glutamicum genome. Then, acetyl-CoA supply was increased by the integration of a terminator in front of gltA and by the heterogeneous overexpression of acetyl-CoA synthetase (ACS) and deacetylase (CobB) derived from Escherichia coli. Next, the transcriptional regulator sugR was deleted to enhance glucose uptake via a phosphotransferase-mediated route. In fed-batch fermentation performed in a 5-L reactor, l-leucine production of 40.11 ± 0.73g L-1 was achieved under the optimized conditions, with an l-leucine yield and productivity of 0.25g g-1 glucose and 0.59g L-1 h-1 , respectively. These results represent a significant improvement in the l-leucine production of C. glutamicum, indicating that the process possesses high potential for industrial application. These strategies can be also expanded to enable the production of other value-added biochemicals derived from the intermediates of central carbon metabolism.

Biotechnological Production of the Sunscreen Pigment Scytonemin in Cyanobacteria: Progress and Strategy.

Scytonemin is a promising UV-screen and antioxidant small molecule with commercial value in cosmetics and medicine. It is solely biosynthesized in some cyanobacteria. Recently, its biosynthesis mechanism has been elucidated in the model cyanobacterium Nostoc punctiforme PCC 73102. The direct precursors for scytonemin biosynthesis are tryptophan and p-hydroxyphenylpyruvate, which are generated through the shikimate and aromatic amino acid biosynthesis pathway. More upstream substrates are the central carbon metabolism intermediates phosphoenolpyruvate and erythrose-4-phosphate. Thus, it is a long route to synthesize scytonemin from the fixed atmospheric CO2 in cyanobacteria. Metabolic engineering has risen as an important biotechnological means for achieving sustainable high-efficiency and high-yield target metabolites. In this review, we summarized the biochemical properties of this molecule, its biosynthetic gene clusters and transcriptional regulations, the associated carbon flux-driving progresses, and the host selection and biosynthetic strategies, with the aim to expand our understanding on engineering suitable cyanobacteria for cost-effective production of scytonemin in future practices.

Exploring the Use of Gas Chromatography Coupled to Chemical Ionization Mass Spectrometry (GC-CI-MS) for Stable Isotope Labeling in Metabolomics.

Isotopic-labeling experiments have been valuable to monitor the flux of metabolic reactions in biological systems, which is crucial to understand homeostatic alterations with disease. Experimental determination of metabolic fluxes can be inferred from a characteristic rearrangement of stable isotope tracers (e.g., 13C or 15N) that can be detected by mass spectrometry (MS). Metabolites measured are generally members of well-known metabolic pathways, and most of them can be detected using both gas chromatography (GC)-MS and liquid chromatography (LC)-MS. In here, we show that GC methods coupled to chemical ionization (CI) MS have a clear advantage over alternative methodologies due to GC's superior chromatography separation efficiency and the fact that CI is a soft ionization technique that yields identifiable protonated molecular ion peaks. We tested diverse GC-CI-MS setups, including methane and isobutane reagent gases, triple quadrupole (QqQ) MS in SIM mode, or selected ion clusters using optimized narrow windows (∼10 Da) in scan mode, and standard full scan methods using high resolution GC-(q)TOF and GC-Orbitrap systems. Isobutane as a reagent gas in combination with both low-resolution (LR) and high-resolution (HR) MS showed the best performance, enabling precise detection of isotopologues in most metabolic intermediates of central carbon metabolism. Finally, with the aim of overcoming manual operations, we developed an R-based tool called isoSCAN that automatically quantifies all isotopologues of intermediate metabolites of glycolysis, TCA cycle, amino acids, pentose phosphate pathway, and urea cycle, from LRMS and HRMS data.

Two-step derivatization for determination of sugar phosphates in plants by combined reversed phase chromatography/tandem mass spectrometry

BackgroundSugar phosphates are important intermediates of central carbon metabolism in biological systems, with roles in glycolysis, the pentose–phosphate pathway, tricarboxylic acid (TCA) cycle, and many other biosynthesis pathways. Understanding central carbon metabolism requires a simple, robust and comprehensive analytical method. However, sugar phosphates are notoriously difficult to analyze by traditional reversed phase liquid chromatography.ResultsHere, we show a two-step derivatization of sugar phosphates by methoxylamine and propionic acid anhydride after chloroform/methanol (3:7) extraction from Populus leaf and developing wood that improves separation, identification and quantification of sugar phosphates by ultra high performance liquid chromatography–electrospray ionization–mass spectrometry (UHPLC–ESI–MS). Standard curves of authentic sugar phosphates were generated for concentrations from pg to ng/μl with a correlation coefficient R2 > 0.99. The method showed high sensitivity and repeatability with relative standard deviation (RSD) < 20% based on repeated extraction, derivatization and detection. The analytical accuracy for Populus leaf extracts, determined by a two-level spiking approach of selected metabolites, was 79–107%.ConclusionThe results show the reliability of combined reversed phase liquid chromatography–tandem mass spectrometry for sugar phosphate analysis and demonstrate the presence of two unknown sugar phosphates in Populus extracts.

Dynamic Metabolic Rewiring Enables Efficient Acetyl Coenzyme A Assimilation in Paracoccus denitrificans.

During growth, microorganisms have to balance metabolic flux between energy and biosynthesis. One of the key intermediates in central carbon metabolism is acetyl coenzyme A (acetyl-CoA), which can be either oxidized in the citric acid cycle or assimilated into biomass through dedicated pathways. Two acetyl-CoA assimilation strategies in bacteria have been described so far, the ethylmalonyl-CoA pathway (EMCP) and the glyoxylate cycle (GC). Here, we show that Paracoccus denitrificans uses both strategies for acetyl-CoA assimilation during different growth stages, revealing an unexpected metabolic complexity in the organism's central carbon metabolism. The EMCP is constitutively expressed on various substrates and leads to high biomass yields on substrates requiring acetyl-CoA assimilation, such as acetate, while the GC is specifically induced on these substrates, enabling high growth rates. Even though each acetyl-CoA assimilation strategy alone confers a distinct growth advantage, P. denitrificans recruits both to adapt to changing environmental conditions, such as a switch from succinate to acetate. Time-resolved single-cell experiments show that during this switch, expression of the EMCP and GC is highly coordinated, indicating fine-tuned genetic programming. The dynamic metabolic rewiring of acetyl-CoA assimilation is an evolutionary innovation by P. denitrificans that allows this organism to respond in a highly flexible manner to changes in the nature and availability of the carbon source to meet the physiological needs of the cell, representing a new phenomenon in central carbon metabolism.IMPORTANCE Central carbon metabolism provides organisms with energy and cellular building blocks during growth and is considered the invariable "operating system" of the cell. Here, we describe a new phenomenon in bacterial central carbon metabolism. In contrast to many other bacteria that employ only one pathway for the conversion of the central metabolite acetyl-CoA, Paracoccus denitrificans possesses two different acetyl-CoA assimilation pathways. These two pathways are dynamically recruited during different stages of growth, which allows P. denitrificans to achieve both high biomass yield and high growth rates under changing environmental conditions. Overall, this dynamic rewiring of central carbon metabolism in P. denitrificans represents a new strategy compared to those of other organisms employing only one acetyl-CoA assimilation pathway.

Transient drug-tolerance and permanent drug-resistance rely on the trehalose-catalytic shift in Mycobacterium tuberculosis

Stochastic formation of Mycobacterium tuberculosis (Mtb) persisters achieves a high level of antibiotic-tolerance and serves as a source of multidrug-resistant (MDR) mutations. As conventional treatment is not effective against infections by persisters and MDR-Mtb, novel therapeutics are needed. Several approaches were proposed to kill persisters by altering their metabolism, obviating the need to target active processes. Here, we adapted a biofilm culture to model Mtb persister-like bacilli (PLB) and demonstrated that PLB underwent trehalose metabolism remodeling. PLB use trehalose as an internal carbon to biosynthesize central carbon metabolism intermediates instead of cell surface glycolipids, thus maintaining levels of ATP and antioxidants. Similar changes were identified in Mtb following antibiotic-treatment, and MDR-Mtb as mechanisms to circumvent antibiotic effects. This suggests that trehalose metabolism is associated not only with transient drug-tolerance but also permanent drug-resistance, and serves as a source of adjunctive therapeutic options, potentiating antibiotic efficacy by interfering with adaptive strategies.

Exploiting High-Resolution Mass Spectrometry for Targeted Metabolite Quantification and 13C-Labeling Metabolism Analysis.

Quantification of targeted metabolites, especially trace metabolites and structural isomers, in complex biological materials is an ongoing challenge for metabolomics. In this chapter, we summarize high-resolution mass spectrometry-based approaches mainly used for targeted metabolite and metabolomics analysis, and then introduce an MS1/MS2-combined PRM workflow for quantification of central carbon metabolism intermediates, amino acids, and shikimate pathway-related metabolites. Major steps in the workflow, including cell culture, metabolite extraction, LC-MS analysis and data processing, are described. Furthermore, we adapt this new approach to a dynamic 13C-labeling experiment and demonstrate its unique advantage in capturing and correcting isotopomer labeling curves to facilitate nonstationary 13C-labeling metabolism analysis.

Abstract P296: High Salt (NaCl) Induces Metabolic Modifications and Affects Classic and Alternative Macrophage Activation

High intake of dietary sodium is a risk factor for cardiovascular disease. Previously, we have shown that an increase in extracellular hypertonic sodium (+40 mM Na + ; HS) inhibits alternatively-activated M2 macrophage (M(IL4+IL13)) gene signature and function, while boosting classic M1 macrophage polarization (M(LPS)) and bacterial killing capacity. M1 macrophages predominantly rely on glycolysis, whereas M2 macrophages use oxidative phosphorylation, fueled by the oxidation of fatty acids, at a late phase of activation (24 h). The aim of this study was to elucidate the role of HS on the metabolic signature of M1 and M2 macrophages. Therefore, we quantified 13 C incorporation into intermediates of central carbon metabolism via GC-MS based pulsed stable isotope resolved metabolomics (pSIRM), and performed Seahorse analyses at varying time points during M1/M2-activation. After 24 h, LPS-induced increase in basal ECAR (extracellular acidification as surrogate parameter for glycolysis) and lactate production were further boosted under HS compared to isotonic salt conditions (NS), whereas mitochondrial respiration (OCR) was not affected. On the other hand, IL4+IL13-induced increase in OCR was decreased under HS compared to NS, while ECAR was not affected at this stage. Regarding TCA-cycle, we have found an early (1 h and 3 h post-stimulation) increase in citrate levels in M1 and M2 macrophages independent of HS, which further increased after 24 h in M1 and M1+HS. Interestingly, both in early and late phase of activation, glucose-derived 13 C incorporation into citrate was lower in HS-treated compared to NS-treated M0, M1 and M2 macrophages, suggesting an alternative replenishing source. Consistently, other TCA-cycle intermediates increased after 24h (especially in M1 and even further in M1+HS), whereas glucose-derived 13 C incorporation was minimal. Our data suggests that under hypertonic sodium macrophages re-direct TCA-cycle intermediates into the production of pro-inflammatory mediators (as described for late-phase M1 macrophages), whereas TCA-cycle itself is replenished by other alternative metabolic substrates. These HS-induced metabolic modifications might play an important role for macrophage activation and function.

Quantitative imaging mass spectroscopy reveals roles of heme oxygenase-2 for protecting against transhemispheric diaschisis in the brain ischemia.

Carbon monoxide-generating heme oxygenase-2 is expressed in neurons and plays a crucial role for regulating hypoxic vasodilation through mechanisms unlocking carbon monoxide-dependent inhibition of H2S-generating cystathionine β-synthase expressed in astrocytes. This study aims to examine whether heme oxygenase-2 plays a protective role in mice against stroke. Focal ischemia was induced by middle cerebral artery occlusion. Regional differences in metabolites among ipsilateral and contralateral hemispheres were analysed by quantitative imaging mass spectrometry equipped with an image-processing platform to optimize comparison of local metabolite contents among different animals. Under normoxia, blood flow velocity in precapillary arterioles were significantly elevated in heme oxygenase-2-null mice vs controls, while metabolic intermediates of central carbon metabolism and glutamate synthesis were elevated in the brain of heme oxygenase-2-null mice, suggesting greater metabolic demands to induce hyperemia in these mice. In response to focal ischemia, heme oxygenase-2-null mice exhibited greater regions of ischemic core that coincide with notable decreases in energy metabolism in the contralateral hemisphere as well as in penumbra. In conclusion, these findings suggest that heme oxygenase-2 is involved in mechanisms by which not only protects against compromised energy metabolism of the ipsilateral hemisphere but also ameliorates transhemispheric diaschisis of the contralateral hemisphere in ischemic brain.

Quantifying the Metabolome of Pseudomonas taiwanensis VLB120: Evaluation of Hot and Cold Combined Quenching/Extraction Approaches.

Absolute quantification of free intracellular metabolites is a valuable tool in both pathway discovery and metabolic engineering. In this study, we conducted a comprehensive examination of different hot and cold combined quenching/extraction approaches to extract and quantify intracellular metabolites of Pseudomonas taiwanensis (P. taiwanensis) VLB120 to provide a useful reference data set of absolute intracellular metabolite concentrations. The suitability of commonly used metabolomics tools including a pressure driven fast filtration system followed by combined quenching/extraction techniques (such as cold methanol/acetonitrile/water, hot water, and boiling ethanol/water, as well as cold ethanol/water) were tested and evaluated for P. taiwanensis VLB120 metabolome analysis. In total 94 out of 107 detected intracellular metabolites were quantified using an isotope-ratio-based approach. The quantified metabolites include amino acids, nucleotides, central carbon metabolism intermediates, redox cofactors, and others. The acquired data demonstrate that the pressure driven fast filtration approach followed by boiling ethanol quenching/extraction is the most adequate technique for P. taiwanensis VLB120 metabolome analysis based on quenching efficiency, extraction yields of metabolites, and experimental reproducibility.

Metabolic determinants in Listeria monocytogenes anaerobic listeriolysin O production

Listeria monocytogenes is a human pathogen and a facultative anaerobe. To better understand how anaerobic growth affects L. monocytogenes pathogenesis, we first showed that anaerobic growth led to decreased growth and changes in surface morphology. Moreover, compared to aerobically grown bacteria, anaerobically grown L. monocytogenes established higher level of invasion but decreased intracellular growth and actin polymerization in cultured cells. The production of listeriolysin O (LLO) was significantly lower in anaerobic cultures—a phenotype observed in wild type and isogenic mutants lacking transcriptional regulators SigB or CodY or harboring a constitutively active PrfA. To explore potential regulatory mechanisms, we established that the addition of central carbon metabolism intermediates, such as acetate, citrate, fumarate, pyruvate, lactate, and succinate, led to an increase in LLO activity in the anaerobic culture supernatant. These results highlight the regulatory role of central carbon metabolism in L. monocytogenes pathogenesis under anaerobic conditions.

Integrating MS1 and MS2 Scans in High-Resolution Parallel Reaction Monitoring Assays for Targeted Metabolite Quantification and Dynamic 13C-Labeling Metabolism Analysis.

Quantification of targeted metabolites, especially trace metabolites and structural isomers, in complex biological materials is an ongoing challenge for metabolomics. Initially developed for proteomic analysis, the parallel reaction monitoring (PRM) technique exploiting high-resolution MS2 fragment ion data has shown high promise for targeted metabolite quantification. Notably, MS1 ion intensity data acquired independently as part of each PRM scan cycle are often underutilized in the PRM assay. In this study, we developed an MS1/MS2-combined PRM workflow for quantification of central carbon metabolism intermediates, amino acids and shikimate pathway-related metabolites on an orthogonal QqTOF system. Concentration curve assessment revealed that exploiting both MS1 and MS2 scans in PRM analysis afforded higher sensitivity, wider dynamic range and better reproducibility than relying on either scan mode for quantification. Furthermore, Skyline was incorporated into our workflow to process the MS1/MS2 ion intensity data, and eliminate noisy signals and transitions with interferences. This integrated MS1/MS2 PRM approach was applied to targeted metabolite quantification in engineered E. coli strains for understanding of metabolic pathway modulation. In addition, this new approach, when first implemented in a dynamic 13C-labeling experiment, showed its unique advantage in capturing and correcting isotopomer labeling curves to facilitate nonstationary 13C-labeling metabolism analysis.

Creating metabolic demand as an engineering strategy in Pseudomonas putida – Rhamnolipid synthesis as an example

Metabolic engineering of microbial cell factories for the production of heterologous secondary metabolites implicitly relies on the intensification of intracellular flux directed toward the product of choice. Apart from reactions following peripheral pathways, enzymes of the central carbon metabolism are usually targeted for the enhancement of precursor supply. In Pseudomonas putida, a Gram-negative soil bacterium, central carbon metabolism, i.e., the reactions required for the synthesis of all 12 biomass precursors, was shown to be regulated at the metabolic level and not at the transcriptional level. The bacterium's central carbon metabolism appears to be driven by demand to react rapidly to ever-changing environmental conditions. In contrast, peripheral pathways that are only required for growth under certain conditions are regulated transcriptionally. In this work, we show that this regulation regime can be exploited for metabolic engineering.We tested this driven-by-demand metabolic engineering strategy using rhamnolipid production as an example. Rhamnolipid synthesis relies on two pathways, i.e., fatty acid de novo synthesis and the rhamnose pathway, providing the required precursors hydroxyalkanoyloxy-alkanoic acid (HAA) and activated (dTDP-)rhamnose, respectively. In contrast to single-pathway molecules, rhamnolipid synthesis causes demand for two central carbon metabolism intermediates, i.e., acetyl-CoA for HAA and glucose-6-phosphate for rhamnose synthesis.Following the above-outlined strategy of driven by demand, a synthetic promoter library was developed to identify the optimal expression of the two essential genes (rhlAB) for rhamnolipid synthesis. The best rhamnolipid-synthesizing strain had a yield of 40% rhamnolipids on sugar [CmolRL/CmolGlc], which is approximately 55% of the theoretical yield. The rate of rhamnolipid synthesis of this strain was also high. Compared to an exponentially growing wild type, the rhamnose pathway increased its flux by 300%, whereas the flux through de novo fatty acid synthesis increased by 50%.We show that the central carbon metabolism of P. putida is capable of meeting the metabolic demand generated by engineering transcription in peripheral pathways, thereby enabling a significant rerouting of carbon flux toward the product of interest, in this case, rhamnolipids of industrial interest.

Assignment of function to a domain of unknown function: DUF1537 is a new kinase family in catabolic pathways for acid sugars

Using a large-scale "genomic enzymology" approach, we (i) assigned novel ATP-dependent four-carbon acid sugar kinase functions to members of the DUF1537 protein family (domain of unknown function; Pfam families PF07005 and PF17042) and (ii) discovered novel catabolic pathways for d-threonate, l-threonate, and d-erythronate. The experimentally determined ligand specificities of several solute binding proteins (SBPs) for TRAP (tripartite ATP-independent permease) transporters for four-carbon acids, including d-erythronate and l-erythronate, were used to constrain the substrates for the catabolic pathways that degrade the SBP ligands to intermediates in central carbon metabolism. Sequence similarity networks and genome neighborhood networks were used to identify the enzyme components of the pathways. Conserved genome neighborhoods encoded SBPs as well as permease components of the TRAP transporters, members of the DUF1537 family, and a member of the 4-hydroxy-l-threonine 4-phosphate dehydrogenase (PdxA) oxidative decarboxylase, class II aldolase, or ribulose 1,5-bisphosphate carboxylase/oxygenase, large subunit (RuBisCO) superfamily. Because the characterized substrates of members of the PdxA, class II aldolase, and RuBisCO superfamilies are phosphorylated, we postulated that the members of the DUF1537 family are novel ATP-dependent kinases that participate in catabolic pathways for four-carbon acid sugars. We determined that (i) the DUF1537/PdxA pair participates in a pathway for the conversion of d-threonate to dihydroxyacetone phosphate and CO2 and (ii) the DUF1537/class II aldolase pair participates in pathways for the conversion of d-erythronate and l-threonate (epimers at carbon-3) to dihydroxyacetone phosphate and CO2 The physiological importance of these pathways was demonstrated in vivo by phenotypic and genetic analyses.

Effects of fluconazole on the metabolomic profile of Candida albicans.

Little is known about the effects of fluconazole on the metabolism of Candida albicans. We performed LC/MS-based metabolomic profiling of the response of C. albicans cells to increasing doses of fluconazole. C. albicans cells were cultured to mid-logarithmic growth phase in liquid medium and then inoculated in replicate on to nitrocellulose filters under vacuum filtration. Organisms were cultured to mid-logarithmic growth phase and treated with 0-4 mg/L fluconazole. Following metabolic quenching at mid-logarithmic growth phase, intracellular metabolites were extracted and analysed by LC/MS. Changes in pool sizes of individual metabolites were verified by Student's t-test, adjusted for multiple hypothesis testing by Benjamini-Hochberg correction. Distribution of metabolites was analysed by the Kyoto Encyclopedia of Genes and Genomes metabolic pathways database. We reproducibly detected 64 metabolites whose identities were confirmed by comparison against a pure standard and a library of accurate mass-retention time pairs. These 64 metabolites were broadly representative of eukaryotic central metabolic pathways. Among them 12 had their mean abundance significantly altered in response to increasing fluconazole concentrations. Pool sizes of four intermediates of central carbon metabolism (α-ketoglutarate, glucose-6-phosphate, phenylpyruvate and ribose-5-phosphate) and mevalonate were increased by 0.5-1.5-fold (P ≤ 0.05). Five amino acids (glycine, proline, tryptophan, aminoisobutanoate and asparagine) and guanine were decreased by 0.5-0.75-fold (P ≤ 0.05). Fluconazole treatment of C. albicans resulted in increased central carbon and decreased amino acid synthesis intermediates, suggesting a rerouting of metabolic pathways. The function of these metabolomic changes remains to be elucidated; however, they may represent previously unrecognized mechanisms of metabolic injury induced by fluconazole against C. albicans.

Metabolic Impact of Redox Cofactor Perturbations on the Formation of Aroma Compounds in Saccharomyces cerevisiae.

Redox homeostasis is a fundamental requirement for the maintenance of metabolism, energy generation, and growth in Saccharomyces cerevisiae. The redox cofactors NADH and NADPH are among the most highly connected metabolites in metabolic networks. Changes in their concentrations may induce widespread changes in metabolism. Redox imbalances were achieved with a dedicated biological tool overexpressing native NADH-dependent or engineered NADPH-dependent 2,3-butanediol dehydrogenase, in the presence of acetoin. We report that targeted perturbation of the balance of cofactors (NAD(+)/NADH or, to a lesser extent, NADP(+)/NADPH) significantly affected the production of volatile compounds. In most cases, variations in the redox state of yeasts modified the formation of all compounds from the same biochemical pathway (isobutanol, isoamyl alcohol, and their derivatives) or chemical class (ethyl esters), irrespective of the cofactors. These coordinated responses were found to be closely linked to the impact of redox status on the availability of intermediates of central carbon metabolism. This was the case for α-keto acids and acetyl coenzyme A (acetyl-CoA), which are precursors for the synthesis of many volatile compounds. We also demonstrated that changes in the availability of NADH selectively affected the synthesis of some volatile molecules (e.g., methionol, phenylethanol, and propanoic acid), reflecting the specific cofactor requirements of the dehydrogenases involved in their formation. Our findings indicate that both the availability of precursors from central carbon metabolism and the accessibility of reduced cofactors contribute to cell redox status modulation of volatile compound formation.

A liquid chromatography–tandem mass spectrometry-based investigation of the lamellar interstitial metabolome in healthy horses and during experimental laminitis induction

Lamellar bioenergetic failure is thought to contribute to laminitis pathogenesis but current knowledge of lamellar bioenergetic physiology is limited. Metabolomic analysis (MA) can systematically profile multiple metabolites. Applied to lamellar microdialysis samples (dialysate), lamellar bioenergetic changes during laminitis (the laminitis metabolome) can be characterised. The objectives of this study were to develop a technique for targeted MA of lamellar and skin dialysates in normal horses, and to compare the lamellar and plasma metabolomic profiles of normal horses with those from horses developing experimentally induced laminitis. Archived lamellar and skin dialysates (n = 7) and tissues (n = 6) from normal horses, and lamellar dialysate and plasma from horses given either 10 g/kg oligofructose (treatment group, OFT; n = 4) or sham (control group, CON; n = 4) were analysed. The concentrations of 44 intermediates of central carbon metabolism (CCM) were determined using liquid chromatography–tandem mass spectrometry. Data were analysed using multivariate (MVA) and univariate (UVA) analysis methods.The plasma metabolome appeared to be more variable than the lamellar metabolome by MVA, driven by malate, pyruvate, aconitate and glycolate. In lamellar dialysate, these metabolites decreased in OFT horses at the later time points. Plasma malate was markedly increased after 6 h in OFT horses. Plasma malate concentrations between OFT and CON at this time point were significantly different by UVA. MA of lamellar CCM was capable of differentiating horses developing experimental laminitis from controls. Lamellar malate, pyruvate, aconitate and glycolate, and plasma malate alone were identified as the source of differentiation between OFT and CON groups. These results highlighted clear discriminators between OFT and CON horses, suggesting that changes in energy metabolism occur locally in the lamellar tissue during laminitis development. The biological significance of these alterations requires further investigation.