Central Carbon Metabolites Research Articles

From CO2to cell: Energetic expense of creating biomass using the Calvin-Benson-Bassham and reductive citric acid cycles based on genome data

The factors driving the dominance of the Calvin-Benson-Bassham cycle (CBB) or reductive citric acid cycle (rCAC) in autotrophic microorganisms in different habitats are debated. Based on costs for synthesizing a few metabolic intermediates, it has been suggested that the CBB poses a disadvantage due to higher metabolic cost. The purpose of this study was to extend this estimate of cost from metabolite synthesis to biomass synthesis. For 12 gammaproteobacteria (CBB) and five epsilonproteobacteria (rCAC), the amount of ATP to synthesize a gram of biomass from CO2 was calculated from genome sequences via metabolic maps. The eleven central carbon metabolites needed to synthesize biomass were all less expensive to synthesize via the rCAC (66%-89% of the ATP needed to synthesize them via CBB). Differences in cell compositions did result in differing demands for metabolites among the organisms, but the differences in cost to synthesize biomass were small among organisms that used a particular pathway (e.g. rCAC), compared to the difference between pathways (rCAC versus CBB). The rCAC autotrophs averaged 0.195 moles ATP per g biomass, while their CBB counterparts averaged 0.238. This is the first in silico estimate of the relative expense of both pathways to generate biomass.

Functional overexpression and characterization of lipogenesis-related genes in the oleaginous yeast Yarrowia lipolytica.

Single cell oil (SCO) is an attractive energy source due to scalability, utilization of low-cost renewable feedstocks, and type of product(s) made. Engineering strains capable of producing high lipid titers and yields is crucial to the economic viability of these processes. However, lipid synthesis in cells is a complex phenomenon subject to multiple layers of regulation, making gene target identification a challenging task. In this study, we aimed to identify genes in the oleaginous yeast Yarrowia lipolytica whose overexpression enhances lipid production by this organism. To this end, we examined the effect of the overexpression of a set of 44 native genes on lipid production in Y. lipolytica, including those involved in glycerolipid synthesis, fatty acid synthesis, central carbon metabolism, NADPH generation, regulation, and metabolite transport and characterized each resulting strain's ability to produce lipids growing on both glucose and acetate as a sole carbon source. Our results suggest that a diverse subset of genes was effective at individually influencing lipid production in Y. lipolytica, sometimes in a substrate-dependent manner. The most productive strain on glucose overexpressed the diacylglycerol acyltransferase DGA2 gene, increasing lipid titer, cellular content, and yield by 236, 165, and 246%, respectively, over our control strain. On acetate, our most productive strain overexpressed the acylglycerol-phosphate acyltransferase SLC1 gene, with a lipid titer, cellular content, and yield increase of 99, 91, and 151%, respectively, over the control strain. Aside from genes encoding enzymes that directly catalyze the reactions of lipid synthesis, other ways by which lipogenesis was increased in these cells include overexpressing the glycerol-3-phosphate dehydrogenase (GPD1) gene to increase production of glycerol head groups and overexpressing the 6-phosphogluconolactonase (SOL3) gene from the oxidative pentose phosphate pathway to increase NADPH availability for fatty acid synthesis. Taken together, our study demonstrates that the overall kinetics of microbial lipid synthesis is sensitive to a wide variety of factors. Fully optimizing a strain for single cell oil processes could involve manipulating and balancing many of these factors, and, due to mechanistic differences by which each gene product investigated here impacts lipid synthesis, there is a high likelihood that many of these genes will work synergistically to further increase lipid production when simultaneously overexpressed.

Glucose feeding recalibrates carbon flux distribution and favours lipid accumulation in Chlorella protothecoides through cell energetic management

Heterotrophic culture of algal cells is now known as an efficient avenue for lipid production. However, the mechanisms involved are not clearly understood, especially when feeding glucose concurrently to autotrophic culture condition. In this work, the time course dynamics of central carbon metabolites of Chlorella protothecoides was studied in autotrophic, heterotrophic and mixotrophic cultures to elucidate the effect of glucose on the cell metabolic reorganization. Results show that assimilated CO2 mainly goes to the synthesis of upstream carbohydrate-based metabolites under autotrophic condition, while supplementing glucose recalibrates the metabolism towards downstream metabolites and lipids, rather than carbohydrates accumulation. The analysis of the lipid class shows, under glucose supplementation, that cells accumulate neutral lipids as storage rather than as membrane polar lipids, while fatty acid composition changes from polyunsaturated to saturated and monosaturated, which shows improving the quality of biodiesel precursors. The metabolic flux rearrangement seemed being regulated by a high cell energetic state that was maintained by a glucose metabolism. A high initial ATP-to-ADP ratio was observed after adding glucose, suggesting cell energetics as a biomarker of a metabolic shift from starch to lipid accumulation. These findings thus bring novel data on the regulation of carbon flow in microalgal cells, and enhance our understanding of microalgae as a lipid production platform.

Carbon roadmap from syngas to polyhydroxyalkanoates in Rhodospirillum rubrum.

The gasification of organic waste materials to synthesis gas (syngas), followed by microbial fermentation, provides a significant resource for generating bioproducts such as polyhydroxyalkanoates (PHA). The anaerobic photosynthetic bacterium, Rhodospirillum rubrum, is an organism particularly attractive for the bioconversion of syngas into PHAs. In this study, a quantitative physiological analysis of R. rubrum was carried out by implementing GC-MS and HPLC techniques to unravel the metabolic pathway operating during syngas fermentation that leads to PHA production. Further, detailed investigations of the central carbon metabolites using (13) C-labelled substrate showed significant CO2 assimilation (of 40%) into cell material and PHA from syngas carbon fraction. By a combination of quantitative gene expression and enzyme activity analyses, the main role of carboxylases from the central carbon metabolism in CO2 assimilation was shown, where the Calvin-Benson-Bassham cycle (CBB) played a minor role. This knowledge sheds light about the biochemical pathways that contribute to synthesis of PHA during syngas fermentation being valuable information to further optimize the fermentation process.

Evidence that 2-hydroxyglutarate is not readily metabolized in colorectal carcinoma cells.

BackgroundTwo-hydroxyglutarate (2HG) is present at low concentrations in healthy mammalian cells as both an L and D enantiomer. Both the L and D enantiomers have been implicated in regulating cellular physiology by mechanisms that are only partially characterized. In multiple human cancers, the D enantiomer accumulates due to gain-of-function mutations in the enzyme isocitrate dehydrogenase (IDH) and has been hypothesized to drive malignancy through mechanisms that remain incompletely understood. While much attention has been dedicated to identifying the route of 2HG synthesis, the metabolic fate of 2HG has not been studied in detail. Yet the metabolism of 2HG may have important mechanistic consequences influencing cell function and cancer pathogenesis, such as modulating redox potential or producing unknown products with unique modes of action.ResultsBy applying our isotope-based metabolomic platform, we unbiasedly and comprehensively screened for products of L- and D-2HG in HCT116 colorectal carcinoma cells harboring a mutation in IDH1. After incubating HCT116 cells in uniformly 13C-labeled 2HG for 24 h, we used liquid chromatography/mass spectrometry to track the labeled carbons in small molecules. Strikingly, we did not identify any products of 2HG metabolism from the thousands of metabolomic features that we screened. Consistent with these results, we did not detect any significant changes in the labeling patterns of tricarboxylic acid cycle metabolites from wild type or IDH1 mutant cells cultured in 13C-labeled glucose upon the addition of L, D, or racemic mixtures of 2HG. A more sensitive, targeted analysis revealed trace levels of isotopic enrichment (<1 %) in some central carbon metabolites from 13C-labeled 2HG. However, we found that cells do not deplete 2HG from the media at levels above our detection limit over a 48 h time period.ConclusionsTaken together, we conclude that 2HG carbon is not readily transformed in the HCT116 cell line. These data indicate that the phenotypic alterations induced by 2HG are not a result of its metabolic products.

Bactericidal Antibiotics Induce Toxic Metabolic Perturbations that Lead to Cellular Damage.

Understanding how antibiotics impact bacterial metabolism may provide insight into their mechanisms of action and could lead to enhanced therapeutic methodologies. Here, we profiled the metabolome of Escherichia coli after treatment with three different classes of bactericidal antibiotics (?-lactams, aminoglycosides, quinolones). These treatments induced a similar set of metabolic changes after 30 min that then diverged into more distinct profiles at later time points. The most striking changes corresponded to elevated concentrations of central carbon metabolites, active breakdown of the nucleotide pool, reduced lipid levels, and evidence of an elevated redox state. We examined potential end-target consequences of these metabolic perturbations and found that antibiotic-treated cells exhibited cytotoxic changes indicative of oxidative stress, including higher levels of protein carbonylation, malondialdehyde adducts, nucleotide oxidation, and double-strand DNA breaks. This work shows that bactericidal antibiotics induce a complex set of metabolic changes that are correlated with the buildup of toxic metabolic by-products.

E1 of α-ketoglutarate dehydrogenase defends Mycobacterium tuberculosis against glutamate anaplerosis and nitroxidative stress

Enzymes of central carbon metabolism (CCM) in Mycobacterium tuberculosis (Mtb) make an important contribution to the pathogen's virulence. Evidence is emerging that some of these enzymes are not simply playing the metabolic roles for which they are annotated, but can protect the pathogen via additional functions. Here, we found that deficiency of 2-hydroxy-3-oxoadipate synthase (HOAS), the E1 component of the α-ketoglutarate (α-KG) dehydrogenase complex (KDHC), did not lead to general metabolic perturbation or growth impairment of Mtb, but only to the specific inability to cope with glutamate anaplerosis and nitroxidative stress. In the former role, HOAS acts to prevent accumulation of aldehydes, including growth-inhibitory succinate semialdehyde (SSA). In the latter role, HOAS can participate in an alternative four-component peroxidase system, HOAS/dihydrolipoyl acetyl transferase (DlaT)/alkylhydroperoxide reductase colorless subunit gene (ahpC)-neighboring subunit (AhpD)/AhpC, using α-KG as a previously undescribed source of electrons for reductase action. Thus, instead of a canonical role in CCM, the E1 component of Mtb's KDHC serves key roles in situational defense that contribute to its requirement for virulence in the host. We also show that pyruvate decarboxylase (AceE), the E1 component of pyruvate dehydrogenase (PDHC), can participate in AceE/DlaT/AhpD/AhpC, using pyruvate as a source of electrons for reductase action. Identification of these systems leads us to suggest that Mtb can recruit components of its CCM for reactive nitrogen defense using central carbon metabolites.

Intensive DNA Replication and Metabolism during the Lag Phase in Cyanobacteria.

Unlike bacteria such as Escherichia coli and Bacillus subtilis, several species of freshwater cyanobacteria are known to contain multiple chromosomal copies per cell, at all stages of their cell cycle. We have characterized the replication of multi-copy chromosomes in the cyanobacterium Synechococcus elongatus PCC 7942 (hereafter Synechococcus 7942). In Synechococcus 7942, the replication of multi-copy chromosome is asynchronous, not only among cells but also among multi-copy chromosomes. This suggests that DNA replication is not tightly coupled to cell division in Synechococcus 7942. To address this hypothesis, we analysed the relationship between DNA replication and cell doubling at various growth phases of Synechococcus 7942 cell culture. Three distinct growth phases were characterised in Synechococcus 7942 batch culture: lag phase, exponential phase, and arithmetic (linear) phase. The chromosomal copy number was significantly higher during the lag phase than during the exponential and linear phases. Likewise, DNA replication activity was higher in the lag phase cells than in the exponential and linear phase cells, and the lag phase cells were more sensitive to nalidixic acid, a DNA gyrase inhibitor, than cells in other growth phases. To elucidate physiological differences in Synechococcus 7942 during the lag phase, we analysed the metabolome at each growth phase. In addition, we assessed the accumulation of central carbon metabolites, amino acids, and DNA precursors at each phase. The results of these analyses suggest that Synechococcus 7942 cells prepare for cell division during the lag phase by initiating intensive chromosomal DNA replication and accumulating metabolites necessary for the subsequent cell division and elongation steps that occur during the exponential growth and linear phases.

Comprehensive bioimaging with fluorinated nanoparticles using breathable liquids.

Fluorocarbons are lipophobic and non-polar molecules that exhibit remarkable biocompatibility, with applications in liquid ventilation and synthetic blood. The unique properties of these compounds have also enabled mass spectrometry imaging of tissues where the fluorocarbons act as a Teflon-like coating for nanostructured surfaces to assist in desorption/ionization. Here we report fluorinated gold nanoparticles (f-AuNPs) designed to facilitate nanostructure imaging mass spectrometry. Irradiation of f-AuNPs results in the release of the fluorocarbon ligands providing a driving force for analyte desorption. The f-AuNPs allow for the mass spectrometry analysis of both lipophilic and polar (central carbon) metabolites. An important property of AuNPs is that they also act as contrast agents for X-ray microtomography and electron microscopy, a feature we have exploited by infusing f-AuNPs into tissue via fluorocarbon liquids to facilitate multimodal (molecular and anatomical) imaging.

Metabolomic and (13)C-metabolic flux analysis of a xylose-consuming Saccharomyces cerevisiae strain expressing xylose isomerase.

Over the past two decades, significant progress has been made in the engineering of xylose-consuming Saccharomyces cerevisiae strains for production of lignocellulosic biofuels. However, the ethanol productivities achieved on xylose are still significantly lower than those observed on glucose for reasons that are not well understood. We have undertaken an analysis of central carbon metabolite pool sizes and metabolic fluxes on glucose and on xylose under aerobic and anaerobic conditions in a strain capable of rapid xylose assimilation via xylose isomerase in order to investigate factors that may limit the rate of xylose fermentation. We find that during xylose utilization the flux through the non-oxidative Pentose Phosphate Pathway (PPP) is high but the flux through the oxidative PPP is low, highlighting an advantage of the strain employed in this study. Furthermore, xylose fails to elicit the full carbon catabolite repression response that is characteristic of glucose fermentation in S. cerevisiae. We present indirect evidence that the incomplete activation of the fermentation program on xylose results in a bottleneck in lower glycolysis, leading to inefficient re-oxidation of NADH produced in glycolysis.

Isotope labeling pattern study of central carbon metabolites using GC/MS

Determination of fluxes by (13)C tracer experiments depends on monitoring the (13)C labeling pattern of metabolites during isotope experiments. In metabolome-based (13)C metabolic flux analysis, liquid chromatography combined with mass spectrometry or tandem mass spectrometry (LC/MS or LC/MS/MS, respectively) has been mainly used as an analytical platform for isotope pattern studies of central carbon metabolites. However, gas chromatography with mass spectrometry (GC/MS) has several advantages over LC/MS, such as high sensitivity, low cost, ease of operation, and availability of mass spectra databases for comparison. In this study, analysis of isotope pattern for central carbon metabolites using GC/MS was demonstrated. First, a proper set of mass ions for central carbon metabolites was selected based on carbon backbone information and structural isomers of mass fragment ions. A total of 34 mass fragment ions was selected and used for the quantification of 25 central carbon metabolites. Then, to quantify isotope fractions, a natural mass isotopomer library for selected mass fragment ions was constructed and subtracted from isotopomer mass spectra data. The results revealed a surprisingly high abundance of partially labeled (13)C intermediates, such as 56.4% of fructose 6-phosphate and 47.6% of dihydroxyacetone phosphate at isotopic steady state, which were generated in the pentose phosphate pathway. Finally, dynamic changes of isotope fragments of central metabolites were monitored with a U-(13)C glucose stimulus response experiment in Kluyveromyces marxianus. With a comprehensive study of isotope patterns of central carbon metabolites using GC/MS, 25 central carbon metabolites and their isotopic fractions were successfully quantified. Dynamic and precise acquisition of isotope pattern can then be used in combination with proper kinetic models to calculate metabolic fluxes.

Targeted profiling of polar intracellular metabolites using ion-pair-high performance liquid chromatography and -ultra high performance liquid chromatography coupled to tandem mass spectrometry: Applications to serum, urine and tissue extracts

The effective analysis of polar ionic metabolites by LC–MS, such as those encountered in central carbon metabolism, represents a major problem for metabolic profiling that is not adequately addressed using strategies based on either reversed-phase or HILIC methods. Here we have compared analysis of central carbon metabolites on optimized methods using HILIC, porous graphitic carbon or ion pair chromatography (IPC) using tributyl ammonium as IP reagent. Of the 3 chromatographic approaches examined only IPC enabled us to obtain a robust analytical methodology. This system was used to profile more than a hundred endogenous metabolic intermediates in urine, serum and tissue samples. However, whilst we found IPC to be the best of the approaches examined considerable care was still needed to obtain robust data. Thus, in excess of 40 of representative biological samples were needed to “condition” a new analytical column and further 10 matrix injections were then required at the beginning of each analytical batch in order to obtain robust and reproducible chromatographic separations. An additional limitation that we have found was that, for a small number of phosphorylated and poly carboxylic acid metabolites, measurement was only possible if the analytes were present in relatively high concentrations. We also found that, whilst this methodology could be used for the analysis of both in vitro cell culture media, cell extracts, tissue, and biological fluids (blood, urine), for the best results columns should only be used to analyze a single matrix. However, despite the need for extensive column conditioning, and the manifold disadvantages resulting from the contamination of the separation system and mass spectrometer with the ion pair reagent, IPC-MS currently provides the best means of analyzing these polar, ionic and problematic metabolites.

Genetic Basis of Metabolome Variation in Yeast

Metabolism, the conversion of nutrients into usable energy and biochemical building blocks, is an essential feature of all cells. The genetic factors responsible for inter-individual metabolic variability remain poorly understood. To investigate genetic causes of metabolome variation, we measured the concentrations of 74 metabolites across 100 segregants from a Saccharomyces cerevisiae cross by liquid chromatography-tandem mass spectrometry. We found 52 quantitative trait loci for 34 metabolites. These included linkages due to overt changes in metabolic genes, e.g., linking pyrimidine intermediates to the deletion of ura3. They also included linkages not directly related to metabolic enzymes, such as those for five central carbon metabolites to ira2, a Ras/PKA pathway regulator, and for the metabolites, S-adenosyl-methionine and S-adenosyl-homocysteine to slt2, a MAP kinase involved in cell wall integrity. The variant of ira2 that elevates metabolite levels also increases glucose uptake and ethanol secretion. These results highlight specific examples of genetic variability, including in genes without prior known metabolic regulatory function, that impact yeast metabolism.

Chemical and technical challenges in the analysis of central carbon metabolites by liquid-chromatography mass spectrometry

This review deals with chemical and technical challenges in the analysis of small-molecule metabolites involved in central carbon and energy metabolism via liquid-chromatography mass-spectrometry (LC-MS). The covered analytes belong to the prominent pathways in biochemical carbon oxidation such as glycolysis or the tricarboxylic acid cycle and, for the most part, share unfavorable properties such as a high polarity, chemical instability or metal-affinity. The topic is introduced by selected examples on successful applications of metabolomics in the clinic. In the core part of the paper, the structural features of important analyte classes such as nucleotides, coenzyme A thioesters or carboxylic acids are linked to "problematic hotspots" along the analytical chain (sample preparation and-storage, separation and detection). We discuss these hotspots from a chemical point of view, covering issues such as analyte degradation or interactions with metals and other matrix components. Based on this understanding we propose solutions wherever available. A major notion derived from these considerations is that comprehensive carbon metabolomics inevitably requires multiple, complementary analytical approaches covering different chemical classes of metabolites.

Regulation of central carbon metabolism in Saccharomyces cerevisiae by metabolic inhibitors

Metabolic inhibitors were applied for chemical regulation of central carbon metabolism in Saccharomyces cerevisiae. S. cerevisiae was treated with 10 metabolic inhibitors with various modes of action, and their activities were evaluated using a growth inhibition assay. Among the 6 active inhibitors, the effects of pyrazole (alcohol dehydrogenase inhibitor) and TTA (2-thenoyltrifluoloacetone, succinate dehydrogenase inhibitor) were analyzed in detail. The flask-scale batch-fermentation test showed that ethanol yield was reduced to 0.10 ± 0.01 g g⁻¹ and glycerol yield increased to 0.26 ± 0.01 g g⁻¹ on treatment with pyrazole at 5.0 g L⁻¹, indicating that multiple isozymes of alcohol dehydrogenase were simultaneously inhibited. The multi-targeted metabolic profiling analysis revealed that, although the TTA and pyrazole treatments affected the profiles of all central carbon metabolites in distinct manners, the level of fructose-1,6-bisphosphate commonly increased in the TTA- and pyrazole-treated S. cerevisiae by an unknown mechanism. These results demonstrate that chemical regulation of the central carbon metabolism could be used as an alternative tool to control microbial cell factories for bioproduction, or as a chemical probe to investigate the metabolic systems of useful microorganisms.

Development of a solid phase extraction protocol coupled with liquid chromatography mass spectrometry to analyze central carbon metabolites in lake sediment microcosms

An ion exchange solid phase extraction (SPE) strategy is developed for application with liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS) for characterization of central carbon metabolites involved in methane assimilation and adjacent pathways in natural mixtures. For this purpose, short-time microcosm samples were obtained from lake sediment known to consume methane. Three SPE procedures were developed for the recovery of 51 targeted metabolites from five compound classes (amino acids, carboxylic acids, sugar phosphates, nucleotides and acyl-CoAs). The three SPE procedures employed were mixed mode (i) strong cation exchange, (ii) strong anion exchange and (iii) weak anion exchange. By spiking stable isotopic labeled standards, validation of the SPE procedures for the sediment extracts demonstrated that a 3 cm(3), 60 mg SPE sorbent bed provided effective loading capacity for targeted metabolites with an analytical variation of 16% RSD. We readily analyzed 32 of the targeted 51 metabolites using LC-MS/MS after sediment sample extraction, cleanup and pre-concentration. The remaining 19 targeted metabolites were either at, or below, the limit of detection. The current approach provides a good workflow for absolute quantification of intermediates in C(1)-carbon metabolism in natural microbial communities.

Collisional fragmentation of central carbon metabolites in LC‐MS/MS increases precision of 13C metabolic flux analysis

Experimental determination of fluxes by (13)C-tracers relies on detection of (13)C-patterns in metabolites or by-products. In the field of (13)C metabolic flux analysis, the most recent developments point toward recording labeling patterns by liquid chromatography (LC)-mass spectrometry (MS)/MS directly in intermediates in central carbon metabolism (CCM) to increase temporal resolution. Surprisingly, the flux studies published so far with LC-MS measurements were based on intact metabolic intermediates-thus neglected the potential benefits of using positional information to improve flux estimation. For the first time, we exploit collisional fragmentation to obtain more fine-grained (13)C-data on intermediates of CCM and investigate their impact in (13)C metabolic flux analysis. For the case study of Bacillus subtilis grown in mineral medium with (13)C-labeled glucose, we compare the flux estimates obtained by iterative isotopologue balancing of (13)C-data obtained either by LC-MS/MS for solely intact intermediates or LC-MS/MS for intact and fragmented intermediates of CCM. We show that with LC-MS/MS data, fragment information leads to more precise estimates of fluxes in pentose phosphate pathway, glycolysis, and to the tricarboxylic acid cycle. Additionally, we present an efficient analytical strategy to rapidly acquire large sets of (13)C-patterns by tandem MS, and an in-depth analysis of the collisional fragmentation of primary intermediates. In the future, this catalogue will enable comprehensive in silico calculability analyses to identify the most sensitive measurements and direct experimental design.

Targeted metabolomic analysis of Escherichia coli by desorption electrospray ionization and extractive electrospray ionization mass spectrometry

Desorption electrospray ionization (DESI) was utilized to monitor the presence of targeted central carbon metabolites within bacterial cell extracts and the quench supernatant of Escherichia coli. The targeted metabolites were identified through tandem mass spectrometry (MS/MS) product ion scans using collision-induced dissociation in the negative ion mode. Picogram detection limits were achieved for a majority of the metabolites during MS/MS analysis of standard metabolite solutions. In a [U-13C]glucose pulse experiment, where uniformly labeled glucose was fed to E. coli, the corresponding fragment ions from labeled metabolites in extracts were generally observed. There was evidence of matrix effects including moderate suppression by other metabolites within the spectra of the labeled and unlabeled extracts. To improve the specificity and sensitivity of detection, optimized in situ ambient chemical reactions using DESI and extractive electrospray ionization (EESI) were carried out for targeted compounds. This study provides the first indication of the potential to perform in situ targeted metabolomics of a bacterial sample via ambient ionization mass spectrometry.