Metabolic flux analysis of a glycerol-overproducing<i>Saccharomyces cerevisiae</i>strain based on GC-MS, LC-MS and NMR-derived<sup>13</sup>C-labelling data

Kleijn, Roelco J.; Geertman, Jan-Maarten A.; Nfor, Beckley K.; Ras, Cor; Schipper, Dick; Pronk, Jack T.; Heijnen, Joseph J.; Maris, Antonius J. A. van; Winden, Wouter A. van

doi:10.1111/j.1567-1364.2006.00180.x

Cited by 60 publications

(42 citation statements)

References 38 publications

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“…Glucose was primarily catabolized through glycolysis and then either secreted as acetate or combusted to CO 2 via the TCA cycle. Similar to previous studies (16,38,39), exchange fluxes for most reactions in the network cannot be accurately estimated because of the sparseness of the 13 C-labeling data from proteinogenic amino acids (supplemental Appendices A). The good correspondence between our and previously reported flux distributions adds to the credibility of deriving and using condition-specific network models.…”

Section: Intracellular Metabolite Concentrations and Reactionmentioning

confidence: 72%

Metabolic Fluxes during Strong Carbon Catabolite Repression by Malate in Bacillus subtilis

Kleijn

Buescher²,

Chat³

et al. 2010

Journal of Biological Chemistry

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103

110

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Commonly glucose is considered to be the only preferred substrate in Bacillus subtilis whose presence represses utilization of other alternative substrates. Because recent data indicate that malate might be an exception, we quantify here the carbon source utilization hierarchy. Based on physiology and transcriptional data during co-utilization experiments with eight carbon substrates, we demonstrate that malate is a second preferred carbon source for B. subtilis, which is rapidly co-utilized with glucose and strongly represses the uptake of alternative substrates. From the different hierarchy and degree of catabolite repression exerted by glucose and malate, we conclude that both substrates might act through different molecular mechanisms. To obtain a quantitative and functional network view of how malate is (co)metabolized, we developed a novel approach to metabolic flux analysis that avoids error-prone, intuitive, and ad hoc decisions on 13 C rearrangements. In particular, we developed a rigorous approach for deriving reaction reversibilities by combining in vivo intracellular metabolite concentrations with a thermodynamic feasibility analysis. The thus-obtained analytical model of metabolism was then used for network-wide isotopologue balancing to estimate the intracellular fluxes. These 13 C-flux data revealed an extraordinarily high malate influx that is primarily catabolized via the gluconeogenic reactions and toward overflow metabolism. Furthermore, a considerable NADPH-producing malic enzyme flux is required to supply the biosynthetically required NADPH in the presence of malate. Co-utilization of glucose and malate resulted in a synergistic decrease of the respiratory tricarboxylic acid cycle flux.Typically, microbes prefer a single carbon source whose presence represses utilization of alternative substrates. The classical example is the diauxic shift of Escherichia coli with subsequent growth on first glucose and then lactose (1). This widespread phenomenon, referred to as carbon catabolite repression, has been intensively studied in many chemoorganotrophic microbes, and in the vast majority of documented cases, the preferred carbon source is glucose (2, 3). Apart from repressing the co-utilization of alternative substrates, preferred carbon sources are normally associated with a high specific growth rate and substantial secretion of overflow metabolites such as ethanol, lactate, or acetate. For the Grampositive model bacterium Bacillus subtilis, glucose has long been recognized to be preferred, but recent data indicated that glucose might not be the only preferred carbon source (4, 5).According to the current model of the global carbon catabolite repression mechanism in B. subtilis, glucose-induced catabolite repression is mediated by the HPr kinase-catalyzed phosphorylation of HPr at its Ser-46 residue (2). This effect is propagated throughout the cell by the pleiotropic transcription factor CcpA (6), whose binding to the target sites for catabolite repression is controlled by the presence of HPr(Ser...

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Section: Intracellular Metabolite Concentrations and Reactionmentioning

confidence: 72%

Metabolic Fluxes during Strong Carbon Catabolite Repression by Malate in Bacillus subtilis

Kleijn

Buescher²,

Chat³

et al. 2010

Journal of Biological Chemistry

Self Cite

103

110

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“…It should be mentioned that the 16:1(n-9) FA corresponds to the derivative of oleic acid (18:1) formed after one cycle of ␤-oxidation. The ratio of C 16 to C 18 acyl chain FA was 0.46, and the ratio of unsaturated to saturated FA reached 6.5 after 24 h of culture.…”

Section: Lipid Accumulation In a Mutant With A Deletion Of G3pdh (⌬Gumentioning

confidence: 96%

“…Localized in LB (2), it is suspected of playing a crucial role in TAG metabolism (16,22,28). There are two different routes to G3P synthesis.…”

mentioning

confidence: 99%

Control of Lipid Accumulation in the Yeast Yarrowia lipolytica

Béopoulos

Mrózová

Thevenieau

et al. 2008

Appl Environ Microbiol

357

266

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A genomic comparison of Yarrowia lipolytica and Saccharomyces cerevisiae indicates that the metabolism of Y. lipolytica is oriented toward the glycerol pathway. To redirect carbon flux toward lipid synthesis, the GUT2 gene, which codes for the glycerol-3-phosphate dehydrogenase isomer, was deleted in Y. lipolytica in this study. This ⌬gut2 mutant strain demonstrated a threefold increase in lipid accumulation compared to the wild-type strain. However, mobilization of lipid reserves occurred after the exit from the exponential phase due to ␤-oxidation. Y. lipolytica contains six acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX6 genes, that catalyze the limiting step of peroxisomal ␤-oxidation. Additional deletion of the POX1 to POX6 genes in the ⌬gut2 strain led to a fourfold increase in lipid content. The lipid composition of all of the strains tested demonstrated high proportions of FFA. The size and number of the lipid bodies in these strains were shown to be dependent on the lipid composition and accumulation ratio.

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“…Overall, the cumulative regulation of glycerol metabolism observed in our study may reflect a flux reconfiguration in B. nigra to sustain mitochondrial activity, which led to the accumulation of glycerol as an intermediate metabolite in the pathway (Kleijn et al, 2007;Morandini, 2013;Gomes de Oliveira Dal'Molin et al, 2015).…”

Section: )mentioning

confidence: 98%

Central Metabolic Responses to Ozone and Herbivory Affect Photosynthesis and Stomatal Closure

et al. 2016

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Plants have evolved adaptive mechanisms that allow them to tolerate a continuous range of abiotic and biotic stressors. Tropospheric ozone (O 3 ), a global anthropogenic pollutant, directly affects living organisms and ecosystems, including plant-herbivore interactions. In this study, we investigate the stress responses of Brassica nigra (wild black mustard) exposed consecutively to O 3 and the specialist herbivore Pieris brassicae. Transcriptomics and metabolomics data were evaluated using multivariate, correlation, and network analyses for the O 3 and herbivory responses. O 3 stress symptoms resembled those of senescence and phosphate starvation, while a sequential shift from O 3 to herbivory induced characteristic plant defense responses, including a decrease in central metabolism, induction of the jasmonic acid/ethylene pathways, and emission of volatiles. Omics network and pathway analyses predicted a link between glycerol and central energy metabolism that influences the osmotic stress response and stomatal closure. Further physiological measurements confirmed that while O 3 stress inhibited photosynthesis and carbon assimilation, sequential herbivory counteracted the initial responses induced by O 3 , resulting in a phenotype similar to that observed after herbivory alone. This study clarifies the consequences of multiple stress interactions on a plant metabolic system and also illustrates how omics data can be integrated to generate new hypotheses in ecology and plant physiology.

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Metabolic flux analysis of a glycerol-overproducingSaccharomyces cerevisiaestrain based on GC-MS, LC-MS and NMR-derived¹³C-labelling data

Cited by 60 publications

References 38 publications

Metabolic Fluxes during Strong Carbon Catabolite Repression by Malate in Bacillus subtilis

Metabolic Fluxes during Strong Carbon Catabolite Repression by Malate in Bacillus subtilis

Control of Lipid Accumulation in the Yeast Yarrowia lipolytica

Central Metabolic Responses to Ozone and Herbivory Affect Photosynthesis and Stomatal Closure

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Metabolic flux analysis of a glycerol-overproducingSaccharomyces cerevisiaestrain based on GC-MS, LC-MS and NMR-derived13C-labelling data

Cited by 60 publications

References 38 publications

Metabolic Fluxes during Strong Carbon Catabolite Repression by Malate in Bacillus subtilis

Metabolic Fluxes during Strong Carbon Catabolite Repression by Malate in Bacillus subtilis

Control of Lipid Accumulation in the Yeast Yarrowia lipolytica

Central Metabolic Responses to Ozone and Herbivory Affect Photosynthesis and Stomatal Closure

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Metabolic flux analysis of a glycerol-overproducingSaccharomyces cerevisiaestrain based on GC-MS, LC-MS and NMR-derived¹³C-labelling data