Phenylacetyl Coenzyme A, Not Phenylacetic Acid, Attenuates CepIR-Regulated Virulence in Burkholderia cenocepacia.

During phenylalanine catabolism, phenylacetic acid (PAA) is converted to phenylacetyl coenzyme A (PAA-CoA) by a ligase, PaaK, and then PAA-CoA is epoxidized by a multicomponent monooxygenase, PaaABCDE, before further degradation through the tricarboxylic acid (TCA) cycle. In the opportunistic pathogen Burkholderia cenocepacia, loss of paaABCDE attenuates virulence factor expression, which is under the control of the LuxIR-like quorum sensing (QS) system, CepIR. To further investigate the link between CepIR-regulated virulence and PAA catabolism, we created knockout mutants of the first step of the pathway (PAA-CoA synthesis by PaaK) and characterized them in comparison to a paaABCDE mutant using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and virulence assays. We found that while loss of PaaABCDE decreased virulence, deletion of the paaK genes resulted in a more virulent phenotype than that of the wild-type strain. Deletion of either paaK or paaABCDE led to higher levels of released PAA but no differences in levels of internal accumulation compared to the wild-type level. While we found no evidence of direct cepIR downregulation by PAA-CoA or PAA, a low-virulence cepR mutant reverted to a virulent phenotype upon removal of the paaK genes. On the other hand, removal of paaABCDE in the cepR mutant did not impact its attenuated phenotype. Together, our results suggest an indirect role for PAA-CoA in suppressing B. cenocepacia CepIR-activated virulence.IMPORTANCE The opportunistic pathogen Burkholderia cenocepacia uses a chemical signal process called quorum sensing (QS) to produce virulence factors. In B. cenocepacia, QS relies on the presence of the transcriptional regulator CepR which, upon binding QS signal molecules, activates virulence. In this work, we found that even in the absence of CepR, B. cenocepacia can elicit a pathogenic response if phenylacetyl-CoA, an intermediate of the phenylacetic acid degradation pathway, is not produced. Instead, accumulation of phenylacetyl-CoA appears to attenuate pathogenicity. Therefore, we have discovered that it is possible to trigger virulence in the absence of CepR, challenging the classical view of activation of virulence by this QS mechanism. Our work provides new insight into the relationship between metabolism and virulence in opportunistic bacteria. We propose that in the event that QS signaling molecules cannot accumulate to trigger a pathogenic response, a metabolic signal can still activate virulence in B. cenocepacia.

Stoichiometry and kinetics of single and mixed substrate uptake in Aspergillus niger.

In its natural environment, the filamentous fungus Aspergillus niger grows on decaying fruits and plant material, thereby enzymatically degrading the lignocellulosic constituents (lignin, cellulose, hemicellulose, and pectin) into a mixture of mono- and oligosaccharides. To investigate the kinetics and stoichiometry of growth of this fungus on lignocellulosic sugars, we carried out batch cultivations on six representative monosaccharides (glucose, xylose, mannose, rhamnose, arabinose, and galacturonic acid) and a mixture of these. Growth on these substrates was characterized in terms of biomass yields, oxygen/biomass ratios, and specific conversion rates. Interestingly, in combination, some of the carbon sources were consumed simultaneously and some sequentially. With a previously developed protocol, a sequential chemostat cultivation experiment was performed on a feed mixture of the six substrates. We found that the uptake of glucose, xylose, and mannose could be described with a Michaelis-Menten-type kinetics; however, these carbon sources seem to be competing for the same transport systems, while the uptake of arabinose, galacturonic acid, and rhamnose appeared to be repressed by the presence of other substrates.

Parent-of-origin tumourigenesis is mediated by an essential imprinted modifier in SDHD-linked paragangliomas: SLC22A18 and CDKN1C are candidate tumour modifiers.

Mutations in SDHD and SDHAF2 (both located on chromosome 11) give rise to hereditary paraganglioma almost exclusively after paternal transmission of the mutation, and tumours often show loss of the entire maternal copy of chromosome 11. The 'Hensen' model postulates that a tumour modifier gene located on chromosome 11p15, a region known to harbour a cluster of imprinted genes, is essential to tumour formation. We observed decreased protein expression of the 11p15 candidate genes CDKN1C, SLC22A18 and ZNF215 evaluated in 60 SDHD-mutated tumours compared to normal carotid body tissue and non-SDH mutant tumours.We then created stable knockdown in vitro models, reasoning that the simultaneous knockdown of SDHD and a maternally expressed 11p15 modifier gene would enhance paraganglioma-related cellular characteristics compared to SDHD knockdown alone. Knockdown of SDHD in SNB19 and SHSY5Y cells resulted in the accumulation of succinate, the stabilization of HIF1 protein and a reduction in cell proliferation.Compared to single knockdown of SDHD, knockdown of SDHD together with SLC22A18 or with CDKN1C led to small but significant increases in cell proliferation and resistance to apoptosis, and to a gene expression profile closely related to the known transcriptional profile of SDH-deficient tumours. Of the 60 SDHD tumours investigated, four tumours showing retention of chromosome 11 showed SLC22A18 and CDKN1C expression levels comparable to levels in tumours showing loss of chromosome 11, suggesting loss of protein expression despite chromosomal retention.Our data strongly suggest that SLC22A18 and/or CDKN1C are tumour modifier genes involved in the tumourigenesis of SDHD-linked paraganglioma.

Inactivation of SDH and FH cause loss of 5hmC and increased H3K9me3 in paraganglioma/pheochromocytoma and smooth muscle tumors.

Succinate dehydrogenase (SDH) and fumarate hydratase (FH) are tricarboxylic acid (TCA) cycle enzymes and tumor suppressors. Loss-of-function mutations give rise to hereditary paragangliomas/pheochromocytomas and hereditary leiomyomatosis and renal cell carcinoma. Inactivation of SDH and FH results in an abnormal accumulation of their substrates succinate and fumarate, leading to inhibition of numerous α-ketoglutarate dependent dioxygenases, including histone demethylases and the ten-eleven-translocation (TET) family of 5-methylcytosine (5 mC) hydroxylases. To evaluate the distribution of DNA and histone methylation, we used immunohistochemistry to analyze the expression of 5 mC, 5-hydroxymethylcytosine (5 hmC), TET1, H3K4me3, H3K9me3, and H3K27me3 on tissue microarrays containing paragangliomas/pheochromocytomas (n = 134) and hereditary and sporadic smooth muscle tumors (n = 56) in comparison to their normal counterparts. Our results demonstrate distinct loss of 5 hmC in tumor cells in SDH- and FH-deficient tumors. Loss of 5 hmC in SDH-deficient tumors was associated with nuclear exclusion of TET1, a known regulator of 5 hmC levels. Moreover, increased methylation of H3K9me3 occurred predominantly in the chief cell component of SDH mutant tumors, while no changes were seen in H3K4me3 and H3K27me3, data supported by in vitro knockdown of SDH genes. We also show for the first time that FH-deficient smooth muscle tumors exhibit increased H3K9me3 methylation compared to wildtype tumors. Our findings reveal broadly similar patterns of epigenetic deregulation in both FH- and SDH-deficient tumors, suggesting that defects in genes of the TCA cycle result in common mechanisms of inhibition of histone and DNA demethylases.

Determination of the Cytosolic NADPH/NADP Ratio in Saccharomyces cerevisiae using Shikimate Dehydrogenase as Sensor Reaction.

Eukaryotic metabolism is organised in complex networks of enzyme catalysed reactions which are distributed over different organelles. To quantify the compartmentalised reactions, quantitative measurements of relevant physiological variables in different compartments are needed, especially of cofactors. NADP(H) are critical components in cellular redox metabolism. Currently, available metabolite measurement methods allow whole cell measurements. Here a metabolite sensor based on a fast equilibrium reaction is introduced to monitor the cytosolic NADPH/NADP ratio in Saccharomyces cerevisiae: NADP + shikimate ⇄ NADPH + H(+) + dehydroshikimate. The cytosolic NADPH/NADP ratio was determined by measuring the shikimate and dehydroshikimate concentrations (by GC-MS/MS). The cytosolic NADPH/NADP ratio was determined under batch and chemostat (aerobic, glucose-limited, D = 0.1 h(-1)) conditions, to be 22.0 ± 2.6 and 15.6 ± 0.6, respectively. These ratios were much higher than the whole cell NADPH/NADP ratio (1.05 ± 0.08). In response to a glucose pulse, the cytosolic NADPH/NADP ratio first increased very rapidly and restored the steady state ratio after 3 minutes. In contrast to this dynamic observation, the whole cell NADPH/NADP ratio remained nearly constant. The novel cytosol NADPH/NADP measurements provide new insights into the thermodynamic driving forces for NADP(H)-dependent reactions, like amino acid synthesis, product pathways like fatty acid production or the mevalonate pathway.

A fast sensor for in vivo quantification of cytosolic phosphate in Saccharomyces cerevisiae.

Eukaryotic metabolism consists of a complex network of enzymatic reactions and transport processes which are distributed over different subcellular compartments. Currently, available metabolite measurement protocols allow to measure metabolite whole cell amounts which hinder progress to describe the in vivo dynamics in different compartments, which are driven by compartment specific concentrations. Phosphate (Pi) is an essential component for: (1) the metabolic balance of upper and lower glycolytic flux; (2) Together with ATP and ADP determines the phosphorylation energy. Especially, the cytosolic Pi has a critical role in disregulation of glycolysis in tps1 knockout. Here we developed a method that enables us to monitor the cytosolic Pi concentration in S. cerevisiae using an equilibrium sensor reaction: maltose + Pi < = > glucose + glucose-1-phosphate. The required enzyme, maltose phosphorylase from L. sanfranciscensis was overexpressed in S. cerevisiae. With this reaction in place, the cytosolic Pi concentration was obtained from intracellular glucose, G1P and maltose concentrations. The cytosolic Pi concentration was determined in batch and chemostat (D = 0.1 h(-1) ) conditions, which was 17.88 µmol/gDW and 25.02 µmol/gDW, respectively under Pi-excess conditions. Under Pi-limited steady state (D = 0.1 h(-1) ) conditions, the cytosolic Pi concentration dropped to only 17.7% of the cytosolic Pi in Pi-excess condition (4.42 µmol/gDW vs. 25.02 µmol/gDW). In response to a Pi pulse, the cytosolic Pi increased very rapidly, together with the concentration of sugar phosphates. Main sources of the rapid Pi increase are vacuolar Pi (and not the polyPi), as well as Pi uptake from the extracellular space. The temporal increase of cytosolic Pi increases the driving force of GAPDH reaction of the lower glycolytic reactions. The novel cytosol specific Pi concentration measurements provide new insight into the thermodynamic driving force for ATP hydrolysis, GAPDH reaction, and Pi transport over the plasma and vacuolar membranes.

Quantitative metabolomics using ID-MS.

Quantitative intracellular metabolite measurements are essential for systems biology and modeling of cellular metabolism. The MS-based quantification is error prone because (1) several sampling processing steps have to be performed, (2) the sample contains a complex mixture of partly compounds with the same mass and similar retention time, and (3) especially salts influence the ionization efficiency. Therefore internal standards are required, best for each measured compound. The use of labeled biomass, (13)C extract, is a valuable tool, reducing the standard deviations of intracellular concentration measurements significantly (especially regarding technical reproducibility). Using different platforms, i.e., LC-MS and GC-MS, a large number of different metabolites can be quantified (currently about 110).

Quantitative analysis of intracellular coenzymes in Saccharomyces cerevisiae using ion pair reversed phase ultra high performance liquid chromatography tandem mass spectrometry.

A fast, sensitive and specific analytical method, based on ion pair reversed phase ultrahigh performance liquid chromatography tandem mass spectrometry, IP-RP-UHPLC-MS/MS, was developed for quantitative determination of intracellular coenzyme A (CoA), acetyl CoA, succinyl CoA, phenylacetyl CoA, flavin mononucleotide, (FMN), flavin adenine dinucleotide, (FAD), NAD, NADH, NADP, NADPH. Dibutylammonium acetate (DBAA) was used as volatile ion pair reagent in the mobile phase. Addition of DBAA to the sample solutions resulted in an enhanced sensitivity for the phosphorylated coenzymes. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP·HCl), was added to keep CoA in the reduced form. Isotope dilution mass spectrometry (IDMS) was applied for quantitative measurements for which culture derived global U-(13)C-labeled cell extract was used as internal standard. The analytical method was validated by determining the limit of detection, the limit of quantification, repeatability and intermediate precision. The method was successfully applied for quantification of coenzymes in the cell extracts of Saccharomyces cerevisiae.

An in vivo data-driven framework for classification and quantification of enzyme kinetics and determination of apparent thermodynamic data.

Kinetic modeling of metabolism holds great potential for metabolic engineering but is hindered by the gap between model complexity and availability of in vivo data. There is also growing interest in network-wide thermodynamic analyses, which are currently limited by the scarcity and unreliability of thermodynamic reference data. Here we propose an in vivo data-driven approach to simultaneously address both problems. We then demonstrate the procedure in Saccharomyces cerevisiae, using chemostats to generate a large flux/metabolite dataset, under 32 conditions spanning a large range of fluxes. Reactions were classified as pseudo-, near- or far-from-equilibrium, allowing the complexity of mathematical description to be tailored to the kinetic behavior displayed in vivo. For 3/4 of the reactions we derived fully in vivo-parameterized kinetic descriptions which can be readily incorporated into models. For near-equilibrium reactions this involved a new simplified format, dubbed "Q-linear kinetics". We also demonstrate, for the first time, systematic estimation of apparent in vivo K(eq) values. Remarkably, comparison with E. coli data suggests they constitute a suitable in vivo interspecies thermodynamic reference.

Analysis of glycolytic intermediates with ion chromatography- and gas chromatography-mass spectrometry.

In this chapter, we describe a method for the quantitative analysis of glycolytic intermediates using ion chromatography-mass spectrometry (IC-MS) and gas chromatography (GC)-MS as complementary methods. With IC-MS-MS, pyruvate, glucose-6-phosphate, fructuse-6-phosphate, fructose-1,6-bisphosphate, phosphoenolpyruvate, and the sum of 2-phosphoglyceraldehyde + 3-phosphoglyceraldehyde can be quantified. With GC-MS using selected ion monitoring, glyceraldehyde-3-phosphate, dihydroxyacetonephosphate, 2-phosphoglyceraldehyde, and 3-phosphoglyceraldehyde can be analyzed. The derivatization for GC-MS is performed in two steps. In the first step, the keto and the aldehyde groups are oximated. In the next step, a silylation with N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) is performed, giving TMS-MOX derivatives of the glycolytic intermediates. The derivatives are separated on a GC column and detected with MS in SIM mode.

Quantitative evaluation of intracellular metabolite extraction techniques for yeast metabolomics.

Accurate determination of intracellular metabolite levels requires well-validated procedures for sampling and sample treatment. Several methods exist for metabolite extraction, but the literature is contradictory regarding the adequacy and performance of each technique. Using a strictly quantitative approach, we have re-evaluated five methods (hot water, HW; boiling ethanol, BE; chloroform-methanol, CM; freezing-thawing in methanol, FTM; acidic acetonitrile-methanol, AANM) for the extraction of 44 intracellular metabolites (phosphorylated intermediates, amino acids, organic acids, nucleotides) from S. cerevisiae cells. Two culture modes were investigated (batch and chemostat) to check for growth condition dependency, and three targeted platforms were employed (two LC-MS and one GC/MS) to exclude analytical bias. Additionally, for the determination of metabolite recoveries, we applied a novel approach based on addition of (13)C-labeled internal standards at different stages of sample processing. We found that the choice of extraction method can drastically affect measured metabolite levels, to an extent that for some metabolites even the direction of changes between growth conditions can be inverted. The best performances, in terms of efficacy and metabolite recoveries, were achieved with BE and CM, which yielded nearly identical levels for the metabolites analyzed. According to our results, AANM performs poorly in yeast and FTM cannot be considered adequate as an extraction method, as it does not ensure inactivation of enzymatic activity.

Simultaneous quantification of free nucleotides in complex biological samples using ion pair reversed phase liquid chromatography isotope dilution tandem mass spectrometry.

A new sensitive and accurate analytical method has been developed for quantification of intracellular nucleotides in complex biological samples from cultured cells of different microorganisms such as Saccharomyces cerevisiae, Escherichia coli, and Penicillium chrysogenum. This method is based on ion pair reversed phase liquid chromatography electrospray ionization isotope dilution tandem mass spectrometry (IP-LC-ESI-ID-MS/MS. A good separation and low detection limits were observed for these compounds using dibutylamine as volatile ion pair reagent in the mobile phase of the LC. Uniformly (13)C-labeled isotopes of nucleotides were used as internal standards for both extraction and quantification of intracellular nucleotides. The method was validated by determining the linearity, sensitivity, and repeatability.

Development and application of a differential method for reliable metabolome analysis in Escherichia coli.

Quantitative metabolomics of microbial cultures requires well-designed sampling and quenching procedures. We successfully developed and applied a differential method to obtain a reliable set of metabolome data for Escherichia coli K12 MG1655 grown in steady-state, aerobic, glucose-limited chemostat cultures. From a rigorous analysis of the commonly applied quenching procedure based on cold aqueous methanol, it was concluded that it was not applicable because of release of a major part of the metabolites from the cells. No positive effect of buffering or increasing the ionic strength of the quenching solution was observed. Application of a differential method in principle requires metabolite measurements in total broth and filtrate for each measurement. Different methods for sampling of culture filtrate were examined, and it was found that direct filtration without cooling of the sample was the most appropriate. Analysis of culture filtrates revealed that most of the central metabolites and amino acids were present in significant amounts outside the cells. Because the turnover time of the pools of extracellular metabolites is much larger than that of the intracellular pools, the differential method should also be applicable to short-term pulse response experiments without requiring measurement of metabolites in the supernatant during the dynamic period.

Isotopic non-stationary 13C gluconate tracer method for accurate determination of the pentose phosphate pathway split-ratio in Penicillium chrysogenum.

Current (13)C labeling experiments for metabolic flux analysis (MFA) are mostly limited by either the requirement of isotopic steady state or the extremely high computational effort due to the size and complexity of large metabolic networks. The presented novel approach circumvents these limitations by applying the isotopic non-stationary approach to a local metabolic network. The procedure is demonstrated in a study of the pentose phosphate pathway (PPP) split-ratio of Penicillium chrysogenum in a penicillin-G producing chemostat-culture grown aerobically at a dilution rate of 0.06h(-1) on glucose, using a tracer amount of uniformly labeled [U-(13)C(6)] gluconate. The rate of labeling inflow can be controlled by using different cell densities and/or different fractions of the labeled tracer in the feed. Due to the simplicity of the local metabolic network structure around the 6-phosphogluconate (6pg) node, only three metabolites need to be measured for the pool size and isotopomer distribution. Furthermore, the mathematical modeling of isotopomer distributions for the flux estimation has been reduced from large scale differential equations to algebraic equations. Under the studied cultivation condition, the estimated split-ratio (41.2+/-0.6%) using the novel approach, shows statistically no difference with the split-ratio obtained from the originally proposed isotopic stationary gluconate tracing method.

A metabolome study of the steady-state relation between central metabolism, amino acid biosynthesis and penicillin production in Penicillium chrysogenum.

The relation between central metabolism and the penicillin biosynthesis pathway in Penicillium chrysogenum was studied by manipulating the steady-state flux in both pathways. A high producing industrial strain was cultivated at a growth rate mu=0.05 h(-1) in glucose-limited chemostat cultures, both under penicillin-G producing and non-producing conditions. Non-producing conditions were accomplished in two ways: (1) by cultivation without addition of the side chain precursor phenylacetic acid and (2) by cultivation of a mutant strain which lost all copies of the gene cluster coding for the penicillin biosynthesis pathway. Manipulation of the fluxes through central metabolism was obtained by cultivation on either glucose or ethanol as sole carbon source. A positive relation was observed between metabolite concentrations and carbon flux in central metabolism. Furthermore, in many cases a positive relation was found between the concentrations of free amino acids and their direct precursors in central metabolism. This corresponds with control of the biosynthesis of these amino acids via feed back inhibition by the end product. With respect to the penicillin production pathway, the flux seems not influenced by two of the three precursor amino acids, namely alphaAAA and valine but is only influenced by cysteine, which requires a large NADPH supply, and the ATP level. An interesting observation is that the absence of penicillin production seems to stimulate storage metabolism (trehalose metabolism). This leads to the final conclusion that the penicillin production flux appears to be mostly influenced by the availability of energy and redox cofactors, where ATP is supposed to exert its influence at ACV-synthetase and NADPH at the cysteine level.

Cytosolic NADPH metabolism in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum.

This study addresses the relation between NADPH supply and penicillin synthesis, by comparing the flux through the oxidative branch of the pentose phosphate pathway (PPP; the main source of cytosolic NADPH) in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum. The fluxes through the oxidative part of the PPP were determined using the recently introduced gluconate-tracer method. Significantly higher oxidative PPP fluxes were observed in penicillin-G producing chemostat cultures, indicating that penicillin production puts a major burden on the supply of cytosolic NADPH. To our knowledge this is the first time direct experimental proof is presented for the causal relationship between penicillin production and NADPH supply. Additional insight in the metabolism of P. chrysogenum was obtained by comparing the PPP fluxes from the gluconate-tracer experiment to oxidative PPP fluxes derived via metabolic flux analysis, using different assumptions for the stoichiometry of NADPH consumption and production.

Metabolic flux analysis of a glycerol-overproducing Saccharomyces cerevisiae strain based on GC-MS, LC-MS and NMR-derived C-labelling data.

This study focuses on unravelling the carbon and redox metabolism of a previously developed glycerol-overproducing Saccharomyces cerevisiae strain with deletions in the structural genes encoding triosephosphate isomerase (TPI1), the external mitochondrial NADH dehydrogenases (NDE1 and NDE2) and the respiratory chain-linked glycerol-3-phosphate dehydrogenase (GUT2). Two methods were used for analysis of metabolic fluxes: metabolite balancing and (13)C-labelling-based metabolic flux analysis. The isotopic enrichment of intracellular primary metabolites was measured both directly (liquid chromatography-MS) and indirectly through proteinogenic amino acids (nuclear magnetic resonance and gas chromatography-MS). Because flux sensitivity around several important metabolic nodes proved to be dependent on the applied technique, the combination of the three (13)C quantification techniques generated the most accurate overall flux pattern. When combined, the measured conversion rates and (13)C-labelling data provided evidence that a combination of assimilatory metabolism and pentose phosphate pathway activity diverted some of the carbon away from glycerol formation. Metabolite balancing indicated that this results in excess cytosolic NADH, suggesting the presence of a cytosolic NADH sink in addition to those that were deleted. The exchange flux of four-carbon dicarboxylic acids across the mitochondrial membrane, as measured by the (13)C-labelling data, supports a possible role of a malate/aspartate or malate/oxaloacetate redox shuttle in the transfer of these redox equivalents from the cytosol to the mitochondrial matrix.

13C-labeled gluconate tracing as a direct and accurate method for determining the pentose phosphate pathway split ratio in Penicillium chrysogenum.

In this study we developed a new method for accurately determining the pentose phosphate pathway (PPP) split ratio, an important metabolic parameter in the primary metabolism of a cell. This method is based on simultaneous feeding of unlabeled glucose and trace amounts of [U-13C]gluconate, followed by measurement of the mass isotopomers of the intracellular metabolites surrounding the 6-phosphogluconate node. The gluconate tracer method was used with a penicillin G-producing chemostat culture of the filamentous fungus Penicillium chrysogenum. For comparison, a 13C-labeling-based metabolic flux analysis (MFA) was performed for glycolysis and the PPP of P. chrysogenum. For the first time mass isotopomer measurements of 13C-labeled primary metabolites are reported for P. chrysogenum and used for a 13C-based MFA. Estimation of the PPP split ratio of P. chrysogenum at a growth rate of 0.02 h(-1) yielded comparable values for the gluconate tracer method and the 13C-based MFA method, 51.8% and 51.1%, respectively. A sensitivity analysis of the estimated PPP split ratios showed that the 95% confidence interval was almost threefold smaller for the gluconate tracer method than for the 13C-based MFA method (40.0 to 63.5% and 46.0 to 56.5%, respectively). From these results we concluded that the gluconate tracer method permits accurate determination of the PPP split ratio but provides no information about the remaining cellular metabolism, while the 13C-based MFA method permits estimation of multiple fluxes but provides a less accurate estimate of the PPP split ratio.

Short-term metabolome dynamics and carbon, electron, and ATP balances in chemostat-grown Saccharomyces cerevisiae CEN.PK 113-7D following a glucose pulse.

The in vivo kinetics in Saccharomyces cerevisiae CEN.PK 113-7D was evaluated during a 300-second transient period after applying a glucose pulse to an aerobic, carbon-limited chemostat culture. We quantified the responses of extracellular metabolites, intracellular intermediates in primary metabolism, intracellular free amino acids, and in vivo rates of O(2) uptake and CO(2) evolution. With these measurements, dynamic carbon, electron, and ATP balances were set up to identify major carbon, electron, and energy sinks during the postpulse period. There were three distinct metabolic phases during this time. In phase I (0 to 50 seconds after the pulse), the carbon/electron balances closed up to 85%. The accumulation of glycolytic and storage compounds accounted for 60% of the consumed glucose, caused an energy depletion, and may have led to a temporary decrease in the anabolic flux. In phase II (50 to 150 seconds), the fermentative metabolism gradually became the most important carbon/electron sink. In phase III (150 to 300 seconds), 29% of the carbon uptake was not identified in the measurements, and the ATP balance had a large surplus. These results indicate an increase in the anabolic flux, which is consistent with macroscopic balances of extracellular fluxes and the observed increase in CO(2) evolution associated with nonfermentative metabolism. The identified metabolic processes involving major carbon, electron, and energy sinks must be taken into account in in vivo kinetic models based on short-term dynamic metabolome responses.

In vivo kinetics of primary metabolism in Saccharomyces cerevisiae studied through prolonged chemostat cultivation.

In this study, prolonged chemostat cultivation is applied to investigate in vivo enzyme kinetics of Saccharomyces cerevisiae. S. cerevisiae was grown in carbon-limited aerobic chemostats for 70-95 generations, during which multiple steady states were observed, characterized by constant intracellular fluxes but significant changes in intracellular metabolite concentrations and enzyme capacities. We provide evidence for two relevant kinetic mechanisms for sustaining constant fluxes: in vivo near-equilibrium of reversible reactions and tight regulation of irreversible reactions by coordinated changes of metabolic effectors. Using linear-logarithmic kinetics, we illustrate that these multiple steady-state measurements provide linear constraints between elasticity parameters instead of their absolute values. Upon perturbation by a glucose pulse, glucose uptake and ethanol excretion in prolonged cultures were remarkably lower, compared to a reference culture perturbed at 10 generations. Metabolome measurements during the transient indicate that the differences might be due to a reduced ATP regeneration capacity in prolonged cultures.