Cor Ras - Academia.edu (original) (raw)

Papers by Cor Ras

Applied Microbiology and Biotechnology, 1999

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures was examined, ... more The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures was examined, using on-line o-gas analyses to measure the oxygen and carbon dioxide consumption rates continuously. A cell suspension from continuous cultures at steady state was used as the inoculum. It was observed that a dynamic phase occurred in the initial phase of the experiment. In this phase the bacterial ferrous iron oxidation and growth were uncoupled. After about 16 h the bacteria were adapted and achieved a pseudo-steady state, in which the speci®c growth rate and oxygen consumption rate were coupled and their relationship was described by the Pirt equation. In pseudo-steady state, the growth and oxidation kinetics were accurately described by the rate equation for competitive product inhibition. Bacterial substrate consumption is regarded as the primary process, which is described by the equation for competitive product inhibition. Subsequently the kinetic equation for the speci®c growth rate, l, is derived by applying the Pirt equation for bacterial substrate consumption and growth. The maximum speci®c growth rate, l max , measured in the batch culture agrees with the dilution rate at which washout occurs in continuous cultures. The maximum oxygen consumption rate, q O 2 Ymax , of the cell suspension in the batch culture was determined by respiration measurements in a biological oxygen monitor at excess ferrous iron, and showed changes of up to 20% during the course of the experiment. The kinetic constants determined in the batch culture slightly dier from those in continuous cultures, such that, at equal ferric to ferrous iron concentration ratios, biomass-speci®c rates are up to 1.3 times higher in continuous cultures.

Biotechnol Bioeng, 2005

In the present work LC-MS/MS was applied to measure the concentrations of intermediates of glycol... more In the present work LC-MS/MS was applied to measure the concentrations of intermediates of glycolysis and TCA cycle during autonomous, cell-cycle synchronized oscillations in aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae. This study complements previously reported oscillations in carbon dioxide production rate, intracellular concentrations of trehalose and various free amino acids, and extracellular acetate and pyruvate in the same culture. Of the glycolytic intermediates, fructose 1,6-bisphosphate, 2-and 3-phosphoglycerate, and phosphoenolpyruvate show the most pronounced oscillatory behavior, the latter three compounds oscillating out of phase with the former. This agrees with previously observed metabolic control by phosphofructokinase and pyruvate kinase. Although individually not clearly oscillating, several intermediates of the TCA cycle, i.e., a-ketoglutarate, succinate, fumarate, and malate, exhibited increasing concentration during the cell cycle phase with high carbon flux through glycolysis and TCA cycle. The average mass action ratios of h-phosphoglucomutase and fumarase agreed well with previously determined in vitro equilibrium constants. Minor differences resulted for phosphoglucose isomerase and enolase. Together with the observed close correlation of the pool sizes of the involved metabolites, this might indicate that, in vivo, these reactions are operating close to equilibrium, whereby care must be taken due to possible differences between in vivo and in vitro conditions. Combining the data with previously determined intracellular amino acid levels from the same culture, a few clear correlations between catabolism and anabolism could be identified: phosphoglycerate/serine and a-ketoglutarate/lysine exhibited correlated oscillatory behavior, albeit with different phase shifts. Oscillations in intracellular amino acids might therefore be, at least partly, following oscillations of their anabolic precursors. B

Oncotarget, Jan 12, 2015

Succinate dehydrogenase (SDH) and fumarate hydratase (FH) are tricarboxylic acid (TCA) cycle enzy... more 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 (5mC) hydroxylases. To evaluate the distribution of DNA and histone methylation, we used immunohistochemistry to analyze the expression of 5mC, 5-hydroxymethylcytosine (5hmC), 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 5hmC in tumor cells in ...

Scientific Reports, 2015

Eukaryotic metabolism is organised in complex networks of enzyme catalysed reactions which are di... more 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.

Biotechnology and Bioengineering, 2015

Eukaryotic metabolism consists of a complex network of enzymatic reactions and transport processe... more 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 þ Pi < ¼ > glucose þ 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 ¼ 0.1 h À 1 ) conditions, which was 17.88 mmol/gDW and 25.02 mmol/gDW, respectively under Pi-excess conditions. Under Pi-limited steady state (D ¼ 0.1 h À 1 ) conditions, the cytosolic Pi concentration dropped to only 17.7% of the cytosolic Pi in Pi-excess condition (4.42 mmol/gDW vs. 25.02 mmol/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. specific substrate uptake rate; qCO 2 , the specific carbon dioxide production rate; qO 2 , the oxygen uptake rate; qGly, the specific glycerol production rate; RQ, respiratory quotient. Correspondence to: J. Zhang and S. A. Wahl fax: þ31 (0)15 2782355

Methods in Molecular Biology, 2014

Quantitative intracellular metabolite measurements are essential for systems biology and modeling... more 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).

New Biotechnology, 2009

Dynamic mathematical models allow a better understanding of the metabolism of microorganisms and ... more Dynamic mathematical models allow a better understanding of the metabolism of microorganisms and the optimization of fermentation conditions for a desired product compared to stoichiometric models. Such dynamic models contain mathematical relations to express reaction rates as a function of enzyme levels, enzyme kinetic properties and intra/extracellular metabolite concentrations. To estimate the parameters of these rate equations experimental information on fluxes, enzyme levels and metabolite concentrations under a number of different conditions are required. Measurement of enzyme levels can be avoided if dynamic perturbation experiments are carried out in such a short time interval that the enzyme levels can be assumed not to change. Such short time experiments were carried out with Escherichia coli. For these purposes two replicate aerobic glucose-limited chemostat cultures of E. coli K12 MG1655 fnr (at D = 0.1 h −1 ) were subjected to a moderate glucose pulse, whereby the extracellular glucose concentration was instantaneously increased from the steady state level of 14 mg/L to 0.5 g/L. Subsequently, rapid sampling and immediate quenching of metabolic activity was applied to capture the fast dynamics of the intracellular and extracellular metabolites directly after the pulse. Intracellular metabolites were measured with a newly developed differential method which avoids metabolite leakage during quenching and subsequent sample treatment. During the transient not only the metabolome response but also the offgas response was monitored. The obtained stimulus response data were shown to be highly reproducible for the two pulse experiments carried out in replicate chemostats.

Molecular Systems Biology, 2006

Within the first 5 min after a sudden relief from glucose limitation, Saccharomyces cerevisiae ex... more Within the first 5 min after a sudden relief from glucose limitation, Saccharomyces cerevisiae exhibited fast changes of intracellular metabolite levels and a major transcriptional reprogramming. Integration of transcriptome and metabolome data revealed tight relationships between the changes at these two levels. Transcriptome as well as metabolite changes reflected a major investment in two processes: adaptation from fully respiratory to respiro-fermentative metabolism and preparation for growth acceleration. At the metabolite level, a severe drop of the AXP pools directly after glucose addition was not accompanied by any of the other three NXP. To counterbalance this loss, purine biosynthesis and salvage pathways were transcriptionally upregulated in a concerted manner, reflecting a sudden increase of the purine demand. The short-term dynamics of the transcriptome revealed a remarkably fast decrease in the average halflife of downregulated genes. This acceleration of mRNA decay can be interpreted both as an additional nucleotide salvage pathway and an additional level of glucose-induced regulation of gene expression.

Metabolomics, 2008

Accurate determination of intracellular metabolite levels requires reliable, reproducible techniq... more Accurate determination of intracellular metabolite levels requires reliable, reproducible techniques for sampling and sample treatment. Quenching in 60% (v/v) methanol at -40°C is currently the standard method for sub-second arrest of metabolic activity in microbial metabolomics but there have been contradictory reports in the literature on whether leakage of metabolites from the cells occurs. We have re-evaluated this method in S. cerevisiae using a comprehensive, strictly quantitative approach. By determining the levels of a large range of metabolites in different sample fractions and establishing mass balances we could trace their fate during the quenching procedure and confirm that leakage of metabolites from yeast cells does occur during conventional cold methanol quenching, to such an extent that the levels of most metabolites have been previously underestimated by at least twofold. In addition, we found that the extent of leakage depends on the time of exposure, the temperature and the properties of the methanol solutions. Using the mass balance approach we could study the effect of different quenching conditions and demonstrate that leakage can be entirely prevented by quenching in pure methanol at B-40°C, which we propose as a new improved method. Making use of improved data on intracellular metabolite levels we also re-evaluated the need of sub-second quenching of metabolic activity and of removing the extracellular medium. Our findings have serious implications for quantitative metabolomics-based fields such as non-stationary 13 C flux analysis, in vivo kinetic modeling and thermodynamic network analysis.

Metabolic Engineering, 2008

The relation between central metabolism and the penicillin biosynthesis pathway in Penicillium ch... more 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 m ¼ 0.05 h À1 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 aAAA 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. r

Metabolic Engineering, 2006

In this study, prolonged chemostat cultivation is applied to investigate in vivo enzyme kinetics ... more 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.

Metabolic Engineering, 2008

Current 13 C labeling experiments for metabolic flux analysis (MFA) are mostly limited by either ... more 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 0:06 h À1 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 ð41:2 AE 0:6%Þ using the novel approach, shows statistically no difference with the split-ratio obtained from the originally proposed isotopic stationary gluconate tracing method.

Metabolic Engineering, 2007

This study addresses the relation between NADPH supply and penicillin synthesis, by comparing the... more 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 Engineering, 2011

Kinetic modeling of metabolism holds great potential for metabolic engineering but is hindered by... more 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.

Journal of Molecular Catalysis B: Enzymatic, 1998

The enzymatic, thermodynamically controlled synthesis of amoxicillin in aqueous solution was meas... more The enzymatic, thermodynamically controlled synthesis of amoxicillin in aqueous solution was measured in order to study the feasibility of a 'solid-to-solid' conversion. In aqueous solution, however, the synthetic yield of amoxicillin remains lower than the amoxicillin solubility. Therefore, a 'solid-to-solid' synthesis of amoxicillin in aqueous solution is not feasible. Synthetic yields in enzymatic condensation reactions can often be improved by adding organic solvents in monophasic systems. Addition of cosolvents is calculated to improve the apparent equilibrium constant of amoxicillin synthesis considerably, but probably not the synthetic yield, due to solubility restrictions of the reactants. q

Journal of Chromatography A, 2013

A fast, sensitive and specific analytical method, based on ion pair reversed phase ultrahigh perf... more 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 (2carboxyethyl) 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.

FEMS Yeast Research, 2007

Biotechnology Progress, 1998

The oxidation of ferrous iron by Leptospirillum bacteria was studied in a continuous culture in t... more The oxidation of ferrous iron by Leptospirillum bacteria was studied in a continuous culture in the dilution rate range 0.009-0.077 h -1 and could be described with a rate equation for competitive ferric iron inhibition kinetics in terms of the ferric/ferrous iron ratio in the solution. The ferrous iron oxidation in the continuous culture was followed by means of oxygen and carbon dioxide concentration analyses in reference air and off-gas. From these measurements the oxygen consumption rate, r O 2 , the carbon dioxide consumption rate, r CO 2 , the biomass concentration, C x , and the biomass specific oxygen consumption rate, q O 2 , in the culture were determined. The ferrous iron concentration in the culture was below accurate levels to determine with the usual titrimetric method and was therefore derived from measuring the solution redox potential. The degree of reduction balance was used to check the theoretically expected relation between the rates of ferrous iron, -r Fe 2+ , oxygen, -r O 2 , and biomass, r x . The maximum biomass yield and maintenance coefficient on oxygen are Y ox max ) 0.047 mol of C/mol of O 2 and m o ) 0.057 mol of O 2 /(mol of C‚h). The maximum specific oxygen consumption rate, q O 2 ,max ) 1.7 mol of O 2 /(mol of C‚h), the affinity coefficient, K s /K i ) 0.0005 mol of Fe 2+ /mol of Fe 3+ , and the maximum specific growth rate, µ max ) 0.069 h -1 , K s /K i ) 0.0004, were fitted from the measured data. For several dilution rates, off-line respiratory measurements with cell suspension from the continuous culture were carried out in dynamic BOM-E h measurements. The dissolved oxygen and redox potential were measured simultaneously and monitored. The measured value of q O 2 ,max varied between 2.3 and 1.7 mol/(mol of C‚h). The value of K s /K i ) 0.0007 was equal in all experiments. The measured values of q O 2 in the continuous culture were well described with the kinetics determined in dynamic BOM-E h measurements. It was concluded that dynamic BOM-E h measurements are a convenient method to determine the kinetics of continuous culture grown Leptospirillum bacteria.

Biotechnology and Bioengineering, 2005

In the present work LC-MS/MS was applied to measure the concentrations of intermediates of glycol... more In the present work LC-MS/MS was applied to measure the concentrations of intermediates of glycolysis and TCA cycle during autonomous, cell-cycle synchronized oscillations in aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae. This study complements previously reported oscillations in carbon dioxide production rate, intracellular concentrations of trehalose and various free amino acids, and extracellular acetate and pyruvate in the same culture. Of the glycolytic intermediates, fructose 1,6-bisphosphate, 2-and 3-phosphoglycerate, and phosphoenolpyruvate show the most pronounced oscillatory behavior, the latter three compounds oscillating out of phase with the former. This agrees with previously observed metabolic control by phosphofructokinase and pyruvate kinase. Although individually not clearly oscillating, several intermediates of the TCA cycle, i.e., a-ketoglutarate, succinate, fumarate, and malate, exhibited increasing concentration during the cell cycle phase with high carbon flux through glycolysis and TCA cycle. The average mass action ratios of h-phosphoglucomutase and fumarase agreed well with previously determined in vitro equilibrium constants. Minor differences resulted for phosphoglucose isomerase and enolase. Together with the observed close correlation of the pool sizes of the involved metabolites, this might indicate that, in vivo, these reactions are operating close to equilibrium, whereby care must be taken due to possible differences between in vivo and in vitro conditions. Combining the data with previously determined intracellular amino acid levels from the same culture, a few clear correlations between catabolism and anabolism could be identified: phosphoglycerate/serine and a-ketoglutarate/lysine exhibited correlated oscillatory behavior, albeit with different phase shifts. Oscillations in intracellular amino acids might therefore be, at least partly, following oscillations of their anabolic precursors. B

Applied Microbiology and Biotechnology, 1999

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures was examined, ... more The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures was examined, using on-line o-gas analyses to measure the oxygen and carbon dioxide consumption rates continuously. A cell suspension from continuous cultures at steady state was used as the inoculum. It was observed that a dynamic phase occurred in the initial phase of the experiment. In this phase the bacterial ferrous iron oxidation and growth were uncoupled. After about 16 h the bacteria were adapted and achieved a pseudo-steady state, in which the speci®c growth rate and oxygen consumption rate were coupled and their relationship was described by the Pirt equation. In pseudo-steady state, the growth and oxidation kinetics were accurately described by the rate equation for competitive product inhibition. Bacterial substrate consumption is regarded as the primary process, which is described by the equation for competitive product inhibition. Subsequently the kinetic equation for the speci®c growth rate, l, is derived by applying the Pirt equation for bacterial substrate consumption and growth. The maximum speci®c growth rate, l max , measured in the batch culture agrees with the dilution rate at which washout occurs in continuous cultures. The maximum oxygen consumption rate, q O 2 Ymax , of the cell suspension in the batch culture was determined by respiration measurements in a biological oxygen monitor at excess ferrous iron, and showed changes of up to 20% during the course of the experiment. The kinetic constants determined in the batch culture slightly dier from those in continuous cultures, such that, at equal ferric to ferrous iron concentration ratios, biomass-speci®c rates are up to 1.3 times higher in continuous cultures.

Biotechnol Bioeng, 2005

In the present work LC-MS/MS was applied to measure the concentrations of intermediates of glycol... more In the present work LC-MS/MS was applied to measure the concentrations of intermediates of glycolysis and TCA cycle during autonomous, cell-cycle synchronized oscillations in aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae. This study complements previously reported oscillations in carbon dioxide production rate, intracellular concentrations of trehalose and various free amino acids, and extracellular acetate and pyruvate in the same culture. Of the glycolytic intermediates, fructose 1,6-bisphosphate, 2-and 3-phosphoglycerate, and phosphoenolpyruvate show the most pronounced oscillatory behavior, the latter three compounds oscillating out of phase with the former. This agrees with previously observed metabolic control by phosphofructokinase and pyruvate kinase. Although individually not clearly oscillating, several intermediates of the TCA cycle, i.e., a-ketoglutarate, succinate, fumarate, and malate, exhibited increasing concentration during the cell cycle phase with high carbon flux through glycolysis and TCA cycle. The average mass action ratios of h-phosphoglucomutase and fumarase agreed well with previously determined in vitro equilibrium constants. Minor differences resulted for phosphoglucose isomerase and enolase. Together with the observed close correlation of the pool sizes of the involved metabolites, this might indicate that, in vivo, these reactions are operating close to equilibrium, whereby care must be taken due to possible differences between in vivo and in vitro conditions. Combining the data with previously determined intracellular amino acid levels from the same culture, a few clear correlations between catabolism and anabolism could be identified: phosphoglycerate/serine and a-ketoglutarate/lysine exhibited correlated oscillatory behavior, albeit with different phase shifts. Oscillations in intracellular amino acids might therefore be, at least partly, following oscillations of their anabolic precursors. B

Oncotarget, Jan 12, 2015

Succinate dehydrogenase (SDH) and fumarate hydratase (FH) are tricarboxylic acid (TCA) cycle enzy... more 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 (5mC) hydroxylases. To evaluate the distribution of DNA and histone methylation, we used immunohistochemistry to analyze the expression of 5mC, 5-hydroxymethylcytosine (5hmC), 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 5hmC in tumor cells in ...

Scientific Reports, 2015

Eukaryotic metabolism is organised in complex networks of enzyme catalysed reactions which are di... more 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.

Biotechnology and Bioengineering, 2015

Eukaryotic metabolism consists of a complex network of enzymatic reactions and transport processe... more 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 þ Pi < ¼ > glucose þ 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 ¼ 0.1 h À 1 ) conditions, which was 17.88 mmol/gDW and 25.02 mmol/gDW, respectively under Pi-excess conditions. Under Pi-limited steady state (D ¼ 0.1 h À 1 ) conditions, the cytosolic Pi concentration dropped to only 17.7% of the cytosolic Pi in Pi-excess condition (4.42 mmol/gDW vs. 25.02 mmol/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. specific substrate uptake rate; qCO 2 , the specific carbon dioxide production rate; qO 2 , the oxygen uptake rate; qGly, the specific glycerol production rate; RQ, respiratory quotient. Correspondence to: J. Zhang and S. A. Wahl fax: þ31 (0)15 2782355

Methods in Molecular Biology, 2014

Quantitative intracellular metabolite measurements are essential for systems biology and modeling... more 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).

New Biotechnology, 2009

Dynamic mathematical models allow a better understanding of the metabolism of microorganisms and ... more Dynamic mathematical models allow a better understanding of the metabolism of microorganisms and the optimization of fermentation conditions for a desired product compared to stoichiometric models. Such dynamic models contain mathematical relations to express reaction rates as a function of enzyme levels, enzyme kinetic properties and intra/extracellular metabolite concentrations. To estimate the parameters of these rate equations experimental information on fluxes, enzyme levels and metabolite concentrations under a number of different conditions are required. Measurement of enzyme levels can be avoided if dynamic perturbation experiments are carried out in such a short time interval that the enzyme levels can be assumed not to change. Such short time experiments were carried out with Escherichia coli. For these purposes two replicate aerobic glucose-limited chemostat cultures of E. coli K12 MG1655 fnr (at D = 0.1 h −1 ) were subjected to a moderate glucose pulse, whereby the extracellular glucose concentration was instantaneously increased from the steady state level of 14 mg/L to 0.5 g/L. Subsequently, rapid sampling and immediate quenching of metabolic activity was applied to capture the fast dynamics of the intracellular and extracellular metabolites directly after the pulse. Intracellular metabolites were measured with a newly developed differential method which avoids metabolite leakage during quenching and subsequent sample treatment. During the transient not only the metabolome response but also the offgas response was monitored. The obtained stimulus response data were shown to be highly reproducible for the two pulse experiments carried out in replicate chemostats.

Molecular Systems Biology, 2006

Within the first 5 min after a sudden relief from glucose limitation, Saccharomyces cerevisiae ex... more Within the first 5 min after a sudden relief from glucose limitation, Saccharomyces cerevisiae exhibited fast changes of intracellular metabolite levels and a major transcriptional reprogramming. Integration of transcriptome and metabolome data revealed tight relationships between the changes at these two levels. Transcriptome as well as metabolite changes reflected a major investment in two processes: adaptation from fully respiratory to respiro-fermentative metabolism and preparation for growth acceleration. At the metabolite level, a severe drop of the AXP pools directly after glucose addition was not accompanied by any of the other three NXP. To counterbalance this loss, purine biosynthesis and salvage pathways were transcriptionally upregulated in a concerted manner, reflecting a sudden increase of the purine demand. The short-term dynamics of the transcriptome revealed a remarkably fast decrease in the average halflife of downregulated genes. This acceleration of mRNA decay can be interpreted both as an additional nucleotide salvage pathway and an additional level of glucose-induced regulation of gene expression.

Metabolomics, 2008

Accurate determination of intracellular metabolite levels requires reliable, reproducible techniq... more Accurate determination of intracellular metabolite levels requires reliable, reproducible techniques for sampling and sample treatment. Quenching in 60% (v/v) methanol at -40°C is currently the standard method for sub-second arrest of metabolic activity in microbial metabolomics but there have been contradictory reports in the literature on whether leakage of metabolites from the cells occurs. We have re-evaluated this method in S. cerevisiae using a comprehensive, strictly quantitative approach. By determining the levels of a large range of metabolites in different sample fractions and establishing mass balances we could trace their fate during the quenching procedure and confirm that leakage of metabolites from yeast cells does occur during conventional cold methanol quenching, to such an extent that the levels of most metabolites have been previously underestimated by at least twofold. In addition, we found that the extent of leakage depends on the time of exposure, the temperature and the properties of the methanol solutions. Using the mass balance approach we could study the effect of different quenching conditions and demonstrate that leakage can be entirely prevented by quenching in pure methanol at B-40°C, which we propose as a new improved method. Making use of improved data on intracellular metabolite levels we also re-evaluated the need of sub-second quenching of metabolic activity and of removing the extracellular medium. Our findings have serious implications for quantitative metabolomics-based fields such as non-stationary 13 C flux analysis, in vivo kinetic modeling and thermodynamic network analysis.

Metabolic Engineering, 2008

The relation between central metabolism and the penicillin biosynthesis pathway in Penicillium ch... more 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 m ¼ 0.05 h À1 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 aAAA 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. r

Metabolic Engineering, 2006

In this study, prolonged chemostat cultivation is applied to investigate in vivo enzyme kinetics ... more 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.

Metabolic Engineering, 2008

Current 13 C labeling experiments for metabolic flux analysis (MFA) are mostly limited by either ... more 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 0:06 h À1 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 ð41:2 AE 0:6%Þ using the novel approach, shows statistically no difference with the split-ratio obtained from the originally proposed isotopic stationary gluconate tracing method.

Metabolic Engineering, 2007

This study addresses the relation between NADPH supply and penicillin synthesis, by comparing the... more 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 Engineering, 2011

Kinetic modeling of metabolism holds great potential for metabolic engineering but is hindered by... more 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.

Journal of Molecular Catalysis B: Enzymatic, 1998

The enzymatic, thermodynamically controlled synthesis of amoxicillin in aqueous solution was meas... more The enzymatic, thermodynamically controlled synthesis of amoxicillin in aqueous solution was measured in order to study the feasibility of a 'solid-to-solid' conversion. In aqueous solution, however, the synthetic yield of amoxicillin remains lower than the amoxicillin solubility. Therefore, a 'solid-to-solid' synthesis of amoxicillin in aqueous solution is not feasible. Synthetic yields in enzymatic condensation reactions can often be improved by adding organic solvents in monophasic systems. Addition of cosolvents is calculated to improve the apparent equilibrium constant of amoxicillin synthesis considerably, but probably not the synthetic yield, due to solubility restrictions of the reactants. q

Journal of Chromatography A, 2013

A fast, sensitive and specific analytical method, based on ion pair reversed phase ultrahigh perf... more 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 (2carboxyethyl) 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.

FEMS Yeast Research, 2007

Biotechnology Progress, 1998

The oxidation of ferrous iron by Leptospirillum bacteria was studied in a continuous culture in t... more The oxidation of ferrous iron by Leptospirillum bacteria was studied in a continuous culture in the dilution rate range 0.009-0.077 h -1 and could be described with a rate equation for competitive ferric iron inhibition kinetics in terms of the ferric/ferrous iron ratio in the solution. The ferrous iron oxidation in the continuous culture was followed by means of oxygen and carbon dioxide concentration analyses in reference air and off-gas. From these measurements the oxygen consumption rate, r O 2 , the carbon dioxide consumption rate, r CO 2 , the biomass concentration, C x , and the biomass specific oxygen consumption rate, q O 2 , in the culture were determined. The ferrous iron concentration in the culture was below accurate levels to determine with the usual titrimetric method and was therefore derived from measuring the solution redox potential. The degree of reduction balance was used to check the theoretically expected relation between the rates of ferrous iron, -r Fe 2+ , oxygen, -r O 2 , and biomass, r x . The maximum biomass yield and maintenance coefficient on oxygen are Y ox max ) 0.047 mol of C/mol of O 2 and m o ) 0.057 mol of O 2 /(mol of C‚h). The maximum specific oxygen consumption rate, q O 2 ,max ) 1.7 mol of O 2 /(mol of C‚h), the affinity coefficient, K s /K i ) 0.0005 mol of Fe 2+ /mol of Fe 3+ , and the maximum specific growth rate, µ max ) 0.069 h -1 , K s /K i ) 0.0004, were fitted from the measured data. For several dilution rates, off-line respiratory measurements with cell suspension from the continuous culture were carried out in dynamic BOM-E h measurements. The dissolved oxygen and redox potential were measured simultaneously and monitored. The measured value of q O 2 ,max varied between 2.3 and 1.7 mol/(mol of C‚h). The value of K s /K i ) 0.0007 was equal in all experiments. The measured values of q O 2 in the continuous culture were well described with the kinetics determined in dynamic BOM-E h measurements. It was concluded that dynamic BOM-E h measurements are a convenient method to determine the kinetics of continuous culture grown Leptospirillum bacteria.

Biotechnology and Bioengineering, 2005

In the present work LC-MS/MS was applied to measure the concentrations of intermediates of glycol... more In the present work LC-MS/MS was applied to measure the concentrations of intermediates of glycolysis and TCA cycle during autonomous, cell-cycle synchronized oscillations in aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae. This study complements previously reported oscillations in carbon dioxide production rate, intracellular concentrations of trehalose and various free amino acids, and extracellular acetate and pyruvate in the same culture. Of the glycolytic intermediates, fructose 1,6-bisphosphate, 2-and 3-phosphoglycerate, and phosphoenolpyruvate show the most pronounced oscillatory behavior, the latter three compounds oscillating out of phase with the former. This agrees with previously observed metabolic control by phosphofructokinase and pyruvate kinase. Although individually not clearly oscillating, several intermediates of the TCA cycle, i.e., a-ketoglutarate, succinate, fumarate, and malate, exhibited increasing concentration during the cell cycle phase with high carbon flux through glycolysis and TCA cycle. The average mass action ratios of h-phosphoglucomutase and fumarase agreed well with previously determined in vitro equilibrium constants. Minor differences resulted for phosphoglucose isomerase and enolase. Together with the observed close correlation of the pool sizes of the involved metabolites, this might indicate that, in vivo, these reactions are operating close to equilibrium, whereby care must be taken due to possible differences between in vivo and in vitro conditions. Combining the data with previously determined intracellular amino acid levels from the same culture, a few clear correlations between catabolism and anabolism could be identified: phosphoglycerate/serine and a-ketoglutarate/lysine exhibited correlated oscillatory behavior, albeit with different phase shifts. Oscillations in intracellular amino acids might therefore be, at least partly, following oscillations of their anabolic precursors. B