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Christoph Wittmann

Publications and source records attributed to Christoph Wittmann.

31 records · Page 2Linked to original sources
PubMed2004

Comparative metabolic flux analysis of lysine-producing Corynebacterium glutamicum cultured on glucose or fructose.

A comprehensive approach to (13)C tracer studies, labeling measurements by gas chromatography-mass spectrometry, metabolite balancing, and isotopomer modeling, was applied for comparative metabolic network analysis of lysine-producing Corynebacterium glutamicum on glucose or fructose. Significantly reduced yields of lysine and biomass and enhanced formation of dihydroxyacetone, glycerol, and lactate in comparison to those for glucose resulted on fructose. Metabolic flux analysis revealed drastic differences in intracellular flux depending on the carbon source applied. On fructose, flux through the pentose phosphate pathway (PPP) was only 14.4% of the total substrate uptake flux and therefore markedly decreased compared to that for glucose (62.0%). This result is due mainly to (i) the predominance of phosphoenolpyruvate-dependent phosphotransferase systems for fructose uptake (PTS(Fructose)) (92.3%), resulting in a major entry of fructose via fructose 1,6-bisphosphate, and (ii) the inactivity of fructose 1,6-bisphosphatase (0.0%). The uptake of fructose during flux via PTS(Mannose) was only 7.7%. In glucose-grown cells, the flux through pyruvate dehydrogenase (70.9%) was much less than that in fructose-grown cells (95.2%). Accordingly, flux through the tricarboxylic acid cycle was decreased on glucose. Normalized to that for glucose uptake, the supply of NADPH during flux was only 112.4% on fructose compared to 176.9% on glucose, which might explain the substantially lower lysine yield of C. glutamicum on fructose. Balancing NADPH levels even revealed an apparent deficiency of NADPH on fructose, which is probably overcome by in vivo activity of malic enzyme. Based on these results, potential targets could be identified for optimization of lysine production by C. glutamicum on fructose, involving (i) modification of flux through the two PTS for fructose uptake, (ii) amplification of fructose 1,6-bisphosphatase to increase flux through the PPP, and (iii) knockout of a not-yet-annotated gene encoding dihydroxyacetone phosphatase or kinase activity to suppress overflow metabolism. Statistical evaluation revealed high precision of the estimates of flux, so the observed differences for metabolic flux are clearly substrate specific.

Carbon Isotopes↗
PubMed2004

Metabolic fluxes in Corynebacterium glutamicum during lysine production with sucrose as carbon source.

Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, 13C metabolic flux analysis with parallel studies on [1-(13C)Fru]sucrose, [1-(13C)Glc]sucrose, and [13C6Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTS(Man) or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.

Carbon Isotopes↗
PubMed2004

In-depth profiling of lysine-producing Corynebacterium glutamicum by combined analysis of the transcriptome, metabolome, and fluxome.

An in-depth analysis of the intracellular metabolite concentrations, metabolic fluxes, and gene expression (metabolome, fluxome, and transcriptome, respectively) of lysine-producing Corynebacterium glutamicum ATCC 13287 was performed at different stages of batch culture and revealed distinct phases of growth and lysine production. For this purpose, 13C flux analysis with gas chromatography-mass spectrometry-labeling measurement of free intracellular amino acids, metabolite balancing, and isotopomer modeling were combined with expression profiling via DNA microarrays and with intracellular metabolite quantification. The phase shift from growth to lysine production was accompanied by a decrease in glucose uptake flux, the redirection of flux from the tricarboxylic acid (TCA) cycle towards anaplerotic carboxylation and lysine biosynthesis, transient dynamics of intracellular metabolite pools, such as an increase of lysine up to 40 mM prior to its excretion, and complex changes in the expression of genes for central metabolism. The integrated approach was valuable for the identification of correlations between gene expression and in vivo activity for numerous enzymes. The glucose uptake flux closely corresponded to the expression of glucose phosphotransferase genes. A correlation between flux and expression was also observed for glucose-6-phosphate dehydrogenase, transaldolase, and transketolase and for most TCA cycle genes. In contrast, cytoplasmic malate dehydrogenase expression increased despite a reduction of the TCA cycle flux, probably related to its contribution to NADH regeneration under conditions of reduced growth. Most genes for lysine biosynthesis showed a constant expression level, despite a marked change of the metabolic flux, indicating that they are strongly regulated at the metabolic level. Glyoxylate cycle genes were continuously expressed, but the pathway exhibited in vivo activity only in the later stage. The most pronounced changes in gene expression during cultivation were found for enzymes at entry points into glycolysis, the pentose phosphate pathway, the TCA cycle, and lysine biosynthesis, indicating that these might be of special importance for transcriptional control in C. glutamicum.

Amino Acids↗
PubMed2003

Free intracellular amino acid pools during autonomous oscillations in Saccharomyces cerevisiae.

In the present work dynamic changes of free intracellular amino acid pools during autonomous oscillations of Saccharomyces cerevisiae were quantified in glucose-limited continuous cultivations. At a dilution rate of D = 0.22 h(-1) cyclic changes with a period of 120 min were found for many variables such as carbon dioxide production rate, dissolved oxygen, pH, biomass content, and various metabolite concentrations. On the basis of the observed dynamic patterns, free intracellular amino acids were classified to show oscillatory, stationary, or chaotic behavior. Amino acid pools such as serine, alanine, valine, leucine, or lysine were subjected to clear oscillations with a frequency of 120 min, identical to that of other described cultivation variables, indicating that there is a direct correlation between the periodic changes of amino acid concentrations and the metabolic oscillations on the cellular level. The oscillations of these amino acids were unequally phase-delayed and had different amplitudes of oscillation. Accordingly, they exhibited different patterns in phase plane plots vs. intracellular trehalose. Despite the complex and marked metabolic changes during oscillation, selected intracellular amino acids such as histidine, threonine, isoleucine, or arginine remained about constant. Concentrations of glutamate and glutamine showed a chaotic behavior. However, the ratio of glutamate to glutamine concentration was found to be oscillatory, with a period of 60 min and a corresponding figure eight-shaped pattern in a plot vs. trehalose concentration. Considering the described diversity, it can be concluded that the observed periodic changes are neither just the consequence of low or high rates of protein biosynthesis/degradation nor correlated to changing cell volumes during oscillation. The ratio between doubling time (189 min) and period of oscillation of intracellular amino acids (120 min) was 1:6. The fact that there is a close relationship between doubling time and period of oscillation underlines that the described autonomous oscillations are cell-cycle-associated.

Amino Acids↗
PubMed2003

Integrated optical sensing of dissolved oxygen in microtiter plates: a novel tool for microbial cultivation.

Microtiter plates with integrated optical sensing of dissolved oxygen were developed by immobilization of two fluorophores at the bottom of 96-well polystyrene microtiter plates. The oxygen-sensitive fluorophore responded to dissolved oxygen concentration, whereas the oxygen-insensitive one served as an internal reference. The sensor measured dissolved oxygen accurately in optically well-defined media. Oxygen transfer coefficients, k(L)a, were determined by a dynamic method in a commercial microtiter plate reader with an integrated shaker. For this purpose, the dissolved oxygen was initially depleted by the addition of sodium dithionite and, by oxygen transfer from air, it increased again after complete oxidation of dithionite. k(L)a values in one commercial reader were about 10 to 40 h(-1). k(L)a values were inversely proportional to the filling volume and increased with increasing shaking intensity. Dissolved oxygen was monitored during cultivation of Corynebacterium glutamicum in another reader that allowed much higher shaking intensity. Growth rates determined from optical density measurement were identical to those observed in shaking flasks and in a stirred fermentor. Oxygen uptake rates measured in the stirred fermentor and dissolved oxygen concentrations measured during cultivation in the microtiter plate were used to estimate k(L)a values in a 96-well microtiter plate. The resulting values were about 130 h(-1), which is in the lower range of typical stirred fermentors. The resulting maximum oxygen transfer rate was 26 mM h(-1). Simulations showed that the errors caused by the intermittent measurement method were insignificant under the prevailing conditions.

Bioreactors↗
PubMed2003

Dynamic calibration and dissolved gas analysis using membrane inlet mass spectrometry for the quantification of cell respiration.

A membrane inlet mass spectrometer connected to a miniaturized reactor was applied for dynamic dissolved gas analysis. Cell samples were taken from 7 mL shake flask cultures of Corynebacterium glutamicum ATCC 13032, and transferred to the 12 mL miniaturized reactor. There, oxygen uptake and carbon dioxide and its mass isotopomer production rates were determined using a new experimental procedure and applying nonlinear model equations. A novel dynamic method for the calibration of the membrane inlet mass spectrometer using first-order dynamics was developed. To derive total dissolved concentration of all carbon dioxide species (C(T)) from dissolved carbon dioxide concentration ([CO(2)](aq)), the ratio of C(T) to [CO(2)](aq) was determined by nonlinear parameter estimation, whereas the mass transfer coefficient of CO(2) was determined by the Wilke-Chang correlation. Subsequently, the suitability of the model equations for respiration measurements was examined using residual analysis and the Jarque-Bera hypothesis test. The resulting residuals were found to be random with normal distribution, which proved the adequacy of the application of the model for cell respiration analysis. Hence, dynamic changes in respiration activities could be accurately analyzed using membrane inlet mass spectrometry with the novel calibration method.

Algorithms↗
PubMed2003

Characterization and application of an optical sensor for quantification of dissolved O2 in shake-flasks.

On-line measurement of dissolved O2 in shake-flasks was realized via immobilized sensor spots containing a fluorophore with an O2-dependent luminescent decay time. An unaffected sensor signal during 80 autoclaving cycles suggests multi-usage of sensor equipped shake-flasks. The sensor had a response time of 6 s. Quantification of gas-liquid mass transfer revealed maximum kLa values of 150 h(-1), from which maximum O2 transfer capacity of 33 mM h(-1) was calculated. Liquid volume and shaking frequency have a strong influence on kLa. Exemplified by cultivations of Corynebacterium glutamicum the importance of shaking rate for O2 supply of bacterial cultures is shown. Sampling of microbial cultures with intermittent shaking of a few minutes can cause O2 limitation. Based on the results of this work a simple and straightforward tool is now available for accurate O2 sensing in shake-flasks, which are widely used in microbial cultivations.

Biosensing Techniques↗
PubMed2002

Correcting mass isotopomer distributions for naturally occurring isotopes.

In one method of metabolic flux analysis, simulated mass spectrometry data is fitted to measured mass distributions of metabolites that are isolated from cultures with defined feeding of (13)C-labeled substrates. Doing so, simulated mass distributions must be corrected for the presence of naturally occurring isotopes. A method that was recently introduced for this purpose consists of consecutive correction steps for each isotope of each element in the considered compound. Here we show that all isotopes of each individual element must, however, be corrected in one single step. Furthermore, it is shown that the source of information with respect to isotopic compositions of the elements needs to be chosen with care.

Biomarkers↗
PubMed2002

Derivatization of small biomolecules for optimized matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is a powerful tool for the measurement of low molecular mass compounds of biological interest. The limitations for this method are the volatility of many analytes, possible interference with matrix signals or bad ionization or desorption behavior of the compounds. We investigated the application of well-known and straightforward one-pot derivatization procedures to circumvent these problems. The derivatizations tested allow the measurement and the labeling of alcohols, aldehydes and ketones, carboxylic acids, alpha-ketocarboxylic acids and amines.

Alcohols↗
PubMed2002

Metabolic physiology of aroma-producing Kluyveromyces marxianus.

Kluyveromyces marxianus has a high potential for industrial production of aroma compounds, such as 2-phenylethanol, which is derived in a bioconversion from L-phenylalanine. In the present work the product yield of K. marxianus in batch cultivation was estimated as 0.65 mol 2-phenylethanol/mol L-phenylalanine and thus significantly below the theoretical optimum of 1 mol/mol. By a comprehensive approach of stoichiometric balancing and GC-MS analysis of various substrates and products of K. marxianus a detailed insight into its metabolism was gained. For this purpose ring-labelled ((13)C(6)) L-phenylalanine and naturally labelled glucose were applied as substrates in tracer studies in batch culture. The produced aroma compounds 2-phenylethanol and 2-phenylethylacetate stem exclusively from the supplied L-phenylalanine, whereas glucose was not converted into these products because of efficient feed-back inhibition of prephenate dehydratase in the L-phenylalanine biosynthetic pathway. It could be further shown that the supplied L-phenylalanine completely covers the anabolic cellular demand for this amino acid. Quantification of (13)CO(2) in the exhaust gas provided clear evidence for catabolic breakdown of L-phenylalanine during cultivation. Metabolic balancing around the pool of free intracellular L-phenylalanine revealed a significant loss of L-phenylalanine into catabolic and anabolic pathways. While 73.3% of L-phenylalanine was converted into 2-phenylethanol or 2-phenylethylacetate, 22.4% was catabolized through the cinnamate pathway and 4.3% was directed towards protein biosynthesis. Catabolic breakdown of L-phenylalanine via hydroxylation to L-tyrosine could be excluded. In addition to an insight into metabolic functioning and regulation of 2-phenylethanol-producing K. marxianus, the approach presented here provides important information on potential targets for genetic optimization of 2-phenylethanol-producing yeasts.

Biotechnology↗
PubMed2002

Genealogy profiling through strain improvement by using metabolic network analysis: metabolic flux genealogy of several generations of lysine-producing corynebacteria.

A comprehensive approach of metabolite balancing, (13)C tracer studies, gas chromatography-mass spectrometry, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and isotopomer modeling was applied for comparative metabolic network analysis of a genealogy of five successive generations of lysine-producing Corynebacterium glutamicum. The five strains examined (C. glutamicum ATCC 13032, 13287, 21253, 21526, and 21543) were previously obtained by random mutagenesis and selection. Throughout the genealogy, the lysine yield in batch cultures increased markedly from 1.2 to 24.9% relative to the glucose uptake flux. Strain optimization was accompanied by significant changes in intracellular flux distributions. The relative pentose phosphate pathway (PPP) flux successively increased, clearly corresponding to the product yield. Moreover, the anaplerotic net flux increased almost twofold as a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes to cover the increased demand for lysine formation; thus, the overall increase was a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes. The relative flux through isocitrate dehydrogenase dropped from 82.7% in the wild type to 59.9% in the lysine-producing mutants. In contrast to the NADPH demand, which increased from 109 to 172% due to the increasing lysine yield, the overall NADPH supply remained constant between 185 and 196%, resulting in a decrease in the apparent NADPH excess through strain optimization. Extrapolated to industrial lysine producers, the NADPH supply might become a limiting factor. The relative contributions of PPP and the tricarboxylic acid cycle to NADPH generation changed markedly, indicating that C. glutamicum is able to maintain a constant supply of NADPH under completely different flux conditions. Statistical analysis by a Monte Carlo approach revealed high precision for the estimated fluxes, underlining the fact that the observed differences were clearly strain specific.

Citric Acid Cycle↗