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8 Publications visible to you, out of a total of 8
Cell size and morphological properties of yeast Saccharomyces cerevisiae in relation to growth temperature

Abstract (Expand)

Cell volume is an important parameter for modelling cellular processes. Temperature-induced variability of cellular size, volume, intracellular granularity, a fraction of budding cells of yeast … Saccharomyces cerevisiae CEN.PK 113–7D (in anaerobic glucose unlimited batch cultures) were measured by flow cytometry and matched with the performance of the biomass growth (maximal specific growth rate (μmax), specific rate of glucose consumption, the rate of maintenance, biomass yield on glucose). The critical diameter of single cells was 7.94 μm and it is invariant at growth temperatures above 18.5°C. Below 18.5°C, it exponentially increases up to 10.2 μm. The size of the bud linearly depends on μmax, and it is between 50% at 5°C and 90% at 31°C of the averaged single cell. The intracellular granularity (side scatter channel (SSC)-index) negatively depends on μmax. There are two temperature regions (5–31°C vs. 33–40°C) where the relationship between SSC-index and various cellular parameters differ significantly. In supraoptimal temperature range (33–40°C), cells are less granulated perhaps due to a higher rate of the maintenance. There is temperature dependent passage through the checkpoints in the cell cycle which influences the μmax. The results point to the existence of two different morphological states of yeasts in these different temperature regions.

Authors: Maksim Zakhartsev, Matthias Reuss

Date Published: 26th Apr 2018

Publication Type: Not specified

Metabolic efficiency in yeast Saccharomyces cerevisiae in relation to temperature dependent growth and biomass yield

Abstract (Expand)

Canonized view on temperature effects on growth rate of microorganisms is based on assumption of protein denaturation, which is not confirmed experimentally so far. We develop an alternative concept, … which is based on view that limits of thermal tolerance are based on imbalance of cellular energy allocation. Therefore, we investigated growth suppression of yeast Saccharomyces cerevisiae in the supraoptimal temperature range (30–40 °C), i.e. above optimal temperature (Topt). The maximal specific growth rate (μmax) of biomass, its concentration and yield on glucose (Yx/glc) were measured across the whole thermal window (5–40 °C) of the yeast in batch anaerobic growth on glucose. Specific rate of glucose consumption, specific rate of glucose consumption for maintenance (mglc), true biomass yield on glucose (View the MathML source), fractional conservation of substrate carbon in product and ATP yield on glucose (Yatp/glc) were estimated from the experimental data. There was a negative linear relationship between ATP, ADP and AMP concentrations and specific growth rate at any growth conditions, whilst the energy charge was always high (~0.83). There were two temperature regions where mglc differed 12-fold, which points to the existence of a ‘low’ (within 5–31 °C) and a ‘high’ (within 33–40 °C) metabolic mode regarding maintenance requirements. The rise from the low to high mode occurred at 31–32 °C in step-wise manner and it was accompanied with onset of suppression of μmax. High mglc at supraoptimal temperatures indicates a significant reduction of scope for growth, due to high maintenance cost. Analysis of temperature dependencies of product formation efficiency and Yatp/glc revealed that the efficiency of energy metabolism approaches its lower limit at 26–31 °C. This limit is reflected in the predetermined combination of View the MathML source, elemental biomass composition and degree of reduction of the growth substrate. Approaching the limit implies a reduction of the safety margin of metabolic efficiency. We hypothesize that a temperature increase above Topt (e.g. >31 °C) triggers both an increment in mglc and suppression of μmax, which together contribute to an upshift of Yatp/glc from the lower limit and thus compensate for the loss of the safety margin. This trade-off allows adding 10 more degrees to Topt and extends the thermal window up to 40 °C, sustaining survival and reproduction in supraoptimal temperatures. Deeper understanding of the limits of thermal tolerance can be practically exploited in biotechnological applications.

Authors: Maksim Zakhartsev, Xuelian Yang, Matthias Reuss, Hans Otto Pörtner

Date Published: 1st Aug 2015

Publication Type: Not specified

Fast sampling for quantitative microbial metabolomics: new aspects on cold methanol quenching: metabolite co-precipitation

Abstract (Expand)

The intra- and extracellular concentrations of 16 metabolites were measured in chemostat (D = 0.1 h−1) anaerobic cultures of the yeast Saccharomyces cerevisiae CEN.PK-113-7D growing on minimal medium. … Two independent sampling workflows were employed: (i) conventional cold methanol quenching and (ii) a differential approach. Metabolites were quantified in different sample fractions (total, extracellular, quenching supernatant, methanol/water extract and pellet) in order to derive their mass balance. The differential method in combination with absolute metabolite quantification by gas-chromatography with isotope dilution mass spectrometry (GC–IDMS) was used as a benchmark to assess quality of the cold methanol quenching procedure. Quantitative comparison of metabolite concentrations in all fractions collected by different quenching techniques indicates asystematic loss of the total mass of various metabolites in course of the cold methanol quenching. Pellet resulting from the cold methanol quenching besides biomass contains considerable amounts of precipitated inorganic salts from the fermentation media. Quantitative analysis has revealed significant co-precipitation of polar extracellular metabolites together with these salts. This phenomenon is especially significant for metabolites with large extracellular mass-fraction. We report that the co-precipitation is a hitherto neglected phenomenon and concluded that its degree strongly linked to culturing conditions (i.e. media composition) and chemical properties of the particular metabolite. Thus, intracellular metabolite levels measured from samples collected by cold methanol quenching might be uncertain and variably biased due to corruption by described phenomena.

Authors: Maksim Zakhartsev, Oliver Vielhauer, Thomas Horn, Xuelian Yang, Matthias Reuss

Date Published: 1st Apr 2015

Publication Type: Not specified

‘Domino’ systems biology and the ‘A’ of ATP

Abstract (Expand)

We develop a strategic ‘domino’ approach that starts with one key feature of cell function and the main process providing for it, and then adds additional processes and components only as necessary to … explain provoked experimental observations. The approach is here applied to the energy metabolism of yeast in a glucose limited chemostat, subjected to a sudden increase in glucose. The puzzles addressed include (i) the lack of increase in ATP upon glucose addition, (ii) the lack of increase in ADP when ATP is hydrolyzed, and (iii) the rapid disappearance of the ‘A’ (adenine) moiety of ATP. Neither the incorporation of nucleotides into new biomass, nor steady de novo synthesis of AMP explains. Cycling of the ‘A’ moiety accelerates when the cell's energy state is endangered, another essential domino among the seven required for understanding of the experimental observations. This new domino analysis shows how strategic experimental design and observations in tandem with theory and modeling may identify and resolve important paradoxes. It also highlights the hitherto unexpected role of the ‘A’ component of ATP.

Editor:

Date Published: 1st Sep 2012

Publication Type: Not specified

Simplified absolute metabolite quantification by gas chromatography–isotope dilution mass spectrometry on the basis of commercially available source material

Abstract (Expand)

In the field of metabolomics, GC–MS has rather established itself as a tool for semi-quantitative strategies like metabolic fingerprinting or metabolic profiling. Absolute quantification of intra- or … extracellular metabolites is nowadays mostly accomplished by application of diverse LC–MS techniques. Only few groups have so far adopted GC–MS technology for this exceptionally challenging task. Besides numerous and deeply investigated problems related to sample generation, the pronounced matrix effects in biological samples have led to the almost mandatory application of isotope dilution mass spectrometry (IDMS) for the accurate determination of absolute metabolite concentrations. Nevertheless, access to stable isotope labeled internal standards (ILIS), which are in many cases commercially unavailable, is quite laborious and very expensive. Here we present an improved and simplified gas chromatography–isotope dilution mass spectrometry (GC–IDMS) protocol for the absolute determination of intra- and extracellular metabolite levels. Commercially available 13C-labeled algal cells were used as a convenient source for the preparation of internal standards. Advantages as well as limitations of the described method are discussed.

Authors: Oliver Vielhauer, , Thomas Horn, Ralf Takors,

Date Published: 1st Dec 2011

Publication Type: Not specified

Miniaturized device for agitating a high-density yeast suspension that is suitable for in vivo nuclear magnetic resonance applications

Abstract (Expand)

In vivo nuclear magnetic resonance (NMR) monitoring requires a high-density cell suspension, where cell precipitation should be avoided. We have designed a miniaturized cell agitator that fits entirely … into an 8-mm NMR probe but that, being mounted into the instrument, is situated outside of the sensitive area. The device consists of two glass tubes connected in a way that, when gas flow is blown through them, creates influx of cell suspension into the device that returns through apertures. This flow creates continuous circular vortex of the cell suspension in the whole sample volume, whereas there are no moving mechanical parts or gas bubbles crossing the instrument’s sensitive area. The gas flow controls conditions of the cell suspension and removes volatile waste metabolites.

Authors: , Christian Bock

Date Published: 1st Feb 2010

Publication Type: Not specified

Computer controlled automated assay for comprehensive studies of enzyme kinetic parameters

Abstract (Expand)

Stability and biological activity of proteins is highly dependent on their physicochemical environment. The development of realistic models of biological systems necessitates quantitative information … on the response to changes of external conditions like pH, salinity and concentrations of substrates and allosteric modulators. Changes in just a few variable parameters rapidly lead to large numbers of experimental conditions, which go beyond the experimental capacity of most research groups. We implemented a computer-aided experimenting framework ("robot lab assistant") that allows us to parameterize abstract, human-readable descriptions of micro-plate based experiments with variable parameters and execute them on a conventional 8 channel liquid handling robot fitted with a sensitive plate reader. A set of newly developed R-packages translates the instructions into machine commands, executes them, collects the data and processes it without user-interaction. By combining script-driven experimental planning, execution and data-analysis, our system can react to experimental outcomes autonomously, allowing outcome-based iterative experimental strategies. The framework was applied in a response-surface model based iterative optimization of buffer conditions and investigation of substrate, allosteric effector, pH and salt dependent activity profiles of pyruvate kinase (PYK). A diprotic model of enzyme kinetics was used to model the combined effects of changing pH and substrate concentrations. The 8 parameters of the model could be estimated from a single two-hour experiment using nonlinear least-squares regression. The model with the estimated parameters successfully predicted pH and PEP dependence of initial reaction rates, while the PEP concentration dependent shift of optimal pH could only be reproduced with a set of manually tweaked parameters. Differences between model-predictions and experimental observations at low pH suggest additional protonation-sites at the enzyme or substrates critical for enzymatic activity. The developed framework is a powerful tool to investigate enzyme reaction specifics and explore biological system behaviour in a wide range of experimental conditions.

Authors: Felix Bonowski, , , Jinda Holzwarth, Igor Kitanovic, Van Ngoc Bui, Elke Lederer,

Date Published: 23rd Dec 2009

Publication Type: Not specified

Systems biology: the elements and principles of life

Abstract (Expand)

Systems Biology has a mission that puts it at odds with traditional paradigms of physics and molecular biology, such as the simplicity requested by Occam's razor and minimum energy/maximal efficiency. … By referring to biochemical experiments on control and regulation, and on flux balancing in yeast, we show that these paradigms are inapt. Systems Biology does not quite converge with biology either: Although it certainly requires accurate 'stamp collecting', it discovers quantitative laws. Systems Biology is a science of its own, discovering own fundamental principles, some of which we identify here.

Authors: , Catherine Winder, , Evangelos Simeonidis, Malgorzata Adamczyk, , Frank J Bruggeman, Warwick Dunn

Date Published: 6th Nov 2009

Publication Type: Not specified

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