Biomedical subjects

Matthias Reuss

Publications and source records attributed to Matthias Reuss.

11 recordsLinked to original sources

PubMed2005

Microbial production of single-cell protein from deproteinized whey concentrates.

Deproteinized sweet and sour cheese whey concentrates were investigated for their suitability as substrates for the production of single-cell protein with Kluyveromyces marxianus CBS 6556 up to a 100-l scale. An important factor for gaining high cell concentrations was the use of the Crabtree-negative strain K. marxianus CBS 6556. Supplements such as trace elements, ammonium and calcium were required for the complete conversion of sweet whey concentrates into biomass, whereas sour whey concentrates had to be supplemented with ammonium, trace elements and vitamins. After improvement, biomass dry concentrations of up to 50 g l-1 could be reached with Yx/s values of 0.52 for sweet whey and of up to 65 g l-1 with Yx/s values of 0.48 for sour whey concentrates. The chemical oxygen demand of the whey concentrates were reduced by 80%. The cells were used for the analysis of amino acid and ash composition, showing a distinct increase of eight out of ten essential amino acids compared to sweet and sour whey protein and exceeding the World Health Organisation guidelines for valine, leucine, isoleucine, threonine, phenylalanine and tyrosine.

Amino Acids↗

PubMed2005

Model-based inference of gene expression dynamics from sequence information.

A dynamic model of prokaryotic gene expression is developed that makes considerable use of gene sequence information. The main contribution arises from the fact that the combined gene expression model allows us to access the impact of altering a nucleotide sequence on the dynamics of gene expression rates mechanistically. The high level of detail of the mathematical model is considered as an important step towards bringing together the tremendous amount of biological in-depth knowledge that has been accumulated at the molecular level, using a systems level analysis (in the sense of a bottom-up, inductive approach). This enables to the model to provide highly detailed insights into the various steps of the protein expression process and it allows us to access possible targets for model-based design. Taken as a whole, the mathematical gene expression model presented in this study provides a comprehensive framework for a thorough analysis of sequence-related effects on the stages of mRNA synthesis, mRNA degradation and ribosomal translation, as well as their nonlinear interconnectedness. Therefore, it may be useful in the rational design of recombinant bacterial protein synthesis systems, the modulation of enzyme activities in pathway design, in vitro protein biosynthesis, and RNA-based vaccination.

Base Sequence↗

PubMed2004

Metabolic design based on a coupled gene expression-metabolic network model of tryptophan production in Escherichia coli.

The presumably high potential of a holistic design approach for complex biochemical reaction networks is exemplified here for the network of tryptophan biosynthesis from glucose, a system whose components have been investigated thoroughly before. A dynamic model that combines the behavior of the trp operon gene expression with the metabolic network of central carbon metabolism and tryptophan biosynthesis is investigated. This model is analyzed in terms of metabolic fluxes, metabolic control, and nonlinear optimization. We compare two models for a wild-type strain and another model for a tryptophan producer. An integrated optimization of the whole network leads to a significant increase in tryptophan production rate for all systems under study. This enhancement is well above the increase that can be achieved by an optimization of subsystems. A constant ratio of control coefficients on tryptophan synthesis rate has been identified for the models regarding or disregarding trp operon expression. Although we found some examples where flux control coefficients even contradict the trends of enzyme activity changes in an optimized profile, flux control can be used as an indication for enzymes that have to be taken into account in optimization.

Escherichia coli↗

PubMed2004

Optimal re-design of primary metabolism in Escherichia coli using linlog kinetics.

This paper examines the validity of the linlog approach, which was recently developed in our laboratory, by comparison of two different kinetic models for the metabolic network of Escherichia coli. The first model is a complete mechanistic model; the second is an approximative model in which linlog kinetics are applied. The parameters of the linlog model (elasticities) are derived from the mechanistic model. Three different optimization cases are examined. In all cases, the objective is to calculate the enzyme levels that maximize a certain flux while keeping the total amount of enzyme constant and preventing large changes of metabolite concentrations. For an average variation of metabolite levels of 10% and individual changes of a factor 2, the predicted enzyme levels, metabolite concentrations and fluxes of both models are highly similar. This similarity holds for changes in enzyme level of a factor 4-6 and for changes in fluxes up to a factor 6. In all three cases, the predicted optimal enzyme levels could neither have been found by intuition-based approaches, nor on basis of flux control coefficients. This demonstrates that kinetic models are essential tools in Metabolic Engineering. In this respect, the linlog approach is a valuable extension of MCA, since it allows construction of kinetic models, based on MCA parameters, that can be used for constrained optimization problems and are valid for large changes of metabolite and enzyme levels.

Computer Simulation↗

PubMed2003

Cyclic AMP mediates the cell cycle dynamics of energy metabolism in Saccharomyces cerevisiae.

We have investigated the role of 3',5'-cyclic-adenosine-monophosphate (cAMP) in mediating the coupling between energy metabolism and cell cycle progression in both synchronous cultures and oscillating continuous cultures of Saccharomyces cerevisiae. For the first time, a peak in intracellular cAMP was shown to precede the observed breakdown of trehalose and glycogen during cell cycle-related oscillations. Measurements in synchronous cultures demonstrated that this peak can be associated with the cell cycle dynamics of cAMP under conditions of glucose-limited growth, which was found to differ significantly from that observed in synchronous glucose-repressed cultures. Our results support the notion that cAMP plays a major role in mediating the integration of energy metabolism and cell cycle progression, both in the single cell and during cell cycle-related oscillations in continuous culture, respectively. Evidence is presented that the dynamic behaviour of intracellular cAMP during the cell cycle is modulated depending on nutrient supply. The implications of these findings regarding the role of cAMP in regulating cell cycle progression and energy metabolism are discussed.

Bioreactors↗

PubMed2003

Integration of physiology and fluid dynamics.

The purpose of strategies for the integration of fluid dynamics and physiology is the development of more reliable simulation tools to accelerate the process of scale-up. The rigorous mathematical modeling of the richly interactive relationship between the dynamic response of biosystems and the physical environment changing in time and space must rest on the link between coupled momentum, energy and mass balances and structured modeling of the biophase. With the exponential increase in massive computer capabilities hard- and software tools became available for simulation strategies based on such holistic integration approaches. The review discusses fundamental aspects of application of computational fluid dynamics (CFD) to three-dimensional two-phase turbulence flow in stirred tank bioreactors. Examples of coupling momentum and material balance equations with simple unstructured kinetic models for the behavior of the biophase are used to illustrate the application of these strategies to the selection of suitable impeller configurations. The examples reviewed in this paper include distribution of carbon and energy source in fed batch cultures as well as dissolved oxygen fields during aerobic fermentations. A more precise forecasting of the impact of the multitude of interactions must, however, rest upon a rigorous understanding of the response of the cell factory to the complex dynamic stimulation due to space- and time-dependent concentration fields. The paper also introduces some ideas for fast and very fast experimental observations of intracellular pool concentrations based on stimulus response methods. These observations finally lead to a more complex integration approach based on the coupling of CFD and structured metabolic models.

Bioreactors↗

PubMed2002

New bioreactor-coupled rapid stopped-flow sampling technique for measurements of metabolite dynamics on a subsecond time scale.

Knowledge of concentrations of intracellular metabolites is important for quantitative analysis of metabolic networks. As far as the very fast response of intracellular metabolites in the millisecond range is concerned, the frequently used pulse technique shows an inherent limitation. The time span between the disturbance and the first sample is constrained by the time necessary to obtain a homogeneous distribution of the pertubation within the bioreactor. For determination of rapid changes, a novel sampling technique based on the stopped-flow method has been developed. A continuous stream of biosuspension leaving the bioreactor is being mixed with a glucose solution in a turbulent mixing chamber. Through computer-aided activation of sequentially positioned three-way valves, different residence times and thus reaction times can be verified. The application of this new sampling method is illustrated with examples including measurements of adenine nucleotides and glucose-6-phosphate in Saccharomyces cerevisiae as well as measurements related to the PTS system in Escherichia coli.

Adenine Nucleotides↗

PubMed2002

Cell population modelling of yeast glycolytic oscillations.

We investigated a cell-population modelling technique in which the population is constructed from an ensemble of individual cell models. The average value or the number distribution of any intracellular property captured by the individual cell model can be calculated by simulation of a sufficient number of individual cells. The proposed method is applied to a simple model of yeast glycolytic oscillations where synchronization of the cell population is mediated by the action of an excreted metabolite. We show that smooth one-dimensional distributions can be obtained with ensembles comprising 1000 individual cells. Random variations in the state and/or structure of individual cells are shown to produce complex dynamic behaviours which cannot be adequately captured by small ensembles.

Glycolysis↗

PubMed2002

Determination of in vivo kinetics of the starvation-induced Hxt5 glucose transporter of Saccharomyces cerevisiae.

We have investigated the role and the kinetic properties of the Hxt5 glucose transporter of Saccharomyces cerevisiae. The HXT5 gene was not expressed during growth of the yeast cells in rich medium with glucose or raffinose. However, it became strongly induced during nitrogen or carbon starvation. We have constructed yeast strains constitutively expressing only Hxt5, Hxt1 (low affinity) or Hxt7 (high affinity), but no other glucose transporters. Aerobic fed-batch cultures at quasi steady-state conditions, and aerobic and anaerobic chemostat cultures at steady-state conditions of these strains were used for estimation of the kinetic properties of the individual transporters under in vivo conditions, by investigating the dynamic responses of the strains to changes in extracellular glucose concentration. The K(m) value and the growth properties of the HXT5 single expression strain indicate that Hxt5 is a transporter with intermediate affinity.

Culture Media↗

PubMedDate not supplied

Integrated sampling procedure for metabolome analysis.

Metabolome analysis, the analysis of large sets of intracellular metabolites, has become an important systems analysis method in biotechnological and pharmaceutical research. In metabolic engineering, the integration of metabolome data with fluxome and proteome data into large-scale mathematical models promises to foster rational strategies for strain and cell line improvement. However, the development of reproducible sampling procedures for quantitative analysis of intracellular metabolite concentrations represents a major challenge, accomplishing (i) fast transfer of sample, (ii) efficient quenching of metabolism, (iii) quantitative metabolite extraction, and (iv) optimum sample conditioning for subsequent quantitative analysis. In addressing these requirements, we propose an integrated sampling procedure. Simultaneous quenching and quantitative extraction of intracellular metabolites were realized by short-time exposure of cells to temperatures < or =95 degrees C, where intracellular metabolites are released quantitatively. Based on these findings, we combined principles of heat transfer with knowledge on physiology, for example, turnover rates of energy metabolites, to develop an optimized sampling procedure based on a coiled single tube heat exchanger. As a result, this sampling procedure enables reliable and reproducible measurements through (i) the integration of three unit operations into a one unit operation, (ii) the avoidance of any alteration of the sample due to chemical reagents in quenching and extraction, and (iii) automation. A sampling frequency of 5 s(-)(1) and an overall individual sample processing time faster than 30 s allow observing responses of intracellular metabolite concentrations to extracellular stimuli on a subsecond time scale. Recovery and reliability of the unit operations were analyzed. Impact of sample conditioning on subsequent IC-MS analysis of metabolites was examined as well. The integrated sampling procedure was validated through consistent results from steady-state metabolite analysis of Escherichia coli cultivated in a chemostat at D = 0.1 h(-)(1).

Adenosine Triphosphate↗

PubMedDate not supplied

Investigations of reaction kinetics for immobilized enzymes--identification of parameters in the presence of diffusion limitation.

A method is proposed for identification of kinetic parameters when diffusion of substrates is limiting in reactions catalyzed by immobilized enzymes. This method overcomes conventional sequential procedures, which assume immobilization does not affect the conformation of the enzyme and, thus, consider intrinsic and inherent kinetics to be the same. The coupled equations describing intraparticle mass transport are solved simultaneously using numerical methods and are used for direct estimation of kinetic parameters by fitting modeling results to time-course measurements in a stirred tank reactor. While most traditional procedures were based on Michaelis-Menten kinetics, the method presented here is applicable to more complex kinetic mechanisms involving multiple state variables, such as ping-pong bi-bi. The method is applied to the kinetic resolution of (R/S)-1-methoxy-2-propanol with vinyl acetate catalyzed by Candida antarctica lipase B. A mathematical model is developed consisting of irreversible ping-pong bi-bi kinetics, including competitive inhibition of both enantiomers. The kinetic model, which fits to experimental data over a wide range of both substrates (5-95%) and temperatures (5-56 degrees C), is used for simulations to study typical behavior of immobilized enzyme systems.

Biotechnology↗