Matthias Reuss

An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.

Physiological responses to mixing in large scale bioreactors

Escherichia coli fed-batch cultivations at 22 m3 scale were compared to corresponding laboratory scale processes and cultivations using a scale-down reactor furnished with a high-glucose concentration zone to mimic the conditions in a feed zone of the large bioreactor. Formate accumulated in the large reactor, indicating the existence of oxygen limitation zones. It is suggested that the reduced biomass yield at large scale partly is due to repeated production/re-assimilation of acetate from overflow metabolism and mixed acid fermentation products due to local moving zones with oxygen limitation. The conditions that generated mixed-acid fermentation in the scale-down reactor also induced a number of stress responses, monitored by analysis of mRNA of selected stress induced genes. The stress responses were relaxed when the cells returned to the substrate limited and oxygen sufficient compartment of the reactor. Corresponding analysis in the large reactor showed that the concentration of mRNA of four stress induced genes was lowest at the sampling port most distant from the feed zone. It is assumed that repeated induction/relaxation of stress responses in a large bioreactor may contribute to altered physiological properties of the cells grown in large-scale bioreactor. Flow cytometric analysis revealed reduced damage with respect to cytoplasmic membrane potential and integrity in cells grown in the dynamic environments of the large scale reactor and the scale-down reactor.

In vivo analysis of metabolic dynamics in Saccharomyces cerevisiae : I. Experimental observations

The goal of this work was to obtain rapid sampling technique to measure transient metabolites in vivo. First, a pulse of glucose was added to a culture of the yeast Saccharomyces cerevisiae growing aerobically under glucose limitation. Next, samples were removed at 2 to 5 s intervals and quenched using methods that depend on the metabolite measured. Extracellular glucose, excreted products, as well as glycolytic intermediates (G6P, F6P, FBP, GAP, 3-PG, PEP, Pyr) and cometabolites (ATP, ADP, AMP, NAD(+), NADH) were measured using enzymatic or HPLC methods. Significant differences between the adenine nucleotide concentrations in the cytoplasm and mitochondria indicated the importance of compartmentation for the regulation of the glycolysis. Changes in the intra- and extracellular levels of metabolites confirmed that glycolysis is regulated on a time scale of seconds. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 305-316, 1997.

In vivo analysis of metabolic dynamics in Saccharomyces cerevisiae: II. Mathematical model

A mathematical model of glycolysis in Saccharomyces cerevisiae is presented. The model is based on rate equations for the individual reactions and aims to predict changes in the levels of intra- and extracellular metabolites after a glucose pulse, as described in part I of this study. Kinetic analysis focuses on a time scale of seconds, thereby neglecting biosynthesis of new enzymes. The model structure and experimental observations are related to the aerobic growth of the yeast. The model is based on material balance equations of the key metabolites in the extracellular environment, the cytoplasm and the mitochondria, and includes mechanistically based, experimentally matched rate equations for the individual enzymes. The model includes removal of metabolites from glycolysis and TCC for biosynthesis, and also compartmentation and translocation of adenine nucleotides. The model was verified by in vivo diagnosis of intracellular enzymes, which includes the decomposition of the network of reactions to reduce the number of parameters to be estimated simultaneously. Additionally, sensitivity analysis guarantees that only those parameters are estimated that contribute to systems trajectory with reasonable sensitivity. The model predictions and experimental observations agree reasonably well for most of the metabolites, except for pyruvate and adenine nucleotides. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 592-608, 1997.

Sharper low-power STED nanoscopy by time gating.

Applying pulsed excitation together with time-gated detection improves the fluorescence on-off contrast in continuous-wave stimulated emission depletion (CW-STED) microscopy, thus revealing finer details in fixed and living cells using moderate light intensities. This method also enables super-resolution fluorescence correlation spectroscopy with CW-STED beams, as demonstrated by quantifying the dynamics of labeled lipid molecules in the plasma membrane of living cells.

A kinetic study of immobilized lipase catalysing the synthesis of isoamyl acetate by transesterification in n-hexane

Isoamyl acetate was synthesized by lipase-catalyzed transesterification of ethyl acetate in n-hexane. The selectivity and rates of ester formation decreased when water content of the immobilized enzyme exceeded 3% (w/w). Experimental observations clearly indicate that the substrates as well as the product (ethanol) act as dead-end inhibitors. A ping-pong bi-bi mechanism with competitive inhibition by substrates and products is proposed that predicts the experimental observation satisfactorily.

Production of sophorolipids in high concentration from deproteinized whey and rapeseed oil in a two stage fed batch process using Candida bombicola ATCC 22214 and Cryptococcus curvatus ATCC 20509

High concentrations of 422 g sophorolipids l−1 were produced using a two-stage cultivation process: first deproteinized whey concentrate (DWC) containing 110 g lactose l−1 was used for cultivation of the yeast Cryptococcus curvatus ATCC 20509, resulting in 34 g dry weight l−1, 20 g single-cell oil l−1 and reducing the chemical oxygen demand (COD) from 159 g l−1 to 35 g oxygen l−1. Afterwards cells were disrupted by passing the cell suspension directly through a high pressure laboratory homogeniser. After autoclavation, the resulting crude cell extract containing the single-cell oil served as substrate for growth of Candida bombicola ATCC 22214 and for sophorolipid production in a second stage. When the single-cell oil was consumed, repeated feeding of 400 g rapeseed oil l−1 was started increasing the yield of sophorolipids to 422 g l−1. A simple technique for product isolation, sedimentation, could be used to harvest the crude sophorolipids.

Multiscale Modelling of Vascular Tumour Growth in 3D: The Roles of Domain Size and Boundary Conditions

We investigate a three-dimensional multiscale model of vascular tumour growth, which couples blood flow, angiogenesis, vascular remodelling, nutrient/growth factor transport, movement of, and interactions between, normal and tumour cells, and nutrient-dependent cell cycle dynamics within each cell. In particular, we determine how the domain size, aspect ratio and initial vascular network influence the tumours growth dynamics and its long-time composition. We establish whether it is possible to extrapolate simulation results obtained for small domains to larger ones, by constructing a large simulation domain from a number of identical subdomains, each subsystem initially comprising two parallel parent vessels, with associated cells and diffusible substances. We find that the subsystem is not representative of the full domain and conclude that, for this initial vessel geometry, interactions between adjacent subsystems contribute to the overall growth dynamics. We then show that extrapolation of results from a small subdomain to a larger domain can only be made if the subdomain is sufficiently large and is initialised with a sufficiently complex vascular network. Motivated by these results, we perform simulations to investigate the tumours response to therapy and show that the probability of tumour elimination in a larger domain can be extrapolated from simulation results on a smaller domain. Finally, we demonstrate how our model may be combined with experimental data, to predict the spatio-temporal evolution of a vascular tumour.

Molecular orientation affects localization accuracy in superresolution far-field fluorescence microscopy.

We investigate the cooperative effect of molecular tilt and defocus on fluorophore localization by centroid calculation in far-field superresolution microscopy based on stochastic single molecule switching. If tilt angle and defocus are unknown, the localization contains systematic errors up to about ±125 nm. When imaging rotation-impaired fluorophores of unknown random orientation, the average localization accuracy in three-dimensional samples is typically limited to about ±32 nm, restricting the attainable resolution accordingly.

rsEGFP2 enables fast RESOLFT nanoscopy of living cells.

The super-resolution microscopy called RESOLFT relying on fluorophore switching between longlived states, stands out by its coordinate-targeted sequential sample interrogation using low light levels. While RESOLFT has been shown to discern nanostructures in living cells, the reversibly photoswitchable green fluorescent protein (rsEGFP) employed in these experiments was switched rather slowly and recording lasted tens of minutes. We now report on the generation of rsEGFP2 providing faster switching and the use of this protein to demonstrate 25–250 times faster recordings. DOI: http://dx.doi.org/10.7554/eLife.00248.001