Vanadium uptake by yeast cells

During incubation with vanadyl, Saccharomyces cerevisiae yeast cells were able to accumulate millimolar concentrations of this divalent cation within an intracellular compartment. The intracellular vanadyl ions were bound to low molecular weight substances. This was indicated by the isotropic nature of the electron paramagnetic resonance (EPR) spectra of the respective samples. Accumulation of intracellular vanadyl was dependent on presence of glucose during incubation. It could be inhibited by various di- and trivalent metal cations. Of these cations lanthanum displayed the strongest inhibitory action. If yeast cells were exposed to more than 50 microM vanadyl sulfate at a pH higher than 4.0, a potassium loss into the medium was detected. The magnitude of this potassium loss suggests a damage of the plasma membrane caused by vanadyl. Upon addition of vanadate to yeast cells surface-bound vanadyl was detectable after several minutes by EPR. This could be the consequence of extracellular reduction of vanadate to vanadyl. The reduction was followed by a slow accumulation of intracellular vanadium, which could be inhibited by lanthanum or phosphate. Therefore, permeation of vanadyl into the cells can be assumed as one mechanism of vanadium accumulation by yeast during incubation with vanadate.     

Engineering antibodies by yeast display

Since its first application to antibody engineering 15years ago, yeast display technology has been developed into a highly potent tool for both affinity maturing lead molecules and isolating novel antibodies and antibody-like species. Robust approaches to the creation of diversity, construction of yeast libraries, and library screening or selection have been elaborated, improving the quality of engineered molecules and certainty of success in an antibody engineering campaign and positioning yeast display as one of the premier antibody engineering technologies currently in use. Here, we summarize the history of antibody engineering by yeast surface display, approaches used in its application, and a number of examples highlighting the utility of this method for antibody engineering.     

Breeding of a distiller's yeast by hybridization with a wine yeast

Summary Hybrids were constructed between auxotrophic mutants of a heterothallic distiller's strain and a homothallic wine yeast. The hybridization resulted in a significant increase in both ethanol production and tolerance against exogenous ethanol. The hybrids were heterogeneous in ploidy, probably due to segregation of aneuploids during culturing. Sporulation of the hybrids broke down the high productivity, producing spore clones that were mostly of various intermediate levels of performance. However, a meiotic product superior to both crossing partners was also found. The results demonstrate that fermentation capacity can be improved by crossing with a low performance strain. Offprint requests to: M. Sipiczki     

Yeast antizyme mediates degradation of yeast ornithine decarboxylase by yeast but not by mammalian proteasome: new insights on yeast antizyme

Mammalian antizyme (mAz) is a central element of a feedback circuit regulating cellular polyamines by accelerating ornithine decarboxylase (ODC) degradation and inhibiting polyamine uptake. Although yeast antizyme (yAz) stimulates the degradation of yeast ODC (yODC), we show here that it has only a minor effect on polyamine uptake by yeast cells. A segment of yODC that parallels the Az binding segment of mammalian ODC (mODC) is required for its binding to yAz. Although demonstrating minimal homology to mAz, our results suggest that yAz stimulates yODC degradation via a similar mechanism of action. We demonstrate that interaction with yAz provokes degradation of yODC by yeast but not by mammalian proteasomes. This differential recognition may serve as a tool for investigating proteasome functions.     

Induction and acceleration of yeast lysis by addition of fresh yeast autolysate

Summary Addition of 15 % v/v of fresh yeast autolysate to the baker's yeast suspension significantly accelerated cell autolysis. The addition of classical initiators of autolysis (NaCl, ethanol) led to further 20 % increase of protein yield.     

Alcoholic fermentation by agar-immobilized yeast cells

Immobilized yeast cells in agar gel beads were used in a packed bed reactor for the production of ethanol from cane molasses at 30°C, pH 4.5. The maximum productivity, 79.5g ethanol/l.h was obtained with 195g/l reducing sugar as feed. Substrate (64.2%) was utilized at a dilution of 1.33h^-1. The immobilized cell reactor was operated continuously at a constant dilution rate of 0.67h^-1 for 100 days. The maximum specific ethanol productivity and specific sugar uptake rate were 0.610g ethanol/g cell.h and 1.275g sugar/g cell.h, respectively.     

Disruption of yeast membranes by methylphenidate

Methylphenidate blocked sorbose uptake and loss by yeast spheroplasts and, at higher concentrations (30 mm), disrupted the spheroplasts. At still higher concentrations (70 mm), methylphenidate also ruptured the membranes of whole yeast cells; sorbose and materials absorbing at 280 nm were lost from the cells, and methylene blue stained them. Intracellular structures were extensively affected, as shown by electron micrographs, and were more sensitive to disruption by methylphenidate than the external membrane. N-ethylmaleimide and Ca(2+) enhanced the rupture of external membranes by methylphenidate.     

Wine yeast typing by MALDI-TOF MS

For the production of wine, the most important industrially used yeast species is Saccharomyces cerevisiae. Years of experience have shown that wine quality and property are significantly affected by the employed strain conducting the fermentation. Consequently, the ability of a strain level differentiation became an important requirement of modern winemaking. In our study, we showed that the differentiation by time-consuming and laborious biochemical and DNA-based methods to enable a constant beverage quality and characteristics can be replaced by matrix-assisted-laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), accompanied by the additional benefit of an application prediction. Mass fingerprints of 33 Saccharomyces strains, which are commonly used for varying wine fermentations, were generated by MALDI-TOF MS upon optimized sample preparation and instrument settings and analyzed by a cluster analysis for strain or ecotype-level differentiation. As a reference method, delta-PCR was chosen to study the genetic diversity of the employed strains. Finally, the cluster analyses of both methods were compared. It could be shown that MALDI-TOF MS, acting at proteome level, provides valuable information about the relationship between yeast strains and their application potential according to their MALDI mass fingerprint.     

Enhanced yeast immobilization by nutrient starvation

Saccharomyces uvarum NRRL Y1347 cells were immobilized in a porous support. Cell loadings of up to 600 mg dry cell/g support or 70 mg dry cell/cm³ support were obtained. Starvation in a marine environment increased the adhesion strength of immobilized cells.     

Harvesting and dewatering yeast by microflotation

Microbubble has been applied for the recovery of yeast cells from their growth medium using the bioflocculant–chitosan. Results reaching 99% cell recovery were obtained under various conditions examined. The result of bubble size distribution showed that mean bubble size increased as microbubble diffuser pore size was increased. Also, cell recovery efficiency was a function of both bubble size and particle size (cell size). For smaller particles (<50 μm), relatively smaller bubbles (<80 μm) were found to be more effective for recovery, otherwise, relatively larger bubbles (80–150 μm) proved to be efficient in recovering larger particles (particle size: ∼250 μm). Acidic and neutral pHs were effective in separation as hydrophobic particles were formed. As pH tends toward alkalinity, flocs become more hydrophilic, leading to low recovery from the aqueous solution. In addition, separation efficiency was dependent on flocculant dose as increase in concentration improved flocculation and consequently, yeast recovery. However, above a critical concentration, overdosing occurred and inadvertently, recovery efficiency decreased. The application of chitosan as a bioflocculant and the subsequent application of microflotation for the separation of yeast cells proved effective and promises several advantages over non-bubble based separation techniques that preclude continuous industrial-scale production.     

Alcohol production by magnetic immobilized yeast

Summary Saccharomyces cerevisiae was immobilized in calcium alginate gel together with varying concentrations of iron oxide, in the form of magnetite or a colloidal ferrite suspension, Ferrofluid. Inclusion of magnetic material apparently had no adverse effect on the yeast cells as judged from their fermentation capacity, their operational stability as well as their ability to propagatein situ in the presence of nutrients. The usefulness of magnetic preparations in viscous or particle containing media is discussed.     

Kinetics of sulfate uptake by yeast

Uptake of sulfate by yeast requires the presence of a metabolic substrate and is dependent on the time during which the cells have been metabolizing in the absence of sulfate. At low concentrations of sulfate, uptake can be described by simple saturation kinetics. Uptake of sulfate is accompanied by a net proton influx of 3 H⁺ and an efflux of 1 K⁺ for each sulfate ion taken up. Divalent cations stimulate sulfate uptake at low concentrations of sulfate; the maximal rate of uptake is not significantly affected but $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ is lowered. Stimulation by divalent cations shows an optimum at a cation concentration of about 4 mM. Monovalent cations are less effective, trivalent cations are more effective in stimulating sulfate uptake. The results are qualitatively in accordance with the notion, that the effect of cations is due to an effect via the surface potential.     

Qualitative yeast enzyme analysis by electrophoresis

Summary Baker's yeast (Saccharomyces cerevisiae) was cultivated under different intensities of aeration on glucose and on ethanol. Seventeen enzymes of the Embden-Meyerhof pathway and the TCA cycle or related reactions were then assayed by starch gel electrophoresis. There were both qualitative and quantitative differences in many enzymes, most notably in glyceraldehyde-3-phosphate dehydrogenase, alcohol dehydrogenase, isocitrate dehydrogenase, malate dehydrogenase, and fumarase. Enzyme electrophoresis seems to offer a promising method for rapidly obtaining information about many yeast enzymes from a large number of samples.