Heterologous expression reveals unique properties of Trk K+ importers from nonconventional biotechnologically relevant yeast species together with their potential to support Saccharomyces cerevisiae growth.

In the model yeast Saccharomyces cerevisiae, Trk1 is the main K+ importer. It is involved in many important physiological processes, such as the maintenance of ion homeostasis, cell volume, intracellular pH, and plasma-membrane potential. The ScTrk1 protein can be of great interest to industry, as it was shown that changes in its activity influence ethanol production and tolerance in S. cerevisiae and also cell performance in the presence of organic acids or high ammonium under low K+ conditions. Nonconventional yeast species are attracting attention due to their unique properties and as a potential source of genes that encode proteins with unusual characteristics. In this work, we aimed to study and compare Trk proteins from Debaryomyces hansenii, Hortaea werneckii, Kluyveromyces marxianus, and Yarrowia lipolytica, four biotechnologically relevant yeasts that tolerate various extreme environments. Heterologous expression in S. cerevisiae cells lacking the endogenous Trk importers revealed differences in the studied Trk proteins' abilities to support the growth of cells under various cultivation conditions such as low K+ or the presence of toxic cations, to reduce plasma-membrane potential or to take up Rb+ . Examination of the potential of Trks to support the stress resistance of S. cerevisiae wild-type strains showed that Y. lipolytica Trk1 is a promising tool for improving cell tolerance to both low K+ and high salt and that the overproduction of S. cerevisiae's own Trk1 was the most efficient at improving the growth of cells in the presence of highly toxic Li+ ions.

Salt and oxidative stress tolerance in Debaryomyces hansenii and Debaryomyces fabryi.

We report the characterization of five strains belonging to the halotolerant highly related Debaryomyces hansenii/fabryi species. The analysis performed consisted in studying tolerance properties, membrane characteristics, and cation incell amounts. We have specifically investigated (1) tolerance to different chemicals, (2) tolerance to osmotic and salt stress, (3) tolerance and response to oxidative stress, (4) reactive oxygen species (ROS) content, (5) relative membrane potential, (6) cell volume, (7) K(+) and Na(+) ion content, and (8) membrane fluidity. Unexpectedly, no direct relationship was found between one particular strain, Na(+) content and its tolerance to NaCl or between its ROS content and its tolerance to H(2)O(2). Results show that, although in general, human origin D. fabryi strains were more resistant to oxidative stress and presented shorter doubling times and smaller cell volume than food isolated D. hansenii ones, strains belonging to the same species can be significantly different. Debaryomyces fabryi CBS1793 strain highlighted for its extremely tolerant behavior when exposed to the diverse stress factors studied.

Monovalent cations regulate expression and activity of the Hak1 potassium transporter in Debaryomyces hansenii.

Debaryomyces hansenii was able to grow in a medium containing residual amounts of K(+), indicating the activity of high affinity K(+) transporters. Transcriptional regulation analysis of the genes encoding the two potassium uptake systems in D. hansenii revealed that while DhTRK1 is not regulated at transcriptional level, expression of DhHAK1 required starvation in the absence of K(+) and Na(+) and was not affected by changes in membrane potential. Rb(+) transport in cells expressing DhHAK1 was activated by external Na(+) or acidic pH and inhibited by high pH. We propose a K(+)-H(+) symporter that, under certain conditions may work as a K(+)-Na(+) transporter, as the mechanism driving K(+) influx mediated by DhHak1p.

The co-action of osmotic and high temperature stresses results in a growth improvement of Debaryomyces hansenii cells.

Debaryomyces hansenii is a salt tolerant yeast species, often isolated from sea water or found among other spoilage yeasts in several types of food. In this work, we examined the influence of temperature and increased osmotic pressure (two parameters also important in food industry) on D. hansenii growth. Several other authors showed that its growth at the normal yeast cultivation temperature (28 to 30 degrees C) is stimulated by the presence of sodium, in contrast to the growth of Saccharomyces cerevisiae, which is inhibited by the presence of sodium under the same experimental conditions. Here we show that the previously reported growth stimulation by sodium is temperature dependent in D. hansenii and can be observed under conditions that already amount to high temperature stress for D. hansenii. At a lower temperature (more convenient for D. hansenii cultivation), we found no significant improvement or even an inhibition of cell growth in the presence of Na(+). The growth of D. hansenii at high temperatures is also improved by the presence of potassium or sorbitol. Moreover, the temperature dependence of stimulatory effects of increased osmotic pressure in media does not seem to be unique for D. hansenii; similar relationships between the growth, cultivation temperature and presence of osmolytes we also observed for S. cerevisiae and Schizosaccharomyces pombe.

The Debaryomyces hansenii NHA1 gene encodes a plasma membrane alkali-metal-cation antiporter with broad substrate specificity.

Debaryomyces hansenii is a yeast species often found in salty environments. Its genome sequence is known completely, but the mechanisms behind its halotolerance are poorly understood. In the D. hansenii genome, there is a gene strongly homologous to the Saccharomyces cerevisiae NHA1 gene (encoding a plasma membrane Na+/H+ antiporter). We isolated this DhNHA1 gene from two D. hansenii strains (CBS 767 and CBS 1793) differing in their osmotolerance. Both DhNHA1 alleles were heterologously expressed in a S. cerevisiae strain lacking its own systems for the efflux of alkali metal cations (BW31a, ena1-4delta nha1delta). D. hansenii Na+/H+ antiporters were localized in the plasma membrane of BW31a cells, their presence increased BW31a tolerance to sodium, potassium, lithium and also rubidium. Measurements of Na+ and K+ efflux from S. cerevisiae cells expressing DhNHA1 alleles show that the D. hansenii antiporters efficiently transported both cations out of cells. The sodium and potassium transport activity of Nha1 antiporters from both D. hansenii strains was almost identical, indicating that plasma membrane antiporter activity is not one of the factors determining the different levels of halotolerance in the two strains.