Whole-Cell Biocatalysis in Seawater: New Halotolerant Yeast Strains for the Regio- and Stereoselectivity Reduction of 1-Phenylpropane-1,2-Dione in Saline-Rich Media.

The application of green chemistry concepts in catalysis has considerably increased in recent years, and the interest in using sustainable solvents in the chemical industry is growing. One of the recent proposals to fall in line with this is to employ seawater as a solvent in biocatalytic processes. This involves selecting halotolerant strains capable of carrying out chemical conversions in the presence of the salt concentrations found in this solution. Recent studies by our group have revealed the interest in using strains belonging to Debaryomyces and Schwanniomyces for catalytic processes run in this medium. In the present work, we select other yeasts based on their halotolerance to widen the scope of this strategy. We consider them for the monoreduction of 1-phenylpropane-1,2-dione, a well-characterized reaction that produces acyloin intermediates of pharmaceutical interest. The results obtained herein indicate that using seawater as a solvent for this reaction is possible. The best ones were obtained for Saccharomyces cerevisiae FY86 and Kluyveromyces marxianus, for which acyloins with different stereochemistry were obtained with good to excellent enantiomeric excess.

Towards an understanding of the adaptation of wine yeasts to must: relevance of the osmotic stress response.

During the transformation of grape must sugars in ethanol, yeasts belonging to Saccharomyces cerevisiae strains are particularly involved. One of the stress conditions that yeast cells have to cope with during vinification, especially at the time of inoculation into must, is osmotic stress caused by high sugar concentrations. In this work, we compare several laboratory and wine yeast strains in terms of their ability to start growth in must. By means of transcriptomic approaches and the determination of glycerol intracellular content, we propose several clues for yeast strains to adapt to the wine production conditions: the high expression of genes involved in both biosynthetic processes and glycerol biosynthesis, and the appropriate levels of intracellular glycerol. Besides, we demonstrate that the pre-adaptation of the wine yeast strains showing growth problems at the beginning of vinification in a rehydration medium containing 2% or 5% glucose (depending on the yeast strain considered) may increase their vitality when inoculated into high sugar media.

Antimicrobial peptides and their superior fluorinated analogues: structure-activity relationships as revealed by NMR spectroscopy and MD calculations.

The conformations of two synthetic pentapeptides with antimicrobial activity and their 4-fluorophenylalanine (Pff)-containing analogues (ArXArXAr-NH(2); Ar=Phe, Pff; X=Lys, Arg) have been studied. NMR experiments carried out both in aqueous fluoroalcohol solutions and SDS micelles permitted their interactions with membrane-like environments to be explored. WaterLOGSY experiments and Mn(2+)-based paramagnetic probes were also applied to assess their orientations with respect to the SDS micelles. In addition, pulse-field gradient (PFG) diffusion NMR spectroscopy studies were conducted, under different experimental conditions (i.e., concentration, temperature) to characterize the possible changes in the peptides' aggregation states as a putative critical factor for their antimicrobial activity. Finally, molecular dynamics simulations on a variety of conformations showed the intrinsic flexibility of these peptides in both aqueous solutions and membrane-mimetic systems.

A genetic screen in Saccharomyces cerevisiae identifies new genes that interact with mex67-5, a temperature-sensitive allele of the gene encoding the mRNA export receptor.

The Mex67p protein, together with Mtr2p, functions as the mRNA export receptor in Saccharomyces cerevisiae by interacting with both mRNA and nuclear pore complexes. To identify genes that interact functionally with MEX67, we used transposon insertion to search for mutations that suppressed the temperature-sensitive mex67-5 allele. Four suppressors are described here. The screen revealed that mutant Mex67-5p, but not wild-type Mex67p, is a target of the nuclear protein quality control mediated by San1p, a ubiquitin-protein ligase that participates in degradation of aberrant chromatin-associated proteins. Our finding that overexpression of the SPT6 gene alleviates the growth defects of the mex67-5 strain, together with the impairment of poly(A)(+) RNA export caused by depletion of Spt6p or the related protein Iws1p/Spn1p, supports the mechanism proposed in mammalian cells for Spt6-mediated co-transcriptional loading of mRNA export factors during transcription elongation. Finally, our results also uncovered genetic connections between Mex67p and the poly(A) nuclease complex and with components of chromatin boundary elements.

Sulfur and adenine metabolisms are linked, and both modulate sulfite resistance in wine yeast.

Sulfite treatment is the most common way to prevent grape must spoilage in winemaking because the yeast Saccharomyces cerevisiae is particularly resistant to this chemical. In this paper we report that sulfite resistance depends on sulfur and adenine metabolism. The amount of adenine and methionine in a chemically defined growth medium modulates sulfite resistance of wine yeasts. Mutations in the adenine biosynthetic pathway or the presence of adenine in a synthetic minimal culture medium increase sulfite resistance. The presence of methionine has the opposite effect, inducing a higher sensitivity to SO(2). The concentration of methionine, adenine, and sulfite in a synthetic grape must influences the progress of fermentation and at the transcriptional level the expression of genes involved in sulfur (MET16), adenine (ADE4), and acetaldehyde (ALD6) metabolism. Sulfite alters the pattern of expression of all these genes. This fact indicates that the response to this stress is complex and involves several metabolic pathways.

Expression of stress response genes in wine strains with different fermentative behavior.

The response to adverse growth conditions in yeast depends on the activation of signal transduction pathways which result in transcriptional changes and synthesis of protective molecules. During wine production, yeast cells are affected by a plethora of stress situations. In this work we have analyzed the fermentative behavior in synthetic must for six different wine yeast strains. In addition, we followed the expression of several stress response genes during the first half of the vinification. Our results indicate that common patterns of stress response are found among all the strains, but also that a subset of genes are differentially expressed according to the fermentative behavior of the various strains. Particularly, in the strains with the most severe fermentative problems, higher (and in some cases maintained) mRNA levels of many genes were found. The relevance of an equilibrium between stress response and growth efficiency during wine fermentation is discussed.

Exposure of Saccharomyces cerevisiae to acetaldehyde induces sulfur amino acid metabolism and polyamine transporter genes, which depend on Met4p and Haa1p transcription factors, respectively.

Acetaldehyde is a toxic compound produced by Saccharomyces cerevisiae cells under several growth conditions. The adverse effects of this molecule are important, as significant amounts accumulate inside the cells. By means of global gene expression analyses, we have detected the effects of acetaldehyde addition in the expression of about 400 genes. Repressed genes include many genes involved in cell cycle control, cell polarity, and the mitochondrial protein biosynthesis machinery. Increased expression is displayed in many stress response genes, as well as other families of genes, such as those encoding vitamin B1 biosynthesis machinery and proteins for aryl alcohol metabolism. The induction of genes involved in sulfur metabolism is dependent on Met4p and other well-known factors involved in the transcription of MET genes under nonrepressing conditions of sulfur metabolism. Moreover, the deletion of MET4 leads to increased acetaldehyde sensitivity. TPO genes encoding polyamine transporters are also induced by acetaldehyde; in this case, the regulation is dependent on the Haa1p transcription factor. In this paper, we discuss the connections between acetaldehyde and the processes affected by this compound in yeast cells with reference to the microarray data.