Fermentation is one of the oldest forms of food preservation in the world. In South "merica, most fermented beverages are nondairy products featuring several other food raw materials such as cereals, fruits, and vegetables. Generally, natural fermentations are carried out by yeast and lactic acid bacteria forming a complex microbiota that acts in cooperation. Yeast have a prominent role in the production of beverages, due to the ability to accumulate high levels of ethanol and to produce highly desirable aroma compounds, but lactic acid bacteria are particularly important in fermentation because they produce desirable acids, flavor compounds, and peptides that inhibit the growth of undesirable organisms. "mong the South "merica beverages based on cereals and vegetables, the fermented beverages chicha, caxiri, cauim and champús, and cachaça, a fermented and distilled beverage, could be cited. Genetic and physiological analyses of Saccharomyces cerevisiae strains isolated from cachaça have been shown to present interesting traits for beer production, such as flocculation and production of aroma compounds, fundamental to high-quality beer. The study of these traditional beverages allows the identification of new microorganism strains displaying enhanced resistance or new flavor and aroma profiles that could lead to applications in several industries and ultimately new products.

BackgroundSaccharomyces cerevisiae multicellular communities are sustained by a scaffolding extracellular matrix, which provides spatial organization, and nutrient and water availability, and ensures group survival. According to this tissue-like biology, the yeast extracellular matrix (yECM) is analogous to the higher Eukaryotes counterpart for its polysaccharide and proteinaceous nature. Few works focused on yeast biofilms, identifying the flocculin Flo11 and several members of the HSP70 in the extracellular space. Molecular composition of the yECM, is therefore mostly unknown. The homologue of yeast Gup1 protein in high Eukaryotes (HHATL) acts as a regulator of Hedgehog signal secretion, therefore interfering in morphogenesis and cell-cell communication through the ECM, which mediates but is also regulated by this signalling pathway. In yeast, the deletion of GUP1 was associated with a vast number of diverse phenotypes including the cellular differentiation that accompanies biofilm formation.MethodsS. cerevisiae W303-1A wt strain and gup1∆ mutant were used as previously described to generate biofilm-like mats in YPDa from which the yECM proteome was extracted. The proteome from extracellular medium from batch liquid growing cultures was used as control for yECM-only secreted proteins. Proteins were separated by SDS-PAGE and 2DE. Identification was performed by HPLC, LC-MS/MS and MALDI-TOF/TOF. The protein expression comparison between the two strains was done by DIGE, and analysed by DeCyder Extended Data Analysis that included Principal Component Analysis and Hierarchical Cluster Analysis. ResultsThe proteome of S. cerevisiae yECM from biofilm-like mats was purified and analysed by Nano LC-MS/MS, 2D Difference Gel Electrophoresis (DIGE), and MALDI-TOF/TOF. Two strains were compared, wild type and the mutant defective in GUP1. As controls for the identification of the yECM-only proteins, the proteome from liquid batch cultures was also identified. Proteins were grouped into distinct functional classes, mostly Metabolism, Protein Fate/Remodelling and Cell Rescue and Defence mechanisms, standing out the presence of heat shock chaperones, metalloproteinases, broad signalling cross-talkers and other putative signalling proteins. The data has been deposited to the ProteomeXchange with identifier PXD001133.ConclusionsyECM, as the mammalian counterpart, emerges as highly proteinaceous. As in higher Eukaryotes ECM, numerous proteins that could allow dynamic remodelling, and signalling events to occur in/and via yECM were identified. Importantly, large sets of enzymes encompassing full antagonistic metabolic pathways, suggest that mats develop into two metabolically distinct populations, suggesting that either extensive moonlighting or actual metabolism occurs extracellularly. The gup1∆ showed abnormally loose ECM texture. Accordingly, the correspondent differences in proteome unveiled acetic and citric acid producing enzymes as putative players in structural integrity maintenance.

BackgroundGUP1 gene was primarily identified in Saccharomyces cerevisiae being connected with glycerol uptake defects in association with osmotic stress response. Soon after, Gup1p was implicated in a complex and extensive series of phenotypes involving major cellular processes. These include membrane and wall maintenance, lipid composition, bud-site selection, cytoskeleton orientation, vacuole morphology, secretory/endocytic pathway, GPI anchors remodelling, and lipid-ordered domains assembly, which is compatible with their inclusion in the Membrane Bound O-acyl transferases (MBOAT) family. In mammals, it has been described as a negative regulator of the Sonic hedgehog pathway involved in morphogenesis, differentiation, proliferation, among other processes.ResultsWe show that Candida albicans Gup1p strongly interferes with the capacity of cells to develop hyphae, to adhere, to invade, and to form a biofilm, all of which are significant virulence factors. Furthermore, the mutant colonies exhibited an aberrant morphology/differentiation pattern. Identically to S. cerevisiae, Cagup1Δ null mutant was more resistant to antifungals like fluconazole, ketoconazole, and clotrimazole, and displayed an abnormal even sterol distribution at the plasma membrane.ConclusionsThis work is the first study in the opportunistic yeast Candida albicans, showing a role for the GUP1 gene in virulence as well as in the mechanisms underlying antifungal resistance. Moreover, its impact is even more significant since these results, taken together with all the knowledge about GUP1 gene (from S. cerevisiae and mammals) give consistence to the possibility that Gup1p may be part of a yeast morphogenic pathway parallel to the mammalian Hedgehog.

Microbial cell factories with the ability to maintain high productivity in the presence of weak organic acids, such as acetic acid, are required in many industrial processes. For example, fermentation media derived from lignocellulosic biomass are rich in acetic acid and other weak acids. The rate of diffusional entry of acetic acid is one parameter determining the ability of microorganisms to tolerance the acid. The present study demonstrates that the rate of acetic acid diffusion in S. cerevisiae is strongly affected by the alcohols ethanol and n-butanol. Ethanol of 40 g/L and n-butanol of 8 g/L both caused a 65% increase in the rate of acetic acid diffusion, and higher alcohol concentrations caused even greater increases. Molecular dynamics simulations of membrane dynamics in the presence of alcohols demonstrated that the partitioning of alcohols to the head group region of the lipid bilayer causes a considerable increase in the membrane area, together with reduced membrane thickness and lipid order. These changes in physiochemical membrane properties lead to an increased number of water molecules in the membrane interior, providing biophysical mechanisms for the alcohol-induced increase in acetic acid diffusion rate. n-butanol affected S. cerevisiae and the cell membrane properties at lower concentrations than ethanol, due to greater and deeper partitioning in the membrane. This study demonstrates that the rate of acetic acid diffusion can be strongly affected by compounds that partition into the cell membrane, and highlights the need for considering interaction effects between compounds in the design of microbial processes.

BackgroundDuring the past years, yeast has been successfully established as a model to study mechanisms of programmed cell death regulation. Saccharomyces cerevisiae commits to cell death showing typical hallmarks of metazoan apoptosis, in response to different stimuli. Gup1p, an O-acyltransferase, is required for several cellular processes that are related to apoptosis development, such as rafts integrity and stability, lipid metabolism including GPI anchor correct remodeling, proper mitochondrial and vacuole function, bud site selection and actin dynamics. Therefore, we hypothesize that apoptotic process would be affected by GUP1 deletion.ResultsIn the present work we used two known apoptosis inducing conditions, chronological aging and acetic acid, to assess several apoptotic markers in gup1∆ mutant strain. We found that this mutant presents a significantly reduced chronological lifespan as compared to Wt and it is also highly sensitive to acetic acid treatment. In addition, it presents extremely high levels of ROS. There were notorious differences on apoptotic markers between Wt and gup1∆ mutant strains, namely on the maintenance of plasma membrane integrity, on the phosphatidylserine externalization, on the depolarization of mitochondrial membrane and on the chromatin condensation. Those suggested that the mutant, under either condition, probably dies of necrosis and not from apoptosis.ConclusionsTo Gup1p has been assigned an important function on lipid rafts assembly/integrity, lipid metabolism and GPI anchor remodeling. Our results provide, for the first time, the connection of the integrity of yeast lipid rafts and apoptosis induction and/or signaling, giving new insights into the molecular mechanisms underlying this process in yeast.