Description: Diet plays a key role in determining the longevity of the organisms since it has been demonstrated that glucose restriction increases lifespan whereas a high-glucose diet decreases it. However, the molecular basis of how diet leads to the aging process is currently unknown. We propose that the quantity of glucose that fuels respiration influences ROS generation and glutathione levels, and both chemical species impact in the aging process. Herein, we provide evidence that mutation of the gene GSH1 in S. cerevisiae diminishes glutathione levels. Moreover, glutathione levels were higher with 0.5% than in 10% glucose in the gsh1Δ and WT strains. Interestingly, the chronological life span (CLS) was lowered in the gsh1Δ strain cultured with 10% glucose but not under dietary restriction. The gsh1Δ strain also showed an inhibition of the mitochondrial respiration in 0.5 and 10% of glucose but only increased the H 2 O 2 levels under dietary restriction. These results correlate well with the GSH/GSSG ratio, which showed a decrease in gsh1Δ strain cultured with 0.5% glucose. Together, these data indicate that glutathione exhaustion impact negatively both the electron transport chain function and the CLS of yeast, the latter occurring when a low threshold level of this antioxidant is reached, independently of the H 2 O 2 levels.

Description: Pichia pastoris expression system has been widely used in recombinant protein production. So far the majority of heterologous proteins are expressed by methanol inducible promoter P AOX1 and constitutive promoter P GAP . The use of other promoters is rather limited. Here we selected 16 potentially efficient and regulatory promoter candidates based on the RNA-seq and RNA folding free energy Δ G data. GFP and recombinant amylase were inserted after these promoters to reveal their strength and efficiency under different carbon sources and culture scales. Two novel promoters were successfully identified and could possibly be applied in recombinant protein expression: the methanol inducible promoter P 0547 and the constitutive promoter P 0472 .

Description: Genome editing is a form of highly precise genetic engineering which produces alterations to an organism's genome as small as a single base pair with no incidental or auxiliary modifications; this technique is crucial to the field of synthetic biology, which requires such precision in the installation of novel genetic circuits into host genomes. While a new methodology for most organisms, genome editing capabilities have been used in the budding yeast Saccharomyces cerevisiae for decades. In this review, I will present a brief history of genome editing in S. cerevisiae , discuss the current gold standard method of Cas9-mediated genome editing (CMGE), and speculate on future directions of the field.

Description: No abstract is available for this article.

Description: No abstract is available for this article.

Description: The cellular changes induced by heterologous protein expression in the yeast Saccharomyces cerevisiae have been analyzed on many levels and found to be significant. However, even though high-level protein production poses a metabolic burden, evaluation of the expression host at the level of the metabolome has often been neglected. We present a comparison of metabolite profiles of a wild-type strain with those of three strains producing recombinant antibody variants of increasing size and complexity: a scFv fragment, a scFv-Fc fusion protein, and a full-length IgG molecule. Under producing conditions, all three recombinant strains showed a clear decrease in growth rate compared to the wild-type strain and the severity of the growth phenotype increased with size of the protein. The levels of 76 intracellular metabolites were determined using a targeted (semi) quantitative mass spectrometry based approach. Based on unsupervised and supervised multivariate analysis of metabolite profiles, together with pathway activity profiling, the recombinant strains were found to be significantly different from each other and from the wild-type strain. We observed the most prominent changes in metabolite levels for metabolites involved in amino acid and redox metabolism. Induction of the unfolded protein response was detected in all producing strains and is considered to be a contributing factor to the overall metabolic burden on the cells.

Description: One of the hallmarks of Parkinson disease is α-synuclein aggregate deposition that leads to ER stress, Golgi fragmentation, and impaired energy metabolism with consequent redox imbalance. In the last decade, many studies have used Saccharomyces cerevisiae as a model in order to explore the intracellular consequences of α-synuclein overexpression. In this study we propose to evaluate the respiratory outcome of yeast cells expressing α-synuclein. Cell viability, or growth on selective media for respiratory activity was mainly affected in the α-synuclein expressing cells if they were also treated with menadione, which stimulates ROS production. We also tested whether melatonin, a natural antioxidant, would counteract the deleterious effects of α-synuclein and menadione. In fact, melatonin addition improved respiratory growth of α-synuclein/menadione challenged cells, presented a general improvement in the enzymatic activity of the respiratory complexes, and finally elevated the rate of mitophagy, an important cellular process necessary for the clearance of damaged mitochondria. Altogether, our data confirms that α-synuclein impairs respiration in yeast, which can be rescued by melatonin addition.

Description: Candida albicans is the most common human fungal pathogen. The ability to undergo the morphological transition from yeast to hyphal growth is critical for its pathogenesis. Farnesol, a precursor in the isoprenoid/sterol pathway, is a quorum-sensing molecule produced by C. albicans that inhibits hyphal growth in this polymorphic fungus. Interestingly, C. albicans can tolerate farnesol concentrations that are toxic to other fungi. We hypothesized that changes in phospholipid composition are one of the factors contributing to farnesol tolerance in C. albicans . In this study, we found that loss of enzymes that synthesize the phospholipids phosphatidylserine (PS) and/or phosphatidylethanolamine (PE) compromise the tolerance of C. albicans to farnesol. Compared to wild-type, the phospholipid mutant cho1∆/∆ (loss of PS and decreased PE synthesis) shows greater inhibition of growth, loss of ATP production, increased consumption of oxygen, and increased formation of reactive oxygen species (ROS) in the presence of farnesol (FOH). The cho1∆/∆ mutant also exhibits decreased sensitivity to mitochondrial ATPase inhibition, suggesting that cells lacking PS and/or downstream PE rely less on mitochondrial function for ATP synthesis. These data reveal that PS and PE play roles in farnesol tolerance and maintaining mitochondrial respiratory function.

Description: Interspecific hybrids among species in the Saccharomyces genus are frequently detected in anthropic habitats and can also be obtained easily in the laboratory. This occurs because the most important genetic barriers among Saccharomyces species are post-zygotic. Depending on several factors, including the involved strains, the hybridisation mechanism and stabilisation conditions, the hybrids that bear differential genomic constitutions, and hence phenotypic variability, can be obtained. In the present study, Saccharomyces cerevisiae x Saccharomyces uvarum hybrids were constructed using genetically and physiologically different S. uvarum parents at distinct temperatures (13°C and 20°C). The effect of those variables on the main oenological features of the wines obtained with these hybrids was evaluated. Hybrids were successfully obtained in all cases. However, genetic stabilisation based on successive fermentations in white wine at 13°C was significantly longer than that at 20°C. Our results evidenced that irrespectively of the S. uvarum parental and temperature used for hybrid generation and stabilisation, similar physicochemical and aromatic features were found in wines. The hybrids generated herein were characterised by low ethanol production, high glycerol synthesis, and by the capacity to grow at low temperature and to produce malic acid with particular aroma profiles. These features make these hybrids useful for the new winemaking industry within the climate change era frame.

Description: The strong development of molecular biology techniques and next-generation sequencing technologies in the last two decades has significantly improved our understanding of the evolutionary history of Saccharomyces yeasts. It has been shown that many strains isolated from man-made environments are not pure genetic lines, but contain genetic materials from different species that substantially increase their genome complexity. A number of strains have been described as interspecies hybrids, implying different yeast species that under specific circumstances exchange and recombine their genomes. Such fusing usually results in a wide variety of alterations at the genetic and chromosomal levels. The observed changes have suggested a high genome plasticity and a significant role of interspecies hybridization in the adaptation of yeasts to environmental stresses and industrial processes. There is a high probability that harsh wine and beer fermentation environments, from which the majority of interspecies hybrids have been isolated so far, influence their selection and stabilization as well as their genomic and phenotypic heterogeneity. The lessons we have learned about geno- and phenotype plasticity and the diversity of natural and commercial yeast hybrids have already had a strong impact on the development of artificial hybrids that can be successfully used in the fermentation-based food and beverage industry. The creation of artificial hybrids through the crossing of strains with desired attributes is a possibility to obtain a vast variety of new, but not genetically modified yeasts with a range of improved and beneficial traits.