Project description:Freeze-thaw stress causes various cellular damages, survival and proliferation defects, and metabolic alterations, although how cells cope with it is poorly understood. In this study, model dough fermentations using two different strains of Saccharomyces cerevisiae baker’s yeast were compared after two-week cell preservation in the refrigerator or in the freezer. As a result, one strain specifically exhibited a decreased fermentation rate after exposed to freeze-thaw stress. A DNA microarray analysis of the cells during fermentation revealed that the genes involved in oxidative phosphorylation were upregulated after the freeze-thawing process in the stress-sensitive strain, suggesting a metabolism switching from glycolysis to respiration. In the identical strain, however, most of the genes that encode the components of the proteasome complex were commonly downregulated, and ubiquitinated proteins were highly accumulated by freeze-thaw stress. In the cells with a laboratory-strain background, treatment with a proteasome inhibitor MG132 or deletion of each transcriptional activator gene for the proteasomal genes (RPN4, PDR1, or PDR3) led to a marked decrease in the rate of model dough fermentation using the frozen cells. Based on these data, degradation of freeze-thaw damaged proteins by proteasome may guarantee the high fermentation performance. Furthermore, a heterozygous dominant-negative PDR3 allele (A148T/A229V/H336R/L541P) was found in the diploid genome of the stress-sensitive baker’s yeast strain, which may be associated with the decreased fermentation rate. Removal of such responsible mutations may improve the freeze-thaw stress tolerance and the fermentation performance of baker’s yeast strains, as well as other industrial S. cerevisiae yeast strains.
Project description:Gene expression profiles of baker’s yeast during initial dough-fermentation were investigated using liquid fermentation media to obtain insights at the molecular level into rapid adaptation mechanisms of baker’s yeast. Results showed that onset of fermentation caused drastic changes in gene expression profiles within 15 min. Genes involved in the tricarboxylic acid (TCA) cycle were down-regulated and genes involved in glycolysis were up-regulated, indicating a metabolic shift from respiration to fermentation. Genes involved in ethanol production (PDC genes and ADH1), in glycerol synthesis (GPD1 and HOR2), and in low-affinity hexose transporters (HXT1 and HXT3) were up-regulated at the beginning of model dough-fermentation. Among genes up-regulated at 15 min, several genes classified as transcription were down-regulated within 30 min. These down-regulated genes are involved in messenger RNA splicing and ribosomal protein biogenesis, in zinc finger transcription factor proteins, and in transcriptional regulator (SRB8, MIG1). In contrast, genes involved in amino acid metabolism and in vitamin metabolism, such as arginine biosynthesis, riboflavin biosynthesis, and thiamin biosynthesis, were subsequently up-regulated after 30 min. Interestingly, the genes involved in the unfolded protein response (UPR) pathway were also subsequently up-regulated. Our study presents the first overall description of the transcriptional response of baker’s yeast during dough-fermentation, and will thus help clarify genomic responses to various stresses during commercial fermentation processes. Keywords: fermentation
Project description:Frozen dough baking is useful method in the modern bread-making industry. However, the fermentation activity of baker’s yeast dramatically decreased after thawing due to freeze injuries, because baker’s yeast cells contained in dough experience freeze injuries during freeze-thaw processes. Here, we performed genome-wide expression analysis to determine genetic response in baker’s yeasts under freeze-thaw condition using a DNA microarray analysis. Functional and clustering analyses in gene expression reveal that genes could be characterized by the term of freeze-thaw stress. Under short-term freeze stress (freeze treatment for 3 day), genes involved in ribosomal protein were up-regulated. Under long-term freeze stress (freeze treatment for longer than 7 day), genes involved in energy synthesis were up-regulated. In each phase, genes involved in protein damage, several stresses and trehalose and glycogen metabolism were also up-regulated. Through these freeze stress, yeast cells may improve reduced efficiency of translation and enhanced cell protection mechanism to survive under freeze stress condition. These regulations of these genes would be controlled by the cAMP-protein kinase A pathway. Keywords: baker’s yeast, freeze-thaw stress, gene expression, freezing period
Project description:In frozen dough baking technology, baker’s yeast Saccharomyces cerevisiae encounter freeze-thaw injury. After thawing, dramatically decrease in cell viability and fermentation activity is caused by freeze-thaw injury. The freezing period is critical factor in freeze-thaw injury, thus we focused and investigated time-dependent gene expression profiles in recovery process from freeze injury. First, changes in gene expression profiles in S. cerevisiae in recovery process from freeze-thaw injury were analyzed using a DNA microarray. The results showed the genes which were involved in homeostasis of metal ions were time-dependent up-regulated 2-fold or more in a series. Then we examined whether these genes were related to tolerance in freeze-thaw injury by using deletion strain. The results showed that deletion of MAC1, CTR1, and PCA1 genes which involved in copper ion transport exhibited freeze-thaw sensitivity in compared with wild type. These genes are involved in copper ion uptake to a cell under a copper deficiency condition or in copper ion homeostasis, suggesting that it may be related between freeze-thaw injury and copper ion transport. To determine the effect of supplementation of copper ion on cells after freeze-thaw treatment, cell viability, intracellular superoxide dismutase (SOD) activity, and intracellular levels of reactive oxygen species (ROS) were examined by various copper ion condition medium. The results showed that intracellular SOD activity was increased and intracellular levels of ROS were decreased by supplementation of copper ion, but there was no significant difference in cell viability. These results of the present study may suggest that copper ion concentration in yeast cell after freeze-thaw treatment is important to recovery from freeze-thaw injury due to redox control of intracellular levels of ROS, but copper ion did not directly affect cell viability.
Project description:During fermentation Saccharomyces yeast produces various aroma-active metabolites determining the different characteristics of aroma and taste in fermented beverages. Amino acid utilization by yeast during brewer´s wort fermentation is seen as linked to flavour profile. To better understand the relationship between the biosynthesis of aroma relevant metabolites and the importance of amino acids, DNA microarrays were performed for Saccharomyces cerevisiae strain S81 and Saccharomyces pastorianus var. carlsbergensis strain S23, respectively. Thereby, changes in transcription of genes were measured, which are associated with amino acid assimilation and its derived aroma-active compounds during fermentation.
Project description:Gene expression profiles of bakerâs yeast during initial dough-fermentation were investigated using liquid fermentation media to obtain insights at the molecular level into rapid adaptation mechanisms of bakerâs yeast. Results showed that onset of fermentation caused drastic changes in gene expression profiles within 15 min. Genes involved in the tricarboxylic acid (TCA) cycle were down-regulated and genes involved in glycolysis were up-regulated, indicating a metabolic shift from respiration to fermentation. Genes involved in ethanol production (PDC genes and ADH1), in glycerol synthesis (GPD1 and HOR2), and in low-affinity hexose transporters (HXT1 and HXT3) were up-regulated at the beginning of model dough-fermentation. Among genes up-regulated at 15 min, several genes classified as transcription were down-regulated within 30 min. These down-regulated genes are involved in messenger RNA splicing and ribosomal protein biogenesis, in zinc finger transcription factor proteins, and in transcriptional regulator (SRB8, MIG1). In contrast, genes involved in amino acid metabolism and in vitamin metabolism, such as arginine biosynthesis, riboflavin biosynthesis, and thiamin biosynthesis, were subsequently up-regulated after 30 min. Interestingly, the genes involved in the unfolded protein response (UPR) pathway were also subsequently up-regulated. Our study presents the first overall description of the transcriptional response of bakerâs yeast during dough-fermentation, and will thus help clarify genomic responses to various stresses during commercial fermentation processes. Experiment Overall Design: Saccharomyces cerevisiae T128 was used as a model of typical commercial bakerâs yeast used in Japan. After 18 h cultivation, cells in stationary phase were collected by centrifugation (2,700 ï g for 5 min). Some of the cell pellets were suspended in 900 ml of sterilized water. Cells for no-fermentation control were harvested after the fed-butch cultivation and stored until RNA extraction. Cell pellets (11,700 OD units) were suspended in 390 ml of lequid fermentation (LF) medium in a 500-ml flask and then fermented for 300 min. To investigate gene expression profiles during initial stages of dough-fermentation, cell samples for DNA microarray analysis were obtained from each culture medium at 15 min, 30 min, and 60 min. Cells in stationary phase were then collected by centrifugation (2,700ï g for 5 min), and stored until RNA extraction.
Project description:Environmental conditions are quite effective during propagation and industrial application of Baker’s yeast (Saccharomyces cerevisiae). The change of temperature is one of the most important conditions which affects the dough-leaving capacity of yeast cells. Accordingly, heat changes play an essential role in the commercial and economic impact of the yeast. Recent technologies in genomics have been used for analysis of global gene expression profiles such as microarray and RNA sequencing to solve the problems related with industrial application of yeast. Hereby, the aim of this study is to explore the effects of heat stresses on global gene expression profiles and to identify the candidate genes for the heat stress response in commercial baker’s yeast by using microarray technology and comparative statistical data analyses. Data from all hybridizations and array normalization were analyzed using the GeneSpringGX 12.1 (Agilent) and the R 2.15.2 program language. In the analysis of this dataset, all required statistical methods are performed comparatively in each step and the best performed ones are used in further computations. Hence, as the first step of the analyses, different background normalization algorithms such as MAS5.0, RMA, MBEI and GC-RMA were applied and the algorithm which gives the most accurate findings was chosen for the normalization procedure. As a result, expression values, computed from CEL files, were processed by Robust Multiarray Analysis (RMA) which is the selected procedure for the normalization of the systematic differences between samples. In order to determine differentially expressed genes under heat stress treatments, two different methods, namely, the fold-change and the hypothesis testing approaches are executed. In the fold-change method, various cut-off values are implemented while different multiple testing procedures are performed for the hypothesis testing. Under the heat shock and temperature-shift stress conditions, up/down regulated differentially expressed probes were functionally categorized into the different groups via the cluster analyses. Transcriptome changes under the heat shock and temperature-shift stress treatments show that the number of differentially up-regulated genes among the heat shock proteins (HSPs) and transcription factors (TFs) changed significantly. Consequently, the identification of thousands of genes related with heat stress treatments via microarray technology and comparative statistical analysis resulted in the creation of big picture which provides the understanding of the importance for the baker’s yeast industrial applications.
Project description:In this study, we focused on air-drying stress and analyzed the changes in gene expression of commercial baker’s yeast during the air-drying process. Changes in gene expression profiles of commercial baker’s yeast during an air-drying process at 37oC that simulated dried yeast production were analyzed using DNA microarrays. Keywords: Stress response