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Functional Genomic Analysis of Commercial Baker’s Yeast during Initial Stages of Model Dough-Fermentation

ABSTRACT: 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

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE3043 | GEO | 2006/02/24

SECONDARY ACCESSION(S): PRJNA93059

REPOSITORIES: GEO

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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:Saccharomyces cerevisiae is exposed to freeze-thaw stress in commercial processes including frozen dough baking. The cell viability and fermentation activity after freeze-thaw were dramatically decreased due to freeze-thaw injury. Because freeze-thaw injury involves complex phenomena, the mechanisms of it are not fully understood. We attempted to analyze the mechanisms of freeze-thaw injury by indirect gene expression analysis during post-thaw incubation after freeze-thaw treatment using DNA microarray profiling. The results showed that a high frequency of the genes involved in the homeostasis of metal ions were up-regulated depending on the freezing period. The phenotype of the deletion mutants of the up-regulated genes extracted by indirect gene expression analysis was assessed. The deletion strains of the MAC1 and CTR1 genes involved in copper ion homeostasis exhibited freeze-thaw sensitivity, suggesting that copper ion homeostasis is required for freeze-thaw tolerance. Supplementation with copper ions during post-thaw incubation increased intracellular superoxide dismutase activity. Inverse correlated with intracellular superoxide dismutase activity, intracellular levels of reactive oxygen species were decreased. Moreover, cell viability increased by supplementation with copper ions under specific assessment conditions. This study suggested that insufficiency of copper ion homeostasis may be one of the causes of freeze-thaw injury. Total RNA was extracted from the stress-treated yeast cells by using a hot phenol method. Poly(A)+ RNA was enriched from total RNA by using an Oligotex dT30 (Super) mRNA purification kit (Takara Bio, Ohtsu, Japan). cDNA synthesis, cRNA synthesis, and labeling were performed according to the Affymetrix user’s manual (Affymetrix, Santa Clara, USA). Biotinyated cRNA was fragmented and then used as a probe.Affimetrix Yeast Genome 2.0 arrays (Affymetrix) were used as DNA microarrays. All experiments were done in triplicate independently.

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Functional Genomic Analysis of Commercial Baker’s Yeast during Initial Stages of Model Dough-Fermentation

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