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: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. 48 samples were used in this experiment
Project description:The aim of this study was to determine how nitrogen repletion affected the genomic cell response of a Saccharomyces cerevisiae wine yeast strain, in particular within the first hour following relief from nutrient starvation. We found almost 4000 genes induced or repressed sometimes within minutes of nutrient changes. Some of the transcriptional responses to nitrogen depended on the TOR pathway which control positively ribosomal protein genes, amino acid and purine biosynthesis or amino acid permease genes and negatively stress-response genes, RTG specific TCA cycle genes and NCR sensitive genes. Some unexpected transcriptional responses concerned all the glycolytic genes, the starch and glucose metabolism and citrate cycle related genes which were down-regulated, as well as genes from the lipid metabolism.
Project description:Its characteristic rose-like aroma makes phenylethanol a popular ingredient in foods, beverages and cosmetics. Microbial production of phenylethanol currently relies on whole-cell bioconversion of phenylalanine with yeasts that harbor an Ehrlich pathway for phenylalanine catabolism. Complete biosynthesis of phenylethanol from a cheap carbon source such as glucose provides an economically attractive alternative for phenylalanine bioconversion. In this study, a Synthetic Genetic Array screening was applied to identify genes involved in regulation of phenylethanol synthesis in Saccharomyces cerevisiae. The screen focused on transcriptional regulation of ARO10, which encodes the major decarboxylase involved in conversion of phenylpyruvate to phenylethanol. A deletion in ARO8, which encodes an aromatic amino acid transaminase, was found to cause a transcriptional upregulation of ARO10 during growth with ammonium sulfate as the sole nitrogen source. Physiological characterization revealed that the aro8 mutation led to substantial changes in the absolute and relative intracellular concentrations of amino acids. Moreover, deletion of ARO8 led to de novo production of phenylethanol during growth on a glucose synthetic medium with ammonium as the sole nitrogen source. The aro8 mutation also stimulated phenylethanol production when combined with other, previously documented mutations that deregulate aromatic amino acid biosynthesis in S. cerevisiae. The resulting engineered S. cerevisiae strain produced over 3 mM of phenylethanol from glucose during growth on a simple synthetic medium. The strong impact of a transaminase deletion on intracellular amino acid concentrations opens new possibilities for yeast-based production of amino acid-derived products.
Project description:Its characteristic rose-like aroma makes phenylethanol a popular ingredient in foods, beverages and cosmetics. Microbial production of phenylethanol currently relies on whole-cell bioconversion of phenylalanine with yeasts that harbor an Ehrlich pathway for phenylalanine catabolism. Complete biosynthesis of phenylethanol from a cheap carbon source such as glucose provides an economically attractive alternative for phenylalanine bioconversion. In this study, a Synthetic Genetic Array screening was applied to identify genes involved in regulation of phenylethanol synthesis in Saccharomyces cerevisiae. The screen focused on transcriptional regulation of ARO10, which encodes the major decarboxylase involved in conversion of phenylpyruvate to phenylethanol. A deletion in ARO8, which encodes an aromatic amino acid transaminase, was found to cause a transcriptional upregulation of ARO10 during growth with ammonium sulfate as the sole nitrogen source. Physiological characterization revealed that the aro8M-oM-^AM-^D mutation led to substantial changes in the absolute and relative intracellular concentrations of amino acids. Moreover, deletion of ARO8 led to de novo production of phenylethanol during growth on a glucose synthetic medium with ammonium as the sole nitrogen source. The aro8 mutation also stimulated phenylethanol production when combined with other, previously documented mutations that deregulate aromatic amino acid biosynthesis in S. cerevisiae. The resulting engineered S. cerevisiae strain produced over 3 mM of phenylethanol from glucose during growth on a simple synthetic medium. The strong impact of a transaminase deletion on intracellular amino acid concentrations opens new possibilities for yeast-based production of amino acid-derived products. The goal of the present study was to identify genes that influence the transcriptional (de)repression of the Ehrlich pathway during growth with ammonium as the nitrogen source. With the aid of Synthetic Genetic Array technology, we constructed a strain collection in which deletions in the non-essential genes in the S. cerevisiae genome were combined with a reporter plasmid comprising the ARO10 promoter fused to a reporter gene (egfp) encoding a fluorescent reporter protein. After screening by flow cytometry, deletion of ARO8 led to a deregulated expression from the ARO10 promoter. The impact of this deletion was further studied by transcriptome and intracellular metabolite analyses. Furthermore, phenylethanol production was measured in strains that combined the aro8 mutation with mutations that were previously shown to deregulate aromatic amino acid biosynthesis.
Project description:Characterization of the connection between protein synthesis and amino acid biosynthesis gene expression to growth rate in budding yeast
Project description:High concenHigh concentration acetic acid in the fermentation medium represses cell growth, metabolism and fermentation efficiency of Saccharomyces cerevisiae, which is widely used for cellulosic ethanol production. Our previous study proved that supplementation of zinc sulfate in the fermentation medium improved cell growth and ethanol fermentation performance of S. cerevisiae under acetic acid stress condition. However, the molecular mechanisms is still unclear. To explore the underlying mechanism of zinc sulfate protection against acetic acid stress, transcriptomic and proteomic analysis were performed. The changed genes and proteins are related to carbon metabolism, amino acid biosynthesis, energy metabolism, vitamin biosynthesis and stress responses. In a total, 28 genes showed same expression in transcriptomic and proteomic data, indicating that zinc sulfate affects gene expression at posttranscriptional and posttranslational levels.tration acetic acid in the fermentation medium represses cell growth, metabolism and fermentation efficiency of Saccharomyces cerevisiae, which is widely used for cellulosic ethanol production. Our previous study proved that supplementation of zinc sulfate in the fermentation medium improved cell growth and ethanol fermentation performance of S. cerevisiae under acetic acid stress condition. However, the molecular mechanisms is still unclear. To explore the underlying mechanism of zinc sulfate protection against acetic acid stress, transcriptomic and proteomic analysis were performed. The changed genes and proteins are related to carbon metabolism, amino acid biosynthesis, energy metabolism, vitamin biosynthesis and stress responses. In a total, 28 genes showed same expression in transcriptomic and proteomic data, indicating that zinc sulfate affects gene expression at posttranscriptional and posttranslational levels.
Project description:The yeast protein kinases Sat4/Hal4 and Hal5 are required for the plasma membrane stability of the K+ transporter Trk1 and some amino acid and glucose permeases. The transcriptomic analysis presented here indicates alterations in the general control of both nitrogen and carbon metabolism. Accordingly, we observed reduced uptake of methionine and leucine in the hal4 hal5 mutant. This decrease correlates with activation of the Gcn2-Gcn4 pathway, as measured by expression of the lacZ gene under the control of the Gcn4 promoter. However, with the exception of methionine biosynthetic genes, few amino acid biosynthetic genes are induced in the hal4 hal5 mutant, whereas several genes involved in amino acid catabolism are repressed. Concerning glucose metabolism, we found that this mutant exhibits derepression of respiratory genes in the presence of glucose, leading to an increased activity of mitochondrial enzymes, as measured by SDH activity. In addition, the reduced glucose consumption in the hal4 hal5 mutant correlates with a more acidic intracellular pH and with low activity of the plasma membrane H+-ATPase. As a compensatory mechanism for the low glycolytic rate, the hal4 hal5 mutant overexpresses the HXT4 high affinity glucose transporter and the hexokinase genes. These results indicate that the hal4 hal5 mutant presents defects in the general control of nitrogen and carbon metabolism, which correlate with reduced transport of amino acids and glucose, respectively. A more acidic intracellular pH may contribute to some defects of this mutant.