Proteome analysis of yeast response to various nutrient limitations

We compared the response of Saccharomyces cerevisiae to carbon (glucose) and nitrogen (ammonia) limitation in chemostat cultivation at the proteome level. Protein levels were differentially quantified using unlabeled and (15)N metabolically labeled yeast cultures. A total of 928 proteins covering a wide range of isoelectric points, molecular weights and subcellular localizations were identified. Stringent statistical analysis identified 51 proteins upregulated in response to glucose limitation and 51 upregulated in response to ammonia limitation. Under glucose limitation, typical glucose-repressed genes encoding proteins involved in alternative carbon source utilization, fatty acids β-oxidation and oxidative phosphorylation displayed an increased protein level. Proteins upregulated in response to nitrogen limitation were mostly involved in scavenging of alternative nitrogen sources and protein degradation. Comparison of transcript and protein levels clearly showed that upregulation in response to glucose limitation was mainly transcriptionally controlled, whereas upregulation in response to nitrogen limitation was essentially controlled at the post-transcriptional level by increased translational efficiency and/or decreased protein degradation. These observations underline the need for multilevel analysis in yeast systems biology

Rapid identification of target genes for 3-methyl-1-butanol production in Saccharomyces cerevisiae

Extracellular conditions determine the taste of fermented foods by affecting metabolite formation by the micro-organisms involved. To identify targets for improvement of metabolite formation in food fermentation processes, automated high-throughput screening and cDNA microarray approaches were applied. Saccharomyces cerevisiae was cultivated in 96-well microtiter plates, and the effects of salt concentration and pH on the growth and synthesis of the fusel alcohol-flavoured substance, 3-methyl-1-butanol, was evaluated. Optimal fermentation conditions for 3-methyl-1-butanol concentration were found at pH 3.0 and 0% NaCl. To identify genes encoding enzymes with major influence on product formation, a genome-wide gene expression analysis was carried out with S. cerevisiae cells grown at pH 3.0 (optimal for 3-methyl-1-butanol formation) and pH 5.0 (yeast cultivated under standard conditions). A subset of 747 genes was significantly induced or repressed when the pH was changed from pH 5.0 to 3.0. Expression of seven genes related to the 3-methyl-1-butanol pathway, i.e. LAT1, PDX1, THI3, ALD4, ILV3, ILV5 and LEU4, strongly changed in response to this switch in pH of the growth medium. In addition, genes involved in NAD metabolism, i.e. BNA2, BNA3, BNA4 and BNA6, or those involved in the TCA cycle and glutamate metabolism, i.e. MEU1, CIT1, CIT2, KDG1 and KDG2, displayed significant changes in expression. The results indicate that this is a rapid and valuable approach for identification of interesting target genes for improvement of yeast strains used in industrial processes.

Genome-wide transcription survey on flavour production in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is widely used as aroma producer in the preparation of fermented foods and beverages. During food fermentations, secondary metabolites like 3-methyl-1-butanol, 4-methyl-2-oxopentanoate, 3-methyl-2-oxobutanoate and 3-methylbutyrate emerge. These four compounds have a major influence on the final taste of fermented foods. Their presence is influenced by the availability of free branched chained amino acids (BCAA). To study the underlying molecular mechanism of the formation of these compounds, we performed genome-wide transcription analyses with cDNA microarrays. The expression profile of yeast during flavour formation, when cultivated on L-leucine, was compared to the expression profile of cells cultivated on ammonia. In addition, the expression profiles of cells cultivated in a batch culture were compared to cells cultivated under continuous growth conditions. Genome-wide gene analysis of these samples revealed a group of 117 genes, which w! ere more than two-fold up- or down-regulated and significantly altered in gene expression (P < 0.001) under both cultivation conditions. This group included genes encoding enzymes of different amino acid metabolism pathways. The group of the BCAA metabolism was not significantly altered in gene expression. Genes identified with altered expression levels, in only batch or continuous culture fermentions, represented functional groups concerning energy, protein fate, cell cycle and DNA processing. Furthermore, clustering of genome-wide data revealed that the type of cultivation overruled the differences in N-source in the gene-expression profiles. This observation emphasizes the importance of sample history in gene expression analysis.

Characterisation of the broad substrate specificity 2-keto acid decarboxylase Aro10p of Saccharomyces kudriavzevii and its implication in aroma development

Background: The yeast amino acid catabolism plays an important role in flavour generation since higher alcohols and acetate esters, amino acid catabolism end products, are key components of overall flavour and aroma in fermented products. Comparative studies have shown that other Saccharomyces species, such as S. kudriavzevii, differ during the production of aroma-active higher alcohols and their esters compared to S. cerevisiae. Results: In this study, we performed a comparative analysis of the enzymes involved in the amino acid catabolism of S. kudriavzevii with their potential to improve the flavour production capacity of S. cerevisiae. In silico screening, based on the severity of amino acid substitutions evaluated by Grantham matrix, revealed four candidates, of which S. kudriavzevii Aro10p (SkAro10p) had the highest score. The analysis of higher alcohols and esters produced by S. cerevisiae then revealed enhanced formation of isobutanol, isoamyl alcohol and their esters when endogenous ARO10 was replaced with ARO10 from S. kudriavzevii. Also, significant differences in the aroma profile were found in fermentations of synthetic wine must. Substrate specificities of SkAro10p were compared with those of S. cerevisiae Aro10p (ScAro10p) by their expression in a 2-keto acid decarboxylase-null S. cerevisiae strain. Unlike the cell extracts with expressed ScAro10p which showed greater activity for phenylpyruvate, which suggests this phenylalanine-derivative to be the preferred substrate, the decarboxylation activities measured in the cell extracts with SkAro10p ranged with all the tested substrates at the same level. The activities of SkAro10p towards substrates (except phenylpyruvate) were higher than of those for ScAro10p. Conclusions: The results indicate that the amino acid variations observed between the orthologues decarboxylases encoded by SkARO10 and ScARO10 could be the reason for the distinct enzyme properties, which possibly lead to the enhanced production of several flavour compounds. The knowledge on the important enzyme involved in higher alcohols biosynthesis by S. kudriavzevii could be of scientific as well as of applied interest.BT/Industriele Microbiologi