Wine yeasts are subjected to stress conditions that change continuously during a dynamic process such as alcoholic fermentation. Due to utility of DNA chips to investigate how gene expression changes within biological processes, several attempts have been made on wine yeasts (Perez-Ortin et al., 2002). In this work microarray technology has been used to investigate metabolic shifts at transcriptional level of yeast strains differing for fermentation performances and production of secondary compounds. In particular, a genome-wide expression analysis was performed comparing two commercial yeasts (Lalvin EC1118 and 71B) under oenological conditions (50 mg/L of sulphites added at the beginning of vinification). As expected, most of the upregulated genes are stress responsive elements, chaperonins or heat shock proteins (data not shown). The most interesting finding from microarray data regards the different gene expression of sulphur metabolism pathway and sulphite resistance. In particular, sulphur amino acids biosynthesis was up-regulated in 71B; sulphite uptake/reduction pathway seemed also to be induced. The sulphite efflux permease Ssu1, involved in sulphite resistance (Park & Bakalinsky, 2000) was also highly expressed. This is consistent with the evidence that yeast can cope with sulphite stress by several mechanisms, such as acetaldehyde production, up-regulation of sulphite uptake and reduction and sulphite efflux from the cell by the membrane pump Ssu1 (Casalone et al., 1992). Since sulphites are widely used preservatives in foods and beverages, these metabolic pathways have been studied for many years because of their technological interest: their crosslink has been demonstrated and protein involved have been identified; this notwithstanding, their regulation is still unclear (Aranda et al., 2006) especially during alcoholic fermentation. For these reasons a further investigation on microarray data concerning sulphur metabolism was carried out. Time course Real-time PCR experiments were performed for confirming previous evidences: expression level of SSU1 was tested, together with its transcriptional regulator FZF1 and two genes belonging to sulphured amino acids biosynthesis pathway (MET10 and MET17). These expression data pointed out that in 71B strain SSU1 is activated during stationary phase, whereas previous works demonstrated its constitutive expression. This is the first report of a regulated SSU1 gene expression in Saccharomyces cerevisiae. Therefore its activation mechanism was further investigated by Real-Time PCR gene expression analysis for determining whether the induction is FZF1 dependent in the two strains. Moreover, sulphite resistance assays were carried out in synthetic must to evaluate physiological effects of the different activation mechanisms. Furthermore, PCR primers were designed to explore the organization SSU1 locus and its position in the genome, since this gene has been previously described as a marker of genome polymorphism and genetic evolution of wine yeasts (Aa et al., 2006). At the moment, SSU1 alleles distribution is under investigation by genetic comparison of the same locus in other wine strains; this work is part of a wider project of genetic characterization of autochthonous yeasts strain that will lead to the complete sequencing of four wine yeast genomes.

Comparison of wine yeasts transcriptome during alcoholic fermentation shows differences in sulphur metabolism

NARDI, TIZIANA;GIACOMINI, ALESSIO;CORICH, VIVIANA
2009

Abstract

Wine yeasts are subjected to stress conditions that change continuously during a dynamic process such as alcoholic fermentation. Due to utility of DNA chips to investigate how gene expression changes within biological processes, several attempts have been made on wine yeasts (Perez-Ortin et al., 2002). In this work microarray technology has been used to investigate metabolic shifts at transcriptional level of yeast strains differing for fermentation performances and production of secondary compounds. In particular, a genome-wide expression analysis was performed comparing two commercial yeasts (Lalvin EC1118 and 71B) under oenological conditions (50 mg/L of sulphites added at the beginning of vinification). As expected, most of the upregulated genes are stress responsive elements, chaperonins or heat shock proteins (data not shown). The most interesting finding from microarray data regards the different gene expression of sulphur metabolism pathway and sulphite resistance. In particular, sulphur amino acids biosynthesis was up-regulated in 71B; sulphite uptake/reduction pathway seemed also to be induced. The sulphite efflux permease Ssu1, involved in sulphite resistance (Park & Bakalinsky, 2000) was also highly expressed. This is consistent with the evidence that yeast can cope with sulphite stress by several mechanisms, such as acetaldehyde production, up-regulation of sulphite uptake and reduction and sulphite efflux from the cell by the membrane pump Ssu1 (Casalone et al., 1992). Since sulphites are widely used preservatives in foods and beverages, these metabolic pathways have been studied for many years because of their technological interest: their crosslink has been demonstrated and protein involved have been identified; this notwithstanding, their regulation is still unclear (Aranda et al., 2006) especially during alcoholic fermentation. For these reasons a further investigation on microarray data concerning sulphur metabolism was carried out. Time course Real-time PCR experiments were performed for confirming previous evidences: expression level of SSU1 was tested, together with its transcriptional regulator FZF1 and two genes belonging to sulphured amino acids biosynthesis pathway (MET10 and MET17). These expression data pointed out that in 71B strain SSU1 is activated during stationary phase, whereas previous works demonstrated its constitutive expression. This is the first report of a regulated SSU1 gene expression in Saccharomyces cerevisiae. Therefore its activation mechanism was further investigated by Real-Time PCR gene expression analysis for determining whether the induction is FZF1 dependent in the two strains. Moreover, sulphite resistance assays were carried out in synthetic must to evaluate physiological effects of the different activation mechanisms. Furthermore, PCR primers were designed to explore the organization SSU1 locus and its position in the genome, since this gene has been previously described as a marker of genome polymorphism and genetic evolution of wine yeasts (Aa et al., 2006). At the moment, SSU1 alleles distribution is under investigation by genetic comparison of the same locus in other wine strains; this work is part of a wider project of genetic characterization of autochthonous yeasts strain that will lead to the complete sequencing of four wine yeast genomes.
2009
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/2442920
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