Davide Ravasio:
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Fungi produce a variety of volatile organic compounds (VOCs) during their primary or secondary metabolism and with a wide range of functions. The main focus of this research work has been put on flavour molecules that are produced during fermentation processes, mainly esters and alcohols derived from the catabolism of amino acids. These compounds are produced by the Ehrlich pathway. The conversion of amino acids into aroma alcohols is accomplished by three enzymatic steps: i) a transamination, ii) a decarboxylation and iii) a dehydration reaction. The transaminase and decarboxylase enzymes are encoded by the ARO gene family which represents a widely conserved set of genes in the Saccharomyces clade. Comparative genomic analysis revealed conservation of these genes also in the riboflavin over producer Ashbya gossypii, a closely related species belonging to the Eremothecium clade. ARO80 is a transcription factor that represents the key regulator of the ARO gene family. The first part of the thesis will unveil the ARO80-dependent regulation of the Ehrlich pathway in both Saccharomyces cerevisiae and A. gossypii.
Promoter analyses of the ARO genes in S. cerevisiae showed that the ScARO9 promoter region is directly regulated by the ScAro80 transcription factor. This interaction has been used to create a lacZ-reporter system to correlate the formation of two volatile compounds, 2- phenylethanol and 2-phenylethyl acetate in yeast with ARO9 expression levels. This indirect genetic assay also provides a tool for the prediction of volatile production in other Saccharomyces sensu stricto species. It can be used to screen a large number of strains for their flavour production within a short time and with low costs and effort.
In Ashbya single mutations in the ARO genes led to a strong reduction in volatile production, especially in the amount of isoamyl alcohol and 2-phenylethanol. In contrast, the overexpression of the transcriptional regulator AgARO80 did only increase the level of isoamyl alcohol but did not enhance the 2-phenylethanol yield. Promoter analyses of the ARO genes in A. gossypii identified both ARO8 and ARO10 to be activated by Aro80. In this study we further analyzed the aroma profile of another Eremothecium species, Eremothecium cymbalariae. This species lacks most of the ARO genes involved in amino acid catabolism. The only ARO gene present in E. cymbalariae is a homolog of the A. gossypii ARO8a, which is a non-syntenic homolog of ARO8 in yeast. We compared the VOC profiles of both species in order to investigate the consequences of their different gene set up on their flavour profiles. Here we found that in contrast to A. gossypii E. cymbalariae does not produce 2-phenylethanol and 2-phenylacetate.
The last part of this thesis presents the initial characterization of twenty non-conventional yeasts (NCY) and their potential application in fermentative processes. These strains have been selected as they have been previously isolated from various fermented food sources. This selection of strains was used in fermentations with the aim of identifying new interesting flavour producers. Fermentation profiles, volatile analyses, off-flavour identification and resistance to osmotic/oxidative stress have been addressed to highlight new candidates to use for industrial applications. This resulted in the identification of Wickerhamomyces anomalus and Pichia kluyveri as high producers of esters fruity compounds, which contribute to enhance the complexity of wine and beer product. In addition the strain Debaromyces subglobosus showed high yields of aldehydes and fruity ketones, which constitute active aroma compounds in dry cured ham.