The species of the genus Saccharomyces, S. kudriavzevii and S. uvarum revealed significant differences during the formation of fermentative aroma as compared to S. cerevisiae. The main goal of this PhD thesis was to obtain a deeper fundamental understanding of the molecular aspects behind these differences. First part of the thesis describes observed differences among Saccharomyces kudriavzevii and Saccharomyces uvarum during the production of aroma-active higher alcohols and acetate esters using their amino acidic precursors comparing to Saccharomyces cerevisiae. Subsequent in silico comparative analysis of the enzymes involved in the branched-chain amino acids catabolism in S. cerevesiae, S. kudriavzevii and S. uvarum released ARO10, ATF1 and ATF2 as the candidates for further experiments. The heterologous expression of the individual ARO10, ATF1 and ATF2 alleles in a host S. cerevisiae resulted in the enhanced production of several higher alcohols and acetate esters. All together the data described and discussed in this thesis indicate that the amino acid variations observed between the decarboxylases encoded by the orthologues ARO10 genes and between the AATases encoded by the orthologues ATF1 and ATF2 genes could be the reason for the distinct enzyme properties, which possibly lead to the enhanced production of several flavour compounds. The main objects of the research presented in this dissertation were S. kudriavzevii and S.uvarum. In previous studies these two species, closely related to S. cerevisiae, showed remarkable differences during the production of higher alcohols and esters when comparing the three species (Gamero et al., 2013; Pérez-Torrado et al., 2015). Higher alcohols and esters formed by yeast belong to key components of overall flavour and aroma in the fermented products. As already mentioned, S. kudriavzevii and S. uvarum revealed significant differences during the formation of these important flavour-active compounds as compared to S. cerevisiae. The main goal of this PhD thesis was to obtain a deeper fundamental understanding of the molecular aspects behind these differences. First part of the thesis investigates how the three Saccharomyces species differ during the production of higher alcohols and acetate esters that derived directly from their amino acidic precursors. This objective was fulfilled by carrying out cultivations of S.cerevisiae, S. uvarum and S. kudriavzevii strains with individual amino acids as the sole nitrogen source, followed by analysis and comparison of the produced higher alcohols and acetate esters. The global volatile compound analysis revealed that S. kudriavzevii produced large amounts of higher alcohols, whereas S. uvarum excelled in the production of acetate esters. Particularly from phenylalanine, S.uvarum produced the largest amounts of 2-phenylethyl acetate, while S. kudriavzevii obtained the greatest 2-phenylethanol formation from this precursor. Altogether, the data discussed in Chapter 1 indicated differences in the amino acid metabolism and subsequent production of flavour-active higher alcohols and acetate esters among these closely related Saccharomyces species. Subsequently, in silico comparative analysis of the enzymes involved in the branched-chain amino acids catabolism in S. cerevesiae, S. kudriavzevii and S. uvarum was performed in order to explore the amino acid divergences among the orthologous sequences. In silico screening was based on multiple sequence alignments and the Grantham scoring. The Grantham scoring provides a quantitative assessment of the biochemical distances between amino acids, on the basis of their composition, polarity and molecular volume, and according to increasing dissimilarity classifies the amino acid substitutions as conservative or radical (Grantham, 1974; Li et al., 1984). The analysis released three sequences that were evaluated with significantly higher Grantham scores: 2-keto acid decarboxylase encoded by ARO10, and two alcohol acetyltransferases encoded by ATF1 and ATF2. The last part of the thesis then investigates the impact of the selected genes on the production of higher alcohols and acetate esters, and the catalytic properties of the corresponding enzymes. The heterologous expression of the individual ARO10, ATF1 and ATF2 alleles from S. kudriavzevii and S. uvarum in a host S. cerevisiae resulted in the enhanced production of several higher alcohols and acetate esters. All together the data described and discussed in this thesis indicate that the amino acid variations observed between the decarboxylases encoded by the orthologues ARO10 genes and between the AATases encoded by the orthologues ATF1 and ATF2 genes could be the reason for the distinct enzyme properties, which possibly lead to the enhanced production of several flavour compounds.
