Abstract
Yeast is used for fermentation in the biotechnology sector and plays a critical role in the production of fermented beverages. The quality of the final product depends on yeast performance and the environmental conditions of fermentation. Combining knowledge on yeast biology and the different fermentation elements allows generation of beverages with new properties and improved processes. Nitrogen is an essential nutrient for yeast, nonetheless, besides the contribution of the nitrogen source to growth, some nitrogen compounds are precursors of volatile molecules that produce aroma. The nitrogen compounds assimilated by yeast are classified as rich or poor nitrogen sources depending on how they support growth. Although Saccharomyces cerevisiae is the most important and most widely used yeast species, other non-traditional yeasts are attracting industrial interest because of the new traits of technological interest that they offer. Saccharomyces uvarum has relevant properties that can be exploited for the production of fermented beverages. Particularly, the cryotolerance and capacity to produce high amounts of volatile compounds offers new opportunities for the fermentation industry. Kluyveromyces marxianus is another food-grade yeast that is of interest for its aroma properties. In S. cerevisiae, nitrogen metabolism is well-understood but less is known about these pathways in non-traditional yeasts, nor whether there are regulatory differences between the yeasts. This thesis explores nitrogen metabolism in non-traditional yeasts and the nitrogen source effect on the metabolome and transcriptome. The main focus was on S. uvarum as this is already used to a certain extent in the fermented beverage sector. First, we established the nitrogen preference of S. uvarum and subsequently performed fermentations with a reference S. uvarum strain in oenological conditions varying the nitrogen source to evaluate the fermentation performance and metabolome. Next, a comparative analysis of gene expression (RNAseq) in ammonium, methionine, phenylalanine and asparagine was performed to2determine how the nitrogen source affects the expression of key genes involved in nitrogen metabolism and aroma production in this species. In overall terms, the pathways used by S. uvarum were equivalent to those used by S. cerevisiae but some detail varied. One such detail was a major difference between S. cerevisiae and S. uvarum in use of asparagine, which we found was a preferred amino acid for S. cerevisiae but not for S. uvarum. This was intriguing, in particular because asparagine is the major source of nitrogen in apple juice, where S. uvarum is traditionally used for cider fermentation. This led us to explore the evolution history and functional role of ASP3, a gene involved in asparagine assimilation. Our data on the use of amino acids by S. uvarum highlighted the role of the Ehrlich pathway in interconversion of nitrogen compounds and in generation of precursors of aroma volatiles. As a complement to this work in S. uvarum, we expanded the study of amino acid metabolism to K. marxianus, specifically focusing on expression of the BAT1 gene encoding a branched chain aminotransferase responsible for both synthesis and catabolism of branched chain amino acids. The intriguing aspect of this was that while Saccharomyces spp. possess separate genes for the mitochondrial and cytosolic forms of the enzyme, Kluyveromyces spp. have a single gene that can be used to produce alternative proteoforms for the same purpose. In addition to uncovering a novel mechanism of transcriptional regulation in K. marxianus, the work also provided information to help explain the evolutionary basis for the retention of two copies of the gene in Saccharomyces spp. Overall, the research reported in this thesis demonstrates that exploring the metabolism and characteristics of non-conventional yeasts opens up possibilities to find interesting traits with potential industrial applications. This study increases understanding of the importance of the nitrogen source in fermentation performance and the flavour and aroma production of yeast and broadens the knowledge on S. uvarum, in particular, for applications in the fermented beverage industry.