Driven by the need of innovation, the application of non-Saccharomyces yeasts for brewing has seen a substantial growth in recent years. Pichia kluyveri, for instance, is a maltose-negative yeast applied for production of non- and low- alcoholic beer with an attractive fruity flavor, due to the enhanced production of aroma compounds that it releases. Despite of its brewing potential, intrinsic physiological and metabolic aspects of this yeast are unknown. In this context, the main aim of this project was to understand and to increase the knowledge on phenotypic and genomic aspects of brewing P. kluyveri strains, and, if possible, to establish a link between these aspects.

Three P. kluyveri strains showed similar fermentation parameters in synthetic wort in terms of growth, carbohydrate metabolism and metabolites, enhanced production of esters, a strong preference for methionine and a low preference for several amino acids including glutamate, glutamine and aspartate. Strain differences were observed regarding the development of volatile sulfur aroma compounds, especially in the release of hydrogen sulfide. Since no variations were detected in terms of absence or presence of related genes among the low and high producers of hydrogen sulfide strains, a distinctive genic regulation affecting sulfur-related enzymes might influence the sulfur compound pattern observed among strains.

Genomic analysis of P. kluyveri revealed the absence of some genes encoding key enzymes associated to the sulfur metabolism in S. cerevisiae. For instance, transsulfuration enzymes responsible of the cysteine formation from homocysteine were not found. However, homolog proteins exclusive from other nonSaccharomyces yeasts that allow the synthesis of cysteine through the o-acetyl serine pathway, and the consequent formation of homocysteine through the reverse-transsulfuration pathways, were found in P. kluyveri. In addition, a high protein homology corresponding to a particular C-S lyase, that is able to yield methanethiol from methionine and mainly from homocysteine, was also identified in P. kluyveri and it might be associated to the considerably high methanethiol detected. On the other hand, the absence of an invertase and enzymes related to the maltose metabolism in P. kluyveri might be the genetic basis behind the maltose- and sucrose-negative phenotype observed at a species-level.

The safety aspect of non-Saccharomyces yeasts has been general poorly addressed by the research community and the industrial sector. In fact, P. kluyveri is the first non-Saccharomyces yeast for brewing application having a GRAS (Generally Recognized As Safe) status since it entails a consistent taxonomic classification, a history of safe use and a safety assessment was conducted. The advances on bioinformatic, sequencing and other technological approaches, alongside the availability of a pipeline for yeast safety assessment might illuminate this subject, thus contributing to more non-Saccharomyces yeasts in the market whose safety use for brewing has been assessed.