Functional genomics of beer-related physiological processes in yeast

Abstract

Since the release of the entire genome sequence of the S. cerevisiae laboratory strain S288C in 1996, many functional genomics tools have been introduced in fundamental and application-oriented yeast research. In this thesis, the applicability of functional genomics for the improvement of yeast in beer-related processes is investigated. To this end, genome-wide analysis was focused on a range of nutritional conditions that are typically encountered in beer fermentation. Ultimately, this should provide the brewer with the information and knowledge required to understand and improve yeast quality and fermentation performance. Fusel alcohols are derived from amino acid catabolism via a pathway that was first proposed a century ago by Ehrlich. In the past decade, many efforts have contributed to a better understanding of the genes involved in this pathway and how their expression is regulated. A ‘centenary’ review of the Ehrlich pathway is given in this thesis. Our growing understanding of the key components of the Ehrlich pathway and their regulation will aid in the design of strains that exhibit specific flavor profiles in foodstuffs as well as in the metabolic engineering of yeast strains for the production of individual Ehrlich pathway products. Transcriptomics can also be used as diagnostic tool for industrial fermentations. For example, the prediction of Zn bioavailability in wort is not always accurate and research has therefore focused on the identification of molecular markers for Zn deficiency. For this purpose, we investigated the transcriptional responses S. cerevisiae at a fixed specific growth rate under limiting and abundant Zn concentrations in chemostat culture. Most surprising was the Zn-dependent regulation of genes involved in storage carbohydrate metabolism. Their concerted down-regulation in Zn-limited cultures was physiologically relevant as revealed by a substantial decrease in glycogen and trehalose cellular content under these conditions. In industrial fermentations, the ability to accumulate substantial amounts of storage carbohydrates is an important criterion in the selection and development of new strains, as they contribute to cellular robustness. An analysis of storage carbohydrate metabolism in anaerobic chemostat cultures grown at a fixed specific growth rate under five different nutrient-limitation regimes revealed that storage carbohydrate accumulation is not a general response to nutrient limitation. Over the conditions tested, glycogen accumulation was most pronounced under nitrogen limited conditions. Although the transcriptional induction of both glycogen and trehalose biosynthesis genes was to a large extent driven by the regulator Msn2/4, the main regulatory control of glycogen biosynthesis was post-translational. The hop plant, Humulus lupulus, contains an exceptionally high content of secondary metabolites, the hop iso-?-acids, which possess a range of beneficial properties including antiseptic action. By applying transcriptome analysis and phenotype screening of the S. cerevisiae gene deletion collection we found that yeast tolerance to hop iso-?-acids involves two major processes: active export of iso-?-acids across the plasma membrane and active proton pumping into the vacuole by the V-ATPase to enable vacuolar sequestration of iso-?-acids. Iso-?-acids were also shown to affect cellular metal homeostasis by acting as strong zinc and iron chelator. The co-localization of iso-?-acids and zinc in the vacuole has important implications for the maintenance and quality of brewing strains. Especially upon serial re-pitching of yeast, hop acid stress in the vacuole may result in decreased viability of the yeast culture.