The hybrid yeast Saccharomyces pastorianus is the microbial ‘workhorse’ responsible for production of lager beer. With an annual production of 1.9 billion hectolitres, this type of beer is the most popular alcoholic beverage worldwide. S. pastorianus ferments sugars present in wort to ethanol and a variety of flavour molecules that contribute to the taste and perception of the beverage. This yeast has emerged from a natural hybridization event in a human-caused environment, which probably occurred in late medieval times, between the mesophilic ale yeast Saccharomyces cerevisiae and the cryotolerant yeast Saccharomyces eubayanus. As a consequence, S. pastorianus strains harbour chromosomes from both these parental species. In addition, prolonged domestication in lager brewing processes has led to many additional changes in S. pastorianus: individual chromosomes underwent extensive recombination and occur in different copy numbers that, moreover, vary between strains. The advent of whole genome sequencing technologies has contributed to insights into the complex genomic ‘blueprints’ of S. pastorianus strains.

The allo-aneupolyploid genomes of currently used brewing yeast strains were shaped in centuries of evolution, with industrial brewing conditions providing selective pressure. Today, targeted genetic engineering enables researchers to alter or add traits beyond what is achievable by solely applying selective pressure. The rapid development of CRISPR-Cas-based tools for genome editing have, particularly in baker’s yeast S. cerevisiae and to a lesser extent in non-conventional yeast species, greatly accelerated strain development. However, applying these tools in heterozygous alloaneupolyploid contexts such as those in S. pastorianus poses substantial technological challenges.

The aim of the research described in this thesis was to develop novel genome editing strategies for yeasts, improving the genetic accessibility of hybrid yeasts and subsequently applying these methodologies to generate novel lager brewing strains with enhanced brewing characteristics...@en

The production of ethanol by the yeast Saccharomyces cerevisiae remains the process in industrial biotechnology with the largest product volume (ca. 100 million litres annually in 2022). Carbon losses occur due to the formation of biomass and glycerol, which can account for at least 8% of the product. Under anaerobic conditions, formation of yeast biomass and glycerol are coupled via redox-cofactor balances, as a net generation of NADH during biomass formation needs to be compensated by NADH-dependent formation of glycerol from sugar. This thesis discusses redox-engineering strategies for maximizing ethanol yields on substrate.@en