European breweries realise 25% of world beer production, representing total sales of 4.5 billion euros, and employ 115.000 people. A process of increasing importance is High-Gravity Brewing which permits more efficient and cleaner production of beer of high quality. It makes use of a higher-density wort with a higher initial sugar level. The higher osmolarity and final alcohol content cause a significant delay in fermentation and drop in yeast viability, resulting in undesirable flavour profiles. This project will isolate mutants in brewer's yeast strains with improved tolerance to the stressful HGB conditions. The mutants will be characterised with genomic approaches. Genetic alterations will be identified and used to construct strains with further improved resistance. Evaluation will be done on lab and pilot scale and in a demonstration project.
The main goal of this project is to solve the problems of delayed fermentation, reduced viability and undesirable flavour profile in High-Gravity Brewing, due to the lack of stress resistance of brewer's yeast. A wort density of e.g. 18°Plato results in a final alcohol content of 7.5%, representing an increase of 50% in productivity. Mutants in brewer's yeast strains will be isolated with improved fermentation under HGB conditions. They will be evaluated for all commercially important properties in lab and pilot scale. Further objectives are their characterisation using genomic approaches, determination of resistance to individual stress factors, identification of the genetic alterations responsible for higher resistance, construction of strains with further improvement and extension of the scope of stress resistance and evaluation in a demonstration project.
- Development of novel genetic tools for investigation of polyploid industrial yeast strains. A transposon insertion mutagenesis library and a constitutive promoter cDNA library will be constructed with a marker selectable in polyploid strains.
- Isolation of mutant and transformant strains with a higher resistance to High-Gravity Brewing (HGB) conditions. Brewer's strains will be mutagenised by classical and transposon mutagenesis and transformed with the cDNA library. Genetic screens will be established for strains with a better fermentation rate and survival on media mimicking HGB and media with individual stress conditions considered to be relevant for HGB: high osmolarity, high ethanol, starvation and forced intracellular acidification. Stress-resistant mutants and transformants will be selected directly in the industrial strains.
- Characterisation of the stress- resistant strains. Genomic approaches with micro-array hybridisation and 2D separation of proteins will be used to identify components responsible for enhanced stress resistance. The strains isolated under HGB conditions will be tested for resistance to the individual stress conditions while the strains isolated under the latter conditions will be tested for cross-resistance to HGB and individual stress conditions.
- Identification of the genetic alterations responsible for enhanced stress resistance. For this purpose the flanking DNA of the transposons and the cDNA on the plasmid in the transformants will be sequenced.
- Construction of strains with further improvement and/or extension of the scope of stress resistance. This will be accomplished by combining specific mutations and/or overexpression constructs.
- Evaluation of the novel strains. The strains will be tested in lab and pilot scale for performance under HGB conditions and the best strains will be evaluated in a demonstration project.