Cellular signalling of bottom fermenting brewing yeast while serial re-pitching

The objective of this research is to address the changes in the proteome of S. pastorianus required to exhibit a flocculation phenotype. For this, the proteome along fermentation of beer as well as over several re-pitching cycles will be quantified. Key kinases, characterized by changing their abundancy at the advent of flocculation, will be further investigated for a direct involvement in flocculation. This will give novel and fundamental insight into the underlying flocculation signaling events in S. pastorianus and would have major impact on understanding and controlling this phenotype. Two research hypotheses are going to be investigated:  1. Changes in flocculation behaviour over re-pitching cycles are not only due to changes in the FLO1 gene, but also due to alterated kinase cell signaling. 2. Translocation of FLO1 onto chromosome VIII in S. pastorianus places flocculation under a different set of cellular control pathways than in S. cerevisiae. Following 5 work packages are therefore defined to achieve the objectives and validate or falsify the research hypotheses: 1. Proteome of flocculation phenotype in S. pastorianus to identify involved key kinases The objective of this work package is to quantify changes in the proteome of S. pastorianus over time during fermentation. This will give insight into key proteins expressed at the advent of a flocculation phenotype as in comparison with prior stages of fermentation. Kinases with the most dramatic change in abundance will be regarded as the most important in establishing this phenotype and will be validated in work package 3 and subsequently functionally characterized in work package 4. This work package will be the first multi-omics approach utilizing deep-proteome investigation into yeast flocculation with a metabolomics approach involving the characterization of yeast fermentation by-products and the fermentation environment. 2. Proteome of altered flocculation behaviour in S. pastorianus over several re-pitching cycles S. pastorianus will be re-pitched over several fermentation cycles until an altered flocculation behaviour is observed and severely manifested. Proteome analysis will be used to quantify gradual changes in protein expression, which will be correlated with flocculation behaviour and cell wall integrity. Further, sequencing of the FLO1 gene will be undertaken to gain insight into the contribution of an altered genotype. Kinases identified in work package 1 as well as kinases whose abundance changes correlate with altered flocculation behaviour will be validated and functionally characterized in work package 5. This work package will be the first to establish the changes in the proteome related to flocculation behaviour changes over re-pitching cycles. 3. Validation of kinases potentially involved in flocculation using additional S. pastorianus and S. cerevisiae strains Validation in additional strains of S. pastorianus and S. cerevisiae will give evidence which of the kinases identified in work package 1 are strain specific. “Universal” kinases will be tested for their involvement in flocculation by gene modification to a catalytically inactive form. Futher, the flocculation triggers (nutrional, environmental) effecting the respective kinase will be investigated using high-throughput phenotyping. The inclusion of S. cerevisiae will validate or falsify research hypothesis 2 and is the first investigation on a global proteome wide level to investigate flocculation signaling differences between S. pastorianus and S. cerevisiae. 4. Substrates of key kinases involved in flocculation of S. pastorianus Substrates of the kinases validated to be involved in flocculation in a non-strain specific way (as established in work package 3) will be identified. In brief, strains expressing wither wild-type or catalytically inactive kinase produced in work package 3 will be used in a deep phospho-proteomics workflow to identify and validate the substrates of these kinases. This will give novel and important insight into the underlying cellular signaling pathways leading to a flocculation phenotype in S. pastorianus and, depending on the outcome of work package 3, also in S. cerevisiae. 5. Substrates of key kinases involved in altered flocculation behaviour in re-pitching Kinases established in work package 2 will be genetically modified to be expressed as catalytically inactive form in S. pastorianus. The influence of specific stress sources onto the flocculation behaviour will be investigated by high-throughput phenotyping. Phospho-proteomics will be applied to identify kinase substrates. The results from this work package will set molecular and cellular foundation for changes in flocculation behaviour of bottom fermenting brewing yeast over re-pitching cycles. This will enable the potential usage of these substrates as surrogate read-out of expected flocculation behaviour, up to today only yeast re-usage guidelines based on observational data are used in the industry.