Using Experimental Evolution to Correct Mother-Daughter Separation Defects in Brewing Yeast

Abstract

The budding yeast, Saccharomyces cerevisiae, is the workhorse of the brewing industry. Brewers have domesticated a vast array of different strains with traits that complement the beers they wish to brew. Yet, some domesticated strains also harbor traits that are undesirable. One example of an undesirable trait in brewing strains is the mother-daughter separation defect (MDSD). MDSDs are a reproductive flaw present in a widely used brewing strain, London Ale III. MDSDs cause cells to form large clusters, possibly leading to the known requirement for more headspace during London Ale III fermentations that result in a lower fermentative yield. Because MDSDs can be caused by mutations to a number of genes, targeted genetic approaches to reduce MDSDs are experimentally challenging, especially for a tetraploid strain like London Ale III. To improve MDSDs in this strain, we employed experimental evolution and passaged populations from three biological replicates for over 200 generations to generate three independent evolved strains that form fewer clusters than the ancestral strain, as seen by microscopy. To confirm these results, we used flow cytometry to measure the average size of the clusters in clones of our evolved replicates and found them to be smaller on average than the ancestor. We also qualitatively assessed the aggregation phenotype using a settling assay and found that our evolved replicates settle slower than the ancestor. Finally, we characterized the mutations in our evolved replicates using whole genome sequencing and identified increased copy numbers of chromosome 1 and chromosome 14 in all three evolved clones. The best-performing strain generated by this project is now available commercially. This project demonstrates how experimental evolution can be used to select against less desirable traits in industrial yeast strains when targeted genetic approaches present considerable challenges. Future research could implement a similar approach to improve other traits in widely used brewing and baking strains.