Genomic saturation mutagenesis in Saccharomyces cerevisiae for isolation of mutants in non-selectable and polygenic traits. Saccharomyces cerevisiae

Isolation of mutants in populations of yeast and other microorganisms, has been a valuable tool in experimental genetics for decades. The main disadvantages of classical mutant hunts are the inability to isolate mutants in non-selectable traits and the difficulty of obtaining multiple mutations affecting a single trait. Most traits of organisms, however, are non-selectable and polygenic . This includes many, if not most, commercially-important traits in industrial strains of yeast and other micro-organisms. The advent of powerful technologies for polygenic analysis of complex traits now allows efficient identification of multiple mutations responsible for a complex trait among many thousands of irrelevant mutations We now show that this is also applicable to strains of which the genome has been saturated with artificial mutations so as to affect non-selectable and/or polygenic traits. We have introduced hundreds of mutations into single yeast strains using multiple rounds of EMS mutagenesis, while maintaining genetic proficiency. Two mutants with about 900 mutations were screened for multiple non-selectable phenotypes. We selected one of the manifold mutants that showed strongly reduced ethyl acetate production in semi-anaerobic fermentations for further analysis since this is a desirable trait in yeast alcoholic beverage production and since much knowledge is lacking about ethyl acetate and flavor compound biosynthesis in general. We have mapped the underlying quantitative trait loci (QTLs) using pooled-segregant whole-genome sequence analysis and an unrelated inferior parent strain as mating partner for the manifold mutant. Comparative sequence analysis of the manifold mutant, its parent strain and the unrelated mating partner revealed candidate causative SNPs for artificially induced mutations and for natural genetic background mutations responsible for the difference in ethyl acetate production between the manifold mutant and the inferior reference strain. Reciprocal hemizygosity analysis and allele exchange identified CEM1 and PMA1 as causative alleles containing artificially induced mutations and TPS1 as a causative allele with a genetic background mutation, causing low ethylacetate production and also linked to the superior parent. Our results show that genomic saturation mutagenesis combined with complex trait polygenic analysis can be used to identify mutations in non-selectable and polygenic traits. A collection of such comprehensively mutated strains would likely be affected in a large number of phenotypes. Our results suggest that this would allow for the first time efficient discovery of mutants in non-selectable and polygenic traits, followed by efficient identification of the causative genes.