Fusion ofSaccharomyces UvarumwithSaccharomyces Cerevisiae:Genetic Manipulation and Reconstruction of a Brewer's Yeast

Abstract

Genetic manipulation of brewer's yeast is hampered by the yeast's inability to mate, low frequency of sporulation, and poor spore viability—all of which are partly because of its polyploid or aneuploid consititution. In the present study, we introduced genetic markers into a brewing strain of Saccharomyces uvarum by conventional mutagenic procedures and then crossed the marked strain with auxotrophic S. cerevisiae haploids, using spheroplast fusion. Fusion of the auxotrophic brewing strain with an a mating type S. cerevisiae produced hybrids that were nonsporulating and nonmating when tested with a and α tester strains. Fusion of the same brewing strain with an α mating type S. cerevisiae produced hybrids that were nonmaters but sporulated well, producing principally four-spored asci of high viability. Tetrad analyses of three hybrids recovered from the latter fusion proved that karyogamy had occurred. Asci from two of these hybrids showed regular segregation of all markers, including mating type. The third hybrid, however, exhibited regular segregation in only four of 18 asci dissected and had from one to three nonviable spores in the remaining 14; of these, seven single ascospores did not mate but sporulated. Genetic data, as well as DNA analyses, were used to estimate ploidy. Haploid ascospores were used to construct triploid brewing strains carrying 0, 1, 2, or 3 maltose genes using spheroplast fusion and conventional mating techniques. These triploid strains exhibited a gene dosage effect with respect to fermentation rate corresponding to the number of maltose genes present in each. These results suggest a model for the construction of genetically improved brewing strains.