Keiko Kanai

An efficient method for isolating mating-competent cells from bottom-fermenting yeast using mating pheromone-supersensitive mutants

Crossbreeding is an effective approach to construct novel yeast strains with preferred characteristics; however, it is difficult to crossbreed strains of brewers yeast, especially the bottom‐fermenting yeast Saccharomyces pastorianus, because of the relative inefficiency of the available methods to obtain mating‐competent cells (MCCs). Here, we describe a productive method for the isolation of MCCs without artificial genetic modification. We focused on the characteristics of two mating pheromone‐supersensitive mutants, Δbar1 and Δsst2, that show a growth defect in the presence of the mating pheromone. When MCCs secreting α‐factor and a‐factor were spotted on to a lawn of MATa Δbar1 and MATα Δsst2, a halo was observed around the respective MCCs. This plate assay was successful in identifying MCCs from bottom‐fermenting yeast strains. Furthermore, by selecting for cells that caused the growth defect in pheromone‐supersensitive cells on cultures plates, 40 α/α‐type and six a/a‐type meiotic segregants of bottom‐fermenting yeast strains were successfully isolated and crossed with tester strains to verify their mating type. This method of isolation is expected to be applicable to other industrial yeast strains, including wine, sake and distillers yeasts, and will enable MCCs without genetic modifications to be obtained. As a result, it will be a useful tool for more convenient and efficient crossbreeding of industrial yeast strains that can be applied to practical brewing. Copyright

Intracellular proteins of ethanol-treated yeast cells involved in iron sorption.

Iron is essential for human health, but it sometimes causes an unpleasant taste, rusty colour and a decrease in the stability of food products. Previously, we found that ethanol-treated yeast (ETY) cells could remove iron from wine and juice, and reduce the fishy aftertaste induced by iron in wine-seafood pairings. However, the mechanism of iron sorption by ETY cells is undefined; thus, there is no indicator that can be used to estimate the iron sorption capacity of these cells. In this study, we showed that cell wall components are not mainly associated with iron sorption by investigating ETY cells with the cell wall removed. Moreover, plasma membrane permeability was correlated with the iron sorbing capacity of the cells. Microscopic analysis showed that iron accumulated within ETY cells. Proteinase-treated ETY cells had no iron sorbing capacity. On the basis of these results, we conclude that intracellular proteins are involved in iron sorption by ETY cells.

Multi-locus genotyping of bottom fermenting yeasts by single nucleotide polymorphisms indicative of brewing characteristics

Yeast plays a capital role in brewing fermentation and has a direct impact on flavor and aroma. For the evaluation of competent brewing strains during quality control or development of novel strains it is standard practice to perform fermentation tests, which are costly and time-consuming. Here, we have categorized DNA markers which enable to distinguish and to screen brewing strains more efficiently than ever before. Sequence analysis at 289 loci in the genomes of six bottom fermenting Saccharomyces pastorianus strains revealed that 30 loci contained single nucleotide polymorphisms (SNPs). By determining the nucleotide sequences at the SNP-loci in 26 other S. pastorianus strains and 20 strains of the top fermenting yeast Saccharomyces cerevisiae, almost all these strains could be discriminated solely on the basis of the SNPs. By comparing the fermentative phenotypes of these strains we found that some DNA markers showed a strong association with brewing characteristics, such as the production of ethyl acetate and hydrogen sulphide (H2S). Therefore, the DNA markers we identified will facilitate quality control and the efficient development of brewing yeast strains.