Continuous crossbreeding of sake yeasts using growth selection systems for a-type and α-type cells

Abstract

Sake yeasts belong to the budding yeast species Saccharomyces cerevisiae and have high fermentation activity and ethanol production. Although the traditional crossbreeding of sake yeasts is a time-consuming and inefficient process due to the low sporulation rates and spore viability of these strains, considerable effort has been devoted to the development of hybrid strains with superior brewing characteristics. In the present work, we describe a growth selection system for a- and α-type cells aimed at the crossbreeding of industrial yeasts, and performed hybridizations with sake yeast strains Kyokai No. 6, No. 7 and No. 9 to examine the feasibility of this approach. We successfully generated both a- and α-type strains from all parental strains, and acquired six types of hybrids by outcrossing. One of these hybrid strains was subjected to continuous crossbreeding, yielding the multi-hybrid strain, which inherited the genetic characteristics of Kyokai No. 6, No. 7 and No. 9. Notably, because all of the genetic modifications of the yeast cells were introduced using plasmids, these traits can be easily removed. The approach described here has the potential to markedly accelerate the crossbreeding of industrial yeast strains with desirable properties.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0216-x) contains supplementary material, which is available to authorized users.

Novel brewing yeast hybrids: creation and application

“…The plasmids also carry drug-resistance markers, which allow for the selection of hybrids. Fukuda et al (2016) describe another approach for selecting diploid cells with either an a or α mating type. In their technique, a / α diploid cells are transformed with a plasmid carrying either the a 1 or α 2 gene from the mating-type locus together with drug-resistance markers with promoters specific to either the a 1 or α 2 gene products.…”

Polyploid engineering by increasing mutant gene dosage in yeasts

“…LOH is a natural event that generates homozygous loci via chromosomal rearrangement in heterozygous loci (Daigaku et al, 2004;Alvaro et al, 2006;Andersen et al, 2008;Takagi et al, 2008). Although the spontaneous frequency of LOH is below 1 9 10 À4 as reported in previous studies (Hiraoka et al, 2000;Fukuda et al, 2016), it is higher than that of a point mutation, approximately 1 9 10 À6 (Hayashi and Umezu, 2017). After cultivating strain BY4743C without cerulenin, we isolated homozygous cells generated from heterozygous cells via LOH of the FAS2 using 25 lM cerulenin (Fig.…”

“…Sake is a traditional Japanese alcoholic beverage made from fermented rice, and sake yeast strains have many characteristics suitable for brewing, such as aromatic production and high ethanol tolerance (Katou et al, 2008). A previous study (Fukuda et al, 2016) has reported that K9 strain maintains a neardiploid DNA content, which was measured using FACS analysis after propidium iodide-staining. Unexpectedly, only homozygous cells were generated via recombination of the FAS2 gene in K9 strain (Fig.…”

Crossbreeding of Yeasts Domesticated for Fermentation: Infertility Challenges

“…MATa/a or MATα/α diploids generated through artificial mating type conversion are easily isolated from the cell population since the abundance ratio of target cells in the cell population reaches much beyond 20% by preventing autopolyploidization [36]. The mating type of the isolated cells can be identified by a mating assay using a and α tester strains [82][83][84]. Hybrid tetraploid strains are generated through cocultivation of MATa/a and MATα/α diploids derived from different strains, and selected from the cell population by using marker genes on episomal vectors introduced into each parental strain.…”

“…However, it is difficult to convert mating types of yeast strains with stuck mutation at the MAT gene as described above. Recently, the MAT gene of haploid cells was successfully substituted with a synthetic DNA containing MATa or MATα, through homologous recombination [84]. Mating type alteration of yeast cells was performed by suppressing the mating ability of parental cells in the same manner shown in Figure 7.…”