Phenotype-Independent Isolation of Interspecies Saccharomyces Hybrids by Dual-Dye Fluorescent Staining and Fluorescence-Activated Cell Sorting

Abstract

Interspecies hybrids of Saccharomyces species are found in a variety of industrial environments and often outperform their parental strains in industrial fermentation processes. Interspecies hybridization is therefore increasingly considered as an approach for improvement and diversification of yeast strains for industrial application. However, current hybridization methods are limited by their reliance on pre-existing or introduced selectable phenotypes. This study presents a high-throughput phenotype-independent method for isolation of interspecies Saccharomyces hybrids based on dual dye-staining and subsequent mating of two strains, followed by enrichment of double-stained hybrid cells from a mating population by fluorescence-activated cell sorting (FACS). Pilot experiments on intra-species mating of heterothallic haploid S. cerevisiae strains showed that 80% of sorted double-stained cells were hybrids. The protocol was further optimized by mating an S. cerevisiae haploid with homothallic S. eubayanus spores with complementary selectable phenotypes. In crosses without selectable phenotype, using S. cerevisiae and S. eubayanus haploids derived from laboratory as well as industrial strains, 10 to 15% of double-stained cells isolated by FACS were hybrids. When applied to rare mating, sorting of double-stained cells consistently resulted in about 600-fold enrichment of hybrid cells. Mating of dual-stained cells and FACS-based selection allows efficient enrichment of interspecies Saccharomyces hybrids within a matter of days and without requiring selectable hybrid phenotypes, both for homothallic and heterothallic strains. This strategy should accelerate the isolation of laboratory-made hybrids, facilitate research into hybrid heterosis and offer new opportunities for non-GM industrial strain improvement and diversification.

Lager-brewing yeasts in the era of modern genetics

“…Indeed, a recent study shows how hybrids could be obtained with this method without the use of any selectable phenotype (Gorter de Vries et al . 2019a). Such laboratory hybrids generally display increased evolvability, which can be beneficial for strain improvement, as illustrated by faster and superior evolution of ethanol tolerance in hybrids during laboratory evolution under high-ethanol conditions (Krogerus, Holmström and Gibson 2018).…”

Hybridization of Saccharomyces cerevisiae Sourdough Strains with Cryotolerant Saccharomyces bayanus NBRC1948 as a Strategy to Increase Diversity of Strains Available for Lager Beer Fermentation

“…As our sourdough strains are able to sporulate and produce viable spores, they were mated with the cryotolerant S. bayanus strain NBRC1948 to generate synthetic hybrids via a marker-less spore-to-spore mating approach. Generally, mass mating is fast procedure that is preferred over the spore-to-spore method and, recently, novel methods such as fluorescence-activated cell sorting (FACS) [ 69 ] were proposed for increasing and assisting marker-less hybrid recovery after rare mating. Here we demonstrated that spore-to-spore mating can produce hybrids without the requirement of any selectable phenotypes for the parental strains and at relatively high frequency if the conditions of mating have been optimized.…”

Beyond Saccharomyces pastorianus for modern lager brews: Exploring non-cerevisiae Saccharomyces hybrids with heterotic maltotriose consumption and novel aroma profile

“…The use of genetic modifications gave a convenient and rapid opportunity to study the fermentative and aromatic potential of such novel de novo hybrids in the lab environment. Still, alternative mating methods without genetic manipulations are strongly preferred if hybrids will be used in large-scale industrial applications in beer (Nakatomi and Gunge, 1972;Alexander et al, 2016;Gorter De Vries et al, 2019).…”