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 preexisting 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.

The lager brewing yeast Saccharomyces pastorianus, an interspecies hybrid of S. eubayanus and S. cerevisiae, ferments maltotriose, maltose, sucrose, glucose and fructose in wort to ethanol and carbon dioxide. Complete and timely conversion ("attenuation") of maltotriose by industrial S. pastorianus strains is a key requirement for process intensification. This study explores a new evolutionary engineering strategy for improving maltotriose fermentation kinetics. Prolonged carbon-limited, anaerobic chemostat cultivation of the reference strain S. pastorianus CBS1483 on a maltotriose-enriched sugar mixture was used to select for spontaneous mutants with improved affinity for maltotriose. Evolved populations exhibited an up to 5-fold lower residual maltotriose concentration and a higher ethanol concentration than the parental strain. Uptake studies with ¹⁴C-labeled sugars revealed an up to 4.75-fold higher transport capacity for maltotriose in evolved strains. In laboratory batch cultures on wort, evolved strains showed improved attenuation and higher ethanol concentrations. These improvements were also observed in pilot fermentations at 1,000-L scale with high-gravity wort. Although the evolved strain exhibited multiple chromosomal copy number changes, analysis of beer made from pilot fermentations showed no negative effects on flavor compound profiles. These results demonstrate the potential of evolutionary engineering for strain improvement of hybrid, alloploid brewing strains.