Phenotype-independent isolation of interspecies Saccharomyces hybrids by dual-dye fluorescent staining and fluorescence-activated cell sorting

Phenotype-independent isolation of interspecies Saccharomyces hybrids by dual-dye fluorescent staining and fluorescence-activated cell sorting

Gorter de Vries, A.R. (TU Delft BT/Industriele Microbiologie)
Koster, C.C. (TU Delft BT/Industriele Microbiologie)
Weening, S.M. (TU Delft BT/Industriele Microbiologie)
Luttik, M.A.H. (TU Delft BT/Industriele Microbiologie)
Kuijpers, N.G.A. (TU Delft BT/Biotechnologie)
Geertman, Jan Maarten A. (Heineken Netherlands Supply)
Pronk, J.T. (TU Delft BT/Biotechnologie)
Daran, J.G. (TU Delft BT/Industriele Microbiologie)

BT/Biotechnologie

2019

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.

FACS
Heterosis
Lager beer brewing
Marker-free mating
Non-GMO
Saccharomyces eubayanus × Saccharomyces cerevisiae hybrids

http://resolver.tudelft.nl/uuid:9bdd4b62-ac11-4614-931b-a6fc247e508f

https://doi.org/10.3389/fmicb.2019.00871

1664-302X

Frontiers in Microbiology, 10 (APR)

Institutional Repository

journal article

© 2019 A.R. Gorter de Vries, C.C. Koster, S.M. Weening, M.A.H. Luttik, N.G.A. Kuijpers, Jan Maarten A. Geertman, J.T. Pronk, J.G. Daran