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.

Highlights

  • Saccharomyces yeasts are used in various biotechnological industries including beer brewing, wine making, biopharmaceutical protein synthesis, and biofuels production (Balat, 2011; Nielsen, 2013; Marsit and Dequin, 2015; Jansen et al, 2017; Krogerus et al, 2017a)

  • A functional protocol for dual staining of parental strains, mating and fluorescence-activated cell sorting (FACS)-based sorting of double-stained cells was developed using the heterothallic haploid S. cerevisiae strains CEN.PK1135A (MATa, His−, Lys−, Trp−) and IMK439 (MATα, Ura−). Due to their complementary auxotrophies, the fraction of mated cells could be quantified before and after FACS-based selection of double-stained cells by measuring the ability to grow on synthetic medium without histidine, lysine, tryptophan, and uracil

  • To minimize dilution of the dye due to cell division, stained cells were mated by co-incubation in YPT medium at 12◦C, which resulted in slow growth of S. cerevisiae

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Summary

Introduction

Saccharomyces yeasts are used in various biotechnological industries including beer brewing, wine making, biopharmaceutical protein synthesis, and biofuels production (Balat, 2011; Nielsen, 2013; Marsit and Dequin, 2015; Jansen et al, 2017; Krogerus et al, 2017a). Saccharomyces hybrids occur in natural contexts such as the guts of wasps (Stefanini et al, 2016), strains with chimeric genomes are most commonly found in domesticated environments (Almeida et al, 2014; Boynton and Greig, 2014). Lager beer is brewed by S. cerevisiae × S. eubayanus hybrids, collectively indicated as S. pastorianus (Libkind et al, 2011), S. uvarum × S. eubayanus hybrids called S. bayanus are used for cider brewing among other applications (Naumov et al, 2001), and various double and triple hybrids between S. cerevisiae, S. kudriavzevii, and S. uvarum play an important role in aroma production during wine fermentation (González et al, 2006). Interspecies hybridization likely contributed to the evolution of domesticated Saccharomyces strains by facilitating horizontal gene transfer (Peter et al, 2018). Genetic admixture contributed to the distinct phenotypes of, for instance, ciderfermenting S. uvarum strains and wine-fermenting S. cerevisiae strains (Naumova et al, 2011; Dunn et al, 2012)

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