Maltotriose consumption by hybridSaccharomyces pastorianusis heterotic and results from regulatory cross-talk between parental sub-genomes

Abstract

S. pastorianus strains are hybrids of S. cerevisiae and S. eubayanus that have been domesticated for several centuries in lager-beer brewing environments. As sequences and structures of S. pastorianus genomes are being resolved, molecular mechanisms and evolutionary origin of several industrially relevant phenotypes remain unknown. This study investigates how maltotriose metabolism, a key feature in brewing, may have arisen in early S. eubayanus × S. cerevisiae hybrids. To address this question, we generated a near-complete genome assembly of Himalayan S. eubayanus strains of the Holarctic subclade. This group of strains have been proposed to be the origin of the S. eubayanus subgenome of current S. pastorianus strains. The Himalayan S. eubayanus genomes harbored several copies of a SeAGT1 α-oligoglucoside transporter gene with high sequence identity to genes encountered in S. pastorianus. Although Himalayan S. eubayanus strains are unable to grown on maltose and maltotriose, their maltose-hydrolase and SeMALT1 and SeAGT1 maltose-transporter genes complemented the corresponding null mutants of S. cerevisiae. Expression, in a Himalayan S. eubayanus strain, of a functional S. cerevisiae maltose-metabolism regulator gene (MALx3) enabled growth on oligoglucosides. The hypothesis that the maltotriose-positive phenotype in S. pastorianus is a result of heterosis was experimentally tested by constructing a S. cerevisiae × S. eubayanus laboratory hybrid with a complement of maltose-metabolism genes that resembles that of current S. pastorianus strains. The ability of this hybrid to consume maltotriose in brewer’s wort demonstrated regulatory cross talk between sub-genomes and thereby validated this hypothesis. These results provide experimental evidence of the evolutionary origin of an essential phenotype of lager-brewing strains and valuable knowledge for industrial exploitation of laboratory-made S. pastorianus-like hybrids.ImportanceS.pastorianus, a S.cerevisiae X S.eubayanus hybrid, is used for production of lager beer, the most produced alcoholic beverage worldwide It emerged by spontaneous hybridization and have colonized early lager-brewing processes. Despite accumulation and analysis of genome sequencing data of S.pastorianus parental genomes, the genetic blueprint of industrially relevant phenotypes remains unknown. Assimilation of wort abundant sugar maltotriose has been postulated to be inherited from S.cerevisiae parent. Here, we demonstrate that although Asian S.eubayanus isolates harbor a functional maltotriose transporter SeAGT1 gene, they are unable to grow on α-oligoglucosides, but expression of S. cerevisae regulator ScMAL13 was sufficient to restore growth on trisaccharides. We hypothesized that S. pastorianus maltotriose phenotype results from regulatory interaction between S.cerevisae maltose transcription activator and the promoter of SeAGT1. We experimentally confirmed the heterotic nature of the phenotype and thus this results provide experimental evidence of the evolutionary origin of an essential phenotype of lager-brewing strains.

Into the wild: new yeast genomes from natural environments and new tools for their analysis

“…This data set confirms the previously proposed population structure (Peris et al 2014(Peris et al , 2016Eizaguirre et al 2018), where two major populations were detected (Patagonia A/Population A/PA and Patagonia B/Population B/PB), which has been further divided into five subpopulations (PA-1, PA-2, PB-1, PB-2 and PB-3) (Eizaguirre et al 2018). Other isolates from outside Patagonia belong to PB, either the PB-1 subpopulation that is also found in Patagonia (Gayevskiy and Goddard 2016;Peris et al 2016), or a Holarctic-specific subpopulation (PB-Holarctic) that includes isolates from Tibet and from North Carolina, USA (Bing et al 2014;Peris et al 2016;Brouwers et al 2019), which represents the closest known wild relatives of the S. eubayanus subgenomes of lager-brewing yeasts (Bing et al 2014;Peris et al 2016). Furthermore, heterosis was recently demonstrated in a S. cerevisiae x Tibetan S. eubayanus hybrid, which showed that regulatory cross talk between the two subgenomes is partly responsible for maltotriose and maltose consumption (Brouwers et al 2019).…”

“…Other isolates from outside Patagonia belong to PB, either the PB-1 subpopulation that is also found in Patagonia (Gayevskiy and Goddard 2016;Peris et al 2016), or a Holarctic-specific subpopulation (PB-Holarctic) that includes isolates from Tibet and from North Carolina, USA (Bing et al 2014;Peris et al 2016;Brouwers et al 2019), which represents the closest known wild relatives of the S. eubayanus subgenomes of lager-brewing yeasts (Bing et al 2014;Peris et al 2016). Furthermore, heterosis was recently demonstrated in a S. cerevisiae x Tibetan S. eubayanus hybrid, which showed that regulatory cross talk between the two subgenomes is partly responsible for maltotriose and maltose consumption (Brouwers et al 2019). Multilocus data suggested that two more lineages from China, West China and Sichuan, diverged very early from all other known S. eubayanus strains, while Holarctic isolates from China had unusually low sequence diversity (Bing et al 2014).…”

Wild Patagonian yeast improve the evolutionary potential of novel interspecific hybrid strains for Lager brewing

“…Recent studies on novel S. cerevisiae x S. eubayanus hybrids suggest that hybrid fermentative vigour at low temperature results from a variety of genetic changes, including loss of heterozygosity (LOH), ectopic recombination, transcriptional rewiring, selection of superior parental alleles (Sipiczki, 2018), heterozygote advantage due to the complementation of loss-of-function mutations in the F1 hybrid genome (Brouwers et al, 2019), and novel structural and single nucleotide variants in the hybrid genome (Krogerus, Holmström, et al, 2018). A recent transcriptome analyses of a laboratory-made Lager hybrid strain under fermentation conditions highlighted that the regulatory 'cross-talk' between the parental subgenomes caused a novel sugar consumption phenotype in the hybrid (maltotriose utilization, essential for Lager fermentation), which was absent in both parental strains (Brouwers et al, 2019). Although these studies have greatly contributed to our understanding of the genetic basis of different lager phenotypes, most studies only considered a single S. eubayanus genetic background (type strain CBS 12357), which alone is not representative of the species-rich genetic diversity.…”

“…Experimental evolution with microbes is a powerful tool to study adaptive responses to selection under environmental constraints (Barrick & Lenski, 2013; Cooper, 2018; Maddamsetti et al, 2015; Payen & Dunham, 2016). Recent studies on novel S. cerevisiae x S. eubayanus hybrids suggest that hybrid fermentative vigour at low temperature results from a variety of genetic changes, including loss of heterozygosity (LOH), ectopic recombination, transcriptional rewiring, selection of superior parental alleles (Sipiczki, 2018), heterozygote advantage due to the complementation of loss-of-function mutations in the F1 hybrid genome (Brouwers et al, 2019), and novel structural and single nucleotide variants in the hybrid genome (Krogerus, Holmström, et al, 2018). A recent transcriptome analyses of a laboratory-made Lager hybrid strain under fermentation conditions highlighted that the regulatory ‘cross-talk’ between the parental subgenomes caused a novel sugar consumption phenotype in the hybrid (maltotriose utilization, essential for Lager fermentation), which was absent in both parental strains (Brouwers et al, 2019).…”