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The lager-brewing yeast Saccharomyces pastorianus is a hybrid between S. cerevisiae and S. eubayanus with an exceptional degree of aneuploidy. While chromosome copy number variation (CCNV) is present in many industrial Saccharomyces strains and has been linked to various industrially-relevant traits, its impact on the brewing performance of S. pastorianus remains elusive. Here we attempt to delete single copies of chromosomes which are relevant for the production of off-flavor compound diacetyl by centromere silencing. However, the engineered strains display CNV of multiple non-targeted chromosomes. We attribute this unintended CCNV to inherent instability and to a mutagenic effect of electroporation and of centromere-silencing. Regardless, the resulting strains displayed large phenotypic diversity. By growing centromere-silenced cells in repeated sequential batches in medium containing 10% ethanol, mutants with increased ethanol tolerance were obtained. By using CCNV mutagenesis by exposure to the mitotic inhibitor MBC, selection in the same set-up yielded even more tolerant mutants that would not classify as genetically modified organisms. These results show that CCNV of alloaneuploid S. pastorianus genomes is highly unstable, and that CCNV mutagenesis can generate broad diversity. Coupled to effective selection or screening, CCNV mutagenesis presents a potent tool for strain improvement.

S. pastorianus, an S. cerevisiae × S. eubayanus hybrid, is used for production of lager beer, the most produced alcoholic beverage worldwide. It emerged by spontaneous hybridization and 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 unresolved. Assimilation of maltotriose, an abundant sugar in wort, has been postulated to be inherited from the 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. cerevisiae regulator MAL13 (ScMAL13) was sufficient to restore growth on trisaccharides. We hypothesized that the S. pastorianus maltotriose phenotype results from regulatory interaction between S. cerevisiae maltose transcription activator and the promoter of SeAGT1. We experimentally confirmed the heterotic nature of the phenotype, and thus these results provide experimental evidence of the evolutionary origin of an essential phenotype of lager brewing strains., Saccharomyces pastorianus strains are hybrids of Saccharomyces cerevisiae and Saccharomyces eubayanus that have been domesticated for centuries in lager beer brewing environments. As sequences and structures of S. pastorianus genomes are being resolved, molecular mechanisms and evolutionary origins 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 nearly complete genome assembly of Himalayan S. eubayanus strains of the Holarctic subclade. This group of strains has been proposed to be the S. eubayanus subgenome origin of current S. pastorianus strains. The Himalayan S. eubayanus genomes harbored several copies of an S. eubayanus AGT1 (SeAGT1) α-oligoglucoside transporter gene with high sequence identity to genes encountered in S. pastorianus. Although Himalayan S. eubayanus strains cannot grow on maltose and maltotriose, their maltose-hydrolase and SeMALT1 and SeAGT1 maltose transporter genes complemented the corresponding null mutants of S. cerevisiae. Expression, in Himalayan S. eubayanus 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 an 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 subgenomes and thereby validated this hypothesis. These results support experimentally the new postulated hypothesis on the evolutionary origin of an essential phenotype of lager brewing strains and valuable knowledge for industrial exploitation of laboratory-made S. pastorianus-like hybrids. IMPORTANCE S. pastorianus, an S. cerevisiae × S. eubayanus hybrid, is used for production of lager beer, the most produced alcoholic beverage worldwide. It emerged by spontaneous hybridization and 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 unresolved. Assimilation of maltotriose, an abundant sugar in wort, has been postulated to be inherited from the 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. cerevisiae regulator MAL13 (ScMAL13) was sufficient to restore growth on trisaccharides. We hypothesized that the S. pastorianus maltotriose phenotype results from regulatory interaction between S. cerevisiae maltose transcription activator and the promoter of SeAGT1. We experimentally confirmed the heterotic nature of the phenotype, and thus these results provide experimental evidence of the evolutionary origin of an essential phenotype of lager brewing strains.

S. pastorianusstrains are hybrids ofS. cerevisiaeandS. eubayanusthat have been domesticated for several centuries in lager-beer brewing environments. As sequences and structures ofS. pastorianusgenomes 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 earlyS. eubayanus × S. cerevisiaehybrids. To address this question, we generated a near-complete genome assembly of HimalayanS. eubayanusstrains of the Holarctic subclade. This group of strains have been proposed to be the origin of theS. eubayanussubgenome of currentS. pastorianusstrains. The HimalayanS. eubayanusgenomes harbored several copies of aSeAGT1α-oligoglucoside transporter gene with high sequence identity to genes encountered inS. pastorianus. Although HimalayanS. eubayanusstrains are unable to grown on maltose and maltotriose, their maltose-hydrolase andSeMALT1andSeAGT1maltose-transporter genes complemented the corresponding null mutants ofS. cerevisiae. Expression, in a HimalayanS. eubayanusstrain, of a functionalS. cerevisiaemaltose-metabolism regulator gene (MALx3) enabled growth on oligoglucosides. The hypothesis that the maltotriose-positive phenotype inS. pastorianusis a result of heterosis was experimentally tested by constructing aS. cerevisiae×S. eubayanuslaboratory hybrid with a complement of maltose-metabolism genes that resembles that of currentS. pastorianusstrains. 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-madeS. pastorianus-like hybrids.ImportanceS.pastorianus, aS.cerevisiaeXS.eubayanushybrid, 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 ofS.pastorianusparental genomes, the genetic blueprint of industrially relevant phenotypes remains unknown. Assimilation of wort abundant sugar maltotriose has been postulated to be inherited fromS.cerevisiaeparent. Here, we demonstrate that although AsianS.eubayanusisolates harbor a functional maltotriose transporterSeAGT1gene, they are unable to grow on α-oligoglucosides, but expression ofS. cerevisaeregulatorScMAL13was sufficient to restore growth on trisaccharides. We hypothesized thatS. pastorianusmaltotriose phenotype results from regulatory interaction betweenS.cerevisaemaltose transcription activator and the promoter ofSeAGT1. 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.

BackgroundThe lager brewing yeast,S. pastorianus, is a hybrid betweenS. cerevisiaeandS. eubayanuswith extensive chromosome aneuploidy.S. pastorianusis subdivided into Group 1 and Group 2 strains, where Group 2 strains have higher copy number and a larger degree of heterozygosity forS. cerevisiaechromosomes. As a result, Group 2 strains were hypothesized to have emerged from a hybridization event distinct from Group 1 strains. Current genome assemblies ofS. pastorianusstrains are incomplete and highly fragmented, limiting our ability to investigate their evolutionary history.ResultsTo fill this gap, we generated a chromosome-level genome assembly of theS. pastorianusstrain CBS 1483 using MinION sequencing and analysed the newly assembled subtelomeric regions and chromosome heterozygosity. To analyse the evolutionary history ofS. pastorianusstrains, we developed Alpaca: a method to compute sequence similarity between genomes without assuming linear evolution. Alpaca revealed high similarities between theS. cerevisiaesubgenomes of Group 1 and 2 strains, and marked differences from sequencedS. cerevisiae strains.ConclusionsOur findings suggest that Group 1 and Group 2 strains originated from a single hybridization involving a heterozygousS. cerevisiaestrain, followed by different evolutionary trajectories. The clear differences between both groups may originate from a severe population bottleneck caused by the isolation of the first pure cultures. Alpaca provides a computationally inexpensive method to analyse evolutionary relationships while considering non-linear evolution such as horizontal gene transfer and sexual reproduction, providing a complementary viewpoint beyond traditional phylogenetic approaches.

Saccharomyces pastorianus lager-brewing yeasts are domesticated hybrids of S. cerevisiae x S. eubayanus that display extensive inter-strain chromosome copy number variation and chromosomal recombinations. It is unclear to what extent such genome rearrangements are intrinsic to the domestication of hybrid brewing yeasts and whether they contribute to their industrial performance. Here, an allodiploid laboratory hybrid of S. cerevisiae and S. eubayanus was evolved for up to 418 generations on wort under simulated lager-brewing conditions in six independent sequential batch bioreactors. Characterization of 55 single-cell isolates from the evolved cultures showed large phenotypic diversity and whole-genome sequencing revealed a large array of mutations. Frequent loss of heterozygosity involved diverse, strain-specific chromosomal translocations, which differed from those observed in domesticated, aneuploid S. pastorianus brewing strains. In contrast to the extensive aneuploidy of domesticated S. pastorianus strains, the evolved isolates only showed limited (segmental) aneuploidy. Specific mutations could be linked to calcium-dependent flocculation, loss of maltotriose utilization and loss of mitochondrial activity, three industrially relevant traits that also occur in domesticated S. pastorianus strains. This study indicates that fast acquisition of extensive aneuploidy is not required for genetic adaptation of S. cerevisiae × S. eubayanus hybrids to brewing environments. In addition, this work demonstrates that, consistent with the diversity of brewing strains for maltotriose utilization, domestication under brewing conditions can result in loss of this industrially relevant trait. These observations have important implications for the design of strategies to improve industrial performance of novel laboratory-made hybrids.

BackgroundThe lager brewing yeast,S. pastorianus, is a hybrid betweenS. cerevisiaeandS. eubayanuswith extensive chromosome aneuploidy.S. pastorianusis subdivided into Group 1 and Group 2 strains, where Group 2 strains have higher copy number and a larger degree of heterozygosity forS. cerevisiaechromosomes. As a result, Group 2 strains were hypothesized to have emerged from a hybridization event distinct from Group 1 strains. Current genome assemblies ofS. pastorianusstrains are incomplete and highly fragmented, limiting our ability to investigate their evolutionary history.ResultsTo fill this gap, we generated a chromosome-level genome assembly of theS. pastorianusstrain CBS 1483 from Oxford Nanopore MinION DNA sequencing data and analysed the newly assembled subtelomeric regions and chromosome heterozygosity. To analyse the evolutionary history ofS. pastorianusstrains, we developed Alpaca: a method to compute sequence similarity between genomes without assuming linear evolution. Alpaca revealed high similarities between theS. cerevisiaesubgenomes of Group 1 and 2 strains, and marked differences from sequencedS. cerevisiae strains.ConclusionsOur findings suggest that Group 1 and Group 2 strains originated from a single hybridization involving a heterozygousS. cerevisiaestrain, followed by different evolutionary trajectories. The clear differences between both groups may originate from a severe population bottleneck caused by the isolation of the first pure cultures. Alpaca provides a computationally inexpensive method to analyse evolutionary relationships while considering non-linear evolution such as horizontal gene transfer and sexual reproduction, providing a complementary viewpoint beyond traditional phylogenetic approaches.

Saccharomyces pastorianuslager-brewing yeasts are domesticated hybrids ofS. cerevisiaexS. eubayanusthat display extensive inter-strain chromosome copy number variation and chromosomal recombinations. It is unclear to what extent such genome rearrangements are intrinsic to the domestication of hybrid brewing yeasts and whether they contribute to their industrial performance. Here, an allodiploid laboratory hybrid ofS. cerevisiaeandS. eubayanuswas evolved for up to 418 generations on wort under simulated lager-brewing conditions in six independent sequential batch bioreactors. Characterization of 55 single-cell isolates from the evolved cultures showed large phenotypic diversity and whole-genome sequencing revealed a large array of mutations. Frequent loss of heterozygosity involved diverse, strain-specific chromosomal translocations, which differed from those observed in domesticated, aneuploidS. pastorianusbrewing strains. In contrast to the extensive aneuploidy of domesticatedS. pastorianusstrains, the evolved isolates only showed limited (segmental) aneuploidy. Specific mutations could be linked to calcium-dependent flocculation, loss of maltotriose utilisation and loss of mitochondrial activity, three industrially relevant traits that also occur in domesticatedS. pastorianusstrains. This study indicates that fast acquisition of extensive aneuploidy is not required for genetic adaptation ofS. cerevisiaexS. eubayanushybrids to brewing environments. In addition, this work demonstrates that, consistent with the diversity of brewing strains for maltotriose utilization, domestication under brewing conditions can result in loss of this industrially relevant trait. These observations have important implications for the design of strategies to improve industrial performance of novel laboratory-made hybrids.

Saccharomyces pastorianuslager-brewing yeasts are aneuploidS. cerevisiaexS. eubayanushybrids, whose genomes have been shaped by domestication in brewing-related contexts. In contrast to mostS. cerevisiaeandS. pastorianusstrains,S. eubayanuscannot utilize maltotriose, a major carbohydrate in brewer’s wort. Accordingly,S. eubayanusCBS 12357⊤harbors fourSeMALTmaltose-transporter genes, but no genes resembling theS. cerevisiaemaltotriose-transporter geneScAGT1or theS. pastorianusmaltotriose-transporter geneSpMTY1. To study the evolvability of maltotriose utilization inS. eubayanusCBS 12357⊤, maltotriose-assimilating mutants obtained after UV mutagenesis were subjected to laboratory evolution in carbon-limited chemostat cultures on maltotriose-enriched wort. An evolved strain showed improved maltose and maltotriose fermentation, as well as an improved flavor profile, in 7-L fermenter experiments on industrial wort. Whole-genome sequencing revealed a novel mosaicSeMALT413gene, resulting from repeated gene introgressions by non-reciprocal translocation of at least threeSeMALTgenes. The predicted tertiary structure of SeMalt413 was comparable to the original SeMalt transporters, but overexpression ofSeMALT413sufficed to enable growth on maltotriose, indicating gene neofunctionalization had occurred. The mosaic structure ofSeMALT413resembles the structure ofS. pastorianusmaltotriose-transporter geneSpMTY1, which has sequences with high similarity to alternatinglyScMALx1andSeMALT3. Evolution of the maltotriose-transporter landscape in hybridS. pastorianuslager-brewing strains is therefore likely to have involved mechanisms similar to those observed in the present study.Author SummaryFermentation of the wort sugar maltotriose is critical for the flavor profile obtained during beer brewing. The recently discovered yeastSaccharomyces eubayanusis gaining popularity as an alternative toS. pastorianusandS. cerevisiaefor brewing, however it is unable to utilize maltotriose. Here, a combination of non-GMO mutagenesis and laboratory evolution of theS. eubayanustype strain CBS 12357⊤was used to enable maltotriose fermentation in brewer’s wort. A resultingS. eubayanusstrain showed a significantly improved brewing performance, including improved maltose and maltotriose consumption and a superior flavor profile. Whole genome sequencing identified a novel transporter gene,SeMALT413, which was formed by recombination between three differentSeMALTmaltose-transporter genes. Overexpression ofSeMALT413in CBS 12357⊤confirmed its neofunctionalization as a maltotriose transporter. The mosaic structure of the maltotriose transporter SpMty1 inS. pastorianusresembles that of SeMalt413, suggesting that maltotriose utilization likely emerged through similar recombination events during the domestication of current lager brewing strains.

The haploid Saccharomyces cerevisiae strain CEN.PK113–7D is a popular model system for metabolic engineering and systems biology research. Current genome assemblies are based on short-read sequencing data scaffolded based on homology to strain S288C. However, these assemblies contain large sequence gaps, particularly in subtelomeric regions, and the assumption of perfect homology to S288C for scaffolding introduces bias. In this study, we obtained a near-complete genome assembly of CEN.PK113–7D using only Oxford Nanopore Technology's MinION sequencing platform. Fifteen of the 16 chromosomes, the mitochondrial genome and the 2-μm plasmid are assembled in single contigs and all but one chromosome starts or ends in a telomere repeat. This improved genome assembly contains 770 Kbp of added sequence containing 248 gene annotations in comparison to the previous assembly of CEN.PK113–7D. Many of these genes encode functions determining fitness in specific growth conditions and are therefore highly relevant for various industrial applications. Furthermore, we discovered a translocation between chromosomes III and VIII that caused misidentification of a MAL locus in the previous CEN.PK113–7D assembly. This study demonstrates the power of long-read sequencing by providing a high-quality reference assembly and annotation of CEN.PK113–7D and places a caveat on assumed genome stability of microorganisms., Whole genome sequencing of Saccharomyces cerevisiae strain CEN.PK113–7D using nanopore technology sequencing yielded a near complete reference genome with 770 Kbp of undisclosed novel genetic information.

Saccharomyces eubayanus is the non-S. cerevisiae parent of the lager-brewing hybrid S. pastorianus. In contrast to most S. cerevisiae and Frohberg-type S. pastorianus strains, S. eubayanus cannot utilize the α-tri-glucoside maltotriose, a major carbohydrate in brewer’s wort. In Saccharomyces yeasts, utilization of maltotriose is encoded by the subtelomeric MAL gene family, and requires transporters for maltotriose uptake. While S. eubayanus strain CBS 12357T harbors four SeMALT genes which enable uptake of the α-di-glucoside maltose, it lacks maltotriose transporter genes. In S. cerevisiae, sequence identity indicates that maltotriose and maltose transporters likely evolved from a shared ancestral gene. To study the evolvability of maltotriose utilization in S. eubayanus CBS 12357T, maltotriose-assimilating mutants obtained after UV mutagenesis were subjected to laboratory evolution in carbon-limited chemostat cultures on maltotriose-enriched wort. An evolved strain showed improved maltose and maltotriose fermentation in 7 L fermenter experiments on industrial wort. Whole-genome sequencing revealed a novel mosaic SeMALT413 gene, resulting from repeated gene introgressions by non-reciprocal translocation of at least three SeMALT genes. The predicted tertiary structure of SeMalT413 was comparable to the original SeMalT transporters, but overexpression of SeMALT413 sufficed to enable growth on maltotriose, indicating gene neofunctionalization had occurred. The mosaic structure of SeMALT413 resembles the structure of S. pastorianus maltotriose-transporter gene SpMTY1, which has high sequences identity to alternatingly S. cerevisiae MALx1, S. paradoxus MALx1 and S. eubayanus SeMALT3. Evolution of the maltotriose transporter landscape in hybrid S. pastorianus lager-brewing strains is therefore likely to have involved mechanisms similar to those observed in the present study., Author summary Fermentation of the wort sugar maltotriose is critical for the flavor profile obtained during beer brewing. The recently discovered yeast Saccharomyces eubayanus is gaining popularity as an alternative to S. pastorianus and S. cerevisiae for brewing, however it is unable to utilize maltotriose. Here, a combination of non-GMO mutagenesis and laboratory evolution of the S. eubayanus type strain CBS 12357T was used to enable maltotriose fermentation and improve brewing performance. The improved strain expressed a novel transporter gene, SeMALT413, which was formed by recombination between three different SeMALT maltose-transporter genes. Overexpression of SeMALT413 in CBS 12357T confirmed its neofunctionalization as a maltotriose transporter. As the S. pastorianus maltotriose transporter SpMty1 has a mosaic structure similar to SeMalT413, maltotriose utilization likely involved similar recombination events during the domestication of current lager brewing strains. Based on a posteriori sequence analysis, the emergence of gene functions has been attributed to gene neofunctionalization in a broad range of organisms. The real-time observation of neofunctionalization during laboratory evolution constitutes an important validation of the relevance and importance of this mechanism for Darwinian evolution.