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 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 12357 T 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 12357 T , 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 Se MalT413 was comparable to the original Se MalT 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.

Saccharomyces pastorianus is an interspecies hybrid between S. cerevisiae and S. eubayanus. The identification of the parental species of S. pastorianus enabled the de novo reconstruction of hybrids that could potentially combine a wide array of phenotypic traits. Lager yeasts are characterized by their inability to decarboxylate ferulic acid present in wort, a phenotype also known as Pof- (phenolic off-flavor). However, all known S. eubayanus strains characterized so far produce clove-like aroma specific of 4-vinyl guaiacol, a decarboxylated form of ferulic acid. This study explored a non-GMO approach to construct Pof- S. eubayanus variants derived from the parental strain S. eubayanus CBS 12357. To rapidly screen a population of UV-mutagenized cells two complementary assays were developed. The first assay was based on the difference of light absorption spectra of ferulic acid and 4-vinyl guaiacol, while the second was based on the difference of sensitivity of Pof- and Pof+ strains to cinnamic acid. The S. eubayanus variant HTSE042 was selected and was confirmed not to produce 4-vinyl guaiacol. Whole genome sequencing revealed that this variant lost the subtelomeric region of the CHRXIII right arm that carried the two clustered genes SePAD1- SeFDC1 whose deletion in a naïve S. eubayanus strain (CBS 12357/FM1318) resulted in an identical phenotype. Subsequently, the Pof- variant was crossed with a Pof- S. cerevisiae partner. The resulting hybrid was not able to convert ferulic acid demonstrating the undisputable value of the mutagenized variant HTSE042 to eventually construct S. cerevisiae × S. eubayanus hybrids with phenotypic characteristics of S. pastorianus.

The archetypical system for regulating heterologous gene expression in mammalian cells involves tetracycline-activated transactivators (rtTA). Binding of such transactivators to tet-operator-controlled promoters induces transcription. Immune responses directed against the transactivator proteins may limit the applicability of this system in immune-competent hosts. To circumvent such immune responses the immune evasion mechanism of the Epstein-Barr virus Nuclear-Antigen 1 was exploited. Our data show that fusion of the rtTA with the EBNA-1 derived Gly-Ala repeat yielded an efficient transactivator with no detectable activity in absence of inducer. Antigenic peptides of the fusion protein were not presented in MHC class I.

28Saccharomyces pastorianus lager-brewing yeasts are aneuploid S. cerevisiae x S. eubayanus hybrids, 29 whose genomes have been shaped by domestication in brewing-related contexts. In contrast to most 30 S. cerevisiae and S. pastorianus strains, S. eubayanus cannot utilize maltotriose, a major 31 carbohydrate in brewer's wort. Accordingly, S. eubayanus CBS 12357 T harbors four SeMALT maltose-32 transporter genes, but no genes resembling the S. cerevisiae maltotriose-transporter gene ScAGT1 or 33 the S. pastorianus maltotriose-transporter gene SpMTY1. To study the evolvability of maltotriose 34 utilization in S. eubayanus CBS 12357 T , maltotriose-assimilating mutants obtained after UV 35 mutagenesis were subjected to laboratory evolution in carbon-limited chemostat cultures on 36 maltotriose-enriched wort. An evolved strain showed improved maltose and maltotriose 37 fermentation, as well as an improved flavor profile, in 7-L fermenter experiments on industrial wort. 38Whole-genome sequencing revealed a novel mosaic SeMALT413 gene, resulting from repeated gene 39 introgressions by non-reciprocal translocation of at least three SeMALT genes. The predicted tertiary 40 structure of SeMalt413 was comparable to the original SeMalt transporters, but overexpression of 41SeMALT413 sufficed to enable growth on maltotriose, indicating gene neofunctionalization had 42 occurred. The mosaic structure of SeMALT413 resembles the structure of S. pastorianus maltotriose-43 transporter gene SpMTY1, which has sequences with high similarity to alternatingly ScMALx1 and 44SeMALT3. Evolution of the maltotriose-transporter landscape in hybrid S. pastorianus lager-brewing 45 strains is therefore likely to have involved mechanisms similar to those observed in the present 46 study. 47 2 Author Summary 48 Fermentation of the wort sugar maltotriose is critical for the flavor profile obtained during beer 49 brewing. The recently discovered yeast Saccharomyces eubayanus is gaining popularity as an 50 alternative to S. pastorianus and S. cerevisiae for brewing, however it is unable to utilize maltotriose. 51Here, a combination of non-GMO mutagenesis and laboratory evolution of the S. eubayanus type 52 strain CBS 12357 T was used to enable maltotriose fermentation in brewer's wort. A resulting S. 53 eubayanus strain showed a significantly improved brewing performance, including improved maltose 54 and maltotriose consumption and a superior flavor profile. Whole genome sequencing identified a 55 novel transporter gene, SeMALT413, which was formed by recombination between three different 56SeMALT maltose-transporter genes. Overexpression of SeMALT413 in CBS 12357 T confirmed its 57 neofunctionalization as a maltotriose transporter. The mosaic structure of the maltotriose 58 transporter SpMty1 in S. pastorianus resembles that of SeMalt413, suggesting that maltotriose 59 utilization likely emerged through similar recombination events during the domestication of current 60 lager brewing strains. 61 62Saccharomyces eubayanus was discovered in Patagoni...