In vivorecombination ofSaccharomyces eubayanusmaltose-transporter genes yields a chimeric transporter that enables maltotriose fermentation

AbstractSaccharomyces 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.