In vivo recombination of Saccharomyces eubayanus maltose-transporter genes yields a chimeric transporter that enables maltotriose fermentation

Saccharomyces pastorianus lager-brewing yeasts are aneuploid S. cerevisiae x S. eubayanus hybrids, whose genomes have been shaped by domestication in brewing-related contexts. In contrast to most S. cerevisiae and S. pastorianus strains, S. eubayanus cannot utilize maltotriose, a major carbohydrate in brewer’s wort. Accordingly, S. eubayanus CBS 12357⊤ harbors four SeMALT maltose-transporter genes, but no genes resembling the S. cerevisiae maltotriose-transporter gene ScAGT1 or the S. pastorianus maltotriose-transporter gene SpMTY1 . To study the evolvability of maltotriose utilization in S. eubayanus CBS 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 mosaic SeMALT413 gene, resulting from repeated gene introgressions by non-reciprocal translocation of at least three SeMALT genes. The predicted tertiary structure of S e Malt413 was comparable to the original S e 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 sequences with high similarity to alternatingly ScMALx1 and 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 12357⊤ was used to enable maltotriose fermentation in brewer’s wort. A resulting S. eubayanus strain 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 different SeMALT maltose-transporter genes. Overexpression of SeMALT413 in CBS 12357⊤ confirmed its neofunctionalization as a maltotriose transporter. The mosaic structure of the maltotriose transporter SpMty1 in S. pastorianus resembles that of S e Malt413, suggesting that maltotriose utilization likely emerged through similar recombination events during the domestication of current lager brewing strains.