Abstract
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.
Highlights
Saccharomyces eubayanus was discovered in Patagonia and identified as the non-S. cerevisiae parental species of hybrid S. pastorianus lager-type beer brewing yeasts [1,2]
Fermentation of the wort sugar maltotriose is critical for the flavor profile obtained during beer brewing
The improved strain expressed a novel transporter gene, SeMALT413, which was formed by recombination between three different SeMALT maltose-transporter genes
Summary
Saccharomyces eubayanus was discovered in Patagonia and identified as the non-S. cerevisiae parental species of hybrid S. pastorianus lager-type beer brewing yeasts [1,2]. Maltose and maltotriose utilization are encoded by genes clustered in the MAL loci, which can be present on up to five different chromosomes [13]. While MALS genes enable hydrolysis of both maltose and maltotriose, the MALT gene family comprises transporters with diverse substrate specificities [11,15]. In S. cerevisiae, most MAL loci harbor an ScMalx transporter (Fig 1), which transports maltose and other disaccharides, such as turanose and sucrose [12,16], but cannot import maltotriose [11]. Growth of the resulting strains S. eubayanus IMX1941 (SeSGA1Δ::ScTEF1p-SeMALT2-ScCYC1t) and IMX1942 (SeSGA1Δ::ScTEF1p-SeMALT413-ScCYC1t), as well as the wild-type strain CBS 12357T and the evolved isolate IMS0750 was tested on SM supplemented with different carbon sources (S7 Fig). IMX1942 grew at 0.03 ± 0.00 h-1 and consumed 45% of the maltotriose after 172 h (Fig 4D), demonstrating the capacity of SeMALT413 to transport maltotriose
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