Non-domesticated, wild Saccharomyces yeasts have promising characteristics for beer diversification, particularly when used in the generation of de novo interspecific hybrids. A major motivation for the current work was the question whether attractive novel Saccharomyces interspecific hybrids can be created for the production of exotic lager beers without using the genomic resources of the ale yeast Saccharomyces cerevisiae. Importantly, maltotriose utilization is an essential characteristic typically associated with domesticated ale/lager brewing strains. A high-throughput screening on nearly 200 strains representing all eight species of the Saccharomyces genus was conducted. Three Saccharomyces mikatae strains were able to aerobically grow on maltotriose as the sole carbon source, a trait until recently unidentified for this species. Our screening also confirmed the recently reported maltotriose utilization of the S. jurei strain D5095^T. Remarkably, de novo hybrids between a maltotriose-utilizing S. mikatae or S. jurei strain and the maltotriose-negative Saccharomyces eubayanus strain CBS 12357^T displayed heterosis and outperformed both parents with regard to aerobically utilizing maltotriose as the sole source of carbon. Indeed, the maximum specific growth rates on this sugar were comparable to the well-known industrial strain, Saccharomyces pastorianus CBS 1513. In lager brewing settings (oxygen-limited), the new hybrids were able to ferment maltose, while maltotriose was not metabolized. Favorable fruity esters were produced, demonstrating that the novel hybrids have the potential to add to the diversity of lager brewing.

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.

The lager brewing yeast Saccharomyces pastorianus, an interspecies hybrid of S. eubayanus and S. cerevisiae, ferments maltotriose, maltose, sucrose, glucose and fructose in wort to ethanol and carbon dioxide. Complete and timely conversion ("attenuation") of maltotriose by industrial S. pastorianus strains is a key requirement for process intensification. This study explores a new evolutionary engineering strategy for improving maltotriose fermentation kinetics. Prolonged carbon-limited, anaerobic chemostat cultivation of the reference strain S. pastorianus CBS1483 on a maltotriose-enriched sugar mixture was used to select for spontaneous mutants with improved affinity for maltotriose. Evolved populations exhibited an up to 5-fold lower residual maltotriose concentration and a higher ethanol concentration than the parental strain. Uptake studies with ¹⁴C-labeled sugars revealed an up to 4.75-fold higher transport capacity for maltotriose in evolved strains. In laboratory batch cultures on wort, evolved strains showed improved attenuation and higher ethanol concentrations. These improvements were also observed in pilot fermentations at 1,000-L scale with high-gravity wort. Although the evolved strain exhibited multiple chromosomal copy number changes, analysis of beer made from pilot fermentations showed no negative effects on flavor compound profiles. These results demonstrate the potential of evolutionary engineering for strain improvement of hybrid, alloploid brewing strains.