Maltotriose consuming S. eubayanus IMS0750 Genome sequencing and assembly. Saccharomyces eubayanus strain:IMS0750

Contrarily to most Saccharomyces cerevisiae and S. pastorianus strains, S. eubayanus isolates are unable to utilize the trisaccharide maltotriose, which limits their industrial performance for lager brewing. The genome of S. eubayanus type strain CBS 12357 harbors four SeMalt maltose transporters. However, CBS 12357 does not encode any transporter with high similarity to the S. cerevisiae maltotriose transporter ScAgt1 or to the S. pastorianus maltotriose transporters SpMty1 and SeAgt1.In this study, S. eubayanus CBS 12357 was evolved to obtain maltotriose utilization. To this end, CBS 12357 was sporulated, UV-mutagenized and grown on synthetic medium with maltotriose as sole carbon source. Since the resulting mutants were unable to consume maltotriose in industrial brewer’s wort, they were submitted to laboratory evolution in a carbon-limited chemostat on wort enriched with maltotriose. The obtained mutants were sequenced and revealed the presence of a new transporter allele SeMALT413 resulting from massive recombination between SeMALT genes. The ORF was undisrupted and structural modelling predicted a normal tertiary structure. When overexpressed in CBS 12357, SeMALT413 conferred growth on maltotriose, confirming its functionality. Finally, the non-GMO evolved mutant IMS0750 showed improved brewing performance at 7-L scale on industrial wort, including improved maltose and maltotriose consumption, and faster degradation of the off-flavor compound diacetyl.The new SeMALT413 gene arose from complex rearrangements that were consistent with gene introgressions by non-reciprocal translocations, a key mechanism to introduce large phenotypic leap such as gene neofunctionalization Moreover, the recombinant structure of SeMALT413 was reminiscent of the maltotriose transporter gene SpMTY1 which alternates sequences with high similarity to ScMALx1 and SeMALT3. Therefore, we postulate that SpMTY1 may have emerged from a similar mechanism as SeMALT413. The industrial performance of IMS0750 clearly demonstrated the value of this laboratory evolution method for industrial strain improvement, particularly when non-GMO methods are required.