GENOMIC ADAPTATION OF AN AUTOCHTHONOUS CIDER YEAST STRAIN TO BUCKWHEAT AND BARLEY WORT UNDER STRESSFUL BREWING CONDITIONS

Abstract

Growing consumer demand for specialty beers with unique flavors and enhanced nutritional properties is driving the development of novel, high-performance industrial yeasts. However, the genetic diversity of beer yeast strains is limited. Traditional spontaneous fermentations are a rich source of new strains that are well adapted to fermentative environments but lack the ability to efficiently convert maltose-based substrates that are rich in polyphenols (e.g., buckwheat wort) or maltotriose-rich substrates (e.g., barley wort). To simulate the selection pressure exerted on beer yeasts during domestication, we used adaptive laboratory evolution (ALE) to yield cider yeast Saccharomyces cerevisiae that can efficiently convert buckwheat and barley wort into beer. To this end, 30 serial transfers of yeast biomass were conducted in high-pressure fermenters simulating industrial-scale stress conditions. This approach resulted in efficient maltose conversion in buckwheat wort and improved maltotriose conversion in barley wort. Three evolved clones from each evolutionary experiment were sequenced using short-read technology and aligned to the chromosome-level assembly of the ancestral cider strain. We observed pronounced genomic changes, including near-complete loss-of-heterozygosity, novel single-nucleotide mutations, and chromosomal aberrations resulting in altered chromosome copy numbers or segmental duplications. Additionally, the clones adapted to buckwheat wort were respiratory-deficient, either lacking or having impaired mitochondrial DNA, whereas clones adapted to barley wort retained a truncated mitochondrial genome. These genetic changes mirror hallmarks of beer yeast domestication and were also reflected phenotypically, including loss of sporulation capacity, decreased fitness under non-brewing conditions, and altered production of aromatic compounds. IMPORTANCEConsumer demand for specialty beers with distinctive flavors and nutritional value is growing and highlights the need for novel, high-performance beer yeasts adapted to stressful industrial conditions. This study demonstrates how adaptive laboratory evolution can be used to domesticate non-traditional yeasts, enabling efficient fermentation of alternative substrates, such as buckwheat and barley worts. The evolved strains not only improved sugar utilization under industrial conditions but also acquired genomic and phenotypic traits characteristic of domesticated beer yeasts. These findings demonstrate a viable strategy for expanding the functional diversity of brewing yeasts and support innovation in craft beer production.