Yeast diversity in open agave fermentations across Mexico

Yeasts are a diverse group of fungal microorganisms that are widely used to produce fermented foods and beverages. In Mexico, open fermentations are used to obtain spirits from agave plants. Despite the prevalence of this traditional practice throughout the country, yeasts have only been isolated and studied from a limited number of distilleries. To systematically describe the diversity of yeast species from open agave fermentations, here we generate the YMX.1.0 culture collection by isolating 4,524 strains from 68 sites in diverse climatic, geographical, and biological contexts. We used MALDI-TOF mass spectrometry for taxonomic classification and a subset of the strains was verified by ITS and D1/D2 sequencing. The most abundant species isolated from all producing regions were Saccharomyces cerevisiae, Pichia kudriavzevii, Pichia manshurica, and Kluyveromyces marxianus. Despite the great diversity of environmental conditions and production practices the composition of yeast communities remained largely homogeneous throughout locations and fermentation stages, even if less abundant but commonly occurring yeasts were considered. Furthermore, ITS and D1/D2 sequencing revealed two candidate new species of Saccharomycetales. To explore the intraspecific variation of the yeasts from agave fermentations, we conducted genome sequencing on four isolates of the non-conventional yeast Kazachstania humilis. The genomes of these four strains were considerably distinct from other genomes of the same species, suggesting that they belong to a different population. Our work contributes to the understanding and conservation of an open fermentation system of great cultural and economic importance, providing a valuable resource to study the biology and genetic diversity of microorganisms living at the interface of natural and human-associated environments. TAKE AWAYO_LIWe isolated and identified 4,524 yeast strains from open agave fermentations in Mexico. C_LIO_LIYeast communities remained largely homogeneous throughout diverse locations. C_LIO_LIKazachstania humilis genomes differed significantly from isolates in other regions of the world. C_LIO_LIWe report two candidate new species related to the Pichia clade. C_LI