Uncovering the hidden yeast diversity in fermented coffee: Insights from a shotgun metagenomic approach.

Yeasts play a pivotal role in coffee fermentation, shaping microbial succession and contributing to the development of final flavor profiles. Despite their importance, yeast taxonomy in this context remains poorly resolved. Traditional classification methods often result in misidentifications due to the limited resolution of classical microbiological techniques and the rapidly evolving taxonomic framework driven by advances in phylogenomic. Moreover, the diversity of budding yeasts in coffee fermentations remains underexplored using high-resolution approaches such as metagenomics. To address this gap, we applied a shotgun metagenomic strategy and reconstructed metagenome-assembled genomes (MAGs) from multiple coffee fermentation samples and, using a robust phylogenomic framework based on 832 conserved single-copy genes. We confidently classified 22 yeast MAGs within the subphylum Saccharomycotina. These included well-known taxa such as Pichia kluyveri, Hanseniaspora spp., Torulaspora delbrueckii, and members of the Kurtzmaniella clade. Most MAGs were placed in strongly supported monophyletic groups (ultrafast bootstrap = 100), with short intra-clade branch lengths indicative of intraspecific variation. Pichia kluyveri emerged as the most abundant and widespread species, detected in all analyzed metagenomes, followed by Hanseniaspora spp. Our results underscore the power of high-resolution phylogenomic for classifying yeast MAGs and highlight the ecological importance of Pichia, Hanseniaspora, Torulaspora, and Kurtzmaniella in spontaneous coffee fermentations.