Metataxonomic and metatranscriptomic analysis reveal microbial succession and metabolic functions activated during spontaneous and inoculated fermentation.

The use of commercial Saccharomyces cerevisiae strains is a well-established practice in winemaking, ensuring fermentation reliability. Recently, there has been growing interest in spontaneous fermentations to enhance wine regional typicity. Despite this, our understanding of how different fermentation strategies shape microbial community and metabolic functions during vinification remains limited. We compared spontaneous and inoculated fermentations of grapes from the Greek cultivar Vidiano. Metataxonomic analyses characterized the composition and succession of fungal and bacterial microbiota, while metatranscriptomics elucidated genes, potential functions and pathways mobilized by the vinification microbiota throughout fermentation. Fermentation stage emerged as the primary determinant of microbiota structure, whereas fermentation type exerted a secondary influence. Fungal communities displayed greater diversity at early stages, converging gradually (spontaneous) or rapidly (inoculated) to communities dominated by Saccharomyces spp. The bacterial community was mainly composed of lactic acid bacteria (LAB), with Oenococcus prevailing. Metatranscriptomic analysis indicated that fermentation stage strongly influenced microbial metabolic activity with S. cerevisiae shifting its metabolic priorities from stress responses to abiotic-biotic stress at the start of fermentation, to sugar transportation, glycolysis and ethanol production at mid-fermentation and to ethanol tolerance at fermentation end. Subsystem-level profiling of metatranscriptomics revealed that functional differences between fermentation strategies were most pronounced at fermentation start, with inoculated fermentations enriched in protein metabolism, regulatory, and stress-response pathways, while spontaneous fermentations showed higher representation of sugar transport, sulfur metabolism, and aromatic compound biosynthetic pathways. These differences diminished toward fermentation end, indicating functional convergence. Overall, early fermentation dynamics shape functional trajectories under different inoculation strategies.