Integrated metatranscriptomic and metabolomic analyses reveal microbial succession and flavor formation pathways...

While microbial community composition during wine fermentation has been extensively characterized, how different vinification strategies shape active microbial metabolic pathways and flavor formation across various cultivation systems remains poorly understood. Moreover, previous studies have largely relied on correlation-based relationships between microbiota and flavor compounds, with limited mechanistic interpretation. To address this gap, we integrated metatranscriptomic (RNA-seq) and metabolomics (GC-MS and UHPLC-Q-TOF/MS) in a factorial design (2 cultivation systems × 2 vinification strategies × 3 biological replicates) based on Cabernet Sauvignon fermentations. Spontaneous fermentation exhibited a higher diversity of active microbes, dominated by Saccharomyces cerevisiae, Kazachstania humilis, Hanseniaspora uvarum, and Leuconostoc pseudomesenteroides. Transcript-level analysis revealed enhanced amino acid and lipid metabolism in indigenous consortia, consistent with elevated concentrations of ethyl acetate, phenethyl alcohol, unsaturated fatty acids, and amino acids. In contrast, controlled fermentation showed higher levels of phenolic compounds, as well as medium- and long-chain fatty acids and their ethyl esters. Multi-omics integration provided mechanistic evidence linking microbial gene expression to flavor formation. Coordinated expression of amino acid transaminases and fatty acid synthases in multispecies consortia was associated with increased production of higher alcohols and fatty acids, whereas elevated alcohol acetyltransferase expression in S. cerevisiae promoted ester biosynthesis in controlled fermentations. These findings establish a functional framework for harnessing indigenous microbiota to modulate natural wine flavor.