Integrated Metaproteomics and Untargeted Metabolomics Reveal Season-Specific Enzyme Expression and Non-Volatile...

Seasonal fluctuations in open solid-state fermentation drive batch-to-batch variability in Chinese Baijiu Daqu; however, how environmental shifts reshape microbial functional expression and non-volatile flavour precursors in medium-high-temperature Daqu remains poorly resolved. In this study, data-independent acquisition (DIA)-based quantitative metaproteomics and untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics were integrated to characterise winter and summer Daqu from Luzhou, Sichuan. Among 2904 annotated non-volatile metabolites, orthogonal partial least squares discriminant analysis (OPLS-DA) revealed clear seasonal separation; 1472 differential metabolites (560 up- and 912 downregulated in winter vs. summer; variable importance in projection [VIP] > 1, p < 0.05) were enriched in glycolysis/gluconeogenesis, the tricarboxylic acid (TCA) cycle, amino acid biosynthesis, and starch/sucrose metabolism. DIA-based quantitative metaproteomics further resolved season-specific enzyme expression: summer Daqu exhibited elevated saccharolytic, glycolytic and amino-acid-converting enzymes (β-glucosidase, 6-phosphofructokinase, pyruvate dehydrogenase), whereas winter Daqu was enriched in glucose oxidase, phosphoenolpyruvate carboxykinase and aldehyde dehydrogenase, consistent with a pattern suggestive of carbon-storage prioritisation. Proteome-metabolome integration established a coherent "enzyme protein abundance-inferred metabolic tendency-metabolite accumulation" correlative framework axis: higher hydrolytic and central-carbon enzyme abundance in summer corresponded to increased maltose, lactate, acetate, L-glutamate and L-aspartate. Therefore, production season reshapes Daqu quality chiefly by corresponding to distinct patterns of in situ enzyme protein abundance, providing a DIA quantitative metaproteome-anchored mechanistic framework for screening high-expression starters and stabilising seasonal Daqu quality.