Unraveling Time-Resolved Transcriptional and Metabolic Shifts in the Mixed Fermentation of <i><i>Saccharomyces cerevisiae</i></i> and <i>Hanseniaspora uvarum</i>

Wine fermentation and flavor formation are shaped by complex biochemical reactions driven by a variety of microorganisms. Non-Saccharomyces yeasts, such as <i>Hanseniaspora uvarum</i> (HU), are often used in mixed fermentation with <i><i>Saccharomyces cerevisiae</i></i> (SC) to enhance wine aroma. However, the lack of systematic knowledge regarding transcriptional responses and metabolic behaviors during fermentation has hindered the rational control of the mixed fermentation processes. To address this, we investigated transcriptional dynamics and metabolic behavior throughout the entire fermentation process, with a particular focus on the roles of microbial metabolism in flavor formation during mixed fermentation with HU. At the transcriptional level, the addition of HU led to significant changes in SC's gene expression, particularly in pathways related to glyoxylate and dicarboxylate metabolism, pyruvate metabolism, and amino sugar and nucleotide sugar metabolism. Furthermore, using genome-scale metabolic modeling, we uncovered key metabolic strategies employed by the two strains in mixed fermentation. These include distinct sugar utilization patterns, ethanol production, fatty acid metabolism, and central carbon allocation strategies. Notably, we identified two metabolic bypasses, from dihydroxyacetone phosphate to glycerol and from glucose-6-phosphate to the pentose phosphate pathway, which were found to reduce ethanol production and maintain the metabolic balance. Flux distribution analysis also revealed connections among organic acids, amino acids, and fermentation products, highlighting the role of a partial TCA cycle during fermentation. Additionally, metabolic interactions between SC and HU were identified, contributing to the enhanced production of volatile compounds, such as 2-phenylethanol and indole-3-ethanol in mixed fermentation. These findings provide a more comprehensive understanding of transcriptional regulation and metabolic strategies under fermentation conditions. They also offer practical targets for future bioengineering efforts aimed at controlling and optimizing the wine flavor.