Stage-Resolved Transcriptional Regulation and Metabolic Interplay during Wine Fermentation with <i>Saccharomyces cerevisiae</i> and <i>Oenococcus oeni</i>

Wine fermentation and flavor development are mediated by complex microbial interactions. Previous studies have shown that coinoculation with <i>Oenococcus oeni</i> can alter acid composition and aroma, yet the underlying transcriptional responses of <i>S. cerevisiae</i> and the metabolic strategies adopted by both species remain poorly understood. In this study, we performed mixed fermentation of <i>S. cerevisiae</i> and <i>O. oeni</i>, dividing the process into 6 representative stages for stage-specific analysis. Comparative evaluation with pure <i>S. cerevisiae</i> fermentation revealed that the presence of <i>O. oeni</i> reshaped both the acid profile and aromatic complexity of wine, while also influencing the growth kinetics of <i>S. cerevisiae</i>. Time-resolved transcriptomics demonstrated that <i>O. oeni</i> markedly altered the transcriptional dynamics of <i>S. cerevisiae</i> through changing the magnitude of transcriptional level while incurring opposite regulatory patterns in a small set of genes involved in stress responses and sulfur metabolism. Through the integration of a community genome-scale metabolic model with stage-resolved transcriptomic constraints, we resolved dynamic flux distributions of the mixed fermentation (MF) system and further revealed the metabolic impact of <i>O. oeni</i> on <i>S. cerevisiae</i> and MF. <i>O. oeni</i> exhibited low competitiveness for glucose but actively utilized fructose to generate energy through the phosphoketolase pathway. Also, it showed reversible reaction fluxes of two reactions involved in acetate formation (ACKr/PTAr), thereby impacting acetate levels. In addition, metabolic interactions including amino acid (e.g., arginine, serine, threonine) and mannitol exchange were identified, reflecting both cooperative and competitive features of mixed fermentation. Collectively, this work provides the first comprehensive depiction of the transcriptional and metabolic interplay between <i>S. cerevisiae</i> and <i>O. oeni</i> during mixed fermentation, offering mechanistic insights into microbial cooperation and strategies for the rational design of wine fermentation and flavor quality.