ABSTRACTSaccharomyces kudriavzeviiis a cold-tolerant species identified as a good alternative for industrial winemaking. AlthoughS. kudriavzeviihas never been found in winemaking, its co-occurrence withSaccharomyces cerevisiaein Mediterranean oaks is well documented. This sympatric association is believed to be possible due to the different growth temperatures of the two yeast species. However, the mechanisms behind the cold tolerance ofS. kudriavzeviiare not well understood. In this work, we propose the use of a dynamic genome-scale model to compare the metabolic routes used byS. kudriavzeviiat two temperatures, 25°C and 12°C, to decipher pathways relevant to cold tolerance. The model successfully recovered the dynamics of biomass and external metabolites and allowed us to link the observed phenotype with exact intracellular pathways. The model predicted fluxes that are consistent with previous findings, but it also led to novel results which we further confirmed with intracellular metabolomics and transcriptomic data. The proposed model (along with the corresponding code) provides a comprehensive picture of the mechanisms of cold tolerance that occur withinS. kudriavzevii. The proposed strategy offers a systematic approach to explore microbial diversity from extracellular fermentation data at low temperatures.IMPORTANCENonconventional yeasts promise to provide new metabolic pathways for producing industrially relevant compounds and tolerating specific stressors such as cold temperatures. The mechanisms behind the cold tolerance ofS. kudriavzeviior its sympatric relationship withS. cerevisiaein Mediterranean oaks are not well understood. This study proposes a dynamic genome-scale model to investigate metabolic pathways relevant to cold tolerance. The predictions of the model would indicate the ability ofS. kudriavzeviito produce assimilable nitrogen sources from extracellular proteins present in its natural niche. These predictions were further confirmed with metabolomics and transcriptomic data. This finding suggests that not only the different growth temperature preferences but also this proteolytic activity may contribute to the sympatric association withS. cerevisiae. Further exploration of these natural adaptations could lead to novel engineering targets for the biotechnological industry.