Stress-induced remodeling of yeast amino acid pools occurs within conserved physicochemical constraints

Industrial fermentation processes frequently expose yeast cells to environmental fluctuations, particularly temperature stress and oxygen-transfer limitation, which can profoundly alter cellular metabolism and fermentation performance. In this study, we quantified the free amino acid (FAA) pool and key fermentation metabolites in Saccharomyces cerevisiae ATCC 9763 under control conditions (30 °C), heat stress (37 °C), and oxygen-limited microaerobic conditions induced by a sterile paraffin overlay (n = 3 biological replicates per condition). Targeted LC-MS/MS analysis quantified 19 amino acids and four fermentation-related metabolites: ethanol, pyruvate, acetaldehyde, and glycerol. Metabolite set enrichment analysis (MSEA) was then used to identify pathway-level metabolic alterations. Heat stress caused broad depletion of FAA concentrations and enrichment of glutathione metabolism and nitrogen-associated pathways. In contrast, oxygen limitation increased the abundance of multiple amino acids and elevated ethanol, acetaldehyde, and glycerol levels. Pyruvate concentrations were highest under control conditions, lowest at 37 °C, and intermediate under oxygen limitation. Despite these condition-specific metabolic shifts, Pearson correlation analysis revealed highly conserved relationships between FAA abundance and 16 physicochemical descriptors across all conditions. FAA abundance showed strong positive correlations with molecular size-related properties, including molecular weight and surface area, and negative correlations with predicted aqueous solubility (ALogS), whereas charge-related descriptors contributed only weakly. Together, these findings suggest that stress-induced remodeling of yeast amino acid pools occurs within robust physicochemical constraints that preserve the overall architecture of intracellular metabolite composition. This framework provides quantitative insight into metabolic organization under stress and may support future strategies for strain engineering and industrial process optimization.