Triparental synthetic yeast hybrids as a platform for higher 2-phenylethanol yields and stress resistance.

2‑Phenylethanol (2‑PE) is a valuable aromatic alcohol widely used in the cosmetic, food, and pharmaceutical industries, yet its cytotoxicity remains a major bottleneck for microbial production. In this study, we evaluated a set of previously constructed triple hybrid yeast strains (H1-H5), derived from S. cerevisiae 10‑170 and the double hybrid II/6, to assess their potential for enhanced 2‑PE biosynthesis and tolerance. Comprehensive phenotypic analysis revealed that the triple hybrids consistently outperformed both parental strains, reaching 2-PE titers of up to 3 g/L within 72 h across different cultivation media. Depending on the parental strain used for comparison and the medium composition, the hybrids showed even up to 9-fold higher 2-PE production. Increase in production was observed under all tested cultivation conditions, indicating a robust and reproducible phenotype. In addition, the hybrids displayed increased tolerance to externally supplied 2-PE; for example, strain H1 retained 61% of its growth relative to untreated controls in the presence of 4 g/L 2-PE. The hybrids effectively combined advantageous traits from both parents, inheriting high production capacity from II/6 and strong tolerance from S. cerevisiae 10‑170, in some cases exceeding both. These findings highlight multi‑parental hybridisation as a scalable, non‑GMO strategy for developing robust yeast cell factories for industrial 2‑PE production and broader biotechnological applications.