Molecular Characterization of Propolis-Resistant Saccharomyces cerevisiae Obtained by Evolutionary Engineering

Propolis is a highly complex, resinous natural product collected by honeybees from tree leaves and buds and mixed with pollen and enzymes. Due to its antimicrobial properties, it has various medical and industrial applications. As a nonconventional strategy, the use of propolis was suggested to control contaminating yeast growth in ethanol fermentations, without significantly affecting the starter yeast of the fermentation, Saccharomyces cerevisiae. In this study, we have developed a highly propolis-resistant S. cerevisiae strain using evolutionary engineering. The evolved strain FD11 had a higher growth rate (µ_max = 0.21 h⁻¹) than the reference strain (µ_max = 0.17 h⁻¹) under propolis stress and showed cross-resistance against caffeine stress. Moreover, it had significantly lower reactive oxygen species levels and higher cell wall integrity than the reference strain. Comparative transcriptomic analysis results revealed that the genes involved in oxidoreductase activity, transmembrane transporter activity, unfolded protein binding and pleiotropic drug resistance were upregulated in FD11. Whole genome re-sequencing analysis revealed mutations in multiple genes including PDR1, encoding a transcription factor regulating pleiotropic drug response. The results imply the importance of pleiotropic drug response and cell wall integrity in propolis resistance and the potential of using propolis-resistant, robust yeast strains in industrial applications.