Rewiring Estrogen Receptor α into Bisphenol Selective Receptors Using Darwin Assembly-Based Directed Evolution (DADE) in <i>Saccharomyces cerevisiae</i>

Bisphenols are widely used in manufacturing plastics and resins, but their environmental persistence raises concerns to human health and ecosystems. Accurate measurements for bisphenols are crucial for effective monitoring and regulation. Analytical methods detect only preselected bisphenols, while bioassays assessing estrogen receptor α activation suffer from poor sensitivity and strong background signals due to estrogenic contaminations. To develop a bioassay in <i>Saccharomyces cerevisiae</i> with increased sensitivity and specificity for bisphenols, we performed multi-site directed mutagenesis and directed evolution of more than 10⁸ stably integrated estrogen receptor variants. By mutating the estrogen receptor α towards recognition of bisphenol A in yeast, we determined the preBASE variant (M421G_V422G_V533D_L536G_Y537S) with elevated bisphenol A sensitivity (EC50:329 nM) and lost estrogen responsiveness (EC50:0,17 mM). Further engineering yielded an off-target mutant, identified as the <b>B</b>isphenol-<b>A</b>ffinity and <b>S</b>pecificity-<b>E</b>nhanced (BASE) variant (M421G_V422G_V533D_L536G_Y537S_L544I) that uses bisphenols as its primary agonist (EC50:32 mM) and impaired estrogen sensitivity (EC50:85M). The rewiring into a bisphenol receptor was confirmed in ligand binding assays to purified ligand binding domains. Taken together, the identified variants form stepping stones for further protein engineering to generate bisphenol specific high-throughput yeast-based bioassays.