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

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 Saccharomyces cerevisiae 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 Bisphenol-Affinity and Specificity-Enhanced (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.