An Artificial Yeast Genetic Circuit Enables Deep Mutational Scanning of an Antimicrobial Resistance Protein

Understanding the molecular mechanisms underlying antibiotic resistance requires concerted efforts in enzymology and medicinal chemistry. Here we describe a new synthetic biology approach to antibiotic development, where the presence of tetracycline antibiotics is linked to a life-death selection in Saccharomyces cerevisiae. This artificial genetic circuit allowed the deep mutational scanning of the tetracycline inactivating enzyme TetX, revealing key functional residues. We used both positive and negative selections to confirm the importance of different residues for TetX activity, and profiled activity hotspots for different tetracyclines to reveal substrate-specific activity determinants. We found that precise positioning of FAD and hydrophobic shielding of the tetracycline are critical for enzymatic inactivation of doxycycline. However, positioning of FAD is suboptimal in the case of anhydrotetracycline, potentially explaining its comparatively poor degradation and potential as an inhibitor for this family of enzymes. By combining artificial genetic circuits whose function can be modulated by antimicrobial resistance determinants, we establish a framework to select for the next generation of antibiotics.