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.

中文翻译：

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人工酵母遗传电路可实现抗微生物耐药蛋白的深层突变扫描

理解抗生素抗性的分子机制需要在酶学和药物化学上共同努力。在这里，我们描述了一种新的合成生物学方法来开发抗生素，其中四环素类抗生素的存在与酿酒酵母的生命死亡选择有关。。这种人工遗传电路允许对四环素灭活酶TetX进行深度突变扫描，从而揭示关键的功能残基。我们使用阳性和阴性选择来确认不同残基对TetX活性的重要性，并分析了不同四环素的活性热点，以揭示底物特异性活性决定因素。我们发现，FAD的精确定位和四环素的疏水性屏蔽对于强力霉素的酶促失活至关重要。然而，在脱水四环素的情况下，FAD的定位不是最佳的，这可能解释了其相对较差的降解以及作为该酶家族的抑制剂的潜力。通过结合其功能可由抗药性决定因素调节的人工遗传电路，