Enzyme engineering for gain of function requires navigating a large combinatorial sequence space efficiently. Typically, many mutations are needed to get significant improvements, while a single “bad” mutation can inactivate the enzyme. To establish high-throughput screening and achieve enhanced resolution between two variants, genetic libraries of the organophosphate hydrolase enzyme paraoxonase 1 (PON1) were rapidly screened via an engineered positive-feedback circuit: a p-nitrophenol (PNP)-specific transcription factor (TF) regulated expression of PON1, which catalyzed paraoxon breakdown and PNP production. Rare active mutant colonies, picked by simple visual fluorescence of a PON1–green fluorescent protein (GFP) fusion, were characterized. In a single screening round, high (library-scale) throughput enabled the discovery of enhanced paraoxon degradation activity in PON1, including structurally unexpected mutations.

中文翻译：

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智能微生物细胞将催化和传感耦合在一起，以提供高通量的有机磷酸水解酶选择。

为了获得功能而进行的酶工程设计需要有效导航较大的组合序列空间。通常，需要许多突变才能获得显着改善，而单个“不良”突变可以使酶失活。建立高通量筛选和实现两个变体之间的增强的分辨率，所述有机磷酸酯水解酶对氧磷酶1（PON1）的遗传文库快速筛选通过工程化的正反馈电路：一个p-硝基苯酚（PNP）特异性转录因子（TF）调节PON1的表达，从而催化对氧磷分解和PNP产生。通过对PON1-绿色荧光蛋白（GFP）进行简单的可视荧光分析，鉴定了稀有的活性突变菌落。在单个筛选回合中，高（库规模）通量使得能够发现PON1中增强的对氧磷降解活性，包括结构上意外的突变。