Directed evolution methods based on high-throughput growth selection enable efficient discovery of enzymes with improved function in vivo. High-throughput selection is particularly useful when engineering oxygenases, which are sensitive to structural perturbations and prone to uncoupled activity. In this work, we combine the principle that reactive oxygen species (ROS) produced by uncoupled oxygenase activity are detrimental to cell fitness with a redox balance-based growth selection method for oxygenase engineering that enables concurrent advancement in catalytic activity and coupling efficiency. As a proof-of-concept, we engineered P450-BM3 for degradation of acenaphthene (ACN), a recalcitrant environmental pollutant. Selection of site-saturation mutagenesis libraries in E. coli strain MX203 identified P450-BM3 variants GVQ-AL and GVQ-D222N, which have both improved coupling efficiency and catalytic activity compared to the starting variant. Computational modeling indicates that the discovered mutations cooperatively optimize binding pocket shape complementarity to ACN, and shift the protein’s conformational dynamics to favor the lid-closed, catalytically competent state. We further demonstrated that the selective pressure on coupling efficiency can be tuned by modulating cellular ROS defense mechanisms.

中文翻译：

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利用氧化应激来调节基于氧化还原平衡的氧化酶工程体内生长选择。

基于高通量生长选择的定向进化方法能够有效地发现具有改进的体内功能的酶。当设计加氧酶时，高通量选择特别有用，因为加氧酶对结构扰动敏感并且容易产生解偶联活性。在这项工作中，我们将非偶联加氧酶活性产生的活性氧（ROS）对细胞健康有害的原理与基于氧化还原平衡的加氧酶工程生长选择方法相结合，从而能够同时提高催化活性和偶联效率。作为概念验证，我们设计了 P450-BM3，用于降解苊 (ACN)（一种顽固的环境污染物）。在大肠杆菌菌株 MX203中选择位点饱和诱变文库，鉴定出 P450-BM3 变体 GVQ-AL 和 GVQ-D222N，与起始变体相比，它们均具有提高的偶联效率和催化活性。计算模型表明，所发现的突变协同优化了与 ACN 的结合袋形状互补性，并改变了蛋白质的构象动力学，以有利于盖子闭合的催化活性状态。我们进一步证明，可以通过调节细胞 ROS 防御机制来调节耦合效率的选择压力。