Directed evolution of non-natural cytochrome P450 enzymes:Developing potent biocatalysts and tracing the determinants of enzyme functionality

酶在温和的条件下以极高的选择性和效率催化化学反应，使其成为生物技术应用和环保绿色化学的卓越工具。然而，许多工业上重要的化学反应不是由任何已知的酶催化的，这严重限制了生物催化生产过程中可获得的化合物的种类。为了克服这种缺乏有用的酶的问题，蛋白质定向进化被应用于获得具有非自然催化活性的新型酶。最近，用这种方法获得了几种催化非自然反应的新型细胞色素P450酶，展示了细胞色素P450酶的多功能性和进化性。然而，由于酶的快速失活，新型P450酶经常表现出低催化活性和低总周转率。这种较低的酶活性限制了它们作为工业生物催化剂的使用，并提出了一个根本问题，即新的、高效的酶在自然界中是如何发展的。因此，我建议使用定向蛋白质进化来设计非天然细胞色素P450酶，以催化在活宿主细胞中的氮杂环化反应，从而获得高催化活性。P450酶突变体文库将通过随机突变和对选定的酶残基进行定点突变而产生。由此产生的突变库将被筛选，以寻找在整个活细胞中表现出更好的催化活性的P450变体。将进行反复几轮的诱变和筛选，以进化出在体内高效和稳定地催化氮杂环化反应的细胞色素P450变体。在第二步中，这些改进的变异体将接受详细的生化和结构分析，以确定体内酶活性的分子决定因素。因此，拟议的研究将服务于两个目标：第一，将获得具有强大体内活性的新的细胞色素P450酶，使它们能够随后用于生物催化生产过程。其次，将深入了解体内酶催化活性的分子决定因素和结构-功能关系，追踪从低效酶到高活性变异体的进化路径，这可能与其他蛋白质和酶家族的工程相关。因此，拟议的研究有望推动生物催化领域的发展，并有助于可持续的化学生产过程。

Enzymes catalyze chemical reactions with exquisite selectivity and efficiency under mild conditions, making them superior tools for biotechnological applications and eco-friendly green chemistry. However, many industrially important chemical reactions are not catalyzed by any known enzyme, severely limiting the repertoire of chemical compounds accessible to biocatalytic production processes. To overcome this lack of useful enzymes, directed protein evolution has been applied to derive novel enzymes with non-natural catalytic activity. Recently, several novel cytochrome P450 enzymes catalyzing non-natural reactions were obtained with this approach, impressively demonstrating the versatility and evolvability of cytochrome P450 enzymes. However, the novel P450 enzymes frequently show low catalytic activity and low total turnover numbers due to fast enzyme inactivation. This low enzymatic activity prohibits their use as industrial biocatalysts and raises the fundamental question how novel, efficient enzymes develop in nature. I therefore propose to employ directed protein evolution to engineer non-natural cytochrome P450 enzymes catalyzing aziridination reactions towards high catalytic activity in live host cells. Libraries of P450 enzyme mutants will be generated by random mutagenesis and site-directed mutagenesis of selected enzyme residues. The resulting mutant libraries will be screened for P450 variants showing improved catalytic activity in whole live cells. Iterative rounds of mutagenesis and screening will be performed to evolve cytochrome P450 variants catalyzing aziridination reactions at high efficiency and stability in vivo. In a second step, these improved variants will be subjected to detailed biochemical and structural analyses to define the molecular determinants of in vivo enzymatic activity. The proposed research would thereby serve two objectives: First, novel cytochrome P450 enzymes with potent in vivo activity would be obtained, enabling their subsequent employment in biocatalytic production processes. Secondly, insights into the molecular determinants and structure-function relationships of enzyme catalytic activity in vivo will be generated, tracing the evolutionary paths leading from an inefficient enzyme to a highly active variant with potential relevance for engineering of other protein and enzyme families. Thus, the proposed research is expected to advance the field of biocatalysis and contribute to sustainable chemical production processes.