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

非天然细胞色素 P450 酶的定向进化：开发有效的生物催化剂并追踪酶功能的决定因素

• 批准号: 278933817
• 负责人: Dr. Oliver Brandenberg
• 金额: --
• 依托单位: Division of Chemistry and Chemical Engineering
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/278933817?language=en
• 关键词: Directed; evolution; non; natural; cytochrome

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.

酶在轻度条件下以精致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。但是，许多已知的酶不会催化许多具有工业意义的化学反应，严重限制了可用于生物催化生产过程的化合物的曲目。为了克服这种缺乏有用的酶，定向蛋白的进化已应用于具有非天然催化活性的新型酶。最近，通过这种方法获得了几种新型的细胞色素P450酶催化非天然反应，这令人印象深刻地证明了细胞色素P450酶的多功能性和可变性。然而，新型的P450酶经常显示出低催化活性和由于快速失活而导致的总周转率。这种低酶促活性禁止它们用作工业生物催化剂，并提出了一个基本的问题，即新颖，有效的酶在自然界中如何发展。因此，我建议将定向蛋白的进化用于设计非天然细胞色素P450酶，催化对活宿主细胞中高催化活性的氮化反应。 P450酶突变体的文库将通过随机的诱变和选定酶残基的位置定向诱变产生。将对P450变体进行筛选所得的突变库，显示整个活细胞中的催化活性的改善。将进行诱变和筛选的迭代弹性，以进化细胞色素P450变体，以高效率和体内稳定性催化氮化反应。在第二步中，这些改进的变体将经过详细的生化和结构分析，以定义体内酶活性的分子决定因素。拟议的研究将提供两个目标：首先，将获得具有强大体内活性的新型细胞色素P450酶，从而使其随后在生物催化生产过程中就业。其次，将产生对体内酶催化活性的分子决定因素和结构功能关系的见解，从而追踪从低效率酶到高度活性变体的进化路径，具有与其他蛋白质和酶家族的工程相关性的高度活性变体。因此，拟议的研究预计将推进生物催化领域，并有助于可持续化学生产过程。