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.

酶在温和的条件下催化化学反应，具有极高的选择性和效率，使其成为生物技术应用和生态友好型绿色化学的上级工具。然而，许多工业上重要的化学反应不被任何已知的酶催化，严重限制了生物催化生产过程可获得的化合物的库。为了克服这种缺乏有用的酶，定向蛋白质进化已被应用于衍生具有非天然催化活性的新酶。最近，几个新的细胞色素P450酶催化非天然反应，获得了这种方法，令人印象深刻地证明了细胞色素P450酶的多功能性和可进化性。然而，新的P450酶经常表现出低的催化活性和低的总周转数，由于快速的酶失活。这种低的酶活性阻止了它们作为工业生物催化剂的使用，并提出了如何在自然界中开发新的，有效的酶的基本问题。因此，我建议采用定向蛋白质进化工程师非天然细胞色素P450酶催化aziridination反应对高催化活性的活宿主细胞。P450酶突变体的文库将通过所选酶残基的随机诱变和定点诱变产生。将筛选所得突变体文库中在全活细胞中显示出改善的催化活性的P450变体。将进行迭代轮的诱变和筛选，以在体内以高效率和稳定性进化催化氮丙啶化反应的细胞色素P450变体。在第二步中，将对这些改进的变体进行详细的生物化学和结构分析，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：首先，将获得具有有效体内活性的新型细胞色素P450酶，使其随后在生物催化生产过程中的就业成为可能。其次，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体的进化路径，这些变体与其他蛋白质和酶家族的工程具有潜在的相关性。因此，拟议的研究有望推动生物催化领域的发展，并为可持续的化学生产过程做出贡献。