Understanding mediated/direct electron transfer and solvent resistance by iterative cycles of directed monooxygenase evolution and refinement of computational models

通过定向单加氧酶进化的迭代循环和计算模型的细化来了解介导/直接电子转移和溶剂耐受性

• 批准号: 5426863
• 负责人: Professor Dr. Ulrich Schwaneberg, Ph.D.
• 金额: --
• 依托单位: Lehrstuhl für Biotechnologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2004
• 资助国家: 德国
• 起止时间: 2003-12-31 至 2009-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5426863?language=en
• 关键词: Understanding; mediated; direct; electron; transfer

We propose to fully develop and explore the potential of a novel mutagenesis method (SeSaM-Sequence Saturation Mutagenesis) for creating diversity on the gene level. The SeSaM method is superior to all existing epPCR method. It solves the fundamental problem of creating diversity on the gene level, and enables us to explore novel directed evolution strategies by minimizing stop codons and allowing high mutation frequencies. As an application we propose to use the SeSaM method for evolving a monooxygenase, P450 BM-3 from Bacillus megaterium. We aim to drive P450 BM-3 directly by electrical current and thereby eliminate the NADPH cofactor requirement. We are particularly interested in understanding and engineering an efficient interface for directly transferring electrons from an electrode surface to P450 BM-3 as well as improving turnover numbers of P450 BM-3 by surmounting the thermodynamic switch mechanism that controls within the protein the electron transfer from NADPH to catalytic heme center. Achieving a direct electrical communication between an electrode and an oxidoreductase is scientifically and economically interesting, especially for in-body applications such as miniaturized medical devices/sensors that do not require a sealing or a toxic mediator.

我们建议充分开发和探索一种新的诱变方法（SeSaM序列饱和诱变）的潜力，在基因水平上创造多样性。SeSaM方法上级所有现有的epPCR方法。它解决了在基因水平上创造多样性的基本问题，并使我们能够通过最小化终止密码子和允许高突变频率来探索新的定向进化策略。作为应用，我们建议使用SeSaM方法从巨大芽孢杆菌中进化出单加氧酶P450 BM-3。我们的目标是通过电流直接驱动P450 BM-3，从而消除NADPH辅因子的需求。我们特别感兴趣的是理解和设计一个有效的界面，用于直接将电子从电极表面转移到P450 BM-3，以及通过克服控制蛋白质内电子从NADPH转移到催化血红素中心的热力学开关机制来提高P450 BM-3的周转数。实现电极和氧化还原酶之间的直接电连通在科学上和经济上是令人感兴趣的，特别是对于体内应用，例如不需要密封或有毒介质的小型化医疗装置/传感器。