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

我们建议充分开发和探索一种新的突变方法(SESAM序列饱和突变)在基因水平上创造多样性的潜力。SESAM方法优于现有的所有epPCR方法。它解决了在基因水平上创造多样性的根本问题，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。我们的目标是直接用电流驱动P450 BM-3，从而消除对NADPH辅因的要求。我们特别感兴趣的是了解和设计一种有效的界面，用于将电子从电极表面直接转移到P450 BM-3，以及通过超越控制蛋白质内部从NADPH到催化血红素中心的电子转移的热力学开关机制来提高P450 BM-3的周转次数。在电极和氧化还原酶之间实现直接的电子通信在科学上和经济上都是有意义的，特别是对于体内应用，如不需要密封或有毒介质的微型医疗设备/传感器。

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.