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

点击翻译按钮获取中文摘要

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.