Bioelectrosynthesis of Cyclopropanes by Genetically Expanded and Wired P450BM3

P450BM3 is a widely studied enzyme that went through directed evolution and modifications to enable its promiscuous catalytic activities. However, its a large and complex enzyme that makes it very hard to engineer for specific desired reactions. We would like tobe able to engineer it in a predictable manner to use it for different reactions, as a case study we will improve its activity for cyclopropane synthesis. However, we will use novel tools that may simplify the ability to manipulate its catalytic activity and improve its efficiency. By two major means: a) we will modify its catalytic site, site-specifically using a unique combination of an encoded unnatural amino acid that is redox active (L-DOPA) and boronic acid derivatives thus creating a hybrid organic-biocatalyst. b) We will attach the redox active subunit of the enzyme to an electrode, thus will control the enzyme intra electron transferring mechanism. Electrons that are required for efficient synthesis will be injected directly from an electrode to which the enzyme will be site-specifically and electronically wired. A matrix of different synthetic conditions will be used to asses and study the efficiencyand yields of catalysis, and the resulting data will be further pursued to improve catalytic efficiency. If successful, this will be the first example of P450 wiring to an electrode for electrobiocatalysis purposes, this achievement could be a new era in biocatalysis. Generating effective biocatalysts that are driven by electrical energy for high energy molecules synthesis could prove of an utmost importance to the Navy as different energy rich molecules could be synthesized in a relatively cheap and clean manner. No US collaborators are included in this study, as this is part of an ONRG call. Desired outcome of this research: a series of several high impact publications will result from this research in catalysis specialized journals such as: Nature Catalysis, ACS Catalysis. As well as in general chemistry journals such as: Nature Chemistry and JACS. The PIs will deliver talks in conferences of several societys that specialize in Electrochemistry (ECS, ISE meetings) as well as Bioelectrochemistry and organic Catalysis meetings. A tool that is based on machine learning algorithm will be developed in order to aid in predictions of bioelectrocatalytic reactions on an electrode surface. In the case that our approach is successful, patent applications will be submitted to protect the IP generated through this research.