Practical application of new synthetic molecules for the betterment of human health depends directly on theefficiency with which these compounds can be synthesized, but this is frequently limited by poor reaction yieldsthroughout long reaction sequences in which intermediate compounds must be isolated and purified.Metabolic engineers have demonstrated that novel biosynthetic pathways can be assembled in order toproduce chemicals in vivo with no isolation of intermediates in an aqueous aerobic environment, but thesesequences are limited to transformations catalyzed by natural enzymes. This proposal describes the design,preparation, and application of a new class of artificial metalloenzymes that combines the scope of chemicalcatalysis with the efficiency of biosynthesis in an unprecedented manner to produce molecules of exceptionalbiological importance. The proposed system offers a number of significant advantages over previous artificialmetalloenzyme constructs, which enable its use for in vivo catalysis and metabolic engineering. This ambitiousproject will be conducted as part of the candidate's long term goals of increasing the efficiency of organicsynthesis, particularly for the production of biologically active molecules. In the mentored phase (K99) of the proposed research, amino acids with catalytically active palladacycleside chains will be synthesized, characterized, and incorporated into a suitable scaffold protein. The catalyticactivity of the resulting metalloenzymes will be evaluated using a variety of C-C bond forming reactions. Theproposed amino acids catalysts could prove highly useful for a variety of applications in their own right, andtheir incorporation into proteins would mark a significant achievement in the fields of UAA incorporation andbiocatalysis with potential applications well beyond the scope of this application. This research will beconducted in the laboratory of Professor Frances Arnold, a leader in the field of protein engineering, at theCalifornia Institute of Technology, a world-renowned research institution. Professor Arnold has a strong recordas a mentor of successful members of industry and academia, and she and the candidate have outlined acareer development plan focusing on mentorship, writing, and research to ensure the candidate continues thistrend. The facilities, faculty, and staff at Caltech are ideal for completion of the proposed research and willcontribute greatly to the candidate's overall development as an independent scientist. Independent (R00) research will focus on directed evolution of artificial metalloenzymes for in vivopalladium catalysis of pharmaceutically important cross-coupling reactions with potential applications in organicsynthesis and bio-orthogonal diagnostics. Optimized metalloenzymes will also be expressed with additionalenzymes in E. coli in order to biosynthesize biologically active molecules, including indolocarbazole naturalproduct derivatives. Success in this venture would greatly expand the scope of molecules available viametabolic engineering and simplify the production of new compounds for the betterment of human health. Thiswork will build directly on the candidate's experiences in the Arnold lab, and should foster the development ofan exciting and collaborative research environment in the candidate's independent laboratory focusing on thedevelopment and application of enzymes for sustainable organic synthesis.
The research outlined in this proposal has the potential to greatly improve public health by creating a new class of artificial metalloenzymes for the synthesis biologically active molecules. This platform will enable inclusion of powerful transition metal catalysts in metabolic pathways in unprecedented fashion in order to efficiently produce chemicals in vivo.
