UC Berkeley

This dissertation presents the development of artificial metalloenzymes (ArMs) for unnatural reactions, the in vivo engineering and catalysis of ArMs, and the integration of ArMs with a natural biosynthetic pathway to produce products previously inaccessible by nature in a microbial host.Chapter 1 provides an overview of ArMs that are generated from incorporation of abiotic metal complexes into protein scaffolds. Assembly strategies and non-natural reactivity of this class of biohybrid catalysts are presented in detail. While a majority of work in this field has focused on the design and application of ArMs in vitro, the overview discusses the emerging trend towards integrating ArMs into whole-cell systems and metabolic networks and the challenges associated with the development of ArMs that function in vivo. Chapter 2 describes the directed evolution of an artificial iridium-containing P450 that catalyzed the cyclopropanation of monocyclic terpenes and their derivatives with diastereoselectivities that are complementary to those attained with chiral transition metal catalysts. The combination of the broad reactivity of iridium porphyrin and the exquisite selectivity provided by proteins generates catalysts with activity and selectivity previously unattainable with natural enzymes or transition metal complexes alone. Chapter 3 describes the application of a nonpathogenic Escherichia coli (E. coli) strain, Nissle 1917, for recombinant expression of P450s containing artificial cofactors. The outer-membrane receptor in Nissle 1917 transports iridium porphyrin into cells for binding to the apo-CYP119 expressed in the cytoplasm. We show that the Ir-CYP119 (CYP119 containing iridium porphyrin) assembled in vivo catalyzes carbene insertions into benzylic C-H bonds with remarkable enantioselectivity and site-selectivity. The application of Nissle 1917 as a whole-cell screening platform eliminates the need for laborious processing procedures, drastically increases the throughput of screening, and accelerates the development of Ir-CYP119 with improved catalytic properties. Chapter 4 describes the integration of artificial metalloenzymes that catalyze unnatural reactions into metabolic networks that could broaden the cache of molecules produced biosynthetically by microorganisms. We report the assembly of an artificial P450 containing an iridium-porphyrin complex in the cytoplasm of an E. coli that produces a terpene by heterologous expression and insertion of this ArM into the biosynthetic pathway to produce an unnatural terpenoid. This work shows that synthetic biology and synthetic chemistry, incorporated together in whole cells, can produce molecules previously inaccessible by natural metabolic pathways.