Exploring Novel Enzyme Activity and Genetic Diversity Through Biocatalytic Engineering and Microbiome Mapping

Nature provides us with a wealth of natural product molecules and biological catalysts that represent a rich source of new life-saving pharmaceutical medicines and biotechnological tools for green chemistry. The synthetic challenges associated with derivatizing natural product molecules to access new and improved analogues present a new opportunity for protein and substrate engineering to develop robust biocatalytic methods. Bacterial iterative cytochrome P450 enzymes can perform late-stage C-H oxidation reactions on complex natural product scaffolds with exquisite levels of regio-, stereo- and chemoselectivity. These biocatalytic systems represent chemically useful tools for selective and multi-step oxidation cascades to facilitate access to structural diversification. Furthermore, tapping into unexplored biologically rich environments can enable the discovery of novel and structurally intriguing natural product scaffolds and the biocatalytic enzymes responsible for their biosynthesis and late-stage functionalization. Hence, discovery, characterization and engineering of new bioactive molecules and biocatalysts can advance medicine and biotechnology for improvement of human health and sustainable chemical transformations. Most of the studies presented in this thesis focus on characterizing and engineering the multifunctional bacterial P450 TamI to elucidate its substrate binding mechanism and apparent stepwise reaction order, as well as alter the step sequence, native regio-, chemo- and stereoselectivity, and number of reactions catalyzed. This was completed successfully with the design of a toolbox of TamI biocatalysts that override the innate substrate reactivity in a catalyst-controlled fashion, catalyzing the biosynthesis of five novel tirandamycin congeners that display previously unreported bioactivities against human pathogens. The ability of P450 TamI for catalyzing selective and mechanistically divergent oxidative pathways prompted us to elucidate TamI’s catalytic cycle. This work revealed that the enzyme uses multiple catalytically active oxidant species for its iterative oxidative cascade towards the formation of two of the new tri-oxidized tirandamycin congeners. This versatility motivated us to explore TamI’s scope for oxidizing unnatural bicyclic scaffolds using a substrate engineering approach. Guided by structural and biochemical results, synthetic analogues were made and tested in enzymatic assays to learn that P450 TamI can recognize, bind and turnover substrates with tirandamycin-like bicyclic moieties that harbor a cyclic ring at the tail of the molecule, even if missing the tetramic acid functionalities, but cannot oxidize a different bicyclic scaffold. The in-depth investigation of P450 TamI-mediated catalysis provides a foundation for future protein and substrate engineering efforts to manipulate iterative P450 enzymes to generate powerful biocatalysts to produce structurally novel natural product molecules. The last part of this work focuses on characterizing the microbial composition of a unique, extreme, and biologically rich ecosystem in the Peruvian Amazon, the Boiling River, for future genome mining of versatile tailoring enzymes, such as P450 TamI, that display thermostable properties for the development of robust biocatalytic systems. Using metagenomics and bioinformatic tools, we elucidate the bacterial and archaeal communities in this river, and identify samples with unique ecological roles that may translate into novel biosynthetic gene clusters. The continued investigation of these meso-, thermo- and hyperthermophiles expands our understanding of the biological diversity of fragile environments and builds the groundwork for future genomics and metabolic mining to discover new natural product molecules and their biosynthetic enzymes.