Biotransformation discovery enabled by high-throughput mass-spectrometric enzyme activity screening and comparative genomics
- Author(s): de Rond, Tristan P. T.
- Advisor(s): Keasling, Jay D
- Klinman, Judith P
- et al.
The potential of replacing unsustainable petroleum feedstocks with the biological production of small molecules is stipulated on access to a wide array of enzyme-catalyzed transformations. In this dissertation, we have taken two distinct approaches to expanding the biocatalytic toolbox. In one case, we develop a high-throughput mass spectrometry-based assay for detecting enzyme activity. We lay out in detail the chemical synthesis, software development, assay optimization and benchmarking that has allowed the assay to become a robust method. We then showcase our technology to the screening of a cytochrome P450 mutant library, allowing us to identify enzyme variants with desired altered substrate specificity. In a complimentary enzyme discovery approach, we turn to the diversity of fascinating reactions already present in nature. Through comparative genomics of the marine bacterium Pseudoalteromonas rubra, we identify an enzyme responsible for catalyzing the impressive feat of regioselective C–H activation and C–C bond-formation in the biosynthesis of cycloprodigiosin.