UC Berkeley

The thesis presented here focuses on the discovery and applications of tailoring enzymes for metabolic engineering goals along two tracks of research. The first track is focused on enzyme discovery and presents a powerful assay called the DNA-Linked Enzyme Coupled Assay (DLEnCA). This assay is readily accessible through common molecular biology techniques, and has the potential be scaled to the level of next generation sequencing and microfluidics. The goal is that DLEnCA will enable any research laboratory to populate enzyme databases (e.g., BRENDA) or have access to a platform for the evolution of novel enzymes and pathways. These capabilities would enable the rapid application of protein engineering for biocatalysts in the tailoring space.

The second track of research presented here is based on the in vivo application of tailoring enzymes for goals in metabolic engineering. On one level, this is focused on attempting to leverage tailoring enzymes’ role in metabolite detoxification for replacing antibiotics in industrial settings; on the other level, bioproducing valuable glucose-conjugates directly from yeast metabolism. This later focus is centered around the biosynthesis of the high value natural pigment betanin, which is found in select group of plants and utilized as a commercial food dye.

Ultimately, the research conducted here seeks to provide tools and lessons that adds to the body of knowledge around the biochemistry and engineering of modified secondary metabolites.