UC Berkeley

Concerns over the sustainability and environmental impact of current processes that rely heavily on nonrenewable petroleum feedstock have created a need for developing alternative processes. Bioprocesses that use enzymes or whole cells to transform renewable biomass feedstock into commercial products are an alternative that could be more sustainable and environmentally friendly. Here, I present three novel frameworks for designing, assembling, and optimizing biological pathways that could be utilized in bioprocesses. The first framework uses enzyme families as libraries for identifying enzymes able to catalyze novel reactions. I used the framework to assemble a synthetic biological pathway that produces 3-methyl-3-butenol, 3-methyl-2-butenol, and 3-methyl-butanol from isopentenyl diphosphate. I also developed a novel colorimetric assay using N-methylbenzothiazolinone-2-hydrazone to rapidly quantify these three five-carbon alcohols. The second framework is called feedback-regulated evolution of phenotype (FREP), and describes a system that dynamically regulates the mutation rate in the cell based on the level of a particular phenotype. I used the FREP framework to engineer increased tyrosine production in Escherichia coli. Finally, the third framework describes the construction of synthetic transcription factors using metabolic enzymes. I used the framework to assemble four different transcription factors, all of which generate a transcriptional change as a result of a change in isopentenyl diphosphate concentration. By combining the FREP and synthetic transcription factor assembly frameworks, I implemented an adaptive control system that increased isopentenyl diphosphate production in Escherichia coli.