UC Berkeley

Microorganisms have enormous potential as catalysts for the conversion of renewable organic material into useful chemicals, such as fuels and polymers. But engineering metabolic pathways in microorganisms requires a greater understanding of the enzymes available to catalyze each reaction of a sequential biosynthetic scheme. Fundamental to the construction of organic molecules is the formation of carbon-carbon bonds. This dissertation describes our work to identify and characterize enzymes from non-canonical carbon metabolism pathways in nature. In particular, we focus on two fermentation pathways found in eukaryotic facultative anaerobes: branched acid fermentation in the roundworm Ascaris suum and wax ester fermentation in the single-celled algae Euglena gracilis. These pathways are based on thiolase enzymes, which catalyze carbon-carbon bond formation through Claisen condensation. Our investigation has led to a greater understanding of the factors that determine substrate and product selectivity in thiolases and thiolase-based pathways and has provided insight into the physiology of E. gracilis. We further demonstrate the use of A. suum enzymes to engineer pathways in bacteria for the production of α-methyl organic acids. Our results help to expand the synthetic capabilities of engineered microorganisms.