Design and Construction of Designer Bioester Libraries for Validation of the Modular Cell Theory

Renewable and sustainable fuels and chemicals are required for mankind to reduce their dependence on petroleum. Metabolic engineering and synthetic biology have provided avenues for production of renewable fuels and chemicals by using waste feedstocks derived from biomass, municipal, and off gases, such as carbon dioxide and methane, using microbial cell factories. However, development of optimal microbial cell factories has been a challenge due to the vast combinations of pathways, genetic parts, and hosts to produce a targeted product. The purpose of this work is for validation of the modular cell theory via rational pathway design and testing for development of optimal microbial cell factories. This dissertation is divided up into four different parts.

Part I focuses on engineering and production of butyrate ester libraries for use as fuels, flavors, fragrances and solvents, specifically ethyl butyrate, isopropyl butyrate, and isobutyl butyrate using a modular chassis cell derived from the modular cell theory. Part II focuses on the synthesis of designer esters from waste organic acids, the carboxylates, as well as characterizing the enzyme responsible for condensing ester precursor molecules for novel activity using the modular chassis cell. Part III focuses on the expansion of part II by modulating an ester precursor molecule for the production of novel esters that can be used as next generation biofuels. Part IV focuses on further validating the modular cell theory by using growth-based selection for ethanol production by varying open reading frames and genetic parts.

The work presented will validate and further provide insights to modular cell theory and ester biosynthesis from fermentable sugars and organic acids.