Rapid Design, Construction, and Validation of Synthetic Metabolic Pathways in a Modular Escherichia coli (chassis) Cell

Current strain development has been hindered by the vast biochemical space in nature. The concept of modular cell design has been invented to enable rapid and predictable construction of multiple optimal production strains for efficient production of a large variety of biochemicals with minimal experimental effort. While modular cell design principles have been successfully validated in some cases, its development is still limited by the small library of the production modules demonstrated.

The goals of this thesis are i) to establish a framework for rapid design, construction, and validation of production modules to explore a large space of molecules (e.g., a library of esters), and ii) to demonstrate the modular cell design principles as a rapid strain development platform for production of molecules of interest. This dissertation consists of five chapters. Part I presents a framework for rapid design and construction of biosynthetic pathways for combinatorial biosynthesis of C4-derived esters including butyl acetate, ethyl butyrate, and butyl butyrate. Part II presents the de novo biosynthesis of lactate esters as green solvents as well as identification and alleviation of the most limiting enzymatic steps in lactate esters biosynthesis. Part III presents the development of a computer-guided rational protein engineering protocol for improved production of designer esters. Part IV presents the establishment of a 96-well plate-based high throughput ester synthesis screening platform. Part V presents the evaluation of growth selection of an efficient butyl acetate production module in a modular (chassis) cell.

Using knowledge gained from ester biosynthesis, the work presented will facilitate future strain development efforts for production of a large space of biofuels and biochemicals in a more systematic and efficient fashion.