Engineering and Restructuring of Complex Genetic Traits in Bacteria

Synthetic genomes hold great potential in industrial solutions for a wide range of challenges facing humanity in the 21st century. However, to unveil this potential, we need a better understanding of what is required to engineer microbes to fit our needs. Microbial features are encoded by their genes, which are structured into chromosomes. We have come far in mapping and understanding the function of single genes. However, the challenge remains in understanding how these genes come together and are regulated to produce complex genetic functions. Synthetic biologists have employed an engineering framework of four phases: design, build, test, and learn, that helps to address this biological complexity. This thesis explores how this framework allows the engineering of two inherently different genetic functions in the bacterium Escherichia coli.

The first function we explore is the endogenous ability to ferment maltose, which is linked to glycogen metabolism and is important for strain fitness during the shift between carbon sources. This link enhances the complexity of its manipulation. In this context, we transferred the maltose utilisation function to different chassis, which gives insight into the technical and biological challenges around mobilising and modularising complex genetic functions.

The second function is the heterologous T7 expression system expanded with oxidative folding factors, which allows the production of industrially relevant disulphide-containing proteins in the cytoplasm of Escherichia coli. This work  explores the reengineering of this established system and highlights the importance of balancing expression levels in polygenetic systems.

In summary, this work explores the effects of the genetic context of genetic modules and the balance of expression in genetic systems.