Applications of experimental evolution: From native to synthetic genetic systems

Synthetic biology, which utilizes microbes as workhorses for various applications, has the potential to provide solutions to many current and future problems faced by our society. By utilizing tools and knowledge from, e.g., molecular biology, synthetic biology attempts to build new, synthetic biological functions, based on the genetic building blocks and foundations already established. It is, however, limited in the availability of these building blocks, and in the understanding of microbial hosts and how they cope with stressors. This thesis aims to improve understanding of the bacterium Escherichia coli, and how it adapts during starvation and ageing, in order to provide an improved foundation for future work in synthetic biology. The work presented in this thesis builds on observations of experimental evolution to elucidate functions of specific regulatory systems in E. coli. A main topic of this thesis is the cyclic AMP receptor protein, Crp; a global regulator of gene expression, which is important for optimal cell function during stressors such as starvation and heat stress. Initially, a thorough review is given of mutations studied in Crp throughout the last 50 years, as this kind of studies has constituted a major guide for elucidating Crp function. Mutants that arise during starvation are then investigated, with focus on a mutation in Crp, which renders it ligand-independent, and on sequential mutations, i.a., in Crp and a metabolic enzyme. The study of these sequential mutations and starvation responses unveils connections between Crpmediated gene regulation and nucleotide metabolism, which implicates Crp as a sensor of DNA and RNA demand. Further work reveals that mutations in other regulatory systems are able to alleviate starvation, including mutations in the DNA supercoiling machinery, indicating a role for Crp in genome organization, and mutations that affect the stability of sigma factors RpoS and RpoH, suggestiing a broader role for RpoH as a protein integrity sigma factor during growth as well as stress. Finally, local cis-acting mutations are shown to affect the interplay of Crp and sigma factors at specific promoters during starvation. In summary, the work of this thesis elucidates crucial aspects of stress responses in E. coli, which may be exploited and applied towards increased stability of microbial hosts in synthetic biology.