Applications of experimental evolution in the bacterium Escherichia coli

In our global attempt to transition our fossil-fuel based economy into a sustainable bioeconomy, the field of biotechnology has become a driving force, delivering biological solutions to the major challenges of the 21st century: plastic waste accumulation, pollution, resource depletion and diseases.

Within the field, microorganisms – so-called cell factories – are employed as biological production platforms to sustainably manufacture a desired product. However, host intolerance – hence the inability of a microbial cell to produce a desired product due to toxicity effects – is frequently hampering the efficacy of bioproduction processes, creating the need to expand our understanding of microbial tolerance and adaptation to stress in order to design more robust microbial production strains.

Laboratory evolution is a promising approach to facilitate this understanding and to elucidate the modes of bacterial adaptation. This thesis, therefore, presents the principles of evolution and their recent application in the field of modern biotechnology and employs natural evolution mechanisms occurring in the structured environment of “ageing” bacterial colonies to advance our knowledge of the native stress response of the model bacterium Escherichia coli to carbon starvation, heat and protein production stress.

The described work reveals new insights into the adaptation of global regulatory networks within the cell and how their perturbation can be compensated by DNA supercoiling – demonstrating further evidence for a histone-like role of the global bacterial regulator Crp (cAMP receptor protein). Moreover, performing laboratory evolution within production strain BL21(DE3), we identify RNA stability as a key factor involved in fitness-loss and impaired growth during recombinant protein production processes, leading to novel strain and plasmid solutions facilitating high yield production of “toxic” proteins.

Finally, a surface display-based screening approach for identifying novel enzyme candidates active on the polyester plastic PET (polyethylene terephthalate) was developed – with this contributing to finding more efficient and sustainable means to facilitate plastic waste degradation.