Towards Control and Containment of Engineered Escherichia coli

Escherichia coli has been a favorite chassis for synthetic biologists since the field arose and continues to be a microbe of choice for new efforts to generate microbes with improved or novel capabilities. More recently, the pursuit of engineered probiotics as live therapeutics in human have been increasingly explored. Despite decades of research focused on this single species, the knowns are still largely outweighed by the unknowns. While relatively wellcharacterized at lab scale, the behavior of engineered microbes in more complex settings is less well-defined. For engineered microbes to become a feasible route to efficient production of value products or as live biomedical devices, a set of requirements to strain performance and predictability becomes necessary.

The overall aim of this thesis was to develop technologies that would further enable the use of Escherichia coli as a cell factory – whether for production of value compounds in large-scale fermentations or as live therapeutic agents in the mammalian gut. In the context of industrial production, we show that evolutionary forces allow engineered cells to escape the burden of imposed production phenotypes, and how such detrimental production escape can be mitigated by coupling production to survival. In the context of the mammalian gut, we modulate the colonization of probiotic Escherichia coli by engineering synthetic prebiotic catabolism to controllably enrich for an engineered microbe in vivo. Lastly, we explore two different strategies for biocontainment of engineered microbes in the mammalian gastrointestinal tract: an environmentally triggered kill switch and engineered oxygen sensitivity to impair growth outside the gut.

While the task of engineering microbes to perform reliably and accurately across many different environmental contexts seems a formidable and almost unsurmountable task, we must simply go at it the same way one would go about eating an elephant: one bite at a time. The work included in this thesis presents strategies for increased control and predictability of engineered Escherichia coli – both in the context of industrial fermentation and in the context of the human body. Both settings present unique challenges to the design and performance of the microbes we engineer. As the field of synthetic biology continues to develop, it is the hope that the work presented here can aid the journey towards feasibility.