Abstract
Probiotics are living microorganisms that are increasingly used as gastrointestinal therapeutics by virtue of their innate or engineered genetic function. Unlike abiotic therapeutics, probiotics can replicate in their intended site, subjecting their genomes and therapeutic properties to natural selection. We exposed the candidate probiotic E. coli Nissle (EcN) to the mouse gastrointestinal tract over several weeks, systematically altering the diet and background microbiota complexity. In-transit EcN accumulates genetic mutations that modulate carbohydrate utilization, stress response, and adhesion to gain competitive fitness, while previous exposure to antibiotics reveals an acquisition of resistance. We then leveraged these insights to generate an EcN strain that shows therapeutic efficacy in a mouse model of phenylketonuria and found that it was genetically stable over 1 week, thereby validating EcN's utility as a chassis for engineering. Collectively, we demonstrate a generalizable pipeline that can be applied to other probiotics to better understand their safety and engineering potential.