Directed Evolution of Acid-Tolerant Phenotype Using an Engineered pH-Riboswitch

Extracellular pH constitutes an environmental signal which directs phenotype evolution and metabolism in living cells. Understanding the molecular triggers and events to which this process is regulated will enable the systematic reprogramming of biological systems with improved tolerance and production of short chain fatty acids for colonic disease prevention. To facilitate the discovery and interrogation of acid-tolerant phenotype during adaptive evolution, we designed and characterized a pH-sensing device that enables programmable gene expression control in prokaryotic hosts. Firstly, a rational learn-by-design approach was employed to enhance performance and invert behavior of a natural pH-riboswitch. The pH-sensing device was then decoupled from cellular context effects using an orthogonal phage RNAP to generate the riboswitch transcript. We also demonstrate the robustness and versatility of our system in a myriad of microbial chassis including probiotics. We envision the flexibility and simplicity of our system to facilitate the development of next generation designer probiotic and nutraceuticals.