Engineered Escherichia coli Nissle with an Inducible Phenylalanine Degrading Cassette and a Multi-input Kill Switch.

Engineered probiotics provide new avenues for disease management through diagnostic sensing, delivery of bioactive molecules, and modulation of host-microbiome interactions. However, successful clinical translation requires rational safeguards preventing unintended environmental release. We report an integrated phenylalanine (Phe) sensor and CRISPR-based kill-switch biocontainment system for phenylketonuria (PKU) management. We engineered KSPAL, a strain of Escherichia coli Nissle (EcN) 1917, to sense gut Phe levels and responsively express phenylalanine ammonia lyase (PAL). The kill switch triggers bacterial death below 33°C or upon anhydrotetracycline (aTc) administration, ensuring post-excretion clearance. In vitro, KSPAL demonstrated dose-dependent degradation, achieving more than 60-fold Phe reduction following a 1000 µmol/L Phe spike, and bacterial death upon aTc or temperature induction. In vivo, KSPAL treatment reduced serum Phe by threefold 2-hours post Phe bolus, and KSPAL death 24-hours post aTc induction. This work presents a tunable therapeutic platform for PKU disease management via engineered probiotics.