Thermal kill-switches based biocontainment for genetically engineered cyanobacteria

Cyanobacteria are promising photosynthetic microbial cell factories for carbon negative biomanufacturing. However, as cyanobacteria can readily survive in natural environments, their large-scale use in research and biotechnological applications raises biosafety concerns about potential environmental escape. Active biocontainment strategies offer potential to prevent their survival and proliferation upon accidental environmental release, but current designs often depend on laboratory inducers, limiting practical application due to cost and operational constraints. Here, we developed a temperature-responsive active biocontainment device (trABS) that integrates a toxin-based module with a cyanobacteria-adapted thermal switch, enabling precise activation of cell-killing under non-permissive temperatures (25 °C and 30 °C, representative of natural aquatic environments) while maintaining normal growth at 37 °C under permissive laboratory conditions. Our results showed that trABS completely inhibits cell proliferation under non-permissive conditions, achieving a killing efficiency below the detection threshold of 1 × 10 −8 within three days. In addition, the genetically engineered cyanobacteria embedded trABS maintained wild-type-like growth and metabolism under permissive conditions, remained genetically stable during long-term and scaled-up cultivation, and showed an escape efficiency below 1 × 10 −8 under the simulated aquatic environments. Together, these results highlight trABS as a robust, autonomous and cost-effective biocontainment system for genetically engineered cyanobacteria, providing stringent protection against unintended environmental survival. • Temperature responsive active biocontainment synthetic-device (trABS) designed in cyanobacteria. • Achieves ≤1 × 10 −8 escape rate at 25–30 °C within three days. • No growth or production burden at 37 °C laboratory conditions. • Meets National Institutes of Health (NIH) escape threshold for outdoor genetically engineered cyanobacterial cultivation. • Enables autonomous, cost-effective environmental-release control.