Description

The fluctuating energy price and recognition of environmental consequences like pollution and climate change caused by fossil fuels have driven interests in replacing petroleum-based fuels and chemicals with renewable green biofuels and chemicals. Due to their capability to absorb solar irradiation with abroad wavelength and CO₂ as sole energy and carbon sources, respectively, photosynthetic cyanobacteria have attracted significant attention as one promising alternative to the traditional biomass-based microbial cell factories. However, accompanying the expanding applications are the survival and dissemination of engineered cyanobacteria in natural environments, posing potential biosafety risks to ecosystems and human health.

To prevent laboratory leakage and potential spread of the engineered cyanobacteria in the environments, our team has developed a temperature-based mobility arresting and logic gate based killing biocontainment system using cyanobacterium Synechococcus elongatus PCC 7942 as a model system. Briefly, a temperature-sensitive regulatory system P_R-cI857 from λ bacteriophage is utilized to construct a sRNA-based mobility arresting system targeting cell pili, coupled with a genetic circuit employing toxin-antitoxin systems, to achieve cell biocontainment at both transcriptional and translational levels, ensuring engineered cyanobacteria not able to survive in non-permissive conditions, such as natural environments with temperature typically below 30°C, but grow normally under laboratory conditions (37°C). Escape experiments conducted under both laboratory conditions and stimulated natural pond conditions in Tianjin, China demonstrate that the system successfully achieves biocontainment, maintaining survival rates below those proposed by the National Institute of Health (NIH) guidelines for organisms containing recombinant DNA molecules (10 ⁻⁸).

E² safe project utilizes a synthetic biology approach to develop a “double-safe” system for mobility arresting and killing switches in cyanobacteria, without the addition of exogenous inducers, which could be implemented in various cyanobacterial chasses readily without major modification. It represents a novel strategy to address the biosafety risks associated with potential laboratory leakage and outdoor application of engineered cyanobacteria.