Development of a New Biocontainment Strategy in Model Cyanobacterium Synechococcus Strains

Recent synthetic biology efforts have raised biosafety concerns for possible release of engineered cyanobacteria into natural environments. To address the issues, we developed a controllable metal ion induced biocontainment system for two model cyanobacteria. First, six ion-inducible promoters were respectively evaluated in both Synechococcus elongatus PCC 7942 and the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973, leading to the identification of an iron ion-repressed promoter PisiAB with low leakage and a reduction-fold of 5.4 and 7.9, respectively. Second, holin-endolysin and nuclease NucA systems were introduced, the inhibition rate of which against two Synechococcus strains varied from 61% to 86.4%. Third, two toxin/antitoxin modules were identified capable of inducing programmed suicide in both Synechococcus strains after induction. Furthermore, an escape experiment was conducted and the results showed that the system was able to achieve an escape frequency below the detection limit of 10^-9 after 3 days' duration, demonstrating the strategy integrating iron ion-inducible promoter PisiAB and that toxin/antitoxin modules could be a useful tool for cyanobacterium biocontainment.