Biosafety of biotechnologically important microalgae: intrinsic suicide switch implementation in cyanobacterium Synechocystis sp. PCC 6803

In recent years, photosynthetic autotrophic cyanobacteria have attracted interest for biotechnological applications for sustainable production of valuable metabolites. Although biosafety issues can have a great impact on public acceptance of cyanobacterial biotechnology, biosafety of genetically modified cyanobacteria has remained largely unexplored. We set out to incorporate biocontainment systems in the model cyanobacteriumSynechocystissp. PCC 6803. Plasmid-encoded safeguards were constructed using the nonspecific nuclease NucA fromAnabaenacombined with different metal-ion inducible promoters. In this manner, conditional lethality was dependent on intracellular DNA degradation for regulated autokilling as well as preclusion of horizontal gene transfer. In cells carrying the suicide switch comprising thenucAgene fused to a variant of thecopMpromoter, efficient inducible autokilling was elicited. Parallel to nuclease-based safeguards, cyanobacterial toxin/antitoxin (TA) modules were examined in biosafety switches. Rewiring ofSynechocystisTA pairsssr1114/slr0664andslr6101/slr6100for conditional lethality using metal-ion responsive promoters resulted in reduced growth, rather than cell killing, suggesting cells could cope with elevated toxin levels. Overall, promoter properties and translation efficiency influenced the efficacy of biocontainment systems. Several metal-ion promoters were tested in the context of safeguards, and selected promoters, including anrsBvariant, were characterized by beta-galactosidase reporter assay.