Abstract
ABSTRACTIn 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 cyanobacterium Synechocystis sp. PCC 6803. Plasmid-encoded safeguards were constructed using the nonspecific nuclease NucA from Anabaena combined 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 the nucA gene fused to a variant of the copM promoter, efficient inducible autokilling was elicited. Parallel to nuclease-based safeguards, cyanobacterial toxin/antitoxin (TA) modules were examined in biosafety switches. Rewiring of Synechocystis TA pairs ssr1114/slr0664 and slr6101/slr6100 for 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 a nrsB variant, were characterized by beta-galactosidase reporter assay.
Highlights
Cyanobacteria display high metabolic versatility and are acquiring interest for biotechnological applications, as they are photosynthetic autotrophs that require little more than water, sunlight and CO2 for their growth and sustainable production of valuable metabolites
We addressed biosafety of genetically modified cyanobacteria through construction of biological containment systems in the model cyanobacterium Synechocystis sp
Some growth hindrance was seen for suicide switch cells without Zn2+-induction (Fig. 1D), reflecting weak leakiness of nuclease expression (Fig. 1B). These results indicate that the kill switch KSPcopM195-bicistronic design (BCD)-nucA is functional
Summary
Cyanobacteria display high metabolic versatility and are acquiring interest for biotechnological applications, as they are photosynthetic autotrophs that require little more than water, sunlight and CO2 for their growth and sustainable production of valuable metabolites. Chair of Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana 1000, Slovenia. Received 20 January 2016; Accepted 9 March 2016 of genetically modified cyanobacteria has remained largely unexplored. While modified cyanobacteria can be efficiently contained within closed photobioreactors, there is always a risk of accidental release, and uncertainty about possible negative ecological effects in case the engineered bacteria survive and establish themselves in the environment. The possibility of horizontal transfer of recombinant genetic material from genetically modified cyanobacteria to endogenous population is concerning, considering cyanobacteria are widely present in nature (Li et al, 2001; Vioque, 2007) and some strains are naturally transformable (Vioque, 2007)
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