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

Abstract

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 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.

Evaluation of promoters and ribosome binding sites for biotechnological applications in the unicellular cyanobacterium Synechocystis sp. PCC 6803

“…An alternative set of regulated promoters are metal ion inducible promoters that have already been used for several applications8910111213. In Synechocystis , a set of genes responsible for Ni 2+ , Co 2+ , and Zn 2+ tolerance are all grouped together in a gene cluster14.…”

Biocontainment of Genetically Engineered Algae

“…A variety of lethal genes have been utilized in cyanobacteria—both for biocontainment and for counterselection including proteins from toxin-antitoxin systems and phage lysis proteins ( Cheah et al, 2013 ; Čelešnik et al, 2016 ; Zhou et al, 2019 ). An important benefit to using toxins from antitoxin systems is that the antitoxin can be co-expressed at a low level (or induced only in the lab/industrial setting) to prevent leaky expression of the toxic protein from reducing growth rates in the lab.…”

Survivability of Wild-Type and Genetically Engineered Thermosynechococcus elongatus BP1 with Different Temperature Conditions

“…Approaches for biocontainment of GE cyanobacteria include additional genetic manipulation to nucleases, CO 2 -concentrating genes, and toxin-antitoxin systems to produce "kill switches." [22][23][24][25][26][27] It is also important to study the growth and survivability of the GE cyanobacteria in natural environments to determine how such species would behave if exposed to the environment. We propose that biocontainment may be contained in the organism itself, by using its natural properties as a growth barrier such as producing alcohol in thermophilic cyanobacterium.…”