Chair of Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana 1000, Slovenia.
Chair of Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana 1000, Slovenia
Biol Open. 2016 Apr 15;5(4):519-28. doi: 10.1242/bio.017129.
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.
近年来，光合自养蓝藻因其可持续生产有价值代谢物的生物技术应用而受到关注。尽管生物安全问题会对公众对蓝藻生物技术的接受程度产生重大影响，但转基因蓝藻的生物安全问题在很大程度上仍未得到探索。我们着手在模式蓝藻Synechocystissp. PCC 6803 中引入生物 containment 系统。使用来自 Anabaena 的非特异性核酸酶 NucA 与不同金属离子诱导启动子结合，构建了质粒编码的保护系统。通过这种方式，条件致死性依赖于细胞内 DNA 降解，以实现调控性自杀以及阻止水平基因转移。在携带自杀开关的细胞中，nucA 基因与 copM 启动子的变体融合，可有效诱导可诱导的自杀。与核酸酶保护系统并行，在生物安全开关中研究了蓝藻毒素/抗毒素 (TA) 模块。使用金属离子响应启动子重新布线 SynechocystisTA 对 slr1114/slr0664 和 slr6101/slr6100 进行条件致死性，导致生长减少，而不是细胞死亡，这表明细胞可以应对升高的毒素水平。总的来说，启动子特性和翻译效率影响生物 containment 系统的功效。在安全保障的背景下测试了几种金属离子启动子，并通过β-半乳糖苷酶报告基因测定对选定的启动子（包括 anrsB 变体）进行了表征。
