Simultaneous overexpression of cyanidase and formate dehydrogenase in Arabidopsis thaliana chloroplasts enhanced cyanide metabolism and cyanide tolerance

• Transgenic Arabidopsis with a cyanide degradation pathway were constructed.
• The synthetic pathway was designed to degrade cyanide to ammonia and CO2.
• Plants showed reduced stress responses and enhanced growth on cyanide.

Cyanide is a strong inhibitor of diverse metabolic reactions and easily absorbed by organisms. However, cyanide is also a byproduct of plant and microbial metabolism. This is why these groups of organisms contain pathways for cyanide detoxification. Large amounts of cyanides are also produced by industries and are today mostly removed by physical and chemical methods. Phytoremediation can provide an alternative to these techniques, but existing cyanide concentrations at contaminated sites often exceed the capacities of plant metabolism. In this study, we overexpressed a bacterial cyanidase together with a plant formate dehydrogenase in Arabidopsis thaliana in order to establish a synthetic pathway for cyanide degradation. Simultaneous overexpression of both enzymes would ultimately result in the formation of CO2 and NH3 from cyanide. Both enzymes were targeted to chloroplasts and shown to be active in planta. When plants were spray-inoculated with cyanide, overexpressors of the synthetic cyanide degradation pathway showed less reduction in leaf pigment contents, lower induction of antioxidant enzymes, and reduced growth retardation compared to controls. Growth on cyanide was also tested for seedlings germinating on agar, plants in hydroponics, and plants growing in sand. In all three assays, plants overexpressing the synthetic pathway for cyanide degradation showed enhanced growth and biomass accumulation compared to controls. Gas exchange measurements confirmed enhanced stress resistance of transgenic plants and suggested that cyanide degradation to CO2 increased the leaf internal CO2 concentration. Results are discussed in comparison to other approaches for cyanide phytoremediation.