Engineering cyanobacterial chassis for improved electron supply toward a heterologous ene-reductase

Cyanobacteria are noteworthy hosts for industrially relevant redox reactions, owing to a light-driven cofactor recycling system using water as electron donor. Customizing Synechocystis sp. PCC 6803 chassis by redirecting electron flow offers a particularly interesting approach to further improve light-driven biotransformations. Therefore, different chassis expressing the heterologous ene-reductase YqjM (namely Delta hoxYH, Delta flv3, Delta ndhD2 and Delta hoxYH Delta flv3) were generated/evaluated. The results showed the robustness of the chassis, that exhibited growth and oxygen evolution rates similar to Synechocystis wild-type, even when expressing YqjM. By engineering the electron flow, the YqjM light-driven stereoselective reduction of 2-methylmaleimide to 2-methylsuccinimide was significantly enhanced in all chassis. In the best performing chassis (Delta hoxYH, lacking an active bidirectional hydrogenase) a 39 % increase was observed, reaching an in vivo specific activity of 116 U g(DCW)(-)1 and an initial reaction rate of 16.7 mM h(-1). In addition, the presence of the heterologous YqjM mitigated substrate toxicity, and the conversion of 2-methylmaleimide increased oxygen evolution rates, in particular at higher light intensity. In conclusion, this work demonstrates that rational engineering of electron transfer pathways is a valid strategy to increase in vivo specific activities and initial reaction rates in cyanobacterial chassis harboring oxidoreductases.

Automated summary

Cyanobacteria have the potential for industrially relevant redox reactions due to their ability to use light as an energy source and water as an electron donor. By modifying the electron flow, the efficiency of biotransformation in cyanobacteria can be significantly improved.