Adaptive Laboratory Evolution And Cadmium Tolerance Mechanism In Synechocystis Sp.PCC 6803

Environmental pollution caused by heavy metal has received widespread attention in recent years.Among them,Cadmium has been a significant threat to environments and human health due to its high toxicity and wide applications in fossil fuel and battery industry.Cyanobacteria are one of the most dominant prokaryotes on earth,and previous studies suggested that they could be valuable in removing Cd²⁺from waste water.However,currently the tolerance of cyanobacteria to cadmium is very low.To further engineer cyanobacteria for environmental application,it is necessary to determine the mechanism of how they respond to high concentration of cadmium.In this study,a robust strain（named as ALE-9.0）tolerant to CdSO₄ up to a concentration of 9.0μM,representing a 190%tolerance improvement,was isolated via adaptive laboratory evolution over 802 days’continuous passages under cadmium stress.Whole-genome re-sequencing was then performed and 9 mutations were identified in the evolved strain compared to the wild type strain.Among these mutations,a truncated slr0454 encoding a cation or drug efflux system protein was found to contribute directly to the resistance to Cd²⁺stress.Additionally,five other mutations were also demonstrated related to the improved Cd²⁺tolerance in ALE-9.0.Moreover,the ALE-9.0 strain was found to obtain cross-tolerance to other heavy metals like zinc and cobalt as well as higher resistance to high light.The work here identified six genes related to Cd²⁺tolerance in Synechocystis,and demonstrated the feasibility of adaptive laboratory evolution in tolerance modifications,which also provided valuable information regarding the cadmium tolerance mechanism in Synechocystis,and useful insights for cyanobacterial robustness and tolerance engineering.