Development of shuttle vectors for rapid prototyping of engineered Synechococcus sp. PCC7002

Cyanobacteria are of particular interest for chemical production as they can assimilate CO₂ and use solar energy to power chemical synthesis. However, unlike the model microorganism of Escherichia coli, the availability of genetic toolboxes for rapid proof-of-concept studies in cyanobacteria is generally lacking. In this study, we first characterized a set of promoters to efficiently drive gene expressions in the marine cyanobacterium Synechococcus sp. PCC7002. We identified that the endogenous cpcBA promoter represented one of the strongest promoters in PCC7002. Next, a set of shuttle vectors was constructed based on the endogenous pAQ1 plasmid to facilitate the rapid pathway assembly. Moreover, we used the shuttle vectors to modularly optimize the amorpha-4,11-diene synthesis in PCC7002. By modularly optimizing the metabolic pathway, we managed to redistribute the central metabolism toward the amorpha-4,11-diene production in PCC7002 with enhanced product titer. Taken together, the plasmid toolbox developed in this study will greatly accelerate the generation of genetically engineered PCC7002. KEY POINTS: • Promoter characterization revealed that the endogenous cpcBA promoter represented one of the strongest promoters in PCC7002 • A set of shuttle vectors with different antibiotic selection markers was constructed based on endogenous pAQ1 plasmid • By modularly optimizing the metabolic pathway, amorpha-4,11-diene production in PCC7002 was improved.