RpaA Overexpression Enhances Bioluminescence Intensity and Elevates Rhythmic Extracellular Vesicle Yield in...

The cyanobacterial circadian clock orchestrates a wide range of physiological processes. However, its involvement in extracellular vesicle (EV) biogenesis remains largely unexplored. In this study, we investigated the role of the master circadian output regulator RpaA in modulating EV production in Synechococcus elongatus PCC 7942. Deletion of rpaA simultaneously abolished the bioluminescence rhythmicity and rhythmic EV production, whereas overexpression of rpaA enhances the output of circadian clock signals in a dose-dependent manner and markedly increases the bioluminescence intensity and EV production while maintaining the normal circadian oscillation patterns. Transcriptional analyses indicated that rpaA positively regulates lpxD to promote membrane synthesis and differentially inhibits glgC transcription to optimize carbon allocation. These findings reveal a previously unrecognized link between circadian output signaling and EV biogenesis and suggest that rpaA overexpression is an effective strategy for improving the yield of EVs. It also provides novel insights and regulatory targets for facilitating the synthesis of other high-value metabolites in cyanobacterial cell factories, and holds certain practical significance for advancing cyanobacterial synthetic biology research and the application of nanocarrier technology.