Autonomous Bioluminescence Systems: From Molecular Mechanisms to Emerging Applications

Autonomous bioluminescence systemsgenetically encoded platforms that integrate luciferase enzymes with complete substrate biosynthetic pathwayshave emerged as transformative tools for real-time, noninvasive imaging in living systems. Unlike conventional substrate-dependent bioluminescence, these systems provide continuous light emission without external substrates, enabling long-term monitoring with minimal phototoxicity compared to the use of fluorescence. Here, we present a critical perspective on recent advances in the two best-characterized autonomous systems: bacterial and fungal bioluminescence systems. We assess their molecular mechanisms, protein engineering strategies, and emerging applications in single-cell imaging, multicolor biosensing, and whole-organism monitoring. By comparing their strengths and limitations, we highlight persistent challenges, such as low quantum yield in bacterial bioluminescence and substrate availability constraints in fungal bioluminescence, and discuss strategies to address themincluding AI-guided mutagenesis, de novo protein design, and metabolic pathway optimization. We conclude by outlining application-driven design targets for the next generation of autonomous bioluminescent systems in biomedical research, environmental monitoring, and synthetic biology.