ATP-Free Fatty Aldehyde Biosynthesis Enables an Autonomous Lux-Based Bioluminescence System

Autonomous bioluminescence systems enable continuous light emission in engineered organisms by genetically encoding both luciferase enzymes and their substrate biosynthetic pathways, offering a powerful platform for non-invasive and long-term monitoring of biological processes. However, bioluminescence output is highly sensitive to substrate availability and host metabolic state, often leading to signal instability under energy-limited conditions. Here, we report an alternative luciferin biosynthetic strategy for the bacterial Lux bioluminescence system in which the adenosine triphosphate (ATP)-dependent LuxEC complex is replaced by -dioxygenase (DOX), an enzyme that directly converts fatty acids into fatty aldehydes without consuming ATP. Using machine learning-guided directed evolution, we engineered DOX variants that markedly enhanced bioluminescence intensity when coupled with bacterial luciferase. The resulting ATP-free Lux bioluminescence system enabled single-cell-level bioluminescence imaging and maintained stable light emission under diverse antibiotic treatments, demonstrating enhanced robustness against metabolic perturbations. SIGNIFICANCEAutonomous bioluminescence imaging has become an attractive method for long-term observation of biological phenomena without the need for exogenous substrate addition. However, the light output of existing autonomous bioluminescence systems, including bacterial and fungal pathways, remains ATP-dependent and often declines when cellular metabolism is perturbed, limiting their reliability for quantitative analysis. Here, we report the development of an ATP-independent substrate biosynthesis pathway for the bacterial luciferase system using DOX. To improve system performance, we applied machine learning- guided directed evolution, which significantly enhanced signal intensity and enabled single-cell bioluminescence imaging. Furthermore, the DOX-based bacterial luciferase system maintained stable luminescence under antibiotic treatments, in contrast to the conventional ATP-dependent bacterial luciferase system. In summary, our findings establish a robust ATP-independent autonomous bioluminescence imaging platform that enables monitoring of cellular events under metabolic perturbations.