ATP-Free Fatty Aldehyde Biosynthesis Enables an Autonomous Lux-Based Bioluminescence System
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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 α-dioxygenase (αDOX), an enzyme that directly converts fatty acids into fatty aldehydes without consuming ATP. Using machine learning–guided directed evolution, we engineered αDOX 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.
SIGNIFICANCE
Autonomous 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 αDOX. To improve system performance, we applied machine learning– guided directed evolution, which significantly enhanced signal intensity and enabled single-cell bioluminescence imaging. Furthermore, the αDOX-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.
