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 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 showed a smaller reduction in light emission than the native LuxCDABE system under several antibiotic treatments, although these experiments do not establish ATP availability as the causal mechanism.