(100b) Engineering ROS Metabolism with Futile Cycles

Reactive oxygen species (ROS) are toxic molecules utilized by the innate immune system and antibiotics to kill bacteria.  In addition to their role in host defense, a basal level of ROS is produced inadvertently in all aerobically growing organisms when reduced electron carriers collide with O₂ and transfer their electrons. For this reason, many bacteria have evolved defense capabilities against these toxic species, such as producing detoxification enzymes and antioxidant metabolites. When the level of ROS within a cell exceeds its capacity for detoxification, bacterial death occurs.  In previous work, we have shown that increased ROS production potentiates the effects of oxidative stress. An important insight from that study was that ROS production increases as inefficient use or production of ATP increases. In this study, we sought to increase ATP inefficiency by engaging futile cycles and determine if such a perturbation increases sensitivity toward oxidative stress. Futile cycles are sets of reactions whose net activity results in ATP hydrolysis. Using mixed-integer linear optimization, we have identified all futile cycles in E. coli metabolism containing ≤ 3 reactions, as well as those that require the expression of only one enzyme in aerobic growth in minimal glucose media. We have experimentally validated the presence of futile cycling and measured an increase in sensitivity to H₂O₂ for strains that are actively futile cycling when compared to strains carrying an inactive form of the cycle-enabling enzyme. The knowledge gained from this project could lead to the development of new therapies to sensitize microbes to immunity and antibiotics.