Abstract This Trailblazer Award application will enable a smart bio-robot to ameliorate mitochondrial dysfunctions by coupling a common mitochondrial disease marker, lactate, to the redox levels inside host cells. Mitochondrial dysfunction is associated with many diseases including, but not limited to, aging, cancer, neurodegeneration and diabetes. The dysfunction of mitochondrial electron transport chain (ETC) is one of the hallmarks of mitochondrial diseases and emerging studies show that the elevated NADH/NAD+ ratio resulting from ETC dysfunction can lead to reductive stress. Recent work by others have demonstrated that systemic delivery of a fusion protein comprising bacterial lactate oxidase (LOX) and catalase (CAT), can convert lactate to pyruvate in the blood, which is coupled to lower the intracellular ratio of NADH/NAD+, and thereby mitigating reductive stress in mitochondria. However, systemic delivery of bacterial enzymes to repair mitochondrial dysfunction can face several challenges: (1) LOX and CAT enzymes are immunogenic, (2) enzymes are susceptible to protease degradation in the blood and (3) LOX and CAT enzymes have short serum half-life, therefore requiring repeated injections to sustain the therapeutic effects. Motivated by the fact that lactate and pyruvate can exchange between the gut lumen, circulation and peripheral tissues, we propose to engineer the probiotic strain, E. coli Nissle (EcN), to express the fusion enzyme LOXCAT in the gastrointestinal tract to convert lactate to pyruvate following oral administration. Notably, EcN has a long track record of safety in humans, and is a popular starting point for engineered therapeutic microbe efforts. Building on naturally derived lactate-responsive elements in E. coli, we will develop a synthetic negative feedback loop in EcN with a large dynamic range to sense and respond to elevated levels of lactate in the blood. We hypothesize that this approach will not only address the above- mentioned problems associated with systemic delivery of bacterial enzymes in the blood, but will also enable a new system that is armed with the sensors, genetic circuits, and output genes necessary for administration of the LOXCAT fusion enzyme in a temporally and dosage-controlled manner. To derisk the proposed work, we have validated the expression of LOX and CAT enzymes in EcN, engineered a luciferase reporter in bacteria to allow for noninvasive in vivo tracking, and performed theoretical calculations to predict the feasibility. Building on our preliminary data, we will first optimize the natural lactate-responsive circuit to sense a physiological concentration range of lactate, and the lead circuit will be identified to drive LOXCAT expression (Aim 1). Next, we will examine pharmacokinetics, biodistribution and safety of engineered EcN in wild-type mice. Finally, the therapeutic efficacy will be evaluated in a mouse model of mitochondrial dysfunction via the loss of the complex I subunit Ndufs4 (Aim 2). The successful completion of this proposal will not only have engineered a novel platform for mitochondria dysfunction, but we will have also developed an innovative approach to modulate metabolites in the circulation as a means to interrogate causal relationships between metabolites and diseases.

Public Health Relevance Statement Mitochondria are known as the powerhouses of the cell, and their dysfunction is associated with many diseases such as aging, cancer, neurodegeneration and diabetes. The proposed work will develop a probiotic-based “robot” that resides in the gastrointestinal tract for autonomous sensing of markers (e.g. elevated serum lactate) evident in mitochondrial diseases, and consequently administers therapeutic enzymes in a temporally and dosage-controlled manner. This proposal will also introduce a new approach to selectively control metabolites as a means to understand causal relationships between metabolites and health.