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.