Project Summary/Abstract Molybdenum cofactor deficiency (MoCD) is a rare metabolic disorder caused by mutations along the molybdenum cofactor (Moco) biosynthetic pathway resulting in a loss of Moco-dependent enzymatic activity. An affected child will appear healthy at birth, but within days symptoms begin to emerge. The disease is characterized by progressive neurological damage, intractable neonatal seizures, and developmental difficulties, ultimately leading to death. Recent work in engineering the microbiome has shown potential in treating human metabolic disease, and microbially-mediated enzyme rescue has proved to be a promising treatment. Rather than an enzymatic rescue, the goal of this proposal is to attempt a microbially-mediated metabolite rescue of MoCD. By engineering the microbiome to restore production of cPMP, the first intermediate in the Moco pathway and the missing metabolite in two thirds of patients, we can side-step the human metabolic deficiency and treat the disease with a probiotic. Leveraging the safety profile of the probiotic Escherichia coli Nissle, we will engineer this chassis organism for production of cPMP. My recent work has focused on the engineering of large biosynthetic gene clusters (BGCs) responsible for the production of bioactive small molecules and these same skills can be applied to the Moco pathway. Using purified cPMP as a control, I will then test the in vitro ability of this engineered strain to produce bioavailable cPMP in a gut simulation assay and complement Moco-dependent enzymatic activity in microorganisms unable to synthesize this molecule. Finally, I propose to move the engineered strain and pure molecule into in vivo transgenic homozygous mice that mirror patients with MoCD. However, it is important to note that the aims proposed here are not linear and mutually exclusive, but rather parts of the iterative design-build-test-learn cycle that is foundational for engineering and the industry standard in this field. One aim cannot preclude the next as they are all fundamental to understanding the activity of the engineered strain. It is only by iterating through this cycle that we will be able to potentially generate a strain capable of performing the first in vivo metabolite rescue of a metabolic disorder. This proposal is designed to supplement my prior research experience in engineering BGCs and provide new challenges by applying these skills towards a biomedical application through interactions with my advisor, Professor Bradley Moore, and project co-sponsors Professor Gleeson and Professor Raffatellu. Furthermore, I have spent the summer interning at Synlogic, a biotech company that has pioneered the field of living medicine, and I have learned the skills necessary to accomplish the work proposed here. This proposal combines microbiology, genetics, synthetic biology, and chemistry in a translational medicine approach to address a disease without suitable treatment options. Training at the...