Project Summary/Abstract The Shao group at Iowa State University aims to leverage their expertise in genetic tool development to assemble a comprehensive mitochondrial genetic toolkit. The causative role of mitochondrial DNA is implicated in a myriad of human diseases and disorders. However, human mitochondrial dysfunction studies suffer from the absence of an ideal eukaryotic model. The well-studied model yeast, S. cerevisiae, is unsuitable due to its mitochondrial physiology deviating too far from humans. We propose developing a new promising model to heighten knowledge of human mtDNA, yielding new insights on mitochondrial dysfunction and pathogenicity. We have identified that Yarrowia lipolytica strikes a perfect balance between practicality (i.e., low cost and quick timescale of genetic manipulations as a low eukaryote) and tractability (i.e., mirroring human's obligate aerobic needs). In addition, the overwhelming majority of current mitochondrial dysfunction studies focus solely on nuclear-encoded mitochondrial gene abnormalities. This is mainly attributed to the fact that the explosive progression of nuclear genome editing technology in the epoch of “post-CRISPR” has yet to translate to mtDNA editing. We propose leveraging a recently discovered stem-loop RNA motif to overcome nucleic acid import limitations – the largest technical barrier to the development of CRISPR-associated technologies. If this strategy proves effective, we envision that many mtDNA manipulation tools will be developed by research labs around the globe, following the same trajectory as the CRISPR nuclear genome manipulation revolution. Over the next five years, in addition to the foundational tool development, we will strive for elucidating mtDNA-phenotype relationships in the new model. The ability of Y. lipolytica to accumulate lipids makes it a particularly suitable model for human adipocytes. Mitochondrial dysfunction in adipose tissue is involved in a broad spectrum of epidemics plaguing human health. Mediated by our development of a mitochondrial genetic toolkit, we will reconstitute mtDNA-associated human pathologies in a precise manner, enabling tailored drug development and all the subsequent mechanistic studies. Moreover, we propose studying the impact of modulating the fluidity of the inner mitochondrial membrane on altering mitochondrial physiology, which will lead to the discovery of potential treatments of obesity-related diseases in the future. Lastly, along a side research branch, we will integrate the developed mitochondrial genetic toolkit with our previous efforts in metabolic engineering. We will leverage the multiplicity of mitochondria in a single cell as well as the high copy number of mtDNA in a single mitochondrion to boost the dosage of the gene encoding the rate-limiting step in a biochemical pathway. This strategy will revolutionize the metabolic engineering design of eukaryotic hosts to produce a wide variety of compounds derived from TC...