Designer Mitochondria for Biotechnology, Healthcare, and Basic Research

University Of South Alabama, Mobile AL

Investigators

Abstract

Life as we know it hinges on the recapture of energy from the environment and its subsequent utilization in cellular processes. Consequently, the task of generating artificial “designer” mitochondria, the power plants in nearly all animal cells, is central to synthetic biology. This project will provide fundamental knowledge that is crucial for the ultimate goal of building artificial mitochondria, which can in turn be used in synthetic cells, and potentially in biotechnology applications and medical interventions. This project will also provide crucial training for the next generation of scientists and the STEM workforce of the future. Mitochondria stand out among animal organelles due to their possession of a cellular genome, similar to the nucleus, known as mitochondrial DNA. This DNA encodes several genes that are crucial for the most efficient cellular process of energy production. Therefore, the function of mitochondria is heavily reliant on their DNA, highlighting the pivotal role of our ability to maintain and manipulate DNA in this organelle for the goals of synthetic biology. Regrettably, our current understanding of mitochondrial DNA is so basic that reconstituting its replication in a test tube or even in a closely related organism (e.g., monkey mitochondrial DNA in human cells) presents insurmountable challenges. This represents a critical gap in our knowledge for creating artificial mitochondria. To address these issues, our preliminary studies introduced the GeneSwap approach, a genetic system enabling in situ reverse genetic analysis of proteins involved in mtDNA replication. Using this approach, the first protein controlling the species-specificity of mtDNA replication was identified. Additionally, for the first time, human/mouse somatic hybrid cells stably maintained human mtDNA. These new tools provide a unique resource to address critical questions in synthetic biology, focusing on the mechanisms of mtDNA replication and the species-specificity of this process. Therefore, in the proposed studies, we aim to build upon this initial success, identifying all factors required for human mtDNA replication and those contributing to its species-specificity as the first step toward generating artificial mitochondria. In the proposed studies, we will focus on three specific Aims: In Aim 1, a double-pronged approach will be used to identify new proteins contributing to the species-specificity of mtDNA replication. In Aim 2, replication of human mtDNA in mouse cells will be reconstituted by engineering them to express a defined set of human genes. In Aim 3, a structure-function analysis of the first identified component of IBMDR will be conducted to get mechanistic insights into IBMDR operation. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.