Based on the endosymbiotic theory, one of the key events that occurred during mitochondrial evolution was an extensive loss of nonessential genes from the protomitochondrial endosymbiont genome and transfer of some of the essential endosymbiont genes to the host nucleus. We have developed an approach to recapitulate various aspects of endosymbiont genome minimization using a synthetic system consisting of Escherichia coli endosymbionts within host yeast cells. As a first step, we identified a number of E. coli auxotrophs of central metabolites that can form viable endosymbionts within yeast cells. These studies provide a platform to identify nonessential biosynthetic pathways that can be deleted in the E. coli endosymbionts to investigate the evolutionary adaptations in the host and endosymbiont during the evolution of mitochondria.