Identification and Characterization of the Mitochondrial Replication Origin for Stable and Episomal Expression in Yarrowia lipolytica

Episomal plasmids are crucial expression tools for recombinant protein production and genome editing. In Saccharomyces cerevisiae, 2-μm artificial plasmids with a high copy number have been developed and used in metabolic engineering and synthetic biology. However, in unconventional yeasts such as Yarrowia lipolytica, episomal expression relies on a chromosome replication system; this system has the disadvantages of genetic instability and low copy numbers. In this study, we identified and characterized replication origins from the mitochondrial DNA (mtDNA) of Y. lipolytica. A 516-bp mtDNA sequence, mtORI, was confirmed to mediate the autonomous replication of circular plasmids with high protein expression levels and hereditary stability. However, the nonhomologous end-joining pathway could interfere with mtORI plasmid replication and engender genetic heterogeneity. In the Po 1f ΔKu70 strain, the homogeneity of the mtORI plasmid was significantly improved, and the highest copy number reached 5.0 per cell. Overall, mitochondrial-origin sequences can be used to establish highly stable and autonomously replicating plasmids, which can be a powerful supplement to the current synthetic biology tool library and promote the development of Y. lipolytica as a microbial cell factory.