Cloning of Thalassiosira pseudonana’s Mitochondrial Genome in Saccharomyces cerevisiae and Escherichia coli

SIMPLE SUMMARY: One of the challenges in the emerging field of synthetic biology is engineering organelle genomes. Creating synthetic organelle genomes can open the door to a wide range of applications, such as improving crop yields, treating mitochondrial diseases, or manufacturing high-value chemicals in an environmentally sustainable way. Organelles are tiny biological machines that work inside of living cells. Mitochondria, for example, are responsible for harvesting sugar to create energy for the cell. In previous work, we demonstrated a method to make copies of an alga mitochondrial genome using yeast and bacteria. Algae are of industrial interest for their potential to produce and store large quantities of biofuels and nutritional ingredients. Here, we applied the same approach to copy the mitochondrial genome of a related alga. Although the cloning of this mitochondrial genome in yeast using the previously developed method was possible, the properties of this genome may make it more susceptible to mutations during propagation in bacteria. This work expands our understanding of potential hurdles that can be encountered when cloning and propagating synthetic organelle genomes in host organisms. ABSTRACT: Algae are attractive organisms for biotechnology applications such as the production of biofuels, medicines, and other high-value compounds due to their genetic diversity, varied physical characteristics, and metabolic processes. As new species are being domesticated, rapid nuclear and organelle genome engineering methods need to be developed or optimized. To that end, we have previously demonstrated that the mitochondrial genome of microalgae Phaeodactylum tricornutum can be cloned and engineered in Saccharomyces cerevisiae and Escherichia coli. Here, we show that the same approach can be used to clone mitochondrial genomes of another microalga, Thalassiosira pseudonana. We have demonstrated that these genomes can be cloned in S. cerevisiae as easily as those of P. tricornutum, but they are less stable when propagated in E. coli. Specifically, after approximately 60 generations of propagation in E. coli, 17% of cloned T. pseudonana mitochondrial genomes contained deletions compared to 0% of previously cloned P. tricornutum mitochondrial genomes. This genome instability is potentially due to the lower G+C DNA content of T. pseudonana (30%) compared to P. tricornutum (35%). Consequently, the previously established method can be applied to clone T. pseudonana’s mitochondrial genome, however, more frequent analyses of genome integrity will be required following propagation in E. coli prior to use in downstream applications.