Rapid method for generating designer algal mitochondrial genomes

Abstract

Using synthetic biology, we can turn algae into bio-factories that produce high-value molecules (e.g. medicines or biofuels) or tackle global challenges (e.g. malnutrition and climate change). This realization has provoked rapid progress towards the creation of genetic tools for multiple algal species, notably Phaeodactylum tricornutum. The power of synthetic biology to generate more useful organisms depends on the ability to produce diverse DNA molecules and rapidly screen them for beneficial variants in chosen hosts. However, it is still relatively expensive to synthesize DNA, and delivering large DNA (>50 kbp) to eukaryotic cellular compartments remains challenging. In this study, we establish a robust system for building designer algal mitochondrial genomes ranging in sizes from approximately 60 to 95 kbp as a practical alternative to DNA synthesis. Our approach permits the inexpensive and rapid generation of mitochondrial derivatives designed for testing targeted DNA delivery. First, we cloned the mitochondrial genome of P. tricornutum into the eukaryotic host Saccharomyces cerevisiae using two different techniques: transformation-associated recombination; and PCR-based cloning. Next, we screened the cloned genomes by multiplex PCR, and transformed correct genomes into the prokaryotic host organism Escherichia coli. These genomes were again analyzed by multiplex PCR, followed by diagnostic digest and complete plasmid sequencing to evaluate the fidelity of each cloning method. Finally, we assessed the burden on eukaryotic and prokaryotic hosts to propagate the cloned genomes. We conclude that our system can reliably generate variants for genome-level engineering of algal mitochondria.