Rapid method for generating designer algal mitochondrial genomes

Abstract

With 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 or productive organisms is contingent on the ability to produce diverse DNA molecules and rapidly screen them for beneficial variants. 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 algal mitochondrial genomes as a practical alternative to DNA synthesis. Our approach permits an 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 organism S. cerevisiae using two different techniques: transformation-associated recombination; and PCR-based cloning. Next, we analyzed the cloned genomes by multiplex PCR, and correct genomes were transferred to prokaryotic host E. coli. Then 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.HIGHLIGHTSTAR-cloned mitochondrial genome of P. tricornutum in yeastDeveloped PCR-based cloning method to create designer algal mitochondrial genomesStably propagated algal mitochondrial genomes in S. cerevisiae and E. coli hosts

Rapid method for generating designer algal mitochondrial genomes

“…DNA from E. coli, yeast, and algae was isolated as described by [16]. Before isolating plasmid DNA from E. coli for diagnostic restriction digests or sequencing, cells were induced with arabinose.…”

“…Due to the industrial and academic interest in this algal species, its genomes (nuclear, mitochondrial, and plastid) were sequenced [8][9][10]. The availability of genome sequences allowed for the development of genetic tools and DNA delivery methods such as biolistic-mediated transformation [11,12], electroporation [13][14][15] and bacterial conjugation [16,17]. Additional tools for P. tricornutum include a method for cloning whole chromosomes in yeast and Escherichia coli [18], characterized centromeres for maintaining episomal DNA [19], and genome-editing technologies [6,[20][21][22].…”