Spheroplasted cells: a game changer for DNA delivery to diatoms

Diatoms produce 20% of the world’s fixed organic carbon annually, making them vital to global carbon fixation and climate change mitigation. Their potential as cell factories for biofuels, proteins, and other high value chemicals remains underutilized due to a lack of genetic engineering tools, with DNA delivery being one of the biggest challenges. Here, we present optimized electroporation and polyethylene glycol transformation methods for delivering DNA and ribonucleoprotein complexes toPhaeodactylum tricornutum, a model diatom species and emerging chassis for algal biotechnology. It was possible to recover transformants with as little as 1 ng of DNA, and to transform linear or circular episomes as large as 55.6 kb. With the optimized electroporation protocol, episomes can be assembled in the algal cellde novothrough diatom in vivo assembly (DIVA), forgoing the need for time-consuming traditional cloning steps inEscherichia coliandSaccharomyces cerevisiae. It was also possible to electroporate a Cas9 ribonucleoprotein complex inP. tricornutum, providing an alternative to biolistics for DNA free genome engineering. We have demonstrated that the PEG approach can be adapted to successfully transformThalassiosira pseudonana, demonstrating the applicability of our methods for engineering other diatom species. These tools can be used to accelerate diatom synthetic biology projects and, therefore, the development of sustainable technologies.