Engineering the plastid genome of Cyclotella cryptica to express heterologous proteins and study photosynthesis in diatoms

Diatoms are unicellular photosynthetic microalgae, originated from a secondary endosymbiosis between an eukaryotic heterotrophic protist and a red alga. Diatoms are among the most species-rich group of phytoplankton, found in many different ecosystems, including fresh and seawater. Furthermore, they account for about 20% of the global carbon fixation, having a fundamental environmental role. Nevertheless, despite their pivotal role as primary producers in ocean ecosystems and the growing biotechnological interest around them, the current knowledge of the regulation of diatom photosynthesis remains yet quite restricted. One reason of this knowledge gap is the obligate phototrophic nature of the current diatom model organisms, for which most genetic tools have been developed, such as Phaeodactylum tricornutum and Thalassiosira pseudonana. In these model species knock-out of fundamental photosynthesis-related genes are lethal. To overcome this limitation, the laboratory is using the centric diatom Cyclotella cryptica as a new model system, as it integrates both the availability of genomic resources and the ability to grow heterotrophically in the dark using glucose as source of reduced carbon, the latter being essential to allow the generation of photosynthetic mutants. So far, mutagenesis of nuclear genes using CRISPR/Cas9 has been successfully achieved and a first photosynthesis-defective mutant has been obtained, paving the way for an in-depth characterisation of plastid biogenesis and regulation of photosynthesis. Targeted transformation of the plastid genome has also been achieved in this diatom species, taking advantage of the naturally occurring homologous recombination in plastids. However transformants do not reach homoplasmy (i.e., having all plastid DNA copies mutated). We verified that reaching homoplasmy is indeed essential to have a mutant phenotype so, during this internship, I focused on new technical strategies to engineer the plastid genome and to improve the generation of homoplasmic mutants in C. cryptica and, in parallel, I also assessed heterotrophic growth in other diatom species (e.g., pennate diatoms that have one or two plastids only vs. ~8 in C. cryptica) to find new candidates for plastid transformation. In addition, as the insertion of genes in the plastid genome was achieved, another goal was also to asses heterologous gene expression in C. cryptica plastids using a reporter gene encoding for a fluorescent protein. This would allow us to easily measure levels of expression by following fluorescence, to follow plastid segregation and biogenesis during cell division, and will serve as a proof of concept of using diatom plastids as “bio-factories” for further biotechnological applications. Finally, I also investigated a novel field in diatom research, which is the study of the regulation of diatom plastid gene-expression using RNA blots.