Harnessing organisms for protein and chemical production is useful to the scientific community and has applications in the fuel, food, and pharmaceutical industries. Biological systems commonly used for industrial chemical production include yeast and bacteria due to their fast growth rates and potential for high product yields. However, biologically active proteins, such as for human therapeutics, usually require production in mammalian and insect systems that are prohibitively expensive to grow at scale. Recently, photoautotrophic microalgae have emerged as promising platforms, as some species can be grown quickly and inexpensively at large scales and have the potential to produce biologically active proteins that mimic those produced in mammalian systems. Thus, they combine advantages from several traditional biological systems, but require further genetic and biotechnological development for their full potential to be unlocked.
Ideal biological production systems generally possess a variety of genetic tools to enable foreign gene expression, and genome editing systems to create desired genetic modifications. Here I present a robust gene editing system, novel genetic tools, and valuable strains for the microalga Phaeodactylum tricornutum. First, I identify novel endogenous regulatory elements that can drive expression of foreign genes in P. tricornutum, and design a plasmid-based Cas9 gene editing system for this species. Next, I demonstrate the utility of these tools with the generation of auxotrophic P. tricornutum strains through the genetic knockout of key enzymes in the uracil and histidine biosynthesis pathways. I complement the phenotype of the auxotrophs by introducing intact versions of these genes on replicating plasmids and demonstrate that these genes function as selective markers for transformation of their respective auxotrophic strain. Finally, I highlight the potential of these tools by creating a P. tricornutum expression system for the production of SARS-CoV-2 antigens. This will potentially address the need for a cheap, scalable source of serologically-active antigens for population-wide serological testing to combat the SARS-CoV-2 pandemic, and this system can be rapidly adapted to tackle future pandemics. I hope that these novel tools and strains will broaden the potential applications of P. tricornutum for industrial production of high-value products, and further the study of diatom biology
