Forward Genetic Screens for the Discovery of Functional Glucan Hydrolases in the Diatom Phaeodactylum Tricornutum

Phaeodactylum tricornutum is a marine diatom that is a promising candidate for advanced biofuel and bioproduct production. In order to further utilize this organism as a feedstock, it is crucial that researchers fully understand all aspects of the metabolic pathways involved in carbon storage and breakdown. Unlike many other species of algae, diatoms store sugars as chrysolaminarin, a polymer of glucose connected by β-1,3 bonds with minimal β-1,6-branching. Presumably, these polymers are hydrolyzed by unknown endo- and exo-1,3-β-glucanases. To identify these putative hydrolases, this project employed a forward genetic screen with a cDNA library of P. tricornutum using Escherichia coli as a surrogate host. We first established that E. coli was not able to utilize laminarin, a practical equivalent of chrysolaminarin, as a sole carbon source. We then transformed E. coli with the cDNA library to pick clones that were able to grow on laminarin-medium. The clones presumably contain the candidate β-glucanases of P. tricornutum. Preliminary findings include optimizing selective M9+Laminarin media, categorizing the growth behavior of E. coli on the media, and developing concrete screening parameters. This work may open doors for scientists interested in further engineering carbon fluxes in P. tricornutum for energy applications, in addition to researchers looking to further develop and expand forward genetics screens not only in this diatom, but other organisms as well.