Novel transformation strategies improve efficiency up to 10-fold in stramenopile algae

Stramenopile algae have the potential to become the light-driven photosynthetic biofactories of the future, but the transformation technologies required to reach this goal remain sub-optimal. Nannochloropsis oceanica and Phaeodactylum tricornutum were used as experimental systems for electroporation-mediated transformation. Two transformation approaches were developed; 1) timed transformation of synchronized cells and 2) addition of saponins as transformation adjuvants. Transformation efficiency was increased ~8 times using synchronized N. oceanica cultures transformed in the G2/M phase, in comparison to state-of-the-art methods based on transformation of non-synchronized cells. For P. tricornutum the transformation was up to 5 times more efficient in non-synchronized conditions. N. oceanica and P. tricornutum responded differently upon exposure to different saponin plant extracts. Saponin treatments enhanced P. tricornutum and N. oceanica transformation efficiencies ~2 and ~2.5 times, respectively. Combining cell synchronization and saponin transformation adjuvant treatment, improves transformation efficiency in N. oceanica, results in a >10-fold improvement of the transformation efficiency for N. oceanica. In addition, a protocol for directed ribonucleoprotein (RNP)-mediated genome engineering of DNA constructs with short flanking arms (50 bp) in N. oceanica was established, enabling improved RNP targeted non-homologous end-joining (NHEJ) gene editing. In conclusion, this study expands the toolbox for stramenopile genome engineering, promoting their use as model organisms and sustainable biofactories.