Impact of culture conditions on the secretion of eGFP driven by the nitrate reductase promoter in the diatom Phaeodactylum tricornutum
Résumé
To date, different microalgae species are under investigation to be used as cell biofactories. Phaeodactylum tricornutum is currently emerging as a key candidate for the production of recombinant therapeutic proteins like antibodies. So far, the expression of antibodies produced in P. tricornutum are driven by the Nitrate Reductase promoter.
In this study, we aimed to explore the impact of varying culture conditions on the secretion of recombinant proteins. We have chosen the enhanced green fluorescent protein (eGFP-Sec), driven by the Nitrate Reductase promoter as a model and expressed it in P. tricornutum cells. We achieved this by varying light intensity (30, 90, 150 and 250 µmol photons/m²/s) and testing different nitrate (NaNO3 equal to 0.1 and 1 g/L) and salinity (33 and 8 g/L) concentrations in a culture medium based on artificial seawater. Additionally, we ensured a stable pH level in the natural seawater medium.
Our results revealed that regardless of light irradiance, nitrate concentration, or salinity, maximal eGFP-Sec fluorescence intensity was observed at the beginning of the logarithmic growth phase, followed by a gradual decline. Furthermore, our investigation revealed enhanced eGFP-Sec secretion at an irradiance of 150 µmol photons/m²/s. Interestingly, regulating the pH to 8 in natural seawater led to a significant accumulation of eGFP-Sec, approximately ten times higher than that observed in unregulated conditions.
This study sheds light on the importance of optimizing culture conditions for maximizing the expression and secretion of recombinant proteins in microalgae-based biorefineries, highlighting the potential of P. tricornutum in biotechnological applications.