Enhancing recombinant eGFP secretion in P. tricornutum by optimizing culture conditions

Phaeodactylum tricornutum is a promising candidate for the production of recombinant therapeutic proteins including antibodies. So far, low secretion yields remain a significant bottleneck for pharmaceutical applications. Towards the objective to increase production yield of secreted recombinant protein, this study investigates the optimization of environmental and nutritional parameters, under laboratory conditions, to maximize the production of a model secreted recombinant protein (eGFPsec) under the control of the Nitrate Reductase promoter (P_NR). We evaluated the interplay between nitrate availability, Photosynthetic Photon Flux Density (PPFD), and pH level within both natural and artificial seawater matrices. Our results demonstrate that while P_NR activity does not increase with nitrate concentrations beyond non-limiting levels, a PPFD of 150 µmol photons m^- 2 s^- 1 significantly enhances eGFPsec accumulation in the medium. Interestingly, cultures grown in Natural Sea Water under pH-regulated conditions (pH = 8) exhibited markedly higher secreted-eGFP fluorescence compared to those in artificial seawater. These findings highlight the importance of optimizing key environmental and nutritional parameters to improve secretion recombinant protein yields. By identifying key drivers of eGFP fluorescence-based productivity and accumulation in the medium, this work provides actionable insights for process optimization, reinforcing the potential of P. tricornutum as a robust and cost-effective platform for industrial microalgae-based biomanufacturing.