Optimising light conditions increases recombinant protein production in Chlamydomonas reinhardtii chloroplasts

The green alga Chlamydomonas reinhardtii provides a platform for cheap, scalable and safe production of complex proteins. Despite the fact that chloroplast gene expression in photosynthetic organisms is tightly regulated by light, most expression studies have analysed chloroplast recombinant protein production under constant light. Here, the influence of light period and intensity on expression of green fluorescent protein (GFP)and a GFP-bacterial-lysin (PlyGBS)fusion protein was analysed. Protein yields were strongly influenced by the light period (6-24?h?d?1), the light intensity (0-450??E?m?2?s?1)and trophic condition. Heterotrophic conditions showed low yields of both recombinant proteins due to low growth rates, despite high protein accumulation per cell. Mixotrophic conditions exhibited the highest yields for GFP (4?mg·L?1·d?1)under constant light at 35??E?m?2?s?1 and GFP-PlyGBS (0.4?mg·L?1·d?1)under a light period of 15?h?d?1 and 35??E?m?2?s?1. This is due to the high growth rates and cellular protein content. For GFP-PlyGBS the maximum increase in cellular protein accumulation was ~24-fold, and in total protein yield ~10-fold, in comparison to constant light conditions (~200??E?m?2?s?1). The highest yields under photoautrophic conditions were obtained under a 9?h?d?1 light period. GFP yielded 1.2?mg·L?1·d?1 and GFP-PlyGBS 0.42?mg·L?1·d?1. This represented a ~5-fold increase in cellular protein accumulation for GFP-PlyGBS in comparison to constant light conditions (~200??E?m?2?s?1). Optimising light conditions to balance growth and protein expression can significantly enhance overall recombinant protein production in C. reinhardtii cultures.