The alga Chlamydomonas reinhardtii is a potential platform for recombinant protein expression in the future due to various advantages. Dozens of C. reinhardtii strains producing genetically engineered recombinant therapeutic protein have been reported. However, owing to extremely low protein expression efficiency, none have been applied for industrial purposes. Improving protein expression efficiency at the molecular level is, therefore, a priority. The 3′-end poly(A) tail of mRNAs is strongly correlated with mRNA transcription and protein translation efficiency. In this study, we identified a canonical C. reinhardtii poly(A) polymerase (CrePAPS), verified its polyadenylate activity, generated a series of overexpressing transformants, and performed proteomic analysis. Proteomic results demonstrated that overexpressing CrePAPS promoted ribosomal assembly and enhanced protein accumulation. The accelerated translation was further verified by increased crude and dissolved protein content detected by Kjeldahl and bicinchoninic acid (BCA) assay approaches. The findings provide a novel direction in which to exploit photosynthetic green algae as a recombinant protein expression platform.
Chlamydomonas reinhardtii is a photosynthetic eukaryote showing great industrial potential. The synthesis and in vivo function of the artificial C. reinhardtii genome not only promotes the development of synthetic biology technology but also supports industries that utilize this algae. Mitochondrial genome (MtG) is the smallest and simplest genome of C. reinhardtii that suits synthetic exploration. In this article, we designed and assembled a synthetic mitochondria left arm (syn-LA) genome sharing >92% similarity to the original mitochondria genome (OMtG) left arm, transferred it into the respiratory defect strain cc-2654, screened syn-LA containing transformants from recovered dark-growth defects using PCR amplification, verified internal function of syn-LA via western blot, detected heteroplasmic ratio of syn-LA, tried promoting syn-LA into homoplasmic status with paromomycin stress, and discussed the main limitations and potential solutions for this area of research. This research supports the functionalization of a synthetic mitochondrial genome in living cells. Although further research is needed, this article nevertheless provides valuable guidance for the synthesis of eukaryotic organelle genomes and opens possible directions for future research.
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