The “replacing surgery” of cpDNA:de novochemical synthesis andin vivofunctional testing ofChlamydomonaschloroplast genome

SUMMARYWe have successfully designed and synthesized the 221,372-bp cpDNA SynCpV1.0 with the native cpDNA ofChlamydomonas reinhardtiias the template. Homoplasmic SynCpv1.0-harboring algal strains were obtained by biolistic transformation and selected with an ascending gradient of antibiotic pressure. Meanwhile, we were pleasantly surprised to find that SynCpV1.0 was able to re-introduce and replicate normally after the total DNA of transplastomic algal strains were transformed toEscherichia coli, it indicated that SynCpV1.0 was able to shuttle betweenC. reinhardtiiandE. coli. Finally, we analyzed the photosynthetic properties of SynCpV1.0-harboring transplastomic strains, the results showed that they exhibited the same photosynthetic efficiency as the wild strain ofC. reinhardtiiCC125, and could rescue the photosynthetic defect in mutant strain ofC. reinhardtiiCC5168. Herein, we have performed the “replacing surgery” of cpDNA and established an ideal platform to complete multiple cycles of “Design-Build-Test” for optimizing the cpDNA of photosynthetic organisms.HighlightAn artificial cpDNA SynCpV1.0 is constructed byde novochemical synthesis.The “replacing surgery” of cpDNA was performed in the chloroplast ofC. reinhardtiiIt is found that artificial cpDNA was able to shuttle betweenChlamydomonaschloroplast andE. coli.Establish an ideal platform to complete multiple cycles of “Design-Build-Test” for optimizing the cpDNA.One-Sentence SummaryThe chloroplast genome can be replaced by a complete synthesized genome and performs the designed biological function inC. reinhardtii.