The "replacing surgery" of cpDNA: de novo chemical synthesis and in vivo functional testing of Chlamydomonas chloroplast genome

Abstract

We have successfully designed and synthesized the 221,372-bp cpDNA SynCpV1.0 with the native cpDNA of Chlamydomonas reinhardtii as 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 to Escherichia coli, it indicated that SynCpV1.0 was able to shuttle between C. reinhardtii and E. 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 of C. reinhardtii CC125, and could rescue the photosynthetic defect in mutant strain of C. reinhardtii CC5168. 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. HighlightO_LIAn artificial cpDNA SynCpV1.0 is constructed by de novo chemical synthesis. C_LIO_LIThe "replacing surgery" of cpDNA was performed in the chloroplast of C. reinhardtii C_LIO_LIIt is found that artificial cpDNA was able to shuttle between Chlamydomonas chloroplast and E. coli. C_LIO_LIEstablish an ideal platform to complete multiple cycles of "Design-Build-Test" for optimizing the cpDNA. C_LI One-Sentence SummaryThe chloroplast genome can be replaced by a complete synthesized genome and performs the designed biological function in C. reinhardtii.