4 Discussion
The chloroplast is a key target in algal and plant biotechnology given both its central role in photosynthesis, and as the site of synthesis for primary metabolites such as fatty acids, terpenoids and tetrapyrroles.[46] The algal chloroplast, specifically that of C. reinhardtii , is well suited for genetic engineering and there is an increasing emphasis on the application of synthetic biology.[5,9,11,12] Many of these approaches will be reliant on the ability to perform a series of plastome edits to the same cell line. However, conventional strategies for selection of transformants largely preclude this: methods based on photosynthetic restoration are restricted to a particular mutant host and specific locus, and can only be performed once,[43] whilst portable markers for engineering WT plastomes are currently limited to just three and these also operate on a single-use basis.[18] Recycling these markers via intramolecular recombination can circumvent this issue and also generate marker-free engineered lines[17,24]. However, these strategies have so far been reliant on passive accumulation of plastome copies that have lost the marker under non-selective conditions, requiring lengthy wait times to generate homoplasmic cell lines and the use of larger intramolecular repeat sequences to increase the rates of recombination.
Our CpPosNeg system addresses both these problems by mediating efficient loss of the dual marker through active counter-selection using 5-FC using repeat elements as small as 258 bp. This allows the 3’ UTR of the marker and linked transgene to be used as the direct repeat thereby avoiding introduction of any unwanted DNA scar and producing a final transgenic line containing just the transgene. This line can then be used for further rounds of engineering. Since the choice of 3’UTR has relatively little influence on transgene expression in C. reinhardtii chloroplasts,[47] then different endogenous or synthetic 3’UTRs could be used beyond the two (rbcLand atpB ) used in this study, thereby avoiding having multiple transgenes with the same 3’UTR in an engineered plastome. Furthermore, the minimum size of the direct repeat could probably be smaller than the 258 bp used here since intra- and inter-molecular recombination has been shown to occur in the C. reinhardtii chloroplast between elements as small as 216 bp and 110 bp, respectively.[26,48].
Since the CodA enzyme retains full activity when fused via a flexible linker to AadA, then it should be possible to develop additional dual systems based on CodA. This could involve fusions to other antibiotic-resistance proteins such as AphA6[20]to create alternative CpPosNeg markers, or to reporter proteins such as GFP[18] allowing rapid fluorescence sorting of individual transformed cells[49] for those that have lost this dual reporter-marker. Finally, both aadA andcodA have been shown to work as selectable markers in tobacco chloroplasts,[50] as have the rbcL andatpB 3’UTRs from C. reinhardtii .[51]It is likely therefore that the dual marker described here could be easily adapted for efficient serial engineering of higher plant chloroplasts. CpPosNeg could also be applied to other plastome engineering strategies based on intramolecular recombination such as marker-free deletion of endogenous genes and introduction of SNPs[25,28] thereby accelerating the field of chloroplast synthetic biology.
5 Acknowledgements: The research was funded by grants BB/R016534/1 and BB/R01860X/1 from the U.K.’s Biotechnology and Biological Sciences Research Council. SK was supported by a British Council award under the Newton Bhabha Ph.D. Placement Programme.
6 Conflict of Interest: The authors declare no commercial or financial conflict of interest.