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.