1 Introduction
The chloroplast of plants and algae is not only the site of
photosynthesis, arguably the most important biological process on the
planet, but is also a major biosynthetic compartment within the cells of
these photoautotrophic organisms.[1] Beyond their
fundamental role in nature, chloroplasts possess many traits that make
them attractive as sub-cellular platforms for industrial biotechnology.
They possess a small polyploid genome (=plastome) derived from their
cyanobacterial ancestor, which has retained only a hundred or so genes,
many of which are highly expressed. Foreign genes can be integrated
precisely into the plastome via a process of homologous recombination
allowing targeting of these genes into neutral loci, thereby avoiding
any position effects. Furthermore, since the chloroplast genetic system
lacks any gene silencing mechanisms, high levels of expression and
recombinant protein accumulation are achievable without the need to
maintain selection.[2,3]
Chloroplast transformation was first achieved in
1988[4] using the unicellular green algaChlamydomonas reinhardtii . Since then, this species has been used
extensively to demonstrate the potential of the algal chloroplast as a
chassis for synthesis of recombinant products including therapeutic
proteins,[5] novel
metabolites,[6] and bioactive
RNAs.[7,8] An ever-growing ‘chloroplast toolkit’
for C. reinhardtii now allows routine insertion of
codon-optimised transgenes into the plastome, and their high-level and
regulated expression.[9] More recently, there has
been a growing emphasis on the utilisation of synthetic biology (SynBio)
approaches to chloroplast engineering.[5,10–12]This has been supported by the availability of robust, well annotated
genomic and transcriptomic data for the C. reinhardtiiplastome[13,14] and the emergence of standardised
DNA assembly methods for rapid and high-throughput design and
construction of transgenes.[15] These enabling
technologies are now facilitating progression from simple genetic
engineering strategies based on one or two transgenes to the integration
and effective regulation of multiple transgenes, allowing the
introduction of novel metabolic pathways into the algal
plastid,[16] and radical refactoring of the
plastome.[17]
These ambitious engineering efforts will likely require several rounds
of engineering of the same strain, either to introduce multiple
transgenes for metabolic engineering, or to perform plastome
rearrangements and deletions.[9] However, such
advanced transplastomics is currently constrained by the paucity of
different selectable markers for chloroplast transformation of C.
reinhardtii .[18] For example, only three
bacterial genes have been developed to-date as portable markers: theaadA cassette conferring spectinomycin
resistance,[19] the aphA6 cassette
conferring kanamycin resistance,[20] and theptxD cassette that allows phosphite
auxotrophy.[21] Moreover, each round of
engineering involves the permanent introduction of a marker into the
plastome as well as the gene(s) of interest. This not only prevents the
re-use of the marker in subsequent transformations of the strain, but
also results in strains carrying unnecessary and undesirable bacterial
genes. Commercial cultivation and utilisation of such strains (e.g. as
oral vaccines[22]) is therefore associated with
risks of horizontal transfer of these genes to other
microorganisms.[23]
Several strategies to circumvent these issues have been developed forC. reinhardtii . Plastome mutants carrying defects in a gene
required for photosynthesis can be used as recipient strains whereby
selection is based on the restoration of phototrophy through repair of
the defective gene, thereby generating a marker-free transgenic line.
However, this limits transformation to a specific strain and such a
selection strategy can be utilised only once.[18]Fischer et al. developed an alternative strategy for generating
marker-free lines by using an aadA cassette that was flanked by
direct repeat sequences.[24] Following integration
into the plastome and selection for homoplasmy of the transformed
plastome, the selective pressure is removed allowing the marker to be
lost from the plastome via intramolecular recombination between the
repeats. Loss of the aadA cassette leaves just a single copy of
the repeat sequence as a DNA ‘scar’ at the site of plastome integration,
and the cassette can be reused in further rounds of transformation. A
similar strategy has been developed for higher plant chloroplasts, with
the issue of the unwanted scar being avoided by creating a direct repeat
using endogenous sequence adjacent to the integration site, rather than
two copies of an exogenous element.[25]
The main limitation of the aadA recycling method is that the
direct repeat needs to be of a significant size (0.42 kb or larger) in
order to achieve sufficient rates of intramolecular recombination in the
absence of active selection. Even so, complete loss of the marker can
still involve time consuming cycles of replating on selective media and
extensive screening[24–26]. The use of larger
direct repeat sequences can increase the rate of intramolecular
recombination[24] but poses several issues. If the
direct repeats are incorporated in the endogenous regulatory elements
used to drive expression of the marker, this may result in unwanted
recombination with the original copy of this element elsewhere of the
plastome, yielding a persistent heteroplasmic state due to deletion of
essential genes[27] or unwanted deletion of
non-essential genes.[28] Alternatively, if the
direct repeat is external to the marker, then a large tract of foreign
DNA is left as a scar, potentially perturbing plastome
function.[24]
To address these issues, we have developed the so-called CpPosNeg system
for scarless recycling of the marker in the C. reinhardtiichloroplast. This system uses a dual selectable marker encoding a
CodA–AadA fusion protein that confers both positive and negative
selection. The marker is linked to a transgene such that both share the
same 3’ untranslated region (3’UTR) thereby creating a direct repeat.
Introduction of the construct into the plastome involves a two-step
process with transformants initially selected for spectinomycin
resistance conferred by the AadA moiety. Recombination between the
repeats would then be promoted by a strong negative selection on
5-fluorocytosine with CodA converting it to the toxic product,
5-fluorouracil.[29]
We demonstrate the utility of the method by creating two marker-free
transgenic lines with a luciferase gene inserted into different loci
within the WT plastome. To demonstrate the iterative capability of the
system, a second round of CpPosNeg is used to introduce an additional
reporter gene into the plastome. Since both aadA and codAhave also been developed as selectable markers for the tobacco
chloroplast,[30,31] the CpPosNeg system could be
easily adapted for engineering of plant plastomes.