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.