Abstract
Chlamydomonas reinhardtii is a model alga of increasing interest as a cell factory for the production of valuable compounds, including therapeutic proteins and bioactive metabolites. Expression of foreign genes in the chloroplast is particularly advantageous as: (i) accumulation of product in this sub-cellular compartment minimises potential toxicity to the rest of the cell; (ii) genes can integrate at specific loci of the chloroplast genome (plastome) by homologous recombination; (iii) the high ploidy of the plastome and the high-level expression of chloroplast genes can be exploited to achieve levels of recombinant protein as high as 5% total cell protein; (iv) the lack of any gene silencing mechanisms in the chloroplast ensures stable expression of transgenes. However, the generation of C. reinhardtii chloroplast transformants requires efficient methods of selection, and ideally methods for subsequent marker removal. Additionally, the use of reporter genes is critical to achieving a comprehensive understanding of gene expression, thereby informing experimental design for recombinant applications. This review discusses currently available selection and reporter systems for chloroplast engineering in C. reinhardtii, as well as those used for chloroplast engineering in higher plants and other microalgae, and looks to the future in terms of possible new markers and reporters that will further advance the C. reinhardtii chloroplast as an expression platform.
Highlights
Since the development of recombinant DNA technology several decades ago, genetically engineered organisms have become an increasingly popular source of industrially-valuable proteins and metabolites
We focus on current selection strategies and markers developed for C. reinhardtii and consider the future application of markers that have been used successfully for chloroplast engineering in other algae or in higher plants
Equivalent mutations in bacteria are often associated with reduced fitness [78], and if the same is true in the chloroplast, the maximum yield of recombinant protein, especially for highly expressed transgenes, might be compromised in transgenic lines carrying such mutations
Summary
Since the development of recombinant DNA technology several decades ago, genetically engineered organisms have become an increasingly popular source of industrially-valuable proteins and metabolites. The chloroplast stroma appears to support the correct folding and disulfide bond formation of recombinant proteins [9], and any cytotoxic effects of the proteins is minimised by their strict confinement to the chloroplast [10] Such engineered algal strains offer the potential for low-cost phototrophic production in which high-value products, such as therapeutic proteins and bioactives, are produced from CO2 and simple nutrients through photosynthesis [2,3]. Microalgae are being explored as industrial biotechnology platforms for the production of metabolites that have applications ranging from biofuels to bioactives [22] Such metabolic engineering requires further advances in the molecular toolkit, allowing precisely regulated expression of multiple transgenes. The acquisition of the GOI by every copy of the plastome—homoplasmy—is subsequently achieved by the application of selective pressure, as cells replicate such that progeny containing more transgenic copies are favoured (Figure 1)
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