(Re)design of the choroplast genome - towards a synthetic organelle

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Plants and algal cells contain a compartment (or organelle) not found in animal cells - the chloroplast. This is the site of photosynthesis and other important biosynthetic processes, and it contains its own genetic system that is a legacy of the chloroplast's evolution from a free-living photosynthetic bacterium. Over evolutionary time, the circular genome of the chloroplast (the 'plastome') has been massively reduced in size, with the loss of most of its genes. What remains is a tiny genome that contains only a hundred-or-so genes. About half of these encode components of the photosynthetic apparatus, whilst the remainder are genes for housekeeping functions such as gene expression. The plastome therefore represents a naturally reduced genome that could be readily re-designed using synthetic biology approaches to gain insights into minimal requirements for an entire genome. At the same time this would optimize the plastome as a platform (a chassis) for future engineering efforts, such as production of high value products in the chloroplast or re-engineering the photosynthesis process. Genetic engineering of the plastome is well-established for several plant species, and for the single-celled green alga Chlamydomonas reinhardtii, which has served for many years as a model system for studying chloroplast biology. C. reinhardtii is particularly suited for a plastome redesign project as, unlike plant cells, it contains just a single chloroplast and can dispense completely with photosynthesis when grown on acetate as a source of carbon. Furthermore, the generation of chloroplast-engineered strains takes weeks rather than months. Recently, we have developed new tools for engineering the C. reinhardtii plastome and will apply these to address the following questions: i) by systematically deleting all regions of the plastome known to contain photosynthetic genes and other dispensable DNA, can we define the minimal size and gene content for the plastome? ii) Can we re-introduce the genes for a particular photosynthetic complex as a single refactored gene cluster, thereby allowing a modular 'plug-and-play' approach to studying how gene changes influence photosynthetic performance. iii) Can large gene clusters be engineered into the plastome to allow the reprogramming of the chloroplast as a site for synthetic of high-value products such as vitamin B12? iv) can we design and build an entirely synthetic minimal plastome and introduce this into the chloroplast, replacing the native plastome and thereby 'rebooting' the DNA software of the organelle? v) can we integrate into our plastome technology the capacity to tune up or down the expression of target genes using different combinations of chemicals in the growth medium, allowing us to control gene clusters or test pairs of gene variants in the same chloroplast by switching expression from one to the other?The project will provide essential basic understanding of the challenges of synthetic reprogramming of organelle genomes in plants and animal cells, and serve as a platform for future "designer organelle" studies.