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

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.

Synthetic biology offers an unprecedented opportunity both to consider (re)designing biological systems for useful outputs, but also the ability to dissect existing biological systems, to establish the minimum requirements of a process, a genome, or even an entire organism. However, to fulfill this promise the system under investigation needs to be tractable in terms of manipulation and analysis, and ideally to be amenable to incremental changes. In this proposal we aim to establish what is needed for a minimal chloroplast genome of the alga Chlamydomonas reinhardtii, and then to design, build and test a synthetic version. In the process we will also redesign it to develop a system for exploring fundamentals of photosynthetic complex assembly and function, and for expression of heterologous genes. C. reinhardtii can live heterotrophically and thus photosynthetic genes are completely dispensable. To establish which other parts of the 204 kb C. reinhardtii plastome (CP) can be removed, we will carry out systematic deletions, and in the process identify any cryptic essential sequences, as well as gaining information on the minimum size needed for CP stability and maintenance. We will test the efficiency of refactoring the five pet genes for the cytochrome b6f complex, and use operons for increasing numbers of enzymes of the biosynthetic pathway for vitamin B12 as proxies for heterologous gene clusters. We will take advantage of a system we have developed using nucleus-encoded trans-acting factors required for stability of chloroplast transcripts to tune expression of the introduced genes. These experiments will inform the design of a completely synthetic minimal CP, lacking all non-essential genes. This will be introduced into a recipient host strain that had been previously pretreated to reduce CP copy number, and use several strategies to facilitate complete substitution of the endogenous plastome with our synCP-v1.0.

The full exploitation of synthetic biology (SynBio) within the rapidly expanding bioeconomy requires an understanding of how SynBio can be applied to organisms beyond the model systems of E. coli and yeast. Importantly, the manipulation of phototrophic organisms (plants, algae and cyanobacteria) is fundamental to globally important areas such as food and feed production, biofuel generation, sustainable production of phytochemicals and novel bioactives, and biological carbon capture. This project aims to define the parameters for minimizing and redesigning the chloroplast genome (plastome), with the overall goal of reprogramming an algal chassis with a completely synthetic plastome. An ability to carry out such reprogramming would open up possibilities for making designer chloroplasts with a plethora of novel properties, and would therefore benefit academic researchers and industries across a wide spectrum. Examples include: i) photosynthesis researchers and those aiming to improve photosynthetic performance in food crops, and in organisms grown for biofuels; ii) industrial biotechnologists developing plants and algae as light-driven platforms for low costs synthesis of recombinant proteins and valuable metabolites; iii) synthetic biologists interested in introducing novel organelle compartments into eukaryotic cells; iv) evolutionary biologists interested in how organelle genomes have been shaped over evolutionary time, and genome researchers investigating the miniaturization of genomes.
The project will also contribute to researcher training and capacity building in algal biotechnology and synthetic biology - two priority areas for BBSRC as attested by its funding of the PHYCONET NIBB and the SynBio Centres. This will help to ensure that there are skilled researchers for UK's growing Industrial Biotech sector. Ultimately, the growth of this sector will create jobs and provide economic benefit to the country.
There is a great interest amongst the general public, students and 'lay scientists' regarding synthetic biology and green technologies, and a growing recognition of the need to develop sustainable solutions to the global challenges of providing food, feed, fuels and pharmaceuticals to an ever-increasing population. Through the various engagement activities embedded within the project (detailed in the Pathway to Impact) we will grow this interest and awareness. Furthermore, the dialogue with these groups will help provide a holistic overview of our research and it relevance to society. Finally, we have established links with Government offices, trade organisations and business support bodies (e.g. InnovateUK), Societies (e.g. Microbiology Society, British Phycological Society) and algal associations (e.g. European Algal Biomass Association), and so are able to contribute positively to the framing of legislation regarding the control and exploitation of algal synthetic biology. Lobbying and involvement in the drafting of roadmaps and policy documents are already a key activity for both PI's and this will continue under this project.