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

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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.