14-ERASynBio Engineering the chloroplast of microalgae as a chassis for the direct production of solar fuels and chemicals

One of the greatest challenges of the 21st century is the sustainable supply of energy and chemicals from renewable resources. Driven by solar energy, chloroplasts function in nature as the most efficient minimal cell factories for generating chemical energy through the oxidation of water, but they are naturally tuned towards the fixation of carbon for building-up cellular components. Our long-term goal is to design a synthetic chloroplast in the "green yeast" Chlamydomonas reinhardtii that can be used as a chassis for the sustainable production of biofuels and chemicals. To achieve such an ambitious goal, we will develop various tools that will be indispensable to construct our chassis from a bottom-up approach. First, we will develop well-defined microalgal BioBricks to allow an efficient plug-in of protein and metabolic circuits in the chloroplast. Secondly, we will generate suitable Chlamydomonas strains by re-directing the photosynthetic electron circuits. Thirdly, we will engineer key players of the photosynthetic chain following the principles of Darwinian evolution for controlling energy delivery. State-of-the-art protein film electrochemistry of the engineered biocatalysts will guide the design processes. As a proof-of-principle, we will then use the chloroplast of optimised strains for assembling the BioBricks with the engineered photosynthetic chain players to produce bio-hydrogen and alkanes as by-products from light and water. This project is to be considered as a proof of principle and will step-up the development of novel biotechnology concepts that will establish "solar-cell chloroplast factories". The design and construction of a chloroplast chassis following synthetic biology principles will allow the sustainable production of biofuels and valuable chemicals, paving down the grounds for a carbon-neutral bio-economy that can supply our society with an increasing energy demand, while mitigating the damaging effects of climate change.

One of the greatest challenges of the 21st century is the sustainable supply of energy and chemicals from renewable resources. Driven by solar energy, chloroplasts function in nature as the most efficient minimal cell factories for generating chemical energy through the oxidation of water, but they are naturally tuned towards the fixation of carbon for building-up cellular components. Our long-term goal is to design a synthetic chloroplast in the "green yeast" Chlamydomonas reinhardtii that can be used as a chassis for the sustainable production of biofuels and chemicals. To achieve such an ambitious goal, we will develop various tools that will be indispensable to construct our chassis from a bottom-up approach. First, we will develop well-defined microalgal BioBricks to allow an efficient plug-in of protein and metabolic circuits in the chloroplast. Secondly, we will generate suitable Chlamydomonas strains by re-directing the photosynthetic electron circuits. Thirdly, we will engineer key players of the photosynthetic chain following the principles of Darwinian evolution for controlling energy delivery. State-of-the-art protein film electrochemistry of the engineered biocatalysts will guide the design processes. As a proof-of-principle, we will then use the chloroplast of optimised strains for assembling the BioBricks with the engineered photosynthetic chain players to produce bio-hydrogen and alkanes as by-products from light and water. This project is to be considered as a proof of principle and will step-up the development of novel biotechnology concepts that will establish "solar-cell chloroplast factories". The design and construction of a chloroplast chassis following synthetic biology principles will allow the sustainable production of biofuels and valuable chemicals, paving down the grounds for a carbon-neutral bio-economy that can supply our society with an increasing energy demand, while mitigating the damaging effects of climate change.

The sustainable supply of renewable energy is a major concern of almost all European Societies. Since our project aims to set down important steps towards the development of an artificial chloroplast that can be used as a chassis for the sustainable production of renewable chemicals and biofuels driven by solar energy, Sun2Chem represents an important step towards meeting European needs not only for the right development of a bio-economy, but also for social justice in terms of environment protection and sustainability.
To reach this goal, we will take a bottom-up engineering approach for the development of suitable algal BioBricks (WP1), which will be deposited in the international Genetic Engineered Machines (iGEM) registry. Thus, our results will be partially disseminated at the famous iGEM competition by undergraduates who will be supervised by doctoral and postdoctoral researchers. We will also have doctoral and postdoctoral students working on the metabolic engineering of the photosynthetic pathway (WP2, WP5) as well as the engineering of the hydrogenase, ferredoxin and FNR (WP3) and their characterisation via protein film electrochemistry (WP4).
Regarding intellectual property rights, it will be important to first patent the improved pathways and biocatalysts obtained in WP2 and WP3 (if the deliverables of WP1 are used for iGEM they cannot be patented) as well as WP5, which is the integration of WP1-4. Patenting issues will be handled according to the rules at each of the participating partners organisations. Upon patenting the findings, therefore, their publication in suitable international well-renowned journals preferentially under an open access scheme will follow. Note that in each WP, every milestone lists a set of deliverables, which themselves correspond to a defined problem that upon resolution can be transformed into concise publications. After publishing, we will disseminate the results not only at the local media but also at scientific meetings. The results obtained in all WPs will be generally disseminated at conferences related to synthetic biology, protein as well as metabolic engineering and more specifically at hydrogenase, electrolytic solar fuel and solar-based renewable energy meetings.
We expect to produce significant results to control electron delivery from Photosystem 1 in the chloroplast of C. reinhardtii towards the production of hydrogen and alkanes. Although we may encounter difficulties in engineering a completely O2-tolerant hydrogenase (at atmospheric oxygen concentration of 21%), preliminary results from Partner 1 indicate that mutating the active site of HydA1 can notably improve its tolerance against oxygen. Achieving this goal will be important for patenting the improved biocatalyst and initiate the first experimental trials both in vitro (WP4) and in vivo (WP5), which may even result in a spin-off company to fully realise the technological potential of H2 production (the strong oxygen sensitivity of hydrogenases hinders their industrial use owing to the high-associated costs of producing the required biocatalyst in large-scale). If we can realise the development of this technological feature, there will be certainly a long-term societal benefit: Solar-driven sustainable production of clean energy by algae that do not compete with agricultural land while mitigating the damaging effects of climate change. Upon reaching such a scenario, we would then assess in more detail biosafety as well as biosecurity issues and explore novel interfaces between science and society for the public acceptance of this special kind of genetically modified microorganisms.