NITROPLAST: A LIGHT-DRIVEN, SYNTHETIC NITROGEN-FIXING ORGANELLE

For optimal yields, crop plants require fixed nitrogen in the form of ammonia or nitrate fertilizers, but this requires large fossil fuel inputs and can also result in runoff which contaminates aquifers and estuaries. Unlike some microbes that have the capacity to fix atmospheric nitrogen, plants do not have this ability. So the goal of this research is to engineer a novel synthetic nitrogen fixing organelle, with the long-term aim of conferring efficient nitrogen fixation in non-leguminous crop plants. However, there are significant hurdles in introducing nitrogen fixation into plants, which includes high metabolic energy costs and overcoming oxygen sensitivity of the process. To reach this goal, tools of synthetic biology will be used to engineer nitrogen fixation into a simple model system. Cyanobacteria are single-celled organisms that are evolutionarily related to plant plastids. In cyanobacteria, the engineering goals should be tractable, constituting a technological stepping stone that would lead to the engineering of nitrogen fixation into plant plastids. For this project to be successful, several objectives need to be met. First, ideal candidate gene clusters required for nitrogen fixation need to be identified. Using this information and coupling it to synthetic biology techniques, tunable nitrogen fixing gene modules, which can be precisely controlled, need to be built. Next, these synthetic nitrogen fixing gene modules need to be moved into cyanobacteria. Finally, to deal with the high metabolic energy costs of the process, a novel strategy will be employed by which extra light absorption capacity is engineered into cyanobacteria. These objectives are complex and multi-faceted, requiring tight coordination between participating laboratories. Successful completion of this research will lead to an engineered synthetic, controllable nitrogen fixing gene cluster linked energetically to light energy, which can ultimately be transferred into plastids of crop plants in the form of a 'nitroplast'.

We intend to engineer a novel synthetic N2 fixing organelle, with the long-term aim of conferring efficient N2 fixation to non-leguminous crop plants. To reach this goal, we will use the tools of synthetic biology to engineer a nitrogen fixation cluster into a cyanobacterium. Several specific objectives need to be attained for this project to be successful. First, we will identify candidate gene clusters for nitrogen fixation and associated processes, from photosynthetic prokaryotes, using appropriate bioinformatic tools. Then we will use synthetic biology techniques to build modular, orthogonal, tunable nitrogen fixing gene clusters that can be mixed and matched, and ultimately transferred into a new host. We will eliminate complex native regulation, gain control and understanding of the gene clusters, facilitate optimization and transfer between hosts and develop synthetic biology tools for cyanobacteria. Next, we will transform
synthetic nitrogen fixing modules into cyanobacteria for which optimal transformation and expression protocols will be developed. We will employ a novel strategy by which a simplified photosystem derived from Heliobacterium modesticaldum will be engineered into the host cyanobacterium, to meet the extra energy demand of N2 fixation. This photosystem is composed of a single gene product and has the benefit of absorbing light in the near-infrared region of the spectrum, not otherwise utilized by the endogenous Photosystem I. These objectives are complex and multi-faceted requiring close coordination between participating laboratories. At the end of this research program, we will have engineered synthetic, optimized, highly controllable nitrogen fixing gene cluster linked energetically to reaction centers which can ultimately be transferred into plastids resulting in the formation of a 'nitroplast'.

One of the major challenges of the twenty first century is to ensure food security for an expanding population. At the core of this challenge is the problem of nitrogen assimilation by non leguminous crop plants. The goal of this project is to build a novel synthetic, controllable nitrogen fixing module into a cyanobacterium. We aim to maximise the impact of our research in all appropriate areas in a timely and effective manner. In doing so we will 1. Ensure that our research will be effectively communicated to beneficiaries e.g. scientists and expert stakeholders in a timely manner. 2. Ensure that the work is appropriately disseminated to industry, IP protected and if appropriate commercialized 3. Ensure that the work contributes to project staff development and training. 4. Disseminate the work to the general public and school pupils. We will achieve our objectives by engaging with the following beneficiaries, in appropriate timeframes, in the ways outlined below.
1. The outcome of this research is expected to benefit basic researchers in academia as well as applied scientists through the developments of new tools for cyanobacteria, and through products such as the introduction of nitrogen fixation into plants. This project is a unique opportunity for methodology exchange between U.S and U.K. scientists and for developing tools that will be freely available to the synthetic biology and cyaobacterial research communities. The impact on academic beneficiaries will be realised by communicating the outputs from this project through regular publication in high impact peer reviewed journals, at conference talks.
2. The new technologies derived from this work should provide tools and knowledge to boost nitrogen fixation capacity that could strongly impact agriculture and will therefore be of significant interest to a wide range of major UK, US and EU companies for example Dow, Syngenta, Monsanto. We will seek interest from companies in application areas such as fertilizer producers, Industrial chemicals and Agriculture via research collaborations and access to IP and know how via licensing deals.
3. Synthetic Biology is revolutionising the way we do biology but in order to reach its full potential there needs to be capacity building in the skills development and training of young scientists. An exciting aspect of Synthetic Biology is that it encourages collaboration and multidisciplinary approaches to research, and it is important that the community develops people who want to engage with these diverse ideas and technologies. This project will provide an excellent training experience for the PDRAs employed on the grant.
4. The PIs are actively involved in a number of outreach programmes, and have delivered talks to diverse audiences including schools, youth organizations such as the cub scouts, lawyers, political parties, journalists and the general public about our research. We plan to continue these outreach activities and look for new opportunities. The PDRAs will participate in school visits, discussing their research and interacting with the students.