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

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Biological Sciences

Abstract

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'.

Technical Summary

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'.

Planned Impact

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.

Publications

10 25 50
 
Description We have developed a new intracellular oxygen sensor

We have developed a range of new tools for engineering cyanobacteria including new promoters and new RNA polymerases.

In addition our project collaborators have engineered a range of new terminators, transposons
Exploitation Route The genetic tools we have developed will be of great use to the cyanobacterial research community. The new RNA polymerase we have identified and characterised is the subject of a patent application that is currently being prepared. It has potential for use in vivo and cell free systems. It could be used in the production of vaccines and in diagnostic devices as well as in the control of gene expression for synthetic biology.
Sectors Agriculture, Food and Drink,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description iCURe (Innovation to Commercialisation of University Research) programme
Amount £35,000 (GBP)
Organisation Department for Business, Energy & Industrial Strategy 
Sector Public
Country United Kingdom
Start 01/2018 
End 03/2018
 
Title New RNA polymerase 
Description A new RNA polymerase identified and characterised. This can be used for production of RNA in vivo or cell free systems. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact A patent is being written and a manuscript prepared. 
 
Description Alain Boussac: Collaboration with CNRS CEA Saclay France 
Organisation Saclay Nuclear Research Centre
Country France 
Sector Public 
PI Contribution Two studies: 1) I provided some some input a good deal of the basic thinking that set the stage for the research. I provided some of framework for the interpretation and the impetus for calculating the overall bioenergetic scheme. I also helped with the interpretation of several aspects. 2) I contributed to the interpretation on an in depth EPR study of the Mn in PSII. This work is a continutation of work that I initated when I was head of this group in France. Some of the EPR phenomena were my own discoveries from my work in the 1980's.
Collaborator Contribution 1) Alain Boussac did the EPR studies, Miwa Sugiura's group made the mutants, Fabrice Rappaport did the UV vis studies and the calculation 2) Alain Boussac performed the EPR experiments, developed the biochemical treatments applied and did the the first level of interpretation.
Impact Two research articles were published in these collaborations. This work is at the boundry of biology chemistry and physics. Further collaborations are under way.
Start Year 2013
 
Description David Kramer Michigan State University 
Organisation Michigan State University
Country United States 
Sector Academic/University 
PI Contribution I provided the background information and theory to help interpret new data on electric field induced photoinhibition in plants. I also provided new data coming from our research on bicarbonate based regulation of photosynthetic electron transfer relevant to the in vivo studies on-going in the Kramer lab.
Collaborator Contribution My extensive studies of photoinhibition and correlated them with pulses of light induced electric field. Applied findings from Rutherford lab to understanding regulation of photosynthetic electron transfer.
Impact Published one article in eLife and another article is in draft from.
Start Year 2015
 
Description Fabrice Rappaport Paris 
Organisation National Center for Scientific Research (Centre National de la Recherche Scientifique CNRS)
Department Institute Of Physico-Chemical Biology
Country France 
Sector Public 
PI Contribution 1) We have a long running discussion on photosynthesis bioenergetics with Fabrice Rappaport 2) We have an established experimental collaboration that uses the state of the art equipment developed in Paris: my students/post docs and myseldf have made regular visits bringng our samples. 3) We have benefited from advice on how to set up at Imperial a specialised spectrophotometer that was designed in the Paris group
Collaborator Contribution Fabrice Rappaport and his colleagues have worked in close collaboration with us to address biological problems and samples that we have brought to them. This has mainly entailed doing sophisticated optical spectroscopy; absorption and fluorescence. As part of a long running calculation Fabrice Rappaport performed a calculation that we hdd discussed for some years since we co-authoured an important theory paper in 2012. This calculation verified predictions made in that article and we used this to test and experimental system generated by my old research colleagues in France and Japan, Boussac and Sugiura.
Impact Two papers were published from this collaboration another is being written and there are several findings that require further experimental study.
Start Year 2011
 
Description Advising government of transport policy 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Provided arguments and citations relevant to Government policy on transport fuels with regard to biofuels.
Year(s) Of Engagement Activity 2014,2015
 
Description Member of the Senses about Science Panel on Plants 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I answer questions associated with energy and photosynthesis posed by members of the general public. These are published on line. I have done two of these in recent years: one on chlorophyll as a sun screen and one on photosynthetic animals.
Year(s) Of Engagement Activity 2014,2015,2016
 
Description Participation on policy discussion on sustainable agriculture: A4S accounting for sustainability 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact I was invited to this 10 year anniversary celebration of the Prince of Wales charity "accounting for sustainability". I met several influencial people in the accounting industry and provided information concerning the viability of biofuels.
Year(s) Of Engagement Activity 2014
 
Description Participation on policy discussion on sustainable agriculture: the insurance industry 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact I have been invited to take part in discussions among policy makers from, insurance companies, actuaries, representatives from government and the city on sustainable agriculture. I have duly provided information on biological energy issues.
Year(s) Of Engagement Activity 2013,2014,2015
 
Description Provided advice on request on the feasibility of biofuels for transport to an organisation involved in setting up green bonds as finacial instruments 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Through contact in sustainable agriculture I was asked to provide information on the feasibility of photosynthetic biofuels to inform the process of setting up "green bonds" as finacial instruments.
Year(s) Of Engagement Activity 2014
 
Description Provided advice on request on the feasibility of biofuels in the aviation industry to a avion industry think tank 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact In discussions about sustainable agriculture I met a member of a panel set up to advise the aviation industry on sustainable aviation. I was susbsequently invited to provide evidence on the feasiblity and scaling of biofuels.
Year(s) Of Engagement Activity 2014