14-PSIL Multiple Approaches to Gain Increased Carbon Dioxide

Lead Research Organisation: University of Exeter
Department Name: Biosciences


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Technical Summary

We are building on progress that includes expression of pHR in E. coli, cyanobacteria and plants. These advances have put us in a strong position to deliver within the next few years. Mathematical modeling has validated the idea of using a light-driven ion pump for concentrating CO2; it now remains to assemble and express these pumps and validate function in chloroplasts. The idea to use scaffolds to concentrate CO2 at RuBisCO remains a goal, but our strategies have changed in light of new understanding of the interplay between diffusion and kinetics. We have successfully expressed scaffold proteins in cyanobacteria and plants, demonstrating that they can be both targeted to specific sites and that they function to recruit their respective substrates. Our mathematical models predict that the original idea of utilizing these constructs to enhance channeling of CO2 to RuBisCO will have negligible impact on CO2 assimilation. We need now to confirm this prediction in our cyanobacterial systems. A rethinking of the problem of concentrating CO2 at RuBisCO in C3 plants leads to development of a new approach. Our mathematical models highlight the poor CO2 capture probability of RuBisCO as a major constraint. Here, we propose designs to slow the diffusion rate of CO2 in the stroma and increase assimilation by introducing transient (stationary) binding sites near RuBisCO (a CO2 'sponge'), effectively enhancing the native characteristics recently identified in photosynthetic systems. We will use the cyanobacterial system to screen and optimize this approach and will use the scaffolds now proven in our hands to translate these to chloroplasts. Finally, we previously lacked the ability to quantify performance, ie. HCO3- concentration gains. This capability is now available through a lipid vesicle technique.

Planned Impact

his proposal is for fundamental research to develop new conceptual approaches relevant to ideas emerging within the international plant, systems and synthetic biology communities. The research will stimulate thinking around strategies for modelling and for applications of synthetic biology in plants, especially in relation to photosynthesis, and it should strengthen methodologies relevant at many levels from cell to crop engineering. Thus, the research is expected to benefit fundamental researchers and, in the longer-term agriculture and industry, through conceptual developments and approaches to improving carbon capture by plants. The research will feed into higher education training programmes through capacity building at the postgraduate and postdoctoral levels. Additional impact is proposed through public displays and the development of teaching resources building on the background work for this proposal. Finally the research will help guide future efforts in applications to agricultural/industrial systems. The applicants have established links with industrial/technology transfer partners and research institutes to take advantage of these developments. Further details of these, and additional impacts will be found in Part 1 of the Case for Support and in the Impact Pathways submitted by the lead PI.


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Description This was a collaboration between 4 UK labs and 2 USA labs to explore an approach to improving photosynthesis by "designing" a light driven pump to concentrate carbon dioxide and trap with within the leaf using a carbon dioxide binding protein in a synthetic complex with the CO2 fixing enzyme RuBisCO. The CO2 binding protein approach was assessed by mathematical modelling and, contrary to the original proposal, proved to be unfeasible. Therefore a modified approach to understanding the basis of enzyme scaffolding was taken. We have constructed a synthetic scaffold protein to which two enzymes in a simple metabolic pathway can be attached. This enables us to test the effetc of tethering enzymes together. We used the model plants tobacco and Arabidopsis thaliana as test organisms and the results are currently under analysis. Additionally to engineer changes in enzyme activity in a cyanobacterium (Synechocystis) we have developed a new gene knockdown system involving dCAS9 (a variant of CRISPR-Cas gene editing).
Exploitation Route The dCAS9 for controlled knockdown of gene expression in Synechocystis will be published when validation is finished and the strains will be made available to the community.
Sectors Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology

Title Targeted gene knockdown in Cyanobacteria 
Description An inducible gene expression knockdown system in the cyanobacterium Synechocystis PCC6803 using an inducible dCAS9 system 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? No  
Impact Development still in progress