Photosynthesis Regulation and cyanobacterial biofuel production – ReCyFuel

We combined molecular biology, biochemical and biophysical techniques. Mutated carotenoid proteins were constructed and expressed in E. coli cells. Cyanobacteria strains with modified photoprotection mechanisms were constructed and characterized. The modified proteins and complexes were isolated and characterized by biophysical methods (absorbance and fluorescence fast and slow kinetics). Finally, two different methods to measure cyanobacterial cyclic electron transport in vivo: 1) the Electrochromic Shift of photosynthetic pigments which allows the
measurement of the transthylakoidal electric field depending on cyclic electron transport; 2) the use of a Klas-NIR spectrophotometer to measure the redox changes of Photosystem I donors and acceptors.

Based on our previous knowledge and expertise on OCP-related NPQ we were able to look for different natural and mutant OCPs inducing a large photoprotection with a fast recovery, which is necessary to increase biofuel conditions. We found good natural candidates and several OCP
mutations presenting these characteristics. Some promising cyanobacteria mutants were constructed. Essential discoveries were done about state transitions mechanism showing that previous hypotheses were wrong and that this mechanism is completely different in plants and cyanobacteria. Finally, the methods developed during the project allowed to better understand the role of cyclic electron transport in cyanobacterial photosynthesis. We were able to show that this mode of transport was not dependent on the nature of the donor to Photosystem I (plastocyanin or
cytochrome c6) and that its amplitude remained low in the absence of linear electron transfer.

This project allowed finding the way to modify the NPQ-photoprotective mechanism and to do big advances in the elucidation of state transitions mechanism and cyclic electron transport role. The construction of cyanobacteria mutant strains and the test of these new strains on biofuel production remain to be probed. Further studies are needed to elucidate the factors and proteins involved in state transitions and cyclic electron transport before engineering these mechanisms.

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Due to the global Earth warming arising from increasing atmospheric levels of CO2 and to the decrease of fossil fuel supply, one of the biggest challenges of our modern society is to design new methods to generate renewable, sustainable and clean energy. In the recent years, the use of cyanobacteria as biotechnological platforms to produce biofuels has gained significant importance. The physiological robustness and adaptability of cyanobacteria to variable and extreme environments gives the potential to cultivate them in environments that are unsuitable for agriculture. They can be easily bioengineered and research efforts are focused on the modification of metabolic pathways to direct the electrons and chemical energy towards the synthesis of key molecules. However, these modifications impact in return the metabolic pathways sharing common metabolites and, in particular, photosynthetic CO2 assimilation. Vice-versa, modifications in photosynthesis influences biofuel production yield. Understanding these feed-back responses will be essential in the design of engineered cyanobacterial strains producing high yields of biofuels.
The aim of our project is to understand and engineer mechanisms involved in photosynthesis regulation and photoprotection to favor biofuel production in cyanobacterial cells under a variety of environmental conditions. We will study on one hand the impact of modification of metabolic pathways on photosynthesis in four different biofuel-producing cyanobacterial strains and on the other hand the influence of changing photosynthesis rates and photoprotection on biofuel production. One key target for improving efficiency of cyanobacterial strains lies in the redesign and control of the cyanobacterial Non-Photochemical-Quenching (NPQ) mechanism. While this mechanism protects cells from photodamage, it involves a decrease of incoming excitation energy and could negatively impact the solar-to-biofuel energy conversion. We will engineer the NPQ mechanism to allow cells to rapidly adjust their response to the wide range of growth conditions which occur in bioreactors. To this purpose, the Orange Carotenoid Protein and the Fluorescence Recovery Protein which are the essential elements of cyanobacterial NPQ will be modified. Our second approach will be to engineer the cyanobacterial photosynthetic apparatus to introduce modifications in the regulation of cyclic electron transport and state transitions mechanisms. The development of these strains requires a further characterization of these photosynthesis regulation mechanisms. This will unravel new regulation points and thus allow addressing the issue as to whether and how they impact production of specific biofuels.
The understanding of different regulations and their effect on biofuel synthesis will lead to engineer cyanobacteria with novel approaches decreasing the energy waste and increasing biofuel production. Thus, the project will have societal and socio-economic impacts participating in the improvement of cyanobacteria biofuel-producing strains which may lead to the patent of cyanobacterial strains carrying modifications in photoprotection for increased biofuel production.
The success of the BioCyFuel project requires the combination of expertise provided by the 2 French partners and their collaboration with two foreign laboratories providing the biofuel-producing cyanobacterial strains. Partner 1 discovered and characterized the cyanobacterial NPQ mechanism and has a long expertise in cyanobacterial molecular biology. Partner 2 is an international known expert specialized in algal state transitions and cyclic electron transport. The two foreign laboratories directed by Dr Jianping YU (USA) and Dr Pia Lindberg (Sweden) which are expert in biofuel production have constructed the biofuel-producing strains and will test the biofuel productions in the mutants created during this project.