Tapping the Unused Potential of Photosynthesis

Photosynthesis is the pivotal biological reaction on the planet, providing the food we eat and the oxygen we breathe, and removing CO2 from the atmosphere. Photosynthesis consists of two reactions: the light reactions absorb light energy from the sun and use this to split water (H2O) into electrons, protons and oxygen, and the dark reactions use the electrons and protons from the light reactions to 'fix' CO2 from the atmosphere into simple sugars that are the basis of the food chain. Importantly, the light reactions have a much higher capacity than the dark reactions, resulting in much of the absorbed light energy being dissipated rather than being used to 'fix' CO2.

In this proposal we will use synthetic-biology methods to engineer an additional enzyme (a cytochrome P450, CYP) in-between the light reactions and the dark reactions. This will 'rewire' photosynthesis such that more absorbed light is used to power useful chemical reactions. This work therefore represents an innovation whereby a range of additional valuable chemical reactions can be powered by the sun in cyanobacteria and plants.

We have previously developed a 'platform' cyanobacterial cell line where the 'wasted' electrons of photosynthesis are rewired (using a CYP) to degrade the pollutant atrazine (a herbicide used in agriculture). Atrazine while banned in the EU is still one of the most prevalent pesticides in some groundwater systems. This cell line may be used in the efficient bioremediation of such polluted wastewater areas.

In this proposal we aim to further develop the use of CYP in rewiring photosynthesis such that we can improve and regulate the yield of the desired products, as well as enhance the diversity of products that can be powered by light. Further, we will fully characterise the physiology of engineered cell lines such that we can interrogate the fundamental mechanism of this photosynthetic electron transfer. In addition, we aim to transfer this technology to the model higher plant Arabidopsis as a first step in improving photosynthetic potential in crop species.

The 'added value' we aim to introduce into cyanobacteria and plants may be a critical step toward the commercial realisation of using photosynthetic species to generate 'biofuels' that may one day replace our current dependence on fossil fuels.

Increasing the efficiency of the conversion of light energy to products by photosynthesis represents a grand challenge in biotechnology. Photosynthesis is limited by the carbon-fixing enzyme RuBisCo, resulting in much of the absorbed energy being wasted as heat or fluorescence or lost as excess reductant via alternative electron dissipation pathways. To harness this wasted reductant we have engineered the model cyanobacterium Synechococcus PCC 7002 to express the mammalian cytochrome P450, CYP1A1, which would serve as an artificial electron sink for excess electrons derived from light-catalysed water-splitting. This improved photosynthetic efficiency by increasing the maximum rate of photosynthetic electron flow by 31.3%. Importantly, a simple fluorescent assay for CYP1A1 activity demonstrated that the CYP is light-dependent and scaled with irradiance in vivo. Furthermore, Synechococcus PCC 7002 expressing CYP1A1 degraded the herbicide atrazine, which is an environmental pollutant.

In this proposal we use this engineered cyanobacteria as a 'platform' to further develop this technology. We will use synthetic biology approaches to increase the flux of photosynthetic electrons to the P450 as well as genetically regulating its activity. Further, we will diversify the type of P450s (and therefore products) that can be powered by light. Full photosynthetic physiological characterisation of these cell lines will enable the impact of an artificial photosynthetic electron sink on cellular metabolism to be determined and will reveal important insights as to the mechanism of the photosynthetic electron transport chain. Lastly, this technology will be transferred to plants with a view to increasing herbicide resistance and potentially tolerance to high-light conditions.

Photosynthesis is a key reaction in the biosphere, providing the oxygen we breathe and food we eat, and removing CO2 from the atmosphere. Further, photosynthetic species offer the potential as carbon-neutral, sustainable sources of fuels and products. Improving 'photosynthetic efficiency' is therefore a central aim of BBSRC research strategy linked to bioenergy, food security and synthetic biology.
This proposal aims to rewire photosynthesis such that the current inefficiency can be used to drive useful reactions and generate useful products. The research will have academic impact in that insights can be made into the fundamental mechanisms of photosynthetic electron transport.

The demonstration that novel products can be generated using light energy by photosynthetic species has important scientific (and potentially commercial) impacts in that this approach 'adds value' to the use of photosynthetic species currently considered as candidates for biofuel production. Currently, the economic feasibility of growing photosynthetic species for biofuel is marginal; however, a co-production model that combines fuel production with the synthesis of high-value products (or valuable 'ecosystems-services' such as bioremediation), could have significant impacts in narrowing the economic gap in realising the potential of photosynthetic species for biofuels. We have established links with biotechnological companies in this sector (Algenuity) and have instigated new links with two companies (see letters of support) who are well placed to advise on the potential commercial applications of this research.

As well as developing cyanobacterial species, this proposal aims to transfer these technologies to higher plants using the model plant Arabidopsis. Here we will aim to generate a herbicide resistant plant which may also be less sensitive to photoinhibition at high-light. To parallel this research we received (in 2016) a small award from the Community Resource for Wheat Transformation project supported by NIAB to explore the feasibility of testing our approach in the crop species wheat.

The 'molecular toolkit' that would be developed in this proposal will expand the range of processes that can be engineered into photosynthetic species. This therefore will have significant impact in the biotechnological development of the use of cyanobacterial species.

Lastly, AB will be employed as a researcher Co-I on this grant. AB has recently moved into the field of photosynthesis research bringing a valuable skill set as a synthetic biologist. This proposal will further integrate AB and his skills into the photosynthesis research community where he has the potential to make a substantial impact.