COLLABORATIVE PROJECT: MAGIC - A multi-tiered approach to generating increased carbon dioxide in the chloroplast

Photosynthesis is at the core of virtually every aspect of society, from food production to industrial construction. Terrestrial photosynthesis is intimately connected with our use of other natural resources, and it exerts major controls on the water, mineral and carbon cycles of the world. For example, plant transpiration is thought to have contributed to recent changes in fresh-water availability associated with the global rise in CO2, and it is at the centre of a crisis in water availability expected over the next 20-30 years. Over this same period it is estimated that a 50% increase in global food production will be required to keep pace with the increase in human population. Crop yields have matched population growth until recently, but the gains from cereal cultivars bred in the Green Revolution were realised in full a decade ago. Thus it is vital that routes to further improvements in photosynthetic efficiency are sought now. In most species, CO2 is fixed by Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCO) in the Calvin-Benson cycle to generate a three-carbon compound. RuBisCO is remarkably poor in its substrate selectivity and promiscuously fixes both CO2 and O2, a fact that makes RuBisCO arguably the most inefficient step in photosynthesis. One way of reducing O2 use by RuBisCO is to raise the partial pressure of CO2 (pCO2). So-called carbon concentrating mechanisms (CCMs) have evolved multiple times in nature, albeit not as a feature of most common crop species. Thus, comparisons suggest roughly a 50% increase in overall yield might be realised if O2 use by RuBisCO were bypassed in crops. Significant resources have gone into engineering RuBisCO for increased CO2 selectivity and introducing a single-celled version of C4 photosynthesis in rice, but these approaches have yet to see a step change in photosynthetic efficiency. One new set of strategies yet to be explored is to co-opt light-driven pumps, anion exchange transport and substrate channelling to supply CO2 to RuBisCO. To date none of these processes is known to facilitate photosynthesis, although all three occur naturally and have been employed synthetically in biology. It is our goal to develop the equivalent of a 'two-stage pump': placing in series (1) a transport mechanism to concentrate HCO3- in the chloroplast powered by the light-driven ion pump halorhodopsin (hR) from the archeon Halobacterium halobium, and (2) substrate channelling within the chloroplast using one or more molecular 'building blocks' from Clostridium or cyanobacteria to carry HCO3- or a four-carbon intermediate to RuBisCO. This two-stage strategy is expected to maximise CCM gain driven independently with light energy absorbed by hR, and it has the added potential for engineering hR to tap the unused asset of light beyond the photosynthetic spectrum. Furthermore, an overarching feature of this approach is in its modular nature: it will be possible to develop each stage of the two-stage pump in parallel, and to assess its functionality separately at molecular, cellular and whole-organismal levels, combining the components thereafter for final validation. This modular approach ensures the maximum efficiency and speed in realising our goal within the three-year period.

We will develop our two-stage pump making use of alternative strategies for both the first and second stages, and testing where appropriate in the photosynthetic model of cyanobacteria before translation to the chloroplasts of the C3 model plant Arabidopsis. For the first stage of engineering a HCO3- transport system driven by hR, two mechanistically-different strategies will be pursued. One strategy will take existing transport functionalities of hR and the mammalian Cl-/HCO3- exchange transporter AE1, each proven in independent heterologous expression studies, to generate a Cl- gradient across the chloroplast inner envelope membrane and to use this gradient to drive Cl-/HCO3- exchange. The second, simpler and possibly more elegant strategy is to engineer hR to select for HCO3- over Cl- or other anionic species in order to pump HCO3- directly across the membrane. For the second stage of the two-stage pump, we will engineer a set of scaffold proteins to capture carbon entering the chloroplast stroma either as HCO3- or as a four-carbon intermediate and deliver it for conversion to CO2 directly at the active site of RuBisCO. Scaffolding alternatives will include constructs positioned at the inner face of the envelope membrane as well as fixed to the soluble RuBisCO within the stroma, and we will make use of either proven building blocks derived from Clostridium or a new set of constructs derived from cyanobacterial carboxysomes. At each step of development we will be able to assess functionality and impact separately at molecular, cellular and whole-plant levels. Once we are satisfied that the components will work, we will then combine them for final validation.

This proposal is for fundamental research to establish novel and synthetic approaches to increasing photosynthetic capacity through mechanisms for capturing CO2. The concepts behind the proposal are at the core of ideas emerging within the international cell and synthetic biology communities and should help stimulate thinking about approaches to the next Green Revolution as well as novel applications in other areas of science at the interfaces between biology, physics and systems analysis. Thus, the research is expected to benefit fundamental researchers as well as industry through conceptual developments as well as the introduction of new technologies for light harvesting and its applications in biology. The research will feed into higher education training programmes through research training at the postgraduate and postdoctoral levels, and through international exchange with our collaborators (JHG, CK) in the USA. Finally it will help guide future efforts in applications to agricultural/industrial systems. MRB and JMH have established links with industrial/technology transfer partners (Agrisera, Dualsystems, Plant Bioscience), research institutes (SCRI and JIC) and relevant international consortia (IRRI and the C4 Rice Consortium,) 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 attached Impact Pathways.