Decreasing the oxygenase activity of Rubisco: a synthetic biology approach

Photosynthesis is the process whereby plants convert atmospheric carbon dioxide to organic matter (biomass) using energy from sunlight. Photosynthesis powers plant growth, crop productivity and all life on earth. The first step in photosynthesis combines carbon dioxide with a sugar containing 5 carbon atoms to make two sugar molecules each containing 3 carbon (C3) atoms in a process called carbon dioxide fixation. The C3 sugars then combine to regenerate the 5 carbon accceptor and also to form glucose. Glucose is used as the feedstock to synthesise all other molecules in the plant. Chemical reactions in plants, such as the first step in photosynthesis, only happen quickly enough if they are speeded up by proteins known as enzymes. Rubisco is the enzyme that speeds up carbon dioxide fixation. Unfortunately this enzyme has built-in inefficiencies. It is slow, so plants need to spend energy producing it in large quantities. It also has a side reaction in which oxygen competes with carbon dioxide to react with the 5C acceptor. The consequence of this side reaction (known as the oxygenase reaction) is that less C3 product is formed and photosynthesis rate is slower than it could be. Some algae and plants have developed elaborate mechanisms to increase the concentration of carbon dioxide near Rubisco, thereby decreasing oxygenase activity and increasing the efficiency of photosynthesis. Unfortunately, many of our major crops (e.g. rice, wheat, potatoes and pulses) do not have this mechanism and various approaches to reducing their oxygenase activity are being intensively investigated. To contribute to this effort, we are proposing a pilot study to assess the potential of physically linking Rubisco to another enzyme (carbonic anhydrase, CA) which, under the right conditions, could deliver a high concentration of carbon dioxide to Rubisco and reduce the wasteful oxygenase activity.

To achieve this aim we propose to reconfigure the carbon dioxide fixing step of photosynthesis by engineering the bacterium Synechocystis. We will prepare a synthetic gene which, when introduced into Synechocystis will cause it to produce a protein scaffold that can bind both Rubisco and CA. The modified strain will also contain forms of Rubisco and CA that have been engineered with tags that allow them to bind to the scaffold protein. This engineered organism will allow us to test the proposal that close proximity of Rubisco and CA increases the efficiency of photosynthesis by decreasing the "wasteful" oxygenase activity.

The oxygenase activity of the primary CO2 fixing enzyme Rubisco introduces inefficiency into photosynthesis giving rise to "wasteful" photorespiration (PR). Some plants and algae have evolved CO2 concentrating mechanisms (e.g. C4 photosynthesis, HCO3- transport) to increase [CO2]/[O2] at the Rubisco active site thus reducing PR. Because of decreased PR, C4 plants can have high productivity and require less water (stomata can be more closed) and nitrogen (less Rubisco is required). Cyanobacteria concentrate CO2 by packaging Rubisco and carbonic nanhydrase (CA) in protein microcompartments (carboxysomes). CA catalyses CO2 release from bicarbonate in the vicinity of Rubisco, thereby out-competing oxygenase. Although there has been speculation about transferring carboxysomes to plants their structural complexity makes this a formidable task. Also, introduction of C4 photosynthesis or production of Rubisco are possible approaches. We propose an alternative approach in which a protein scaffold tethers Rubisco and CA in close proximity. The scaffold is a synthetic polypeptide which contains concatenated protein-peptide interaction domains that will bind Rubisco and CA that have been tagged with the appropriate interaction peptides. This method has been used to produce a three enzyme metabolon that greatly improves local substrate concentration, resulting in greater flux through the mevalonate pathway in E. coli. Although the ultimate aim is to engineer plants, it is more convenient to carry out proof of concept in a cyanobacterium. The carboxysome-based CO2 concentrating mechanism of Synechocystis sp. PCC 6803 will be disrupted and the synthetic Rubisco/CA complex introduced. The effectiveness of the Rubisco/CA complex in reducing the oxygenase reaction will be assessed by measuring the photosynthetic characteristics of the cells. In parallel, the kinetic properties of the complex will be investigated in vitro.

This project is curiosity-driven, and as such will be of primary interest to the scientific community interested in developing strategies to improve photosynthetic efficiency in crop plants. The oxygenase activity of the primary CO2-fixing enzyme Rubisco in C3 plants introduces inefficiency into photosynthesis giving rise to "wasteful" photorespiration. Considerable research is being conducted using C4 plant biochemistry, but this approach is scientifically extremely challenging. In this 18 month project we will provide proof-of-principle information regarding an alternative and novel approach - the use of protein scaffolds to hold Rubisco and carbonic anhydrase (CA) in close proximity, thereby promoting pathway activity and in principle largely eliminating photorespiration in C3 plants. The scaffold is a synthetic polypeptide which contains concatenated protein peptide interaction domains that bind pathway enzymes (Rubisco and CA) that have been tagged with the appropriate interaction peptides. This method has been used previously for other biochemical pathways.

In principle, this approach could deliver a step change in the efficiency of photosynthesis within crop plants, thereby increasing their yield and providing enhanced food security. Introduction of a carbon dioxide concentrating mechanism into C3 crop plants would increase the efficiency of photosynthesis by eliminating photorespiration and provide the potential for increased water and nitrogen use efficiency. This pilot project is a feasibility study for a longer term programme which would address the issues of replacing Rubisco in chloroplasts with the Rubisco/CA scaffold. If successful, this approach has considerable potential for engineering traits in crops and cyanobactria that improve their growth characteristics. As a 'blue-skies' project we wish to focus our specific-user activities on management of intellectual assets, scientific advancement, skills and knowledge - the long term outcomes potentially providing a new approach to help deliver food security for our growing global population.