Bilateral NSF/BIO-BBSRC Synthesis of Microcompartments in Plants for Enhanced Carbon Fixation

Global demand for food and fuel is steadily increasing, while gains in yield of many major food crops through traditional breeding have leveled off in recent years. The natural variation that has been the source of substantial crop improvement is becoming exhausted, so that new efforts, including input from synthetic biology will be needed to improve photosynthetic efficiency. More than 90% of biomass is derived directly from photosynthetic products. The properties of the carbon-fixing enzyme Rubisco (ribulose-1:5-bisphosphate carboxylase/oxygenase) limit the efficiency of photosynthesis in land plants. Rubisco can catalyze the combination of RuBP (ribulose-1,5-bisphophate) with CO2, but also can catalyze the reaction of RuBP with oxygen, leading to photorespiration, a process in which previously fixed CO2 is lost. Cyanobacteria and some land plants have evolved to deal with an increase of oxygen in the atmosphere by developing mechanisms that concentrate CO2 near Rubisco. However, many of the globally important crop plants lack this ability; instead, they utilise Rubisco enzymes that have higher CO2 affinity but are slower than Rubisco enzymes in plants with carbon-concentrating mechanisms such as maize. Consequently, these plants must devote considerable amounts of protein, and thereby, nitrogen, to allow Rubisco to carry out adequate amounts of carbon fixation, reducing yield and biomass production.

One of the outstanding millenial goals is to find ways to improve photosynthetic yields for enhanced biomass production. Replacing endogenous Rubisco with a faster enzyme with less CO2 specificity, along with a carbon concentrating mechanism, is one way to significantly improve CO2 fixation, according to published computational models. We propose to undertake work to this end. We will install a novel cyanobacterial-based carbon-concentrating mechanism in a land plant chloroplast and provide the necessary molecular machinery to facilitate its operation. Regulatory modules will be produced to express components of the cyanobacterial carboxysome from the chloroplast genome and chloroplast membrane-targeted bicarbonate pumps from the nuclear genome. As a proof of principle, this work will be carried out in tobacco, a species in which chloroplast transformants can be most rapidly obtained. We have already established the ground work for this engineering feat, demonstrating that several of the components can be introduced; for example, to generate novel microcompartments within the tobacco chloroplast.

We expect the knowledge gained from the project will inform subsequent efforts to enhance photosynthesis in other species, for example by introducing synthetic microcompartments into species such as soybean, in which technology is already available for chloroplast and nuclear transformation. Knowledge will be gained through biochemical and microscopic studies, for example about the effect of stoichiometry of cyanobacterial proteins on microcompartment size, morphology, and function in carbon concentration and photosynthesis. We will examine features of the gene regulatory sequences on the synthetic chloroplast operons needed to express proteins in the amounts and ratios needed for assembly of microcompartments. We expect the findings to be valuable also for future projects and additions of other capabilities to plants by synthesis of artificial microcompartments.

Experimental data and modeling indicate that increasing photosynthesis increases crop yields, provided that other constraints do not become limiting. One approach to enhancing the efficiency of photosynthesis is to introduce carbon-concentrating mechanism (CCM), which increases the CO2 substrate available for assimilation by Rubisco (ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase) and suppresses a competing reaction with O2. The oxygenation of RuBP is also catalysed by Rubisco and initiates photorespiration, with the consequent loss of fixed carbon, consumption of energy and release of nitrogen. CCMs limit photorespiration by altering the proportion of the two gaseous substrates, CO2 and O2, in the vicinity of Rubisco. While CCMs have evolved in a variety of organisms, including certain crop species such as maize, they are lacking in many globally important, so-called C3 crop species such as soybean and wheat. We propose to apply the methods of synthetic biology to engineer a CCM derived from cyanobacteria into the chloroplast in a model C3 plant, tobacco, with potential for future application to crop plants. The cyanobacterial Beta-carboxysome will be constructed in the tobacco chloroplast through expression of component proteins from the chloroplast genome. We will optimize chloroplast vectors for modular cloning that will allow rapid design of new operons to introduce carboxysomal proteins in the proper ratio for assembly of the microcompartment. Our efforts to adjust protein levels will be guided by assays of transgene expression, electron and fluorescence microscopy, and immunolocalization. Incorporating the complete system into chloroplasts will also require introduction of a nuclear-encoded bicarbonate transporter and genetic removal of carbonic anhydrase from the chloroplast stroma. Promising transgenic lines will be assayed for carbon assimilation, stomatal conductance, Rubisco amount and activity, and growth rate and biomass accumulation.

This 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 to develop a new micro-compartment 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 not only improve carbon capture by plants but also for other applications (e.g. packaging bioactive molecules). 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 attached Pathways to Impact document.