BBSRC-NSF/BIO: Engineering an algal pyrenoid into higher plants to enhance yields

Global food demand is projected to double by 2050. To meet this demand while minimizing ecological damage, new agricultural solutions are needed that allow production of significantly more food from the same amount of land. A major opportunity for enhancing the yields of major global crops such as rice and wheat lies in enhancing their ability to take up CO2 by photosynthesis, from which they make sugar. Here, as a key step towards enhancing crop productivity, we propose to enhance CO2 uptake of the model plant Arabidopsis by engineering it with a minimal synthetic CO2 uptake mechanism with components of the pyrenoid from green algae. Pyrenoids enhance CO2 fixation in nearly all eukaryotic algae on the planet and play a key role in the global carbon cycle. The project consists of three aims that are each targeted at engineering one of the key components of the pyrenoid into Arabidopsis, and one computational aim that will develop a quantitative model of pyrenoid function to support the other three aims. These four aims will synergize to produce a minimal, functional pyrenoid in Arabidopsis. To support our plant engineering efforts, we will perform targeted research in algae and in vitro. The project benefits from an outstanding international team with a strong track record of collaboration in advancing both our basic knowledge of the pyrenoid and our ability to engineer algal components into higher plants. The collaboration has recently yielded key insights into the principles underlying pyrenoid structure and biogenesis, and has made significant preliminary advances in expressing algal components in higher plants. The project will use synthetic biology-based approaches to contribute to our basic understanding of an algal mechanism that is of ecological and biogeochemical importance, and will advance our ability to improve plant growth using advanced engineering strategies. If we succeed in enhancing CO2 uptake in Arabidopsis, our work will lay the foundations for significant increases in global crop yields, and will contribute to meeting the 2050 global food demand with minimal ecological impacts.

This project aims to enhance the growth of the model C3 plant Arabidopsis by introducing a minimal CO2-concentrating mechanism (CCM) based on the pyrenoid of the green alga Chlamydomonas reinhardtii. CCMs enhance growth by delivering a high concentration of CO2 to the primary carboxylation enzyme Rubisco, which increases the catalytic rate of CO2 uptake by Rubisco and suppresses photorespiration. The algal CCM enhances CO2 uptake in nearly all eukaryotic algae and works by actively pumping CO2 into the pyrenoid matrix, a dense aggregate of Rubisco. Pyrenoids are traversed by modified photosynthetic thylakoid membranes, which are called pyrenoid tubules. These pyrenoid tubules are thought to deliver concentrated CO2 to Rubisco through the activity of a specialised carbonic anhydrase that leverages the low pH inside the thylakoid lumen to convert bicarbonate into CO2. The bicarbonate enters the tubules from the surrounding stroma via bicarbonate transporters in the thylakoid.

Together our labs have recently discovered the core components required for correct pyrenoid function and we aim to engineer these into Arabidopsis with the aim to reconstitute a functional synthetic pyrenoid. The project consists of three experimental aims and one computational modelling aim, with each aim supported by targeted experiments in Chlamydomonas (see details in Objectives). We aim to i) reconstitute Rubisco aggregation by expressing the algal protein EPYC1, which links Rubisco to form the pyrenoid matrix, ii) cluster the matrix around thylakoid membranes containing bicarbonate transporters by expressing two membrane proteins that bind Rubisco, and iii) express and localise a carbonic anhydrase to the thylakoid membranes that traverse the pyrenoid matrix. Our efforts to engineer a pyrenoid into Arabidopsis will push the boundaries of plant synthetic biology and will advance our fundamental understanding of the principles that underpin the functioning of the algal CCM.

Who will benefit from this research? In the short to medium term, the fundamental aspects of the research will benefit academics and researchers in all fields of plant research based in the UK, US and internationally. Particularly, this work will be of interest to researchers focused on enhancing food security by improving photosynthesis, including members of the international RIPE and C4 Rice Project consortia. It will also be of considerable interest to metabolic engineers and metabolic modellers. Medium to long term benefiters will include members of the agro-industry including biotechnologists and plant breeders seeking to increase plant productivity and/or harvest index, along with Multinational and Government Agencies who can use results to strategize future funding in areas of high potential impact (e.g. GCRF). During the research, the PDRAs and undergraduates working on the project will benefit considerably from training. The general public will also benefit from planned outreach activities.

How will they benefit from the research? Academics and researchers will receive comprehensive new information about the CCM of algae, requirements for CO2 concentration in higher plant chloroplasts, and mechanisms of assembly of supra-molecular complexes. Researchers will have access at the point of publication to new Chlamydomonas and plant expression constructs and lines, novel Arabidopsis material with altered primary carbon assimilation and models describing the relationship between the spatial distributions of inorganic carbon substrates and enzymes and the process of CO2 assimilation in algae. Agro-industry will receive information to underpin rational approaches to increase plant productivity, and relevant new genes and modelling methodologies. The agricultural community will benefit in the longer term from sustainable crop improvements enabled by our research. The PDRAs will receive a wide range of training in molecular biology, plant physiology, synthetic biology, and professional skills, with opportunities to attend training courses and opportunities to interact closely with researchers on an international scale. They will also receive training in transferable skills such as presentation and dissemination of results, and grant-writing. Our research findings relate to issues of public interest including sustainable crop production, global food security and atmospheric and climate change. The research also has wide educational value, at all levels through schools and Universities.

How will we ensure they benefit from the research? We will publish results in high-impact journals in a timely fashion with open access. We will present research results at international meetings and institutions and use social media and lab websites to promote new findings ahead of and during publication. We will submit materials, data and models to relevant international depositories. We will exploit extensive existing contacts of the PIs with other academics with relevant research interests as soon as any exploitable results/materials are generated. We will make informal contacts with biotechnologists as soon as exploitable results are generated; recognise and protect IP to ensure wise and fruitful exploitation. Collectively, we have vibrant contacts with relevant industries. We will provide mentoring to ensure uptake of PDRA training schemes, including regular progress reviews and career development plans, participation in the dissemination of results, and understanding of the wider implications and applications of the research. Results will be used as part of our regular engagement with non-academic audiences, e.g. local interest groups, schools, local and national science showcases, media. We will involve undergraduate students by providing laboratory summer secondments and discussing our research in teaching material. We will seek opportunities to inform the work of charitable bodies and governmental agencies.