22BBSRC-NSF/BIO: A synthetic pyrenoid to guide the engineering of enhanced crops

Meeting future global food demands will require novel approaches for creating higher-yielding crops that are robust in the face of climate change. Synthetic and engineering biology approaches have huge potential to deliver on this challenge. A major opportunity for increasing the yields and resilience of major global crops such as rice and wheat lies in enhancing their ability to assimilate CO2, from which plants make sugars and starch for growth. We propose to enhance CO2 assimilation in crops by endowing them with a specialised cellular compartment called the pyrenoid that has naturally evolved in eukaryotic algae and some lower land plants but is not present in crops. Here, as a key step towards this goal, we will advance our basic understanding of the principles that underlie the assembly and architecture of pyrenoids and will leverage this understanding to build a functional synthetic pyrenoid-based CO2-concentrating mechanism into the model land plant Arabidopsis.

The project has three aims, each of which combines wet-lab based experimentation on synthetic pyrenoids in test tubes and complementary model-based analyses to push forward the engineering efforts in plants. The project builds on the combined outputs of an outstanding international team with a strong track record of collaboration in advancing both the knowledge of pyrenoid biology and the ability to engineer algal components into land plants. The collaboration has previously identified and characterised key pyrenoid components, gleaned fundamental insights into how the pyrenoid is assembled, generated the first computational model to describe how a functional pyrenoid-based CO2-concentrating mechanism works, and successfully assembled a prototype pyrenoid in Arabidopsis. This project will leverage this knowledge to generate a step-change in our basic understanding of an algal mechanism that is of ecological and biogeochemical importance and will significantly advance our ability to engineer improved plant growth.

In this project we will use in vitro reconstitution and modelling to guide and rapidly accelerate the engineering of a functional pyrenoid-based CO2-concentrating mechanism (CCM) into the model C3 plant Arabidopsis. One of the key growth bottlenecks in C3 crop plants is the slow rate of photosynthetic CO2 capture by the CO2-fixing enzyme Rubisco. Pyrenoid-based CCMs overcome this shortcoming by condensing Rubisco into a spherical compartment called the pyrenoid, wherein Rubisco is fed with a high concentration of CO2 to maximise carboxylation rates and minimise the competing oxygenase activity of Rubisco. Pyrenoids in the green alga Chlamydomonas reinhardtii are traversed by modified photosynthetic thylakoid membranes known as pyrenoid tubules, which are understood 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 channels in the thylakoid membranes bordering the pyrenoid.

Our labs have recently discovered the identities of the key components required for Rubisco matrix assembly, tubule biogenesis, and the supply of CO2 to Rubisco. Together, these findings will allow us to advance our basic understanding of the principles that underlie the pyrenoid-based CCM, build a cell-free minimal pyrenoid-based CCM, test a functional pyrenoid-based CCM in Arabidopsis, and lay the groundwork for a high-efficiency pyrenoid supported by a CO2 diffusion barrier. The project has three experimental aims each supported by computational modelling (see Objectives). Our efforts to reconstitute a pyrenoid assembly in vitro and to engineer a plant-based pyrenoid will push the boundaries of plant engineering biology and will advance our fundamental understanding of the principles that underpin the functioning of pyrenoid-based CCMs.