EAGER: Leveraging Chaperones to Escape the Plant RuBisCO Catalytic Catch-22

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Photosynthetic organisms use carbon dioxide from the atmosphere to produce sugar and biomass that support all other life. The enzyme RuBisCO is responsible for this chemical capture of carbon dioxide. Unfortunately, it carries out this process only very slowly – to the extent that carbon dioxide capture often limits plant growth. Moreover, RuBisCO regularly and mistakenly reacts with oxygen instead of carbon dioxide, wasting energy and further limiting plant growth. An improved Rubisco, if translated into agriculture, would lead to a significant increase in crop yields, improved food security, and better climate resilience, and improving Rubisco function has long been viewed as an agricultural technology holy grail. With the potential value of such an enzyme in mind, researchers have asked whether it is possible to substantially improve these areas of RuBisCO function, since natural evolution has failed to do so thus far. One widely touted possibility is that RuBisCO is caught in an evolutionary trap and cannot be improved. An alternative view is that subjecting the enzyme to new-to-nature types of selection pressures and environments would enable evolutionary innovations that actually can unlock a better RuBisCO. The goal of this project is to test the latter idea, by integrating fundamental biophysical concepts with state-of-the-art techniques for laboratory evolution. The proposed research will also help to train the next generation of scientists and engineers by involving high school interns each summer in the work. Molecular chaperones interact with and assist other proteins (clients) in the cell and, in doing so, can profoundly influence the evolutionary trajectories accessible to their clients. In plants, RuBisCO requires nearly a dozen of these molecular chaperones to assist its production, regulation, and maintenance. How do the sequences and activities of these chaperones impact the evolution of plant RuBisCO, and are there ways to optimize the chaperones to open new regions of sequence space for RuBisCO? By applying laboratory evolution to RuBisCO and its chaperones in a microbial host, this project will seek to answer these questions. Next-generation mutagenesis technologies and novel screening systems will be used to achieve two key goals: (1) Demonstrating high-throughput in vivo directed evolution of plant RuBisCO and its chaperones and (2) Testing the idea that co-evolution of RuBisCO and its chaperones can lead to better evolutionary outcomes and enhanced enzymatic properties relative to evolving just RuBisCO alone.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.