pro2neo-RUBISCO · Resurrecting, re-evolving and re-inventing Rubisco: From the evolutionary past of Earth’s No. 1 CO2-fixing enzyme to its synthetic future

Can we re-construct the engine of natural CO2-fixation? Rubisco catalyzes the key reaction in photosynthesis: the capture and conversion of atmospheric CO2 into biomass. However, despite billions of years of evolution the enzyme is (still) constrained by a trade-off between CO2-fixation activity and CO2-specificity, which limits photosynthetic carbon capture, and thus directly agricultural yield. In pro2neo-RUBISCO, we will overcome this fundamental limitation of photosynthetic CO2-fixation with a completely fresh approach. We will use an evolutionary-synthetic biology strategy to study the evolutionary history of Rubisco, derive a molecular understanding for the enzyme’s mechanism, and develop highly efficient, new-to-nature solutions that we will validate in a real-world context: the living chloroplast. We will use ancestral reconstruction to replay crucial steps in the evolutionary history of the enzyme. We will resurrect the very first scaffolds that just learned to fix CO2 (“proto-Rubiscos”), and study Rubisco ancestors that started to loosely interact with an accessory small subunit. We will re-evolve these ancestral enzymes and provide them with artificial small subunits to open up new evolutionary paths leading to improved variants. These (r)evolutionary studies will be complemented by synthetic biology efforts: We will liberate Rubisco from its entropic constraints by separating the complex multi-step reaction onto different proteins that work in tandem, and go even one step further by re-inventing the enzyme’s scaffold, finding a new home for the Rubisco reaction and create a truly ”neo-Rubisco”.Compared to many efforts in the field, pro2neo-RUBISCO does not screen for existing Rubiscos with improved kinetics or improving existing Rubiscos. In pro2neo-RUBISCO, we aim at (re)exploring the history and evolutionary landscape of Rubisco, and delivering completely novel architectures for the key driver of the global carbon cycle.