Rubisco catalyzes the fixation of CO2 and is the primary site of entry for carbon into the biosphere. The enzyme is slow and subject to competitive inhibition by oxygen. The oxygenation reaction increases with temperature, reducing the efficiency of photosynthesis, and will become increasingly problematic with global warming. Rubisco is therefore a key target for improving crop productivity. Despite decades of research, Rubisco engineering efforts have seen limited success. This led to proposals of strong tradeoffs between Rubisco kinetic parameters due to inherent catalytic constraints. However, recent phylogenetic analysis has called this assumption into question. This proposal aims to take a fresh approach in probing how Rubisco evolvability and catalytic parameters are influenced by protein sequence, enzyme stability and oligomeric state. This will be achieved by using advances in artificial intelligence to transplant the Rubisco catalytic site into novel protein backbone structures inspired by diverse Rubisco’s from nature. Using characterized Form I’, II and III Rubisco sequences from Candidatus Promineofilum breve, Rhodospirillum rubrum and Thermococcus kodakarensis as starting points, novel sequences will be designed with altered structures. Solubility and activity of 25 sequences will be tested at EMSL using a cell free expression pipeline. A subset of 5 of the most active sequences will be used to complement a Rubisco deficient strain of the green alga Chlamydomonas reinhardtii, to provide a basis for future directed evolution experiments to explore the potential to vary catalytic parameters. In depth biochemical analysis will be performed on selected variants to determine key parameters including rate of carboxylation (KcatC) and specificity for CO2 or O2 (Sc/o). Finally, cryo-electron microscopy will be performed at EMSL to provide insight into the relationship between protein structure and function. Understanding these relationships has the potential the inform strain improvement efforts for photosynthetic biomass production with importance for energy security and bioproduction. The work is supported by PI Burgess’ start-up funds, preliminary designs have been made and there are graduate students to conduct de novo design and directed evolution work.