Improving Oryza sativa Ribulose-biphosphate carboxylase (Rubisco) Kinetics using a novel directed evolution approach

Abstract

To decrease land sacrifice to agriculture and prevent a sixth mass extinction, increasing yields in agricultural crops is essential. Photosynthetic assimilation of CO2 by green plants starts with the carboxylation of ribulose-1,5-biphosphate catalysed by the holoenzyme Rubisco. On average, Rubisco is slow and suffers from a low specificity producing glycolate in an oxygenation reaction. Rubisco consist of two subunits: RbcL (large), where the catalytic site is retained, and RbcS (small). Improvement of RbcL has yielded promising results with increases of specificity towards CO2 and increased Kcat. Despite indications for a significant role of RbcS in holoenzyme catalysis and stability, improvement of Rubisco small subunit in plants has been lagging over the past two decades. Here, we present a high-throughput technique to test Rubisco variants in a directed evolution screen using Rubisco-dependent E. coli strains. We explore the Oryza sativa RbcS protein sequence bioinformatically to determine residues of interest involved in holoenzyme structure and functioning. These residues will be randomly mutated and variant libraries are then screened in Rubisco Dependent E. coli strains explicitly designed to assemble rice Rubisco. Improved variants are studied to quantify active sites per sample and carboxylation efficiency after which these variants are tested in vivo in rice plants. Lastly, rice plants are selected for improved phenotypes with increased biomass production and decreased oxygenation rates. These plants could potentially display a larger 4 percent increase in yield and, with the addition of other traits such as water usage, this surge could increase further.