Laboratory evolution of Rubisco solubility and catalytic switches to enhance plant productivity

A new Escherichia coli laboratory evolution screen for detecting plant ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) mutations with enhanced CO2-fixation capacity has identified substitutions that can enhance plant productivity. Selected were a large subunit catalytic (Met-116-Leu) mutation that increases the kcatc of varying plant Rubiscos by 25% to 40% and a solubility (Ala-242-Val) mutation that improves plant Rubisco biogenesis in E. coli 2- to 10-fold. Plastome transformation of either mutation into the tobacco plastome rbcL gene had no impact on leaf Rubisco production, photosynthesis or plant growth. However, tobacco transformed with low-abundance hybrid Arabidopsis Rubisco coding M116L improved plant exponential growth rate by ~75% relative to unmutated hybrid enzyme, with the A242V substitution increasing both hybrid Rubisco production and plant growth by ~50%. Our identification of mutations with the potential to enhance plant growth bodes well for broadening the survey of Rubisco sequence space for catalytic switches that can impart more substantive plant productivity improvements. This study demonstrates the power of directed evolution to unlock latent functional potential in plant Rubisco. By identifying mutations that enhance CO2 fixation and solubility, it advances avenues for improving crop photosynthesis and productivity.