Abstract: Rubisco is the most abundant protein on earth, catalysing photosynthetic CO₂ fixation to provide all usable carbon in the biosphere. However, its slow and non-specific catalytic activity limits crop productivity and its resultant over-production represents a huge nitrogen cost. Improving the CO₂ fixing properties of Rubisco has been a longstanding bioengineering goal, hampered by the complex biogenesis and functional requirements of the enzyme.

The work presented here first outlines the development of a novel, high-resolution screening strategy for the directed evolution of Rubisco from the model photosynthetic species Nicotiana tabacum (tobacco). By utilising new site saturation variant library (SSVL) technology from TWIST Bioscience, the screening platform provides access to new evolutionary sequence space that has not been explored in previous directed evolution studies or in nature, uncovering novel single amino acid mutations that provide the largest ever improvements in the CO₂-fixing properties of plant Rubisco.

Second, the work then focuses on establishing a system for expressing and testing canola Rubisco in E. coli. This is the first ever oilseed crop Rubisco expression system and enables the discovery of new carboxylase-improved mutants by directed evolution.

Biography: I completed my undergraduate degree in Genetics in 2019 before undertaking an honours project in the Whitney Lab in 2020. My Honours project focused on providing a new evolutionary starting point for the directed evolution of plant Rubisco. My continuation in the Whitney Lab for my PhD then focused on developing a new workflow for identifying catalytically improved crop Rubisco mutants by directed evolution and expanding Rubisco bioengineering capabilities to canola, Australia’s second largest agricultural export.