title: Engineering Plant Rubisco
Creators: name: Rhodes, Timothy
Publisher: name: The Australian National University
Year: 2025

description: Rubisco, the primary enzyme in the Calvin cycle responsible for atmospheric CO2 fixation, plays a crucial role in the global carbon cycle and plant productivity. However, its catalytic efficiency is hindered by slow turnover rates and less-than-optimal specificity between carboxylase and oxygenase reactions. These limitations have driven efforts to enhance Rubisco kinetics through genetic engineering, focusing on two main strategies: leveraging the natural diversity of Rubisco kinetics from various plants, often associated with Carbon Concentrating Mechanisms (CCM), and advancing laboratory-based protein engineering, especially notable with the success of expressing plant Rubisco in Escherichia coli. Research has predominantly involved model species like tobacco and Arabidopsis, constrained by the challenges of expressing Rubiscos from other plants. This thesis aimed to deepen understanding of plant Rubisco kinetic diversity and develop new synthetic biology tools and E. coli expression systems to enhance Rubisco catalysis. Investigations involved studying the temperature kinetics of Rubisco from six plant species exhibiting the Crassulacean Acid Metabolism (CAM) photosynthetic CCM (Chapter 3) and trialling a new directed evolution method for engineering tobacco Rubisco in E. coli that integrated nanopore-assisted mutation scanning to explore new evolutionary paths for catalytic enhancement (Chapter 4). Further development led to a system for mutagenic testing of maize Rubisco, analyzing multiple plant Rubisco E. coli expression systems and evolving the chaperonin protein folding cages from chloroplasts (Chapter 5). Rubisco activities were measured, including CO2-fixing speed and CO2/O2 specificity at six temperature intervals from 10 to 37C. The Rubiscos from Portulaca oleracea and Mesembryanthemum crystallinum, primarily undertaking C4 and C3 photosynthesis respectively, showed faster rates across temperatures. Challenges persisted in producing CAM Rubisco in E. coli due to limitations in expressing Rubisco assembly machinery from tobacco chloroplast chaperones, hindering mutagenic validation tests. Further kinetic characterization and directed evolution tests focused on using a catalytically impaired tobacco Rubisco mutant to explore novel evolutionary trajectories. A 'new-to-nature' pathway in plant Rubisco evolution was pursued by combining a tobacco chloroplast compatible Rubisco-dependent E. coli screen and Nanopore-assisted mutation scanning (NaMS). This approach effectively identified nine single amino acid substitutions that enhanced activity, illustrating the potential of NaMS for rapid and sensitive mutant detection. The final chapter discusses the ongoing challenge of expressing maize and wheat L8S8 Rubisco in E. coli. Using a dual-plasmid expression system, individual substitutions of each tobacco RAP with a wheat homolog differentially impacted Rubisco production, with heterologous chaperonin replacement mostly impacting L8S8 biogenesis. Directed evolution on maize chaperonin identified a mutation that improved maize Rubisco production, and was used for mutational testing of a mutant maize Rubisco. In summary, this thesis highlights the critical strategies for plant Rubisco engineering. Natural kinetic variability offers examples of improved Rubisco, yet current expression systems remain inadequate. The sensitivity and simplicity of NaMS provide a more reliable approach for mutant detection in directed evolution screens. Additionally, modifications to the RAP components in these screens offer new opportunities for understanding sequence complementarity requirements with Rubisco and accessing new evolutionary spaces in plant Rubisco sequence within E. coli, now including maize. These advancements aim to deepen our understanding of Rubisco functionality and pave the way for significant enhancements in Rubisco function and the photosynthetic efficiency of key agricultural crops.