Redesigning phosphoenolpyruvate carboxylase for improved catalysis in C4 photosynthesis
Creators: name: Leith, Rowarne
Publisher: name: The Australian National University
Year: 2023

description: C4 photosynthesis, a carbon concentrating mechanism, evolved as an adaptation to improve photosynthetic CO2 assimilation in terrestrial plants under conditions of low CO2, increased temperatures and varying rainfall patterns. Understanding the C4 photosynthetic cycle could prove invaluable when trying to engineer C3 crop species with better photosynthetic rates or to improve the photosynthetic rates of C4 crop species. Phosphoenolpyruvate carboxylase (PEPC), the primary carboxylase of the C4 photosynthetic cycle, operates without any oxygenase activity, and at catalytic rates 3-8 times that of Rubisco. PEPC is responsible for catalysing the irreversible beta-carboxylation of PEP by HCO3- to form oxaloacetate (OAA), which is then converted to a 4-carbon carboxylic acid. The identity of this 4C carboxylic acid is dependent on the C4 photosynthetic subtype to which the plant belongs. Regardless of C4 subtype, these organic acids contribute to the cytosolic mesophyll environment, where PEPC is located, and are important in driving the carbon flux to the BSC. In this thesis numerous properties of the C4 PEPC enzyme have been examined and this research has provided valuable insights into avenues for redesigning PEPC for improved catalysis in C4 photosynthesis. Firstly, a method of PEPC expression and purification using E. coli as an expression system was established and optimised. Establishing and optimising this method showed that PEPC purified from E. coli purifies as a dimer and raised questions about whether the tetrameric form of the PEPC enzyme is more prone to dissociation then often presented in the literature. A C4 subtype-specific sequence that confers differences in PEPC kinetic properties was identified. This sequence, although not directly involved in allosteric regulator binding, has been shown to affect inhibition of PEPC activity by malate. Purification of C4 PEPCs and chimera PEPCs expressed in E. coli, have allowed for a direct comparison between the kinetic properties of different PEPC enzymes and the effect of this C4 subtype-specific sequence. As well as insights into the function of this subtype-specific region, this work also has shown that the U. Panicoides PEPC enzyme has superior catalytic properties, with Vmax and kcat values far greater than any of those reported in the literature for PEPC enzymes from C3 or C4 plants. This thesis has also shown that oxaloacetate might be a more important C4 PEPC inhibitor, and aspartate a less important inhibitor, than reported in the literature. Lastly, an attempt was made to overexpress PEPC in the model plant C4 grass, Setaria viridis and to investigate the effects of expressing a malate-insensitive PEPC enzyme in Setaria viridis. Although the PEPC overexpression experiments were ultimately unsuccessful due to range of unforeseen factors including cross reactivity of the AcV5 antibody in the MEO34V S. viridis ecotype, this research has provided valuable information on how to redesign this experiment for future PEPC overexpression studies. The attempts to investigate the expressing a malate-insensitive PEPC enzyme in Setaria viridis were also unsuccessful for the same reasons. Given the recent success of the CRISPR/Cas9 technology in many plant species, however, revisiting this experiment using the CRISPR/Cas9 technology will allow for a more sophisticated approach. The overall results of this thesis have shown that there is still more to learn about the regulatory properties of the C4 PEPC enzyme. This thesis has also highlighted that there are C4 PEPC enzymes with superior catalytic properties available for transformation into plants. Therefore, increasing photosynthesis through PEPC modification is a prime opportunity and will allow plants to function more efficiently in an increasingly hot, dry climate.