Examining the interactions of cyanobacterial Rubisco
Creators: name: Nguyen, Nghiem
Publisher: name: The Australian National University
Year: 2021

description: By 2050, a global food shortage is expected based on projected population growth and food availability (Long et al., 2015). A proposed solution is to genetically modify crop plants by introducing the kinetically faster cyanobacterial Rubisco components of the cyanobacterial CO2 concentrating mechanism (CCM) into crop plants, which could theoretically increase crop yields by up to 60% (Parry and Hawkesford, 2010; Parry et al., 2011; Price et al., 2013; McGrath and Long, 2014; Hanson et al., 2016; Hennacy and Jonikas, 2020). Central to this system is the carboxysome, a proteinaceous structure encapsulating Rubisco. While attempts to date at incorporating a carboxysome into tobacco chloroplasts have shown promise (Lin et al., 2014b; Long et al., 2018; Orr et al., 2020), questions remain regarding the biogenesis of these complex protein compartments and the requirements to generate correctly functional carboxysomes in plant chloroplasts. The biogenesis of beta-freshwater cyanobacteria is currently understood to be contingent upon three key interactions; the assembly Form IB Rubisco into an active holoenzyme, its interaction with a carboxysome binding partner and, its successful encapsulation. In this thesis each of these processes is examined in detail. Chapters 2 and 5 examine the roles of Rubisco chaperons RbcX and Raf1 and show that both are required for maximum Rubisco assembly in E. coli. In combination with the carboxysomal Rubisco binding protein CcmM-47, correctly folded Rubisco holoenzymes form insoluble complexes which phase-separate from the bulk medium to form dynamic condensates with quantifiable interaction kinetics (Chapter 4). The kinetics of this interaction were found to be dependent upon salt, temperature and oxidation state (Chapter 4) and provide a novel understanding of the first steps in beta-carboxysome biogenesis. To resolve the Rubisco:CcmM interaction, conserved residues in the S. elongatus PCC7942 CcmM Rubisco small subunit-like (SSUL) domain were mutated and the phenotypic response at air was observed and presented in Chapter 3. Conserved CcmM cysteine residues aided efficient carboxysome assembly, however, conserved CcmM arginine residues were essential for the Rubisco:CcmM interaction. This in vivo analysis of the Rubisco:CcmM interaction was complemented by further work published by Wang et al., (2019b, Appendix 3) which elucidates key electrostatic attractions that drive the Rubisco:CcmM interaction. Using conditions which lead to maximum Rubisco holoenzyme formation (Chapters 2-4), the compelling possibility that fast Form1B Rubisco enzymes could be incorporated into easily formed alpha carboxysomes is addressed in Chapter 5. Rubisco, The representative Form IB beta-carboxysomal Rubisco from T. elongatus BP-1 was found to interact with the Cyanobium PCC7001 alpha-carboxysome binding partner protein, CsoS2, and be imperfectly encapsulated within rod-like carboxysome structures. However, abnormal internal substructure within these simple carboxysome suggests that other protein-protein interactions within carboxysomes must be resolved for the successful construction of a hybrid carboxysome which might be used in future chloroplastic CCM bio-engineering designs. Taken together, the results presented in this thesis provide an overall view of the interaction processes involved in Rubisco folding, assembly and incorporation into phase-separated bodies which establish the basis of carboxysome assembly. The results presented here highlight that a greater understanding of Rubiscos protein-interaction surfaces, involved in a myriad of protein interactions with chaperones, activases and condensate binding partners, will assist future attempts to incorporate the complete and functional carboxysome into the chloroplasts of crop plants, leading to not only important gains in yield, but find-tuned maximization of potential outputs.