Exploring novel in planta and in vitro approaches for bioengineering Rubisco

The CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) represents the major point of carbon entry into the biosphere. Rubisco has a slow catalytic rate and poorly discriminates between substrate CO2 and O2. Recycling the undesired product of O2-fixation consumes energy and releases fixed CO2. To maintain adequate levels of carbon assimilation, higher plants invest 30% of leaf nitrogen into Rubisco and require larger stomatal apertures, increasing water loss via transpiration. Rubisco catalysis can be light-limited, particularly in lower canopy leaves. Poor substrate specificity, slow catalysis and associated limitations to nitrogen-, water- and radiation-use efficiency (NUE, WUE, RUE) mean that Rubisco catalysis often limits plant growth. Rubisco is thus a major direct (Rubisco catalysis) and indirect (e.g. RUE) engineering target to improve photosynthetic carbon assimilation and yield potential. Engineering higher plant L8S8 Rubisco, comprised of eight large- (LSu, plastome-synthesised) and eight small- (SSu, cytosol-synthesised) subunits, is complicated by disparate subunit synthesis locations and the reliance on many ancillary proteins for correct folding/assembly. Investigated in this study is the feasibility of several complementary Rubisco engineering strategies to improve the yield potential of higher plants. Regulated expression of catalytically desirable Rubisco isoforms in alternative parts of the plant canopy could improve photosynthetic carbon assimilation. This study established the feasibility of stably expressing two kinetically divergent Rubisco isoforms within the same tobacco chloroplast. Improved yield potential from "dual Rubisco" expression will rely on harnessing natural catalytic diversity, identifying evolutionary constraints on Rubisco evolution, overcoming chaperone incompatibilities and differentially controlling Rubisco expression. The kinetics of certain non-green algal Rubisco isoforms surpass those of higher plants, but tobacco chloroplasts cannot satisfy their folding/assembly requirements. This thesis demonstrated that insoluble red-type Rubisco subunits only accumulate in metabolically competent plants and algal Rubisco assembly limitations do not extend to the bacterial Rubisco isoform from Rhodobacter sphaeroides whose catalysis is altered by substituting in the Rubisco SSu from algal species. The R. sphaeroides Rubisco backbone will be utilised for phylogenetic grafting of catalysis-enhancing elements from related algal Rubiscos to directly improve Rubisco catalysis. The assembly complexity of algal Rubiscos in tobacco chloroplasts extends to higher plant Rubiscos. This thesis utilised chimeric engineering strategies to identify regions of wheat Rubisco LSu that impede assembly with tobacco SSus into L8S8 complexes and demonstrated that, similar to previously identified residues, codon 371 in the tobacco LSu influences Rubisco biogenesis and catalysis consistent with biogenesis- and performance-debilitating residue changes constraining Rubisco's evolutionarily potential to select for functional improvements. Chaperone incompatibilities stymie folding/assembly of many Rubisco isoforms in heterologous expression hosts. This thesis demonstrates that cyanobacteria and higher plant RbcX (a cyanobacteria Rubisco-specific chaperone) homologs bind tobacco LSu forming stable complexes comprising RbcX and the tobacco LSu and SSu in planta. Addition of soluble tobacco SSu and a synthetic peptide to these complexes in vitro mildly stimulated Rubisco activity. Further refinement may yield a reliable high throughput approach for in vitro reconstitution of higher plant Rubisco. The experimental undertakings of this thesis provide vital insights into the future challenges for bioengineering Rubisco in leaf chloroplasts to modulate plant photosynthesis and growth