Abstract
The carboxysome is a versatile paradigm of prokaryotic organelles and is a proteinaceous self-assembling microcompartment that plays essential roles in carbon fixation in all cyanobacteria and some chemoautotrophs. The carboxysome encapsulates the central CO2-fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), using a polyhedral protein shell that is selectively permeable to specific metabolites in favor of Rubisco carboxylation. There is tremendous interest in repurposing carboxysomes to boost carbon fixation in heterologous organisms. Here, we develop the design and engineering of α-carboxysomes by coexpressing the Rubisco activase components CbbQ and CbbO with α-carboxysomes in Escherichia coli. Our results show that CbbQ and CbbO could assemble into the reconstituted α-carboxysome as intrinsic components. Incorporation of both CbbQ and CbbO within the carboxysome promotes activation of Rubisco and enhances the CO2-fixation activities of recombinant carboxysomes. We also show that the structural composition of these carboxysomes could be modified in different expression systems, representing the plasticity of the carboxysome architecture. In translational terms, our study informs strategies for engineering and modulating carboxysomes in diverse biotechnological applications.
Highlights
Cells exploit the physical and chemical nature of molecules to generate self-assembling supramolecular complexes, membrane domains, and organelles, which provides a means for segregating specific functions into different subcellular regions to modulate metabolic reactions in space and in time.[1,2] While the emergence of compartmentalization and confinement in the cell is widely accepted as a key event in the evolution of eukaryotic cells, more recent work has documented that compartmentalization is ubiquitous in prokaryotes
Previous studies have shown that expressing the H. neapolitanus α-carboxysome cso operon could lead to the formation of catalytically functional α-carboxysome structures in E. coli[20,21,26,41] and a Gram-positive bacterium.[22]
Stoichiometric plasticity has been recently assessed as a general feature of natural and recombinant bacterial microcompartment (BMC), including βcarboxysomes,[13,14,46] the propanediol utilization metabolosome,[47] and several recombinant shell structures.[46,48,49]
Summary
Cells exploit the physical and chemical nature of molecules to generate self-assembling supramolecular complexes, membrane domains, and organelles, which provides a means for segregating specific functions into different subcellular regions to modulate metabolic reactions in space and in time.[1,2] While the emergence of compartmentalization and confinement in the cell is widely accepted as a key event in the evolution of eukaryotic cells, more recent work has documented that compartmentalization is ubiquitous in prokaryotes. A versatile paradigm is the bacterial microcompartment (BMC). That encapsulates diverse metabolic enzymes within the nanoscale compartments using a polyhedral protein shell.[3−9]. BMCs are widespread in the bacterial phyla and are of paramount importance microbial ecology.[10−12] for CO2 fixation, pathogenesis, and Carboxysomes are the canonical BMCs found in all cyanobacteria and some chemoautotrophs. Carboxysomes encapsulate the key CO2-fixing enzymes ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) and carbonic anhydrase (CA), using a protein shell made of numerous protein paralogs (Figure 1a).[8,13,14] Rubisco is the central enzyme in the Calvin−Benson−Bassham cycle of photosynthesis, mediating CO2 fixation by catalyzing the carboxylation of its substrate ribulose-1,5-bisphosphate (RuBP).
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