Engineering CO₂-fixing modules in Escherichia coli via efficient assembly of cyanobacterial Rubisco and carboxysomes

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) is the central enzyme for conversion of atmospheric CO₂ into organic molecules, playing a crucial role in the global carbon cycle. In cyanobacteria and some chemoautotrophs, Rubisco complexes, together with carbonic anhydrase, are enclosed within specific proteinaceous microcompartments known as carboxysomes. The polyhedral carboxysome shell ensures the dense packaging of Rubisco and creates a high-CO₂ internal environment to facilitate CO₂ fixation. Rubisco and carboxysomes have been popular targets for bioengineering, with the intent of enhancing plant photosynthesis, crop yields, and biofuel production. However, efficient generation of Form 1B Rubisco and cyanobacterial β-carboxysomes in heterologous systems remains a challenge. Here, we developed genetic systems to efficiently engineer functional cyanobacterial Form 1B Rubisco in Escherichia coli by incorporating Rubisco assembly factor Raf1 and modulating the RbcL/S stoichiometry. We then reconstituted catalytically active β-carboxysomes in E. coli with cognate Form 1B Rubisco by fine-tuning the expression levels of individual β-carboxysome components. In addition, we investigated the mechanism of Rubisco encapsulation into carboxysomes by constructing hybrid carboxysomes; this was achieved by creating a chimeric encapsulation peptide incorporating small sub-unit-like domains, which enabled the encapsulation of Form 1B Rubisco into α-carboxysome shells. Our study provides insights into the assembly mechanisms of plant-like Form 1B Rubisco and the principles of its encapsulation in both β-carboxysomes and hybrid carboxysomes, highlighting the inherent modularity of carboxysome structures. These findings lay the framework for rational design and repurposing of CO₂-fixing modules in bioengineering applications, e.g., crop engineering, biocatalyst production, and molecule delivery.