The carboxysome is a complex, proteinaceous organelle that plays essential roles in carbon assimilation in cyanobacteria and chemoautotrophs. It comprises hundreds of protein homologs that self-assemble in space to form an icosahedral structure. Despite its significance in enhancing CO₂ fixation and potentials in bioengineering applications, the formation of carboxysomes and their structural composition, stoichiometry, and adaptation to cope with environmental changes remain unclear. Here we use live-cell single-molecule fluorescence microscopy, coupled with confocal and electron microscopy, to decipher the absolute protein stoichiometry and organizational variability of single β-carboxysomes in the model cyanobacterium Synechococcus elongatus PCC7942. We determine the physiological abundance of individual building blocks within the icosahedral carboxysome. We further find that the protein stoichiometry, diameter, localization, and mobility patterns of carboxysomes in cells depend sensitively on the microenvironmental levels of CO₂ and light intensity during cell growth, revealing cellular strategies of dynamic regulation. These findings, also applicable to other bacterial microcompartments and macromolecular self-assembling systems, advance our knowledge of the principles that mediate carboxysome formation and structural modulation. It will empower rational design and construction of entire functional metabolic factories in heterologous organisms, for example crop plants, to boost photosynthesis and agricultural productivity.

The biosynthesis and spatial positioning of β-carboxysomes, the carbon fixation machinery in cyanobacteria, are modulated by light intensity and redox state of

Significance: Cyanobacteria are keystone organisms in global carbon fixation. Their great carbon-assimilation capability arises from a specialized virus-like protein organelle, the carboxysome, which comprises hundreds of proteins that form a shell to encapsulate the CO ₂ -fixing enzymes Rubisco and carbonic anhydrase. How do these proteins self-assemble to construct the defined architecture? Here we explore the significance of one assembly factor, Raf1, in Rubisco assembly and carboxysome formation. We show that Raf1 mediates Rubisco assembly; without Raf1, carboxysome proteins are prone to form intermediate assemblies and small carboxysome-like structures rather than intact carboxysomes. Our results suggest a model of the Raf1-mediated biogenesis of carboxysomes and provide advanced knowledge of carboxysome assembly and function, informing synthetic engineering of functional CO ₂ -fixing organelles for biotechnological applications.

Rubisco is the essential enzyme mediating the fixation of atmospheric CO₂ during photosynthesis. In cyanobacteria, Rubisco enzymes are densely packed and encapsulated in a specialized organelle known as the carboxysome. Well-defined Rubisco assembly and carboxysome formation are pivotal for efficient CO₂ fixation. Numerous chaperone proteins, including RbcX, are essential for proper protein folding and Rubisco assembly. In this study, we investigated the in vivo function of RbcX in the cyanobacterium Synechococcus elongatus PCC 7942 (Syn7942) using molecular, biochemical, and live-cell fluorescence imaging approaches. Our results show that genetic deletion of the rbcX gene affects Rubisco abundance, as well as carboxysome formation and spatial distribution. Moreover, RbcX appears as one component of the carboxysome and shows a dynamic interaction with Rubisco enzymes. These in vivo observations provide insight into the role of RbcX from Syn7942 in mediating carboxysome assembly. Understanding the molecular mechanism underlying Rubisco assembly and carboxysome biogenesis will provide essential information required for engineering functional CO₂-fixing complexes in heterogeneous organisms, especially plants, with the aim of boosting photosynthesis and agricultural productivity.

Carboxysomes are the specific CO₂-fixing microcompartments in all cyanobacteria. Although it is known that the organization and subcellular localization of carboxysomes are dependent on external light conditions and are highly relevant to their functions, how carboxysome organization and function are actively orchestrated in natural diurnal cycles has remained elusive. Here, we explore the dynamic regulation of carboxysome positioning and carbon fixation in the model cyanobacterium Synechococcus elongatus PCC 7942 in response to diurnal light-dark cycles, using live-cell confocal imaging and Rubisco assays. We found that carboxysomes are prone to locate close to the central line along the short axis of the cell and exhibit a greater preference of polar distribution in the dark phase, coupled with a reduction in carbon fixation. Moreover, we show that deleting the gene encoding the circadian clock protein KaiA could lead to an increase in carboxysome numbers per cell and reduced portions of pole-located carboxysomes. Our study provides insight into the diurnal regulation of carbon fixation in cyanobacteria and the general cellular strategies of cyanobacteria living in natural habitat for environmental acclimation.