Understanding carboxysomes to enhance carbon fixation in crops

Abstract

Carboxysomes are bacterial microcompartments that enhance photosynthetic CO2 fixation by encapsulating ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) within a high-CO2 environment. Their modular, self-assembling nature makes them attractive for synthetic biology applications, particularly their transplantation alongside functional bicarbonate (HCO3-) transporters into plant chloroplasts to achieve improved photosynthetic efficiency. Recent advances have deepened our understanding of carboxysome biogenesis, Rubisco organisation and shell function. However, key questions remain, including the precise shell mechanistic action, which is critical for functional integration into new hosts. Addressing these questions, as well as identifying suitable bicarbonate transporters and fine-tuning expression levels, will be essential to utilising carboxysomes and the cyanobacterial CO2-concentrating mechanism for enhanced photosynthetic efficiency in crops.

Keywords: Rubisco; carbon fixation; cyanobacteria; photosynthesis; synthetic biology.

Figure 1:. Schematic representation of cyanobacterial CO₂-concentrating mechanism (CCM) in both α- and β-cyanobacteria.

(A) Membrane-bound bicarbonate (HCO₃ ^-) transporters (coloured in light blue) facilitate accumulation of HCO₃ ^- within the cyanobacterial cytosol. This accumulated HCO₃ ^- subsequently diffuses into specialised microcompartments known as carboxysomes. In α-cyanobacteria, α-carboxysomes (pink) are typically smaller and more numerous than those found in β-cyanobacteria (green) [23]. (B) Despite structural differences between α- and β-carboxysomes, both serve similar functional roles. Across both carboxysome types, HCO₃ ^- and ribulose-1,5-bisphosphate (RuBP) must pass through the selectively permeable carboxysome protein shell. Within the carboxysome, the carbonic anhydrase (CA) enzyme (yellow/green CsoSCA in α-carboxysomes; and blue CcaA/CcmM in β-carboxysomes) interconverts HCO₃ ^- and CO₂, raising the internal CO₂ concentration for rapid fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Here, the α-carboxysome Rubisco is shown in red, pink and blue. The β-carboxysome Rubisco is shown in green and yellow. The resulting product, 3-phosophoglycerate (PGA) exits the carboxysome for conversion into sugars within the Calvin cycle [5].

Figure 2:. Schematic of the α- and β-carboxysome biogenesis pathways.

Both α- and β-carboxysomes encapsulate a shared core of proteins within their lumen: ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), carbonic anhydrase (CA), the Rubisco-binding partner (CsoS2 in α-carboxysomes and CcmM in β-carboxysomes). Form IA Rubisco (found in α-carboxysomes; top) is shown in red and pink; Form IB Rubisco (found in β-carboxysomes; bottom) in green and yellow. The α-carboxysomal CA is depicted as a green and yellow hexameric unit, while the β-carboxysomal CA is shown as a blue hexameric unit. The α-carboxysome Rubisco-binding partner (CsoS2) is represented as a continuous ribbon of yellow, green, and blue; the β-carboxysomal binding partner (CcmM) appears as a red line with yellow globular domains. (A) α-carboxysome biogenesis involves the concomitant assembly of Rubisco condensates (formed between Rubisco, CsoS2 and CA, alongside shell assembly (hexameric shell proteins are coloured grey, while trimeric shell proteins are coloured blue and pentameric shell proteins are coloured pink) to produce intact carboxysomes. (B) For β-carboxysomes, this condensate formation appears to be a key step prior to shell protein recruitment (a so-called ‘inside-out’ formation pathway, hexameric shell proteins are coloured grey, while trimeric shell proteins are coloured blue and pentameric shell proteins are coloured pink).

Figure 3:. Schematic of the proposed mechanistic action of the carboxysome shell.

The carboxysome face is composed of hexameric shell proteins (coloured dark grey and light blue) and pentameric shell proteins (coloured in brown), with pentamers occupying the vertices. The carboxysome shell protein encapsulates ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), carbonic anhydrase (CA) and Rubisco activase. Central pores in the shell proteins are thought to facilitate the selective transport of small molecules, with the hexameric pores implicated in the movement of bicarbonate (HCO₃ ^-), CO₂ and O₂. Notably, gaps between shell proteins have the capacity to allow small molecular passage [36,39,40,101]. Protons are expected to have easy passage across the shell [102,103] and play a significant role in modulating carboxysome function (not shown [10,104]). The mechanisms by which the Rubisco reaction products phosphoglycerate (PGA) and 2-phosphoglycolate (2 PG) traverse the carboxysome shell remains unknown. Additionally, key metabolites such as ribulose-1,5-bisphosphate (RuBP), adenosine triphosphate (ATP) and adenosine diphosphate (ADP) which are required by Rubisco and Rubisco activase, must cross the carboxysome shell but the transport routes for these substrates require further elucidation. To date, there is no evidence that there is selective discrimination of CO₂ and O₂ at the shell, while modelling suggests the elevation of CO₂ within the carboxysome by CA action is sufficient to reduce Rubisco oxygenation [10]. Coupled shell proteins may act as gated pores, allowing shuttling of larger molecules in and out of the carboxysome [97,105,106].