Part:BBa_K5052311

Carbonic Anhydrase (CA)

About

Carbonic anhydrase (CA) in the carboxysomes of cyanobacteria catalyzes the dehydration of HCO₃^- to CO₂ ¹. Carbonic anhydrase is a critical component of the carbon concentrating mechanism, ensuring that the carboxysome remains saturated with CO₂ for RubisCO to use as a substrate to react with ribulose 1,5-bisphosphate to produce two molecules of 3-phosphoglycerate, a crucial intermediate in the Calvin-Benson cycle. We sought to improve the fitness of our modified strains to be suitable for use in a wide range of environments from intense light to intense temperatures, and to do this we chose to modify carbonic anhydrase. Carbonic anhydrase in N. vulgaris is thermally robust and is being explored as an extracellular addition to carbon capture and sequestration systems that seek to remove CO₂ from flue gas ².

In order to ensure localization of N. vulgaris CA to the carboxysomes of our cyanobacterial strains, we identified the localization sequence of CcaA in Synechococcus elongatus sp. PCC 7942 to the carboxysome. The last 17 residues on CcaA associates with the scaffolding protein CcmM58 and get assembled into the carboxysome ³. These 17 residues were then attached to the C-terminus of N. vulgaris carbonic anhydrase via a linker sequence to act as a localization tag that will allow N. vulgaris CA to be incorporated into the carboxysomes of the cell during carboxysome assembly.

Once localized in the carboxysome, the thermal stability of N. vulgaris carbonic anhydrase provides the cell the ability to convert HCO₃^- into CO₂ at a wider range of temperatures and therefore the ability to continue to metabolize under more extreme conditions.

References

(1) Badger, M. R.; Price, G. D. CO2 Concentrating Mechanisms in Cyanobacteria: Molecular Components, Their Diversity and Evolution. J. Exp. Bot. 2003, 54 (383), 609–622. doi.org/10.1093/jxb/erg076.

(2) Alvizo, O.; Nguyen, L. J.; Savile, C. K.; Bresson, J. A.; Lakhapatri, S. L.; Solis, E. O. P.; Fox, R. J.; Broering, J. M.; Benoit, M. R.; Zimmerman, S. A.; Novick, S. J.; Liang, J.; Lalonde, J. J. Directed Evolution of an Ultrastable Carbonic Anhydrase for Highly Efficient Carbon Capture from Flue Gas. Proc. Natl. Acad. Sci. 2014, 111 (46), 16436–16441. doi.org/10.1073/pnas.1411461111.

(3) Zang, K.; Wang, H.; Hartl, F. U.; Hayer-Hartl, M. Scaffolding Protein CcmM Directs Multiprotein Phase Separation in β-Carboxysome Biogenesis. Nat. Struct. Mol. Biol. 2021, 28 (11), 909–922. doi.org/10.1038/s41594-021-00676-5.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]