Fig. 3.

Expression of carboxysome genes without other CCM components does not improve the growth of an engineered rubisco-dependent E. coli in any CO₂ level tested. We recently reconstituted a functional H. neapolitanus α-carboxysome CCM in a rubisco-dependent E. coli strain, CCMB1, by expressing 20 CCM genes from 2 plasmids (2). Here, we generated plasmid variants to test whether carboxysome expression improves rubisco-dependent growth in any of the four CO₂ partial pressures during growth in a gas-controlled plate reader (Methods). Each diamond gives the end point optical density (600 nm) after 4 d of cultivation for one technical replicate of four biological replicates. The CCMB1 strain grows in elevated CO₂ (1.5 and 5%) when rubisco is expressed (“Rubisco Alone”, Left). As previously reported, expressing the full complement of CCM genes from the pCB’ and pCCM’ plasmids (“Full CCM”) enabled growth in all CO₂ levels. By replacing pCCM’ with a vector control and making an inactivating mutation to the carboxysomal CA (CsosCA C173S), we were able to express rubisco in a carboxysome without CA or Ci transport activities (“Encap. Rub.”). This strain grew similarly to the reference “Rubisco Alone” in all conditions. When the CA active site was left intact (“En. Rub.+CA”), growth improved above the “Rubisco Alone” baseline in 0.5% and 1.5% CO₂. A negative control strain carrying inactive rubisco (“En. Rub.⁻”, CbbL K194M) failed to grow in all CO₂ conditions. (B) Focusing on the growth in 1.5% CO₂ highlights the contribution of CA activity to rubisco-dependent growth. See SI Appendix, Tables S4 and S5 for description of strains and plasmids, and see SI Appendix, Figs. S4 and S5 for growth curves and analysis of statistical significance.