Towards assembling functional cyanobacterial β-carboxysomes in Oryza sativa chloroplasts

The major limiting factor of photosynthesis in C3 plants is the enzyme, rubisco which inadequately distinguishes between carbon dioxide and oxygen. To overcome catalytic deficiencies of Rubisco, cyanobacteria utilize advanced protein microcompartments, called the carboxysomes which envelopes the enzymes, Rubisco and Carbonic Anhydrase (CA). These microcompartments facilitate the diffusion of bicarbonate ions which are converted to CO₂ by CA, following in an increase in carbon flux near Rubisco boosting CO₂ fixation process. Inspired by this mechanism, our study aims to improve photosynthetic efficiency in the C₃ model crop, rice (Oryza sativa), by stably engineering the genetic components of the β-carboxysome of Synechococcus elongatus PCC 7942 (hereafter, Syn7942) in the rice genome. We demonstrated this proof of concept by developing two types of transgenic rice plants. The first type involved targeting the chloroplasts with three key carboxysome structural proteins (ccmL, ccmO, and ccmK) and a chimeric protein (ccmC), which integrates domains from four distinct carboxysome proteins. The second type combined these proteins with the introduction of cyanobacterial Rubisco targeted to chloroplasts. Additionally, in the second transgenic background, RNA interference was employed to silence the endogenous rice Rubisco along with stromal carbonic anhydrase gene. The transgenic plants exhibited the assembly of carboxysome-like compartments and aggregated proteins in the chloroplasts and the second type demonstrated reduced plant growth and yield. We have followed a bottom-up approach for targeting the cyanobacterial CCM in rice chloroplast which would help in stacking up the components further required for increasing the photosynthetic efficiency in future.