Hybrid Rubisco with Complete Replacement of Rice Rubisco Small Subunits by Sorghum Counterparts Confers C4 Plant-like High Catalytic Activity

Photosynthetic rate at the present atmospheric condition is limited by the CO2 -fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) because of its extremely low catalytic rate (k(cat)) and poor affinity for CO2 (K-c) and specificity for CO2 (S-c(/o)). Rubisco in C-4 plants generally shows higher k(cat) than that in C-3 plants. Rubisco consists of eight large subunits and eight small subunits (RbcS). Previously, the chimeric incorporation of sorghum C-4-type RbcS significantly increased the k(cat) of Rubisco in a C-3 plant, rice. In this study, we knocked out rice RbcS multigene family using the CRISPR-Cas9 technology and completely replaced rice RbcS with sorghum RbcS in rice Rubisco. Obtained hybrid Rubisco showed almost C-4 plant-like catalytic properties, i.e., higher k(cat), higher K-c, and lower S-c/0. Transgenic lines expressing the hybrid Rubisco accumulated reduced levels of Rubisco, whereas they showed slightly but significantly higher photosynthetic capacity and similar biomass production under high CO2 condition compared with wild-type rice. High-resolution crystal structural analysis of the wild-type Rubisco and hybrid Rubisco revealed the structural differences around the central pore of Rubisco and the beta C-beta D hairpin in RbcS. We propose that such differences, particularly in the beta C-beta D hairpin, may impact the flexibility of Rubisco catalytic site and change its catalytic properties.