Designing Stacked Assembly of Type III Rubisco for CO2 Fixation with Higher Efficiency

The slow catalytic rate of the carboxylation enzyme d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a major barrier to increasing the rate of carbon assimilation from the atmosphere into the biosphere. It is of great importance to establish a method to improve the carboxylation efficiency of Rubisco. Inspired by the assembly of Rubisco in carboxysomes, herein, we presented a rational protein engineering approach for the construction of one-dimensional (1D) protein arrays of type III Rubisco through designed π-π stacking interactions by using crystal structural information as a guide. In aqueous solutions, the dimensions of these 1D protein arrays collectively span nearly the entire nano- and micrometer scale (200 nm to 5.0 μm) by adjusting protein and NaCl concentrations. As a result, the stacked Rubisco assemblies increase by 40% in the carboxylase activity, while their turnover number (kcat) is around twofold larger than that of wild-type III Rubisco. Notably, upon heat treatment at temperature up to 75 °C for 30 min, most of the assembled nanostructures and the enzyme activity are retained. More importantly, the initial relative activity of stacked assemblies retained 91% after 10 cycles of reuse. This work provides a simple, effective solution for the improvement of the CO2 carboxylation efficiency of Rubisco.