Effects of chaperone selectivity on the assembly of plant Rubisco orthologs in <i>E. coli</i>

Engineering improved Rubisco has the potential for enhancing photosynthesis in crops, but this endeavor faces challenges from the dependence of plant Rubisco biogenesis on selective assembly factors. Here we examine the ability of different assembly factors to assemble plant Rubiscos in a recently developed Escherichia coli (E. coli) expression system with high potential utility for plant Rubisco engineering. We tested combinations of Cpn60α/Cpn60β/Cpn20 chaperonins and Raf1/Raf2/RbcX/BSD2 chaperones from different plant species for the ability to assemble different plant Rubisco orthologs. We observe that Raf1 is a source of selectivity that recognizes Rubisco from phylogenetically related species but not more distant species. We show that Raf1 compatibility alone can enable assembly of several dicot Rubisco orthologs with non-cognate dicot assembly factors. By analyzing the Raf1-Rubisco interface, we identify the RbcL βC-βD loop as a selective recognition site for Raf1. Finally, we demonstrate that assembly of a more phylogenetically divergent Rubisco from the monocot Oryza sativa requires a greater number of assembly factors to be of monocot origin. Our results contribute insights into the species-specific interactions between Rubisco and its assembly factors, and provide a strategy for assembling different plant Rubiscos, including several from monocot species, in the E. coli expression system.