Replacement of large subunit N terminus enabled biogenesis of different plant Rubiscos in <i>E. coli</i>

The efforts of engineering plant ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) with the goal of improving plant photosynthetic efficiency and crop yield have existed for long. However, the directed evolution of plant Rubisco has not been widely explored because its biogenesis in a heterologous host such as Escherichia coli remains challenging. Recent breakthroughs in developing the Arabidopsis five-auxiliary-chaperone package and optimizing the chaperone origins have enabled the functional assembly of several plant Rubisco large subunits with their native or other plant small subunits in E. coli. But tedious and unpredictable optimization of chaperone origins might still be required for the assembly of another plant Rubisco. Here, we identified several residues at the N terminus of the large subunit that were critical for Rubisco assembly in E. coli by comparative sequential and structural analysis of cyanobacterial and plant Rubiscos. These residues in cyanobacterial Rubisco showed intensive molecular interactions with other residues within this and neighbouring large subunits. The replacement of these residues of plant Rubisco by their cyanobacterial counterparts, in combination with co-expression of the six auxiliary chaperones, enabled/improved the assembly of Rubiscos from Flaveria bidentis, Spinacia oleracea, Nicotiana tabacum and Arabidopsis thaliana in E. coli. These chimeric plant Rubiscos exhibited similar carboxylation kinetics as their native enzyme, indicating they can serve as a starting point for molecular engineering to identify those activity-improving amino acid substitutions. This work may facilitate the development of a universal biogenesis platform for plant Rubiscos, where only some N-terminal residues of a plant Rubisco are replaced by the cyanobacterial ones, whereas no complex chaperone optimization is needed.