Grafting Rhodobacter sphaeroides with red algae Rubisco to accelerate catalysis and plant growth

Improving the carboxylation properties of Rubisco has primarily arisen from unforeseen amino acid substitutions remote from the catalytic site. The unpredictability has frustrated rational design efforts to enhance plant Rubisco towards the prized growth-enhancing carboxylation properties of red algae Griffithsia monilis Gm Rubisco. To address this, we determined the crystal structure of Gm Rubisco to 1.7 Å. Three structurally divergent domains were identified relative to the red-type bacterial Rhodobacter sphaeroides Rs Rubisco that, unlike Gm Rubisco, are expressed in Escherichia coli and plants. Kinetic comparison of 11 Rs Rubisco chimaeras revealed that incorporating C329A and A332V substitutions from Gm Rubisco Loop 6 (corresponding to plant residues 328 and 331) into Rs Rubisco increased the carboxylation rate ( k cat c ) by 60%, the carboxylation efficiency in air by 22% and the CO 2 /O 2 specificity ( S c/o ) by 7%. Plastome transformation of this Rs Rubisco Loop 6 mutant into tobacco enhanced photosynthesis and growth up to twofold over tobacco producing wild-type Rs Rubisco. Our findings demonstrate the utility of Rs Rubisco for the identification and in planta testing of amino acid grafts from algal Rubisco that can enhance the enzyme’s carboxylase potential.