Selection of Cyanobacterial ( Synechococcus sp. Strain PCC 6301) RubisCO Variants with Improved Functional Properties That Confer Enhanced CO 2 -Dependent Growth of Rhodobacter capsulatus, a Photosynthetic Bacterium

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a ubiquitous enzyme that catalyzes the conversion of atmospheric CO 2 into organic carbon in primary producers. All naturally occurring RubisCOs have low catalytic turnover rates and are inhibited by oxygen. Evolutionary adaptations of the enzyme and its host organisms to changing atmospheric oxygen concentrations provide an impetus to artificially evolve RubisCO variants under unnatural selective conditions. A RubisCO deletion strain of the nonsulfur purple photosynthetic bacterium Rhodobacter capsulatus was previously used as a heterologous host for directed evolution and suppressor selection studies that led to the identification of a conserved hydrophobic region near the active site where amino acid substitutions selectively impacted the enzyme's sensitivity to O 2 In this study, structural alignments, mutagenesis, suppressor selection, and growth complementation with R. capsulatus under anoxic or oxygenic conditions were used to analyze the importance of semiconserved residues in this region of Synechococcus RubisCO. RubisCO mutant substitutions were identified that provided superior CO 2 -dependent growth capabilities relative to the wild-type enzyme. Kinetic analyses of the mutant enzymes indicated that enhanced growth performance was traceable to differential interactions of the enzymes with CO 2 and O 2 Effective residue substitutions also appeared to be localized to two other conserved hydrophobic regions of the holoenzyme. Structural comparisons and similarities indicated that regions identified in this study may be targeted for improvement in RubisCOs from other sources, including crop plants. IMPORTANCE RubisCO catalysis has a significant impact on mitigating greenhouse gas accumulation and CO 2 conversion to food, fuel, and other organic compounds required to sustain life. Because RubisCO-dependent CO 2 fixation is severely compromised by oxygen inhibition and other physiological constraints, improving RubisCO's kinetic properties to enhance growth in the presence of atmospheric O 2 levels has been a longstanding goal. In this study, RubisCO variants with superior structure-functional properties were selected which resulted in enhanced growth of an autotrophic host organism ( R. capsulatus ), indicating that RubisCO function was indeed growth limiting. It is evident from these results that genetically engineered RubisCO with kinetically enhanced properties can positively impact growth rates in primary producers.