Selection of Cyanobacterial (Synechococcus sp. Strain PCC 6301) RubisCO Variants with Improved Functional Properties That Confer Enhanced CO2-Dependent Growth of Rhodobacter capsulatus, a Photosynthetic Bacterium

Abstract

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a ubiquitous enzyme that catalyzes the conversion of atmospheric CO₂ 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 O₂ 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 CO₂-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 CO₂ and O₂ 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 CO₂ conversion to food, fuel, and other organic compounds required to sustain life. Because RubisCO-dependent CO₂ fixation is severely compromised by oxygen inhibition and other physiological constraints, improving RubisCO's kinetic properties to enhance growth in the presence of atmospheric O₂ 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.

Keywords: RubisCO; carbon dioxide fixation; directed evolution; enzyme engineering; selection.

FIG 1

Hydrophobic region adjacent to the active site in the X-ray crystal structure of activated Synechococcus form I RubisCO (yellow; PDB ID 1RBL). Relevant residues are shown in stick representation and labeled. The transition state analog carboxyarabinitol-1,5-bisphosphate (CABP) is colored gray, and the active-site residues are colored black. Gray dotted lines represent van der Waals interactions between the active-site residues and CABP. Residues Ala³⁷⁵, Thr³²⁷, Phe³⁹¹, and Leu³⁹⁷ (red) are within 4 Å of each other and were targeted for directed mutagenesis. Residues in this region that were identified via suppressor selection are colored green. For better clarity, the terminal atoms are colored based on electronegativities (oxygen, red; nitrogen, blue, phosphorus, orange; sulfur, yellow).

FIG 2

Growth phenotypes of R. capsulatus wild type (strain SB1003) and the RubisCO deletion mutant strains that had been complemented with wild type (WT) or site-directed mutants (labeled with respective residue substitutions) of Synechococcus RubisCO. Mixotrophic growth was assessed on rich (peptone-yeast extract) medium supplemented with tetracycline (to select for plasmid-complemented strains) and a gas mixture comprising 5% CO₂ and 95% H₂. CO₂-dependent growth was assessed on minimal medium supplemented with a gas mixture comprising either 5% CO₂ and 95% H₂ gas mixture (photoautotrophy) or 5% CO₂, 45% H₂, and 50% air (chemoautotrophy). The RubisCO deletion mutant strain that had been complemented with an empty plasmid was used as a negative control (“-ve ctrl”).

FIG 3

Distribution of residues identified via suppressor selection (green) in the holoenzyme structure of Synechococcus form I RubisCO (PDB ID 1RBL). (A) Residues that were targeted for mutagenesis (red) are shown in one of the large subunits (yellow). Two neighboring small subunits (blue) come in contact with a large subunit in the holoenzyme. Some residues are labeled to illustrate the position of the respective regions relative to the active site. (B) The boxed region in panel A showing interactions involving Val¹⁸⁶ (green) is enlarged and shown with a few additional residues (yellow sticks) that are within 4 Å of the side chain of Val¹⁸⁶. Active-site residues (black) in this region and the transition state analog CABP (light gray) are displayed in both panels. Terminal atoms in CABP and the amino acid side chains are colored based on electronegativities (oxygen, red; nitrogen, blue; phosphorus, orange; sulfur, yellow).

FIG 4

Growth responses of suppressor mutants in liquid cultures placed under anoxic (A and C) or oxic (B and D) CO₂-dependent autotrophic growth conditions. Strain names indicate the mutant substitutions encoded by the large (rbcL) or small (rbcS) subunit genes of Synechococcus form I RubisCO. Large subunit mutant substitutions are separated by a shill, and the substitutions following a double shill are in the small subunit. Each curve was plotted with mean absorbance values measured from triplicate cultures, and the error bars represent the standard deviations for each data point. Data are representative of several independent growth experiments.

FIG 5

Position of residue Met²⁵⁹ in the X-ray crystal structure of Synechococcus form I RubisCO (PDB ID 1RBL). (A) The side chain of Met²⁵⁹ (red sticks) is at the interface of large (yellow/gray) and small (blue/gray) subunits, lining the central solvent channel in the holoenzyme. Its position in one of the large subunits (yellow) is indicated with an arrow. The position of CABP, the transition state analog (shown as spheres), which is present in all eight active sites, is labeled in one of them (yellow). (B) A closeup view of the region surrounding Met²⁵⁹, showing hydrophobic interactions (within 4 Å) with other nonpolar residues (yellow; stick representation).