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

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.

Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction

“…Prior RDE-directed evolution studies have mistakenly reported Se RbcL amino acid substitutions which improve the folding and assembly (solubility) of Se Rubisco as mutations that enhance catalysis. As summarized in Table 1, the commonly selected M262T Se RbcL substitution initially reported to enhance catalysis [36] was subsequently shown to enhance Se Rubisco solubility in E. coli [29] and may also account for the same mutation enhancing fitness in R. capsulatus selection [28]. Similarly the Se RbcL F140L and V189I mutations were initially characterized a catalytic enhancers of RDE fitness [27] but subsequently shown to improve Se Rubisco solubility by nearly 5-fold in E. coli [22].…”

“…As in any directed protein evolution experiment, the level of success is dependent on the fidelity and throughput of the selection system employed. For evolving Rubisco, the use of the photosynthetic R. capsulitis bacterial screen is hampered by low throughput (screening is limited to a few thousand mutants) and hindered in its ability to characterize mutants with alterations in solubility [28,31,43,44]. As summarized in Figure 1, while RDE screens have higher throughput, their fidelity can be compromised by false positives.…”

Highly active rubiscos discovered by systematic interrogation of natural sequence diversity

“…their alternative biological function may have allowed for alternative evolutionary adaptation in terms of their CO 2 and O 2 fixation properties. It would be interesting to test whether other heterologous systems and/or alternative codon-usage approaches enable the biochemical characterization of this diverse group (Mignon et al, 2018;Satagopan et al, 2019).…”

Recent Advances in Developing Artificial Autotrophic Microorganism for Reinforcing CO2 Fixation

“…Moreover, remarkable kinetic enhancement was not obtained, likely due to an improper library screening platform, such as the host. In addition to model heterotrophs, Rhodobacter capsulatus has been used as an autotrophic host for directed evolution and rational design of Rubisco (Satagopan et al, 2019). In the R. capsulatus Rubisco deletion strain, complementation of a foreign Rubisco gene allowed the selection of substitutions.…”