High-Throughput Detection of Cyanobacterial Form I Rubisco Assembly

Rubisco catalyzes the CO₂ fixation step in the dark reactions of photosynthesis. Transgenic expression of better-performing Rubisco orthologs in plants or discovery of improved mutants of Rubisco via protein engineering could theoretically accelerate plant growth and improve crop yields. However, efforts to heterologously express or engineer Rubisco are frequently stymied by the chaperone-dependent folding and assembly of the Rubisco holoenzyme, a process that can be disrupted by changes to Rubisco's sequence. Elucidation of the effects that alterations to Rubisco's sequence impose upon its biogenesis is hampered by reliance upon low-throughput methods for verification of Rubisco assembly. Here, we report the engineering of a genetically encoded biosensor to sense the assembly of Form I Rubiscos in <i>E. coli</i>. We show that the biosensor can detect the RbcS-dependent assembly of cyanobacterial Rubisco orthologs, the formation of chaperone-stabilized RbcL oligomeric assembly intermediates, and differences in assembly caused by mutations to the RbcL sequence. Additionally, we perform a large-scale examination of the relative assembly levels of a ∼7500-member <i>Halothiobacillus neapolitanus</i> RbcL mutant library by adapting the biosensor for use with phage-assisted noncontinuous selection. Our experiment predicts that the majority (>90%) of examined RbcL mutations exert a negative effect on assembly, lending support to the hypothesis that Rubisco biogenesis constrains both its natural evolution and improvement by protein engineering.