# High-Throughput Detection of Cyanobacterial Form I Rubisco Assembly

## TL;DR

Scientists developed a biosensor to study how changes in Rubisco protein affect its assembly, finding that most mutations hinder the process.

## Contribution

A genetically encoded biosensor was engineered to enable high-throughput detection of cyanobacterial Rubisco assembly in E. coli.

## Key findings

- The biosensor detects RbcS-dependent assembly of cyanobacterial Rubisco orthologs and chaperone-stabilized RbcL intermediates.
- Most RbcL mutations in a large mutant library negatively affect Rubisco assembly, constraining its evolution and engineering.
- The biosensor was adapted for phage-assisted noncontinuous selection to screen thousands of RbcL mutants efficiently.

## Abstract

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 E. coli. 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 Halothiobacillus neapolitanus 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.