High-Throughput Detection of Cyanobacterial Form I Rubisco Assembly

Area of Science:

• Biochemistry
• Molecular Biology
• Plant Science

Background:

• Rubisco is crucial for photosynthesis, catalyzing CO2 fixation.
• Improving Rubisco through protein engineering could enhance crop yields.
• Rubisco assembly is complex and sensitive to sequence alterations, hindering engineering efforts.

Purpose of the Study:

• To engineer a genetically encoded biosensor for detecting Form I Rubisco assembly in E. coli.
• To use the biosensor to analyze the impact of Rubisco sequence mutations on holoenzyme biogenesis.
• To assess Rubisco assembly across a large mutant library to understand evolutionary constraints.

Main Methods:

• Engineering a Rubisco assembly biosensor in E. coli.
• Utilizing the biosensor to detect cyanobacterial Rubisco ortholog assembly.
• Adapting the biosensor with phage-assisted noncontinuous selection for large-scale mutant library screening.

Main Results:

• The biosensor successfully detected RbcS-dependent assembly of cyanobacterial Rubisco and chaperone-stabilized intermediates.
• The biosensor identified assembly differences caused by RbcL sequence mutations.
• Analysis of a ~7500-member RbcL mutant library predicted that >90% of mutations negatively affect Rubisco assembly.

Conclusions:

• Rubisco biogenesis is a significant constraint on its natural evolution and protein engineering.
• The developed biosensor is a valuable tool for high-throughput analysis of Rubisco assembly and engineering.
• Understanding Rubisco assembly is critical for efforts to improve photosynthesis and crop productivity.