Directed evolution of a plant Rubisco chaperone with altered client recognition

Area of Science:

• Plant molecular biology
• Biochemistry
• Photosynthesis research

Background:

• The enzyme Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is crucial for photosynthesis and crop productivity.
• Chaperone proteins, like Raf1, mediate Rubisco assembly but exhibit high selectivity for their native Rubisco clients.
• This selectivity limits Rubisco protein engineering and the expression of foreign Rubisco variants in plants.

Purpose of the Study:

• To investigate if a plant Rubisco chaperone can be reprogrammed via directed evolution to assemble non-native Rubisco.
• To overcome the selectivity roadblock in Rubisco engineering for enhanced photosynthesis.

Main Methods:

• Developed a high-throughput selection strategy to screen Rubisco assembly factor activity.
• Employed directed evolution to generate mutants of Arabidopsis thaliana Raf1 (AtRaf1).
• Assessed the ability of evolved AtRaf1 variants to assemble Nicotiana tabacum Rubisco.

Main Results:

• Directed evolution successfully generated AtRaf1 variants with significantly enhanced ability to assemble N. tabacum Rubisco compared to wild-type AtRaf1.
• Evolved AtRaf1 variants retained their native client assembly function.
• The reprogrammed chaperones demonstrated the ability to assemble other non-cognate dicot Rubisco orthologs.

Conclusions:

• Directed evolution is a viable strategy to reprogram plant Rubisco chaperones for non-native clients.
• This approach can overcome chaperone selectivity limitations in Rubisco engineering.
• The findings offer a potential pathway to improve plant photosynthesis and crop yields through Rubisco modification.