A Cyanobacterial Screening Platform for Rubisco Mutant Variants.

Area of Science:

• Biochemistry
• Enzyme Engineering
• Synthetic Biology

Background:

• Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is crucial for carbon fixation but has limitations like low activity and oxygenation.
• Enzyme engineering of Rubisco aims to improve its efficiency for enhanced carbon capture and global carbon cycling.
• Cyanobacteria, like *Synechocystis* sp. PCC 6803, serve as model organisms for studying and engineering Rubisco.

Purpose of the Study:

• To develop and validate an enzyme engineering and screening platform for Rubisco in *Synechocystis*.
• To assess the functional replacement of native Form I Rubisco with heterologous Form II Rubisco from *Gallionella*.
• To engineer improved Rubisco variants with enhanced stability and catalytic efficiency for accelerated carbon fixation.

Main Methods:

• Established an enzyme engineering platform in *Synechocystis* sp. PCC 6803.
• Replaced native Form I Rubisco with *Gallionella* Form II Rubisco and assessed growth responses to CO2 and O2.
• Utilized phylogenetically guided EV mutation and in silico evolution to create a multisite mutagenesis library.
• Employed competitive growth experiments with deep sequencing to screen Rubisco variants under varying gas conditions.

Main Results:

• Demonstrated successful replacement of native Rubisco with *Gallionella* Rubisco in *Synechocystis*, altering CO2/O2 sensitivity.
• Identified a specific amino acid substitution in *Gallionella* Rubisco that enhances thermostability.
• Observed that this amino acid exchange confers resilience against detrimental mutations.
• Developed a platform for high-throughput screening of Rubisco variants in a cyanobacterial system.

Conclusions:

• The developed platform enables efficient screening of Rubisco variants in *Synechocystis*.
• Engineering efforts successfully improved thermostability and resilience of *Gallionella* Rubisco.
• This work represents a significant step towards optimizing Rubisco for accelerated carbon fixation in cyanobacteria and potentially chloroplasts.