Robust Synthetic Biology Toolkit to Advance Carboxysome Study and Redesign

Area of Science:

• Biochemistry
• Synthetic Biology
• Microbiology

Background:

• Carboxysomes are protein organelles essential for carbon dioxide assimilation in microorganisms.
• Their modular structure offers potential for metabolic engineering, but size and complexity pose challenges.
• Efficient expression and purification systems are needed to study and engineer carboxysomes.

Purpose of the Study:

• To design and validate a plasmid set, the pXpressome toolkit, for robust expression of functional alpha-carboxysomes.
• To investigate the impact of genetic modifications on carboxysome structure and performance.
• To provide a platform for carboxysome engineering for enhanced carbon fixation and nanoencapsulation.

Main Methods:

• Development of the pXpressome toolkit for alpha-carboxysome expression.
• Introduction of mutations affecting carboxysome structure (vertex-capping and facet-forming genes).
• Purification and characterization of expressed carboxysomes, including fluorescent labeling.

Main Results:

• The pXpressome toolkit enables robust expression, intactness, and functionality of purified alpha-carboxysomes.
• Deletion of vertex-capping genes increased carboxysome size, while deletion of facet-forming genes decreased size.
• The toolkit promotes uniform expression and improved cell health compared to previous systems.

Conclusions:

• The pXpressome toolkit facilitates the study and rational redesign of carboxysomes.
• Adjusting the ratio of specific proteins can control carboxysome morphology.
• This toolkit supports efforts in enhancing carbon fixation and developing novel nanoencapsulation platforms.