A designed bacterial microcompartment shell with tunable composition and precision cargo loading.

Area of Science:

• Synthetic biology
• Biochemistry
• Structural biology

Background:

• Bacterial microcompartments (BMCs) are protein-based organelles that enhance microbial metabolism by encapsulating enzymes.
• BMC shells are self-assembled from various protein subunits and regulate molecule transport.
• Engineering BMCs with non-native enzymes is a key goal in synthetic biology for creating efficient nanoreactors.

Purpose of the Study:

• To design and characterize a synthetic BMC shell protein with altered N- and C-terminal residue orientation.
• To enable targeted encapsulation of protein cargo within engineered BMCs.
• To develop tools for precise control over cargo loading in BMC-based systems.

Main Methods:

• Designed and determined the crystal structure of a synthetic BMC shell protein.
• Utilized genetic fusion to link protein cargo to the synthetic shell protein.
• Employed a tetracycline-inducible promoter to control cargo expression and encapsulation levels.

Main Results:

• A synthetic BMC shell protein with inverted sidedness was successfully designed and structurally characterized.
• Genetically fused protein cargo was effectively targeted and encapsulated into the lumen of the synthetic BMC shell.
• The quantity of encapsulated fluorescent protein cargo was demonstrated to be controllable via an inducible promoter.

Conclusions:

• The developed synthetic shell protein expands the toolkit for engineering BMC-based nanoreactors.
• This approach facilitates the precise targeting and controlled encapsulation of enzymes for biotechnological applications.
• The findings support the development of sophisticated multi-enzyme pathways within engineered BMCs.