Encapsulation of multiple cargo proteins within recombinant Eut nanocompartments

Area of Science:

• Synthetic biology
• Biochemistry
• Protein engineering

Background:

• Spatial organization of enzymes in nanocompartments can enhance multienzyme cascade efficiency.
• Previous work demonstrated single cargo protein encapsulation in Escherichia coli using Salmonella enterica LT2 ethanolamine utilization bacterial microcompartment (BMC) shell proteins.
• Understanding the encapsulation mechanism is crucial for optimizing systems to target multiple cargo proteins.

Purpose of the Study:

• To elucidate the mechanism of cargo encapsulation within recombinant bacterial microcompartments (BMCs).
• To identify the interactions between signal sequences and BMC shell proteins for efficient cargo targeting.
• To provide a foundation for engineering BMCs for colocalizing multiple enzymes in synthetic biology.

Main Methods:

• Coexpression of Salmonella enterica LT2 ethanolamine utilization (Eut) BMC shell proteins (EutS, EutSMNLK) with signal sequence-cargo protein fusions (EutC1-19).
• Investigating the interaction of signal sequence EutC1-19 with Eut BMC shell proteins using biochemical assays.
• Analyzing the interaction of a second signal sequence, EutE1-21, with EutS shell protein.

Main Results:

• The signal sequence EutC1-19 targets cargo to the BMC interior via hydrophobic interaction with a helix on the EutS shell protein.
• EutC1-19 does not interact with other Eut BMC shell proteins (EutMNLK).
• A second signal sequence, EutE1-21, also interacts specifically with the same helix on EutS, indicating competition for binding.
• Simultaneous colocalization of two cargo proteins is achieved through competition for the EutS binding site.

Conclusions:

• This study provides the first insights into signal sequence-shell protein interactions governing cargo sequestration within Eut BMCs.
• The findings reveal a mechanism for dual cargo targeting mediated by specific helix interactions on the EutS protein.
• This work lays the groundwork for engineering Eut BMCs as a platform for multienzyme cascade applications in synthetic biology.