Advanced and Safe Synthetic Microbial Chassis with Orthogonal Translation System Integration

Area of Science:

• Synthetic Biology
• Genetic Engineering
• Biochemistry

Background:

• Conventional plasmid vectors present limitations in genetic stability and scalability for synthetic biology applications.
• Precise genome engineering is essential for creating synthetic cells and orthogonalizing cellular processes like metabolism and protein translation.
• Developing safe and robust synthetic protein producers requires stable integration of novel genetic systems.

Purpose of the Study:

• To engineer a safe Escherichia coli (E. coli) chassis with an integrated orthogonal translation system (OTS) for enhanced synthetic protein production.
• To enable site-specific incorporation of non-canonical amino acids for advanced biomaterial development.
• To establish a stable and safe synthetic auxotroph with a genetic firewall.

Main Methods:

• CRISPR-assisted transposition was employed for simultaneous integration of the OTS into multiple E. coli chromosomal loci.
• Metabolic engineering, including the introduction of proline auxotrophy, was utilized to enhance bioglue expression.
• A synthetic auxotroph reliant on caged Dopa was engineered to create a genetic firewall.

Main Results:

• A stable, synthetic auxotrophic E. coli chassis with an integrated OTS was successfully created.
• Site-specific incorporation of m-oNB-Dopa was achieved through amber stop codon readthrough, enabling the expression of Dopa-Lysine motif-containing bioglues.
• Enhanced bioglue production was demonstrated by incorporating scaffold-stabilizing fluoroprolines, and a caged Dopa-dependent auxotroph was established for increased safety.

Conclusions:

• The engineered E. coli chassis provides a reliable and robust platform for synthetic protein production, overcoming limitations of traditional methods.
• The developed system facilitates the expression of advanced biomaterials, such as underwater bioglues with applications in wound healing and tissue regeneration.
• The integration of a genetic firewall significantly enhances the stability and safety of synthetic cells.