A fully orthogonal system for protein synthesis in bacterial cells

Area of Science:

• Molecular Biology
• Synthetic Biology
• Biochemistry

Background:

• Ribosome engineering offers potential for expanding protein synthesis capabilities.
• Engineered ribosomes must be functionally isolated from native cellular translation machinery to prevent detrimental effects.
• Previous solutions like Ribo-T tethered ribosome subunits for selective protein production.

Purpose of the Study:

• To design a cell with two orthogonal protein synthesis systems.
• To utilize a dissociable ribosome for specific mRNA translation, distinct from the Ribo-T system.
• To explore ribosome engineering by creating and analyzing mutations in the peptidyl transferase center.

Main Methods:

• Development of a cell with two independent protein synthesis systems.
• Utilizing the Ribo-T system for general proteome production.
• Employing a dissociable ribosome for targeted mRNA translation.
• Systematic mutagenesis of rRNA nucleotides within the peptidyl transferase center.
• Isolation and characterization of gain-of-function ribosome variants.

Main Results:

• Successful implementation of a dual-system cell with orthogonal protein synthesis.
• Generation of a comprehensive library of peptidyl transferase center rRNA mutants.
• Identification of ribosome variants capable of overcoming translation arrest peptides.
• Demonstration of engineered ribosomes with enhanced functional properties.

Conclusions:

• The developed dual-system cell design provides functional isolation for engineered ribosomes.
• This system facilitates the exploration of ribosome function and engineering through targeted mutagenesis.
• Gain-of-function variants were isolated, expanding the capabilities of the synthetic translation machinery.
• This approach advances synthetic biology by enabling precise control over protein synthesis.