Engineering the Genetic Code in Cells and Animals: Biological Considerations and Impacts.

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Genetic code expansion allows site-specific incorporation of unnatural amino acids (Uaas) into proteins.
• Orthogonal tRNA and aminoacyl-tRNA synthetase (aaRS) systems decode unique codons (e.g., UAG) for Uaa incorporation.
• This methodology has been successfully applied in various organisms, from bacteria to mammals.

Purpose of the Study:

• To address biological challenges in rewriting the genetic code.
• To describe the impact of code expansion on cell physiology.
• To discuss implications for studying genetic code evolution.

Main Methods:

• Development of a method for efficient expression of prokaryotic tRNAs in eukaryotic cells using a type-3 polymerase III promoter.
• Engineering of Uaa-specific aaRS for eukaryotic cells and animals, enhancing substrate specificity for diverse Uaas.
• Strategies to increase Uaa bioavailability, inhibit nonsense-mediated mRNA decay (NMD), and overcome release factor one (RF1) competition in bacteria.

Main Results:

• Engineered aaRSs accommodate structurally diverse Uaas, expanding stereochemical diversity.
• Inhibition of NMD enhances Uaa incorporation efficiency in yeast and C. elegans.
• Knocking out RF1 in E. coli significantly increases Uaa incorporation efficiency and enables multi-site incorporation, creating a model for directed evolution and code evolution studies.

Conclusions:

• Genetic code expansion is feasible across diverse organisms, including yeast, mammalian cells, and mice.
• RF1 knockout E. coli provides a novel model for real-time genetic code evolution studies.
• Genetically encoding Uaas in live systems opens new avenues for in vivo biology, code investigation, and synthetic biology applications.