Evolving Bacterial Fitness with an Expanded Genetic Code.

Area of Science:

• Synthetic Biology
• Molecular Biology
• Genetics

Background:

• The genetic code has been largely fixed with 20 proteinogenic amino acids throughout evolution.
• Orthogonal translation systems offer a way to incorporate noncanonical amino acids, expanding the genetic code.
• Existing cellular machinery is optimized for the canonical code, posing challenges for incorporating new amino acids.

Purpose of the Study:

• To create and evolve a 21 amino acid genetic code.
• To enable the specific incorporation of 3-nitro-L-tyrosine (3-N-L-Y) using amber codons.
• To assess the adaptability and fitness of cells with an expanded genetic code.

Main Methods:

• Directed evolution experiments were performed over 2000 generations.
• An orthogonal translation system was used to insert 3-nitro-L-tyrosine opposite amber codons.
• An essential beta-lactamase gene dependent on 3-N-L-Y incorporation was employed to enforce the expanded code.

Main Results:

• The fitness deficit of the initial strain was significantly repaired through acquired mutations.
• Mutations arose that reduced the toxicity associated with the noncanonical amino acid.
• Despite ambiguous coding of the amber codon, cellular fitness improvements allowed for new amber codons in protein-coding sequences.

Conclusions:

• Directed evolution can adapt cells to utilize an expanded 21 amino acid genetic code.
• The incorporation of noncanonical amino acids can be made less toxic through evolutionary adaptation.
• Expanded genetic codes show potential for increased biological complexity and functional diversity.