Chemical Evolution of a Bacterial Proteome

Area of Science:

• Synthetic Biology
• Microbial Evolution
• Genetic Code Engineering

Background:

• The genetic code relies on a standard set of 20 amino acids.
• Incorporating noncanonical amino acids into proteins is a challenge in synthetic biology.
• Escherichia coli is a model organism for genetic and metabolic engineering.

Purpose of the Study:

• To evolve Escherichia coli to utilize a synthetic amino acid, L-β-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa), as a replacement for L-tryptophan (Trp).
• To demonstrate the feasibility of altering the amino acid composition of a living organism's proteome.
• To explore methods for creating synthetic cells with alternative biochemical building blocks.

Main Methods:

• Long-term cultivation of Escherichia coli in defined synthetic media.
• Directed evolution to select for cells incorporating [3,2]Tpa.
• Genetic analysis to confirm Tryptophan (Trp) to [3,2]Tpa substitutions in the proteome.
• Growth assays in the absence of Trp.

Main Results:

• Evolved E. coli cultures demonstrated robust growth using [3,2]Tpa as the sole surrogate for Trp.
• Successful substitution of Trp with [3,2]Tpa occurred at all 20,899 TGG codons in the E. coli W3110 genome.
• The evolved bacteria exhibited a new-to-nature amino acid composition.
• The engineered cells grew effectively in a complete absence of Trp.

Conclusions:

• It is possible to evolve microorganisms to incorporate synthetic amino acids into their proteomes, replacing essential canonical amino acids.
• This study provides a proof-of-concept for engineering alternative biochemical building blocks in cells.
• The approach offers a pathway for developing synthetic cells with fundamentally altered biological properties.