Directed Evolution Pipeline for the Improvement of Orthogonal Translation Machinery for Genetic Code Expansion at...

Area of Science:

• Synthetic biology
• Protein engineering
• Genetic code expansion

Background:

• Expanding the genetic code with noncanonical amino acids (ncAAs) is limited by inefficient sense codon reassignment machinery.
• Directed evolution for improving sense codon reassignment is challenging due to heterogeneous protein mixtures resulting from fractional incorporation.

Purpose of the Study:

• To develop and validate a directed evolution workflow for enhancing ncAA incorporation via sense codon reassignment.
• To improve the efficiency of incorporating a ncAA in response to the arginine AGG sense codon.

Main Methods:

• Utilized a fluorescence-based screen to select for improved ncAA incorporation machinery.
• Employed directed evolution targeting the anticodon loop of Methanocaldococcus jannaschii tyrosyl-tRNA and the anticodon binding domain of its cognate aminoacyl-tRNA synthetase (aaRS).
• Transplanted beneficial mutations to an aaRS evolved for incorporating a different ncAA (para-azidophenylalanine).

Main Results:

• Identified variants of the orthogonal machinery that efficiently incorporated tyrosine in response to the AGG codon, achieving levels comparable to endogenous translation.
• Demonstrated improved ncAA incorporation using fluorescence and mass spectrometry reporters after transplanting beneficial mutations.
• Found that genomically engineered E. coli strains with reduced competing tRNAs did not outperform standard strains in this specific sense codon reassignment system.

Conclusions:

• The described directed evolution workflow is generalizable for tailoring orthogonal machinery to incorporate diverse ncAAs at targeted sense codons.
• This approach facilitates the rapid development of tools for expanding the genetic code.
• Further optimization of host strains may be necessary for maximal efficiency in specific sense codon reassignment applications.