High-Throughput Aminoacyl-tRNA Synthetase Engineering for Genetic Code Expansion in Yeast

Area of Science:

• Synthetic Biology
• Biochemistry
• Molecular Biology

Background:

• Genetic code expansion using noncanonical amino acids (ncAAs) is limited by the availability of orthogonal translation systems (OTSs).
• Evolved aminoacyl-tRNA synthetases (aaRSs) from bacteria are not always orthogonal in eukaryotes, hindering ncAA incorporation.
• Efficient genetic code expansion requires robust OTSs for repurposed stop codons.

Purpose of the Study:

• To develop and apply a yeast display-based reporter platform for high-throughput screening of aaRS libraries.
• To discover novel aaRS variants for ncAA incorporation in yeast, including those for previously unencoded ncAAs.
• To engineer aaRSs with improved performance, enhanced selectivity, and broader polyspecificity for diverse ncAAs.

Main Methods:

• Utilized a yeast display platform coupled with fluorescence-activated cell sorting for screening aaRS libraries.
• Screened for aaRSs capable of incorporating new ncAAs, improving existing aaRS performance, and achieving high selectivity or polyspecificity.
• Applied high-throughput screening to identify functional aaRS variants for genetic code expansion in yeast.

Main Results:

• More than doubled the number of translationally active aaRSs available for genetic code manipulation in yeast.
• Identified numerous previously undiscovered aaRS variants with diverse functionalities.
• Demonstrated the ability to engineer OTSs for specific or polyspecific incorporation of structurally diverse ncAAs.

Conclusions:

• The developed screening platform significantly expands the toolkit for genetic code expansion in yeast.
• The newly discovered aaRS variants enhance the potential for protein engineering, chemical biology, and synthetic biology.
• Understanding aaRS evolvability is crucial for advancing genetic code manipulation technologies.