# Expanding the genetic code: phage-driven evolution of pyrrolysyl-synthetase for site-specific incorporation of synthetic phenylalanine and tyrosine derivatives

## TL;DR

Scientists evolved an enzyme to better incorporate synthetic amino acids into proteins, improving the efficiency and specificity of the process.

## Contribution

A polymorphic mutation cluster in the binding pocket of PylRS was identified, enabling efficient incorporation of tyrosine and phenylalanine derivatives.

## Key findings

- Evolved PylRS variants showed orders-of-magnitude increases in ncAA incorporation efficiency.
- Mutations clustered in the amino acid binding pocket correlate with improved substrate recognition.
- PANCE proved effective for engineering PylRS with desired ncAA specificity.

## Abstract

Pyrrolysyl-tRNA synthetase (PylRS) is a key enzyme for the site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. However, its native form has a limited substrate scope. This study aimed to evolve PylRS from Methanosarcina mazei to enhance recognition and incorporation of tyrosine and phenylalanine derivatives.

We used phage-assisted non-continuous evolution (PANCE) to generate a library of PylRS variants under selective pressure for the target ncAAs. Evolved variants were sequenced to identify mutations. Their aminoacylation efficiency and specificity were quantitatively assessed using fluorescence-based incorporation assays and mass spectrometry.

Sequencing revealed a polymorphic population of mutations, with a significant cluster located within the enzyme's amino acid binding pocket. Several evolved variants showed an orders-of-magnitude increase in the efficiency of incorporating their target ncAAs compared to the wild-type enzyme.

Our findings confirm that PANCE is a highly effective method for engineering PylRS variants with strong and specific activity towards desired non-canonical amino acids. The identified mutations, particularly those in the binding pocket, provide a basis for understanding and further optimising substrate specificity in synthetic biology applications.