ORIGINAL RESEARCH article
Front. Mol. Biosci.
Sec. Protein Biochemistry for Basic and Applied Sciences
Expanding the genetic code: phage-driven evolution of pyrrolysyl-synthetase for site-specific incorporation of synthetic phenylalanine and tyrosine derivatives
1. Biotechnology department, Sirius University, Sochi, Russia
2. Scientific Center of Genetics and Life Sciences, Sirius University, Sochi, Russia
Select one of your emails
You have multiple emails registered with Frontiers:
Notify me on publication
Please enter your email address:
If you already have an account, please login
You don't have a Frontiers account ? You can register here
Abstract
Pyrrolizyl-tRNA synthetase (PylRS) is frequently used for the site-specific incorporation of non-canonical amino acids (ncAA) into proteins. Native PylRS structures have a limited ability to incorporate non-proteinogenic amino acids. The activity of PylRS is mediated by specific amino acids within the active site, therefore precise changes in the protein sequence can increase the efficiency of aminoacylation. In this study, we used phage-assisted non-continuous evolution (PANCE) to evolve PylRS from Methanosarcina mazei into a variant that selectively recognises tyrosine and phenylalanine derivatives. Sequencing of the evolved variants revealed a polymorphic population of mutations, many of which were located within the amino acid binding pocket. We examined the efficiency of inclusion of the mutants towards the target amino acids using fluorescence and mass spectrometry analyses. We identified a number of mutations that improved the efficiency of incorporation of the corresponding ncAAs by orders of magnitude compared to non-mutant clones. Thus, these results confirm that PANCE is an effective method for designing PylRS variants with strong substrate specificity for target non-canonical amino acids.
Summary
Keywords
Directed Evolution, Noncanonical amino acid, PANCE, PylRS, Pyrrolysyl-tRNA synthetase, tRNA
Received
02 November 2025
Accepted
09 January 2026
Copyright
© 2026 Anastasia, Miasoutova, Iliushin, Rozanov and Ivanov. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Dakhnevich Anastasia; Roman Ivanov
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.