Engineering posttranslational proofreading to discriminate nonstandard amino acids

Proceedings of the National Academy of Sciences of the United States of America
Aditya M KunjapurGeorge M Church

Abstract

Incorporation of nonstandard amino acids (nsAAs) leads to chemical diversification of proteins, which is an important tool for the investigation and engineering of biological processes. However, the aminoacyl-tRNA synthetases crucial for this process are polyspecific in regard to nsAAs and standard amino acids. Here, we develop a quality control system called "posttranslational proofreading" to more accurately and rapidly evaluate nsAA incorporation. We achieve this proofreading by hijacking a natural pathway of protein degradation known as the N-end rule, which regulates the lifespan of a protein based on its amino-terminal residue. We find that proteins containing certain desired N-terminal nsAAs have much longer half-lives compared with those proteins containing undesired amino acids. We use the posttranslational proofreading system to further evolve a Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) variant and a tRNATyr species for improved specificity of the nsAA biphenylalanine in vitro and in vivo. Our newly evolved biphenylalanine incorporation machinery enhances the biocontainment and growth of genetically engineered Escherichia coli strains that depend on biphenylalanine incorporation. Finally, we show t...Continue Reading

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Citations

Jan 25, 2019·Applied Microbiology and Biotechnology·Jennifer TullmanJohn P Marino
Feb 20, 2019·International Journal of Molecular Sciences·Yusuke Kato
May 12, 2019·International Journal of Molecular Sciences·Ana CrnkovićDieter Söll
Aug 28, 2019·Proceedings of the National Academy of Sciences of the United States of America·Xiaohui GaoEduardo A Groisman
Feb 6, 2020·Chembiochem : a European Journal of Chemical Biology·Jeffery M TharpDieter Söll
Feb 15, 2018·Proceedings of the National Academy of Sciences of the United States of America·Timothy M WannierGeorge M Church
Jul 31, 2018·Current Opinion in Chemical Biology·Oscar Vargas-RodriguezAna Crnković
Apr 12, 2019·Current Opinion in Biotechnology·Arlinda RezhdoJames A Van Deventer
Mar 24, 2021·Journal of the American Chemical Society·Miglena ManandharFloyd E Romesberg
Nov 12, 2020·Journal of the American Chemical Society·Lorenzo LafranchiSimon J Elsässer

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Methods Mentioned

BETA
electrophoresis
PCR
fluorescence-activated cell sorting
aminoacylation

Software Mentioned

ClpS
BipARS

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