Engineered Aminoacyl-tRNA Synthetases with Improved Selectivity toward Noncanonical Amino Acids.

A wide range of noncanonical amino acids (ncAAs) can be incorporated into proteins in living cells by using engineered aminoacyl-tRNA synthetase/tRNA pairs. However, most engineered tRNA synthetases are polyspecific; that is, they can recognize multiple rather than one ncAA. Polyspecificity of engineered tRNA synthetases imposes a limit to the use of genetic code expansion because it prevents specific incorporation of a desired ncAA when multiple ncAAs are present in the growth media. In this study, we employed directed evolution to improve substrate selectivity of polyspecific tRNA synthetases by developing substrate-selective readouts for flow-cytometry-based screening with the simultaneous presence of multiple ncAAs. We applied this method to improve the selectivity of two commonly used tRNA synthetases, p-cyano-l-phenylalanyl aminoacyl-tRNA synthetase ( pCNFRS) and Nε-acetyl-lysyl aminoacyl-tRNA synthetase (AcKRS), with broad specificity. Evolved pCNFRS and AcKRS variants exhibit significantly improved selectivity for ncAAs p-azido-l-phenylalanine ( pAzF) and m-iodo-l-phenylalanine ( mIF), respectively. To demonstrate the utility of our approach, we used the newly evolved tRNA synthetase variant to produce highly pure proteins containing the ncAA mIF, in the presence of multiple ncAAs present in the growth media. In summary, our new approach opens up a new avenue for engineering the next generation of tRNA synthetases with improved selectivity toward a desired ncAA.

[1]  G. Georgiou,et al.  Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[2]  J. Mendenhall,et al.  Display of β-lactamase on the Escherichia coli surface: outer membrane phenotypes conferred by Lpp′–OmpA′–β-lactamase fusions , 1996 .

[3]  G. Georgiou,et al.  Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface. , 1999, Protein engineering.

[4]  Andrew B. Martin,et al.  Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[5]  P. Schultz,et al.  Expanding the genetic code. , 2002, Chemical communications.

[6]  Peter G Schultz,et al.  An Expanded Eukaryotic Genetic Code , 2003, Science.

[7]  George Georgiou,et al.  Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Jennifer A. Prescher,et al.  A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems. , 2004, Journal of the American Chemical Society.

[9]  Peter G Schultz,et al.  Control of protein phosphorylation with a genetically encoded photocaged amino acid. , 2007, Nature chemical biology.

[10]  T. Steitz,et al.  Structure of pyrrolysyl-tRNA synthetase, an archaeal enzyme for genetic code innovation , 2007, Proceedings of the National Academy of Sciences.

[11]  Andrew E. Firth,et al.  GLUE-IT and PEDEL-AA: new programmes for analyzing protein diversity in randomized libraries , 2008, Nucleic Acids Res..

[12]  P. Schultz,et al.  Addition of an α-Hydroxy Acid to the Genetic Code of Bacteria† , 2008 .

[13]  Jason W. Chin,et al.  Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome , 2010, Nature.

[14]  J. Noel,et al.  Stereochemical Basis for Engineered Pyrrolysyl-tRNA Synthetase and the Efficient in Vivo Incorporation of Structurally Divergent Non-native Amino Acids , 2011, ACS chemical biology.

[15]  P. Schultz,et al.  An evolved aminoacyl-tRNA synthetase with atypical polysubstrate specificity. , 2011, Biochemistry.

[16]  D. Söll,et al.  Expanding the Genetic Code of Escherichia coli with Phosphoserine , 2011, Science.

[17]  D. Söll,et al.  N‐Acetyl lysyl‐tRNA synthetases evolved by a CcdB‐based selection possess N‐acetyl lysine specificity in vitro and in vivo , 2012, FEBS letters.

[18]  J. Chin,et al.  Genetic Encoding of Bicyclononynes and trans-Cyclooctenes for Site-Specific Protein Labeling in Vitro and in Live Mammalian Cells via Rapid Fluorogenic Diels–Alder Reactions , 2012, Journal of the American Chemical Society.

[19]  J. Noel,et al.  Expanding the Library and Substrate Diversity of the Pyrrolysyl‐tRNA Synthetase to Incorporate Unnatural Amino Acids Containing Conjugated Rings , 2013, Chembiochem : a European journal of chemical biology.

[20]  P. Schultz,et al.  A genetically encoded fluorescent probe in mammalian cells. , 2013, Journal of the American Chemical Society.

[21]  T. Steitz,et al.  Polyspecific pyrrolysyl-tRNA synthetases from directed evolution , 2014, Proceedings of the National Academy of Sciences.

[22]  Jeffery M. Tharp,et al.  Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool. , 2014, Biochimica et biophysica acta.

[23]  Chunaram Choudhary,et al.  The growing landscape of lysine acetylation links metabolism and cell signalling , 2014, Nature Reviews Molecular Cell Biology.

[24]  Alexandra M. E. Jones,et al.  Site Specific Genetic Incorporation of Azidophenylalanine in Schizosaccharomyces pombe , 2015, Scientific Reports.

[25]  D. Söll,et al.  Probing the active site tryptophan of Staphylococcus aureus thioredoxin with an analog , 2015, Nucleic acids research.

[26]  Dieter Söll,et al.  Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids , 2015, Nature Biotechnology.

[27]  D. Söll,et al.  Expanding the genetic code of Escherichia coli with phosphotyrosine , 2016, FEBS letters.

[28]  Scott J. Miller,et al.  Dual Genetic Encoding of Acetyl-lysine and Non-deacetylatable Thioacetyl-lysine Mediated by Flexizyme. , 2016, Angewandte Chemie.

[29]  Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage , 2017, Nature.

[30]  P. Schultz,et al.  Genetically encoding phosphotyrosine and its nonhydrolyzable analog in bacteria , 2017, Nature chemical biology.

[31]  D. Söll,et al.  Rewriting the Genetic Code. , 2017, Annual review of microbiology.

[32]  A. Chatterjee,et al.  Defining the current scope and limitations of dual noncanonical amino acid mutagenesis in mammalian cells† †Electronic supplementary information (ESI) available: Experimental details, supplementary data, figures and references. See DOI: 10.1039/c7sc02560b Click here for additional data file. , 2017, Chemical science.

[33]  Zhipeng A. Wang,et al.  A Versatile Approach for Site-Specific Lysine Acylation in Proteins. , 2017, Angewandte Chemie.

[34]  Dieter Söll,et al.  Continuous directed evolution of aminoacyl-tRNA synthetases , 2017, Nature chemical biology.

[35]  Frances H. Arnold,et al.  Anti-Markovnikov alkene oxidation by metal-oxo–mediated enzyme catalysis , 2017, Science.

[36]  Aditya M. Kunjapur,et al.  Engineering posttranslational proofreading to discriminate nonstandard amino acids , 2018, Proceedings of the National Academy of Sciences.