Abstract
Genetic code expansion involves introducing non-canonical amino acids (ncAAs) with unique functional groups into proteins to broaden their applications. Orthogonal aminoacyl tRNA synthetase (aaRS), essential for genetic code expansion, facilitates the charging of ncAAs to tRNA. In this study, we developed a new aaRS mutant from Methanosaeta concilii tyrosyl-tRNA synthetase (Mc TyrRS) to incorporate para-azido-l-phenylalanine (AzF). The development involved initial site-specific mutations in Mc TyrRS, followed by random mutagenesis. The new aaRS mutant with amber suppression was isolated through fluorescence-activated cell sorting. The M. concilii aaRS mutant structure was further analyzed to interpret the effect of mutations. This research provides a novel orthogonal aaRS evolution pipeline for highly efficient ncAA incorporation that will contribute to develo** novel aaRS from various organisms.
Similar content being viewed by others
Introduction
Non-canonical amino acids (ncAAs) with unique functional groups can diversify protein structure and activity, overcoming the structural or functional limitations of canonical amino acids. In particular, the site-specific binding of ncAA is currently researched in synthetic biology, proteomics, and medicine (Bain et al. 1989). As an example, introducing ncAAs is garnering attention as an innovative method for increasing the half-life of therapeutic proteins. Contrasting PEGylation via cysteine or lysine, incorporating site-specific ncAAs with certain residues allows site-specific PEGylation and large-scale synthesis of homogeneous proteins (Nguyen et al. 2009; Yang et al. 2016).
Genetic code expansion is a representative method to incorporate ncAAs by constructing novel translation systems that do not cross-act with the host cell. The vital elements of this technology are (1) a codon capable of encoding ncAA (a stop codon or a quadruplet codon), (2) a tRNA that recognizes the codon (suppressor tRNA), and (3) an aminoacyl-tRNA synthetase (aaRS) that aminoacylates the ncAA to the tRNA (Wang et al. 2001). An essential factor is that the tRNA, aaRS, and ncAA must work orthogonally to avoid interfering with the host cell's translation system.
Therefore, aaRS/tRNA pairs were imported from several organisms into Escherichia coli to fulfill these requirements. For example, the tyrosyl-tRNA synthetase/tRNATyr pair from Methanococcus jannaschii (Wang et al. 2001) or Archaeoglobus fulgidus (Cervettini et al. 2020) and the pyrrolysyl-tRNA synthetase/tRNAPyl pair from Methanosarcina barkeri (Srinivasan et al. 2002) or Methanosarcina mazei (Odoi et al. 2013) were developed to incorporate ncAA into proteins in E. coli. Although several mutant aaRS/tRNA pairs were developed, additional aaRS/tRNA pairs are still being required to broaden the usage of ncAAs. However, most aaRSs used to incorporate ncAAs are derived from M. jannaschii (Chin et al. 2002a, 2002b; Deiters and Schultz 2005; Guo et al. 2008; Schultz et al. All authors declare that the data supporting the findings of this study are available within the article. The sequencing data of Methanosaeta concilii tyrosyl-tRNA synthetase were deposited at NCBI((https://www.ncbi.nlm.nih.gov/) under accession number (PE011898.1). Bain JD, Diala ES, Glabe CG, Dix TA, Chamberlin AR (1989) Biosynthetic site-specific incorporation of a non-natural amino acid into a polypeptide. J Am Chem Soc 111:8013–8014. https://doi.org/10.1021/ja00202a052 Cervettini D, Tang S, Fried SD, Willis JCW, Funke LFH, Colwell LJ, Chin JW (2020) Rapid discovery and evolution of orthogonal aminoacyl-tRNA synthetase–tRNA pairs. Nat Biotechnol 38:989–999. https://doi.org/10.1038/s41587-020-0479-2 Chin JW, Martin AB, King DS, Wang L, Schultz PG (2002a) Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. Proc Natl Acad Sci USA 99:11020–11024. https://doi.org/10.1073/pnas.172226299 Chin JW, Santoro SW, Martin AB, King DS, Wang L, Schultz PG (2002b) Addition of p-Azido-l-phenylalanine to the Genetic Code of Escherichia coli. J Am Chem Soc 124:9026–9027. https://doi.org/10.1021/ja027007w Deiters A, Schultz PG (2005) In vivo incorporation of an alkyne into proteins in Escherichia coli. Bioorg Med Chem Lett 15:1521–1524. https://doi.org/10.1016/j.bmcl.2004.12.065 Eberhardt J, Santos-Martins D, Tillack AF, Forli S (2021) AutoDock Vina 1.2.0: new docking methods, expanded force field, and python bindings. J Chem Inf Model 61:3891–3898. https://doi.org/10.1021/acs.jcim.1c00203 Guo J, Wang J, Anderson JC, Schultz PG (2008) Addition of an α-Hydroxy Acid to the Genetic Code of Bacteria. Angew Chem Int Ed 47:722–725. https://doi.org/10.1002/anie.200704074 Kaiser F, Krautwurst S, Salentin S, Haupt VJ, Leberecht C, Bittrich S, Labudde D, Schroeder M (2020) The structural basis of the genetic code: amino acid recognition by aminoacyl-tRNA synthetases. Sci Rep 10:12647. https://doi.org/10.1038/s41598-020-69100-0 Kim J, Choi J-i (2022) Expression of green fluorescence proteins with non-canonical amino acids in different Escherichia coli Strains. Korean Soc Biotechnol Bioeng 37:58–63. https://doi.org/10.7841/ksbbj.2022.37.2.58 Lee D, Kim M-K, Choi J-i (2023) Development of orthogonal aminoacyl tRNA Synthetase mutant with enhanced incorporation ability with Para-azido-L-phenylalanine. Biotechnol Bioprocess Eng. https://doi.org/10.1007/s12257-022-0252-0 Madeira F, Pearce M, Tivey ARN, Basutkar P, Lee J, Edbali O, Madhusoodanan N, Kolesnikov A, Lopez R (2022) Search and sequence analysis tools services from EMBL-EBI in 2022. Nucleic Acids Res 50:W276–W279. https://doi.org/10.1093/nar/gkac240 Nguyen DP, Lusic H, Neumann H, Kapadnis PB, Deiters A, Chin JW (2009) genetic encoding and labeling of aliphatic azides and alkynes in recombinant proteins via a Pyrrolysyl-tRNA Synthetase/tRNACUA pair and click chemistry. J Am Chem Soc 131:8720–8721. https://doi.org/10.1021/ja900553w Odoi KA, Huang Y, Rezenom YH, Liu WR (2013) Nonsense and sense suppression abilities of original and derivative Methanosarcina mazei pyrrolysyl-tRNA synthetase-tRNA(Pyl) pairs in the Escherichia coli BL21(DE3) cell strain. PLoS ONE 8:e57035. https://doi.org/10.1371/journal.pone.0057035 Robert X, Gouet P (2014) Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 42:W320–W324. https://doi.org/10.1093/nar/gku316 Schrodinger LLC (2021) The PyMOL molecular graphics system. Version 2:5 Schultz KC, Supekova L, Ryu Y, **e J, Perera R, Schultz PG (2006) A genetically encoded infrared probe. J Am Chem Soc 128:13984–13985. https://doi.org/10.1021/ja0636690 Srinivasan G, James CM, Krzycki JA (2002) Pyrrolysine encoded by UAG in Archaea: charging of a UAG-Decoding specialized tRNA. Science 296:1459–1462. https://doi.org/10.1126/science.1069588 Turner JM, Graziano J, Spraggon G, Schultz PG (2005) Structural characterization of a p-Acetylphenylalanyl aminoacyl-tRNA synthetase. J Am Chem Soc 127:14976–14977. https://doi.org/10.1021/ja0549042 Wang L, Schultz PG (2001) A general approach for the generation of orthogonal tRNAs. Chem Biol 8:883–890. https://doi.org/10.1016/S1074-5521(01)00063-1 Wang L, Brock A, Herberich B, Schultz PG (2001) Expanding the genetic code of Escherichia coli. Science 292:498–500. https://doi.org/10.1126/science.1060077 Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L, Lepore R, Schwede T (2018) SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 46:W296-w303. https://doi.org/10.1093/nar/gky427 **e J, Wang L, Wu N, Brock A, Spraggon G, Schultz PG (2004) The site-specific incorporation of p-iodo-L-phenylalanine into proteins for structure determination. Nat Biotechnol 22:1297–1301. https://doi.org/10.1038/nbt1013 Yang B, Lim SI, Kim JC, Tae G, Kwon I (2016) Site-Specific Albumination as an alternative to PEGylation for the enhanced serum half-life in vivo. Biomacromol 17:1811–1817. https://doi.org/10.1021/acs.biomac.6b00238 Young TS, Ahmad I, Yin JA, Schultz PG (2010) An enhanced system for unnatural amino acid mutagenesis in E. coli. J Mol Biol 395:361–374. https://doi.org/10.1016/j.jmb.2009.10.030 Not applicable. The work was funded by the Chonnam Natinal University Supporting Program and was supported by "Regional Innovation Strategy (RIS)" through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE) (2021RIS-002). DL: Writing–original draft. JGK: Methodology, Investigation. TWK: Investigation, Writing–original draft. JC: Writing–review & editing. Not applicable. Not applicable. Authors declare that there is no conflict of interest. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Lee, D., Kim, J.G., Kim, T.W. et al. Development of orthogonal aminoacyl-tRNA synthetase mutant for incorporating a non-canonical amino acid.
AMB Expr 14, 60 (2024). https://doi.org/10.1186/s13568-024-01706-3 Received: Accepted: Published: DOI: https://doi.org/10.1186/s13568-024-01706-3Availability of data and materials
References
Acknowledgements
Funding
Author information
Authors and Affiliations
Contributions
Corresponding authors
Ethics declarations
Ethics approval and consent to participate
Consent for publication
Competing interests
Additional information
Publisher's Note
Rights and permissions
About this article
Cite this article
Keywords
