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Expanding the amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes

Nature Chemistry volume 8, pages 317–325 (2016)Cite this article

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Abstract

In ribosomal polypeptide synthesis the library of amino acid building blocks is limited by the manner in which codons are used. Of the proteinogenic amino acids, 18 are coded for by multiple codons and therefore many of the 61 sense codons can be considered redundant. Here we report a method to reduce the redundancy of codons by artificially dividing codon boxes to create vacant codons that can then be reassigned to non-proteinogenic amino acids and thereby expand the library of genetically encoded amino acids. To achieve this, we reconstituted a cell-free translation system with 32 in vitro transcripts of transfer RNASNN (tRNASNN) (S = G or C), assigning the initiator and 20 elongator amino acids. Reassignment of three redundant codons was achieved by replacing redundant tRNASNNs with tRNASNNs pre-charged with non-proteinogenic amino acids. As a demonstration, we expressed a 32-mer linear peptide that consists of 20 proteinogenic and three non-proteinogenic amino acids, and a 14-mer macrocyclic peptide that contains more than four non-proteinogenic amino acids.

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Figure 1: Schematic representation of the artificial division of codon boxes.
Figure 2: Decoding by custom-made FIT systems that consisted of in vitro tRNA transcripts.
Figure 3: Artificial division of the Val GUN codon box.
Figure 4: Assignments of two different Naa in the Arg CGN codon box.
Figure 5: Simultaneous division of multiple codon boxes.
Figure 6: Artificial division of three codon boxes to express a peptide that contains a library of 23 amino acids and a non-standard N-methyl-macrocyclic peptide.

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Acknowledgements

We thank M. E. Harris and M. Ohuchi for a gift of plasmids that coded E. coli M1 RNA and C5 protein and their products. We thank S. Jongkees and J. Rogers for proofreading the manuscript. This research was supported by the Japan Science and Technology Agency (JST) Core Research for Evolutional Science and Technology (CREST) of Molecular Technologies to H.S., Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Young Scientists (B) to Y.G. (22750145) and Grants-in-Aid for JSPS Fellows to Y.I. (26-9576).

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Authors and Affiliations

  1. Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Yoshihiko Iwane, Takayuki Katoh, Yuki Goto & Hiroaki Suga

  2. Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Azusa Hitomi

  3. Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8603, Japan

    Hiroshi Murakami

  4. JST-CREST, Tokyo, 113-0033, Japan

    Hiroaki Suga

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  1. Yoshihiko Iwane
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Contributions

Y.I., A.H., H.M., T.K., Y.G. and H.S. designed the experiments and analysed the data. Y.I. and A.H. performed the experiments. Y.I., T.K., Y.G. and H.S. wrote the paper.

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Correspondence to Hiroaki Suga.

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Iwane, Y., Hitomi, A., Murakami, H. et al. Expanding the amino acid repertoire of ribosomal polypeptide synthesis via the artificial division of codon boxes. Nature Chem 8, 317–325 (2016). https://doi.org/10.1038/nchem.2446

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