Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids

Miriam Amiram; Adrian D. Haimovich; Chenguang Fan; Yane Shih Wang; Hans Rudolf Aerni; Ioanna Ntai; Daniel W. Moonan; Natalie J. Ma; Alexis J. Rovner; Seok Hoon Hong; Neil L. Kelleher; Andrew L. Goodman; Michael C. Jewett; Dieter Söll; Jesse Rinehart; Farren J. Isaacs

doi:10.1038/nbt.3372

Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids

Miriam Amiram, Adrian D. Haimovich, Chenguang Fan, Yane Shih Wang, Hans Rudolf Aerni, Ioanna Ntai, Daniel W. Moonan, Natalie J. Ma, Alexis J. Rovner, Seok Hoon Hong, Neil L. Kelleher, Andrew L. Goodman, Michael C. Jewett, Dieter Söll, Jesse Rinehart, Farren J. Isaacs^*

^*Corresponding author for this work

Research output: Contribution to journal › Article

106 Scopus citations

Abstract

Expansion of the genetic code with nonstandard amino acids (nsAAs) has enabled biosynthesis of proteins with diverse new chemistries. However, this technology has been largely restricted to proteins containing a single or few nsAA instances. Here we describe an in vivo evolution approach in a genomically recoded Escherichia coli strain for the selection of orthogonal translation systems capable of multi-site nsAA incorporation. We evolved chromosomal aminoacyl-tRNA synthetases (aaRSs) with up to 25-fold increased protein production for p-acetyl-L-phenylalanine and p-azido-L-phenylalanine (pAzF). We also evolved aaRSs with tunable specificities for 14 nsAAs, including an enzyme that efficiently charges pAzF while excluding 237 other nsAAs. These variants enabled production of elastin-like-polypeptides with 30 nsAA residues at high yields (â 1/450 mg/L) and high accuracy of incorporation (>95%). This approach to aaRS evolution should accelerate and expand our ability to produce functionalized proteins and sequence-defined polymers with diverse chemistries.

Original language	English (US)
Pages (from-to)	1272-1279
Number of pages	8
Journal	Nature biotechnology
Volume	33
Issue number	12
DOIs	https://doi.org/10.1038/nbt.3372
State	Published - Dec 1 2015

ASJC Scopus subject areas

Biotechnology
Bioengineering
Applied Microbiology and Biotechnology
Molecular Medicine
Biomedical Engineering

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Amiram, M., Haimovich, A. D., Fan, C., Wang, Y. S., Aerni, H. R., Ntai, I., Moonan, D. W., Ma, N. J., Rovner, A. J., Hong, S. H., Kelleher, N. L., Goodman, A. L., Jewett, M. C., Söll, D., Rinehart, J., & Isaacs, F. J. (2015). Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids. Nature biotechnology, 33(12), 1272-1279. https://doi.org/10.1038/nbt.3372

Amiram, Miriam ; Haimovich, Adrian D. ; Fan, Chenguang ; Wang, Yane Shih ; Aerni, Hans Rudolf ; Ntai, Ioanna ; Moonan, Daniel W. ; Ma, Natalie J. ; Rovner, Alexis J. ; Hong, Seok Hoon ; Kelleher, Neil L. ; Goodman, Andrew L. ; Jewett, Michael C. ; Söll, Dieter ; Rinehart, Jesse ; Isaacs, Farren J. / Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids. In: Nature biotechnology. 2015 ; Vol. 33, No. 12. pp. 1272-1279.

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abstract = "Expansion of the genetic code with nonstandard amino acids (nsAAs) has enabled biosynthesis of proteins with diverse new chemistries. However, this technology has been largely restricted to proteins containing a single or few nsAA instances. Here we describe an in vivo evolution approach in a genomically recoded Escherichia coli strain for the selection of orthogonal translation systems capable of multi-site nsAA incorporation. We evolved chromosomal aminoacyl-tRNA synthetases (aaRSs) with up to 25-fold increased protein production for p-acetyl-L-phenylalanine and p-azido-L-phenylalanine (pAzF). We also evolved aaRSs with tunable specificities for 14 nsAAs, including an enzyme that efficiently charges pAzF while excluding 237 other nsAAs. These variants enabled production of elastin-like-polypeptides with 30 nsAA residues at high yields ({\^a} 1/450 mg/L) and high accuracy of incorporation (>95%). This approach to aaRS evolution should accelerate and expand our ability to produce functionalized proteins and sequence-defined polymers with diverse chemistries.",

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Amiram, M, Haimovich, AD, Fan, C, Wang, YS, Aerni, HR, Ntai, I, Moonan, DW, Ma, NJ, Rovner, AJ, Hong, SH, Kelleher, NL, Goodman, AL, Jewett, MC, Söll, D, Rinehart, J & Isaacs, FJ 2015, 'Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids', Nature biotechnology, vol. 33, no. 12, pp. 1272-1279. https://doi.org/10.1038/nbt.3372

Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids. / Amiram, Miriam; Haimovich, Adrian D.; Fan, Chenguang; Wang, Yane Shih; Aerni, Hans Rudolf; Ntai, Ioanna; Moonan, Daniel W.; Ma, Natalie J.; Rovner, Alexis J.; Hong, Seok Hoon; Kelleher, Neil L.; Goodman, Andrew L.; Jewett, Michael C.; Söll, Dieter; Rinehart, Jesse; Isaacs, Farren J.

In: Nature biotechnology, Vol. 33, No. 12, 01.12.2015, p. 1272-1279.

Research output: Contribution to journal › Article

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AU - Amiram, Miriam

AU - Haimovich, Adrian D.

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AU - Ntai, Ioanna

AU - Moonan, Daniel W.

AU - Ma, Natalie J.

AU - Rovner, Alexis J.

AU - Hong, Seok Hoon

AU - Kelleher, Neil L.

AU - Goodman, Andrew L.

AU - Jewett, Michael C.

AU - Söll, Dieter

AU - Rinehart, Jesse

AU - Isaacs, Farren J.

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