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

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. 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 (~50 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.

Authors:
 [1];  [1];  [2]; ORCiD logo [2]; ORCiD logo [3];  [4];  [1];  [1];  [1]; ORCiD logo [5];  [4];  [6];  [5];  [7]; ORCiD logo [3];  [1]
  1. Yale Univ., New Haven, CT (United States). Dept.of Molecular, Cellular, and Developmental Biology and Systems Biology Inst.
  2. Yale Univ., New Haven, CT (United States). Dept. of Molecular Biophysics and Biochemistry
  3. Yale Univ., New Haven, CT (United States). Systems Biology Inst. and Dept. of Cellular and Molecular Physiology
  4. Northwestern Univ., Evanston, IL (United States). Dept. of Chemistry
  5. Northwestern Univ., Evanston, IL (United States). Dept. of Chemical and Biological Engineering
  6. Yale Univ. School of Medicine, New Haven, CT (United States). Dept. of Microbial Pathogenesis and Microbial Sciences Inst.
  7. Yale Univ., New Haven, CT (United States). Dept. of Chemistry and Dept. of Molecular Biophysics and Biochemistry
Publication Date:
Research Org.:
Harvard Univ., Cambridge, MA (United States)
Sponsoring Org.:
USDOE; Defense Advanced Research Projects Agency (DARPA); National Institutes of Health (NIH); US Army Research Office (ARO); David and Lucile Packard Foundation; Camille Dreyfus Teacher-Scholar Awards Program; Arnold and Mabel Beckman Foundation
OSTI Identifier:
1466973
Grant/Contract Number:  
FG02-02ER63445; N66001-12-C-4020; N66001-12-C-4211; GM22854; GM67193; T32GM007205; 1F30CA196191; W911NF- 11-1-0445
Resource Type:
Accepted Manuscript
Journal Name:
Nature Biotechnology
Additional Journal Information:
Journal Volume: 33; Journal Issue: 12; Journal ID: ISSN 1087-0156
Publisher:
Springer Nature
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY

Citation Formats

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, and Isaacs, Farren J. Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids. United States: N. p., 2015. Web. doi: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. United States. doi: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, and Isaacs, Farren J. Mon . "Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids". United States. doi:https://doi.org/10.1038/nbt.3372. https://www.osti.gov/servlets/purl/1466973.
@article{osti_1466973,
title = {Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids},
author = {Amiram, Miriam and Haimovich, Adrian D. and Fan, Chenguang and Wang, Yane-Shih and Aerni, Hans-Rudolf and Ntai, Ioanna and Moonan, Daniel W. and Ma, Natalie J. and Rovner, Alexis J. and Hong, Seok Hoon and Kelleher, Neil L. and Goodman, Andrew L. and Jewett, Michael C. and Söll, Dieter and Rinehart, Jesse and Isaacs, Farren J.},
abstractNote = {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. 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 (~50 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.},
doi = {10.1038/nbt.3372},
journal = {Nature Biotechnology},
number = 12,
volume = 33,
place = {United States},
year = {2015},
month = {11}
}

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