@article{c31091e6806540958661aa3836982bca,
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. 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.",
author = "Miriam Amiram and Haimovich, {Adrian D.} and Chenguang Fan and Wang, {Yane Shih} and Aerni, {Hans Rudolf} and Ioanna Ntai 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 Dieter S{\"o}ll and Jesse Rinehart and Isaacs, {Farren J.}",
note = "Funding Information: We thank K. Bilguvar and J. Knight (Yale Center for Genome Analysis) for conducting next-generation sequencing experiments; T. Wu (Yale West Campus Analytical Core facility) for conducting intact MS experiments; B. Gassaway for assistance with shotgun MS experiments. We are grateful to members of the Isaacs laboratory, J. Ling and G. Church for critical discussions and feedback. This work was supported by the Defense Advanced Research Projects Agency contracts N66001-12-C-4020 and N66001-12-C-4211 to (F.J.I., J.R., D.S., and M.C.J.), U.S. Department of Energy (DE-FG02-02ER63445 to F.J.I.) grants GM22854 to D.S. and GM67193 to N.L.K. from the National Institute for General Medical Sciences, T32GM007205 and 1F30CA196191 (A.D.H.), Army Research Office (W911NF-11-1-0445 to M.C.J.), the David and Lucile Packard Foundation (M.C.J.), the Camille Dreyfus Teacher-Scholar Program (M.C.J.), DuPont, Inc. (F.J.I.) and the Arnold and Mabel Beckman Foundation (F.J.I.). Publisher Copyright: {\textcopyright} 2015 Nature America, Inc.",
year = "2015",
month = dec,
day = "1",
doi = "10.1038/nbt.3372",
language = "English (US)",
volume = "33",
pages = "1272--1279",
journal = "Nature Biotechnology",
issn = "1087-0156",
publisher = "Nature Publishing Group",
number = "12",
}