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Nat Biotechnol. 2015 Dec;33(12):1272-1279. doi: 10.1038/nbt.3372. Epub 2015 Nov 16.

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

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Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, USA.
Systems Biology Institute, Yale University, West Haven, Connecticut, USA.
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.
Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut, USA.
Department of Chemistry, Northwestern University, Evanston, Illinois, USA.
Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois, USA.
Department of Microbial Pathogenesis and Microbial Sciences Institute, Yale University School of Medicine, New Haven, Connecticut, USA.
Department of Chemistry, Yale University, New Haven, Connecticut, USA.


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 (∼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.

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