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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 (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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Figure 1: Evaluation of multi-site nsAA incorporation and expression profiles on the activity of M. jannaschii derived pAcF orthogonal translation system (OTS).
Figure 2: Evolution of chromosomally integrated aaRS variants.
Figure 3: Evolution and characterization of chromosomally integrated pAcFRS and pAzFRS variants with improved efficiencies.
Figure 4: Evaluation of multi-site nsAA incorporation by evolved aaRS variants expressed on multi-copy plasmids.
Figure 5: Quantitative MS evaluation of the purity of multi-site nsAA incorporation by evolved aaRS variants expressed on multi-copy plasmids.

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Acknowledgements

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.).

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Contributions

M.A. designed ELP constructs, and conducted and interpreted multi-site nsAA incorporation experiments. M.A., A.D.H. and F.J.I. designed, conducted and interpreted synthetase evolution experiments. H.-R.A. and J.R. conducted and interpreted MS experiments. I.N. and N.L.K. performed and interpreted top-down MS experiments. A.D.H, A.L.G. and F.J.I. analyzed NextGen sequencing experiments. C.F., D.W.M. and D.S. conducted and interpreted biochemical experiments. Y.-S.W. executed nsAA screens. S.H.H. tested ELP expression in vitro. M.A., A.D.H. and A.J.R. performed crystal structure analysis and target selection. N.J.M. constructed and characterized the tolC variant used for negative selection. F.J.I., J.R. and D.S. directed the studies and interpreted data. M.A. and F.J.I. wrote the paper with assistance from A.D.H., N.J.M., D.S., M.C.J., I.N., N.L.K. and J.R.

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Correspondence to Dieter Söll, Jesse Rinehart or Farren J Isaacs.

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M.A., A.D.H. and F.J.I. have filed a provisional application with the US Patent and Trademark Office on this work.

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Amiram, M., Haimovich, A., Fan, C. et al. Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids. Nat Biotechnol 33, 1272–1279 (2015). https://doi.org/10.1038/nbt.3372

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