Published on Web 10/06/2010 Expanding the Genetic Code of Yeast for Incorporation of Diverse Unnatural Amino Acids via a Pyrrolysyl-tRNA Synthetase/tRNA Pair Susan M. Hancock,† Rajendra Uprety,‡ Alexander Deiters,‡ and Jason W. Chin†,* Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, U.K., and North Carolina State UniVersity, Department of Chemistry, Raleigh, North Carolina 27695 Received May 27, 2010; E-mail: chin@mrc-lmb.cam.ac.uk Abstract: We report the discovery of a simple system through which variant pyrrolysyl-tRNA synthetase/ tRNACPyUlA pairs created in Escherichia coli can be used to expand the genetic code of Saccharomyces cerevisiae. In the process we have solved the key challenges of producing a functional tRNACPyUlA in yeast and discovered a pyrrolysyl-tRNA synthetase/tRNACPyUlA pair that is orthogonal in yeast. Using our approach we have incorporated an alkyne-containing amino acid for click chemistry, an important post-translationally modiﬁed amino acid and one of its analogs, a photocaged amino acid and a photo-cross-linking amino acid into proteins in yeast. Extensions of our approach will allow the growing list of useful amino acids that have been incorporated in E. coli with variant pyrrolysyl-tRNA synthetase/tRNAPCyUlA pairs to be site-speciﬁcally incorporated into proteins in yeast. Introduction The pyrrolysyl-tRNA synthetase/tRNAPCyUlA (PylRS/tRNAPCyUlA) pairs from Methanosarcina barkeri (Mb) and M. mazei (Mm) are orthogonal in Escherichia coli.1 These pairs have been evolved to direct the site-speciﬁc incorporation of a range of unnatural amino acids, including amino acids that are post- translationally modiﬁed, amino acids containing bio-orthogonal chemical handles, and amino acids protected with light- and acid-sensitive groups, into proteins in E. coli in response to the amber codon.1-6 In contrast to other aminoacyl-tRNA syn- thetase/tRNA pairs for the incorporation of unnatural amino acids, which are orthogonal in either eukaryotic or prokaryotic hosts, the PylRS/tRNACPyUlA pairs are orthogonal in both E. coli † Medical Research Council Laboratory of Molecular Biology. ‡ North Carolina State University. (1) Neumann, H.; Peak-Chew, S. Y.; Chin, J. W. Nat. Chem. Biol. 2008, 4, 232. (2) Gautier, A.; Nguyen, D. P.; Lusic, H.; An, W.; Deiters, A.; Chin, J. W. J. Am. Chem. Soc. 2010, 132, 4086. (3) Neumann, H.; Hancock, S. M.; Buning, R.; Routh, A.; Chapman, L.; Somers, J.; Owen-Hughes, T.; van Noort, J.; Rhodes, D.; Chin, J. W. Mol. Cell 2009, 36, 153. (4) (a) Zhao, S.; Xu, W.; Jiang, W.; Yu, W.; Lin, Y.; Zhang, T.; Yao, J.; Zhou, L.; Zeng, Y.; Li, H.; Li, Y.; Shi, J.; An, W.; Hancock, S. M.; He, F.; Qin, L.; Chin, J.; Yang, P.; Chen, X.; Lei, Q.; Xiong, Y.; Guan, K. L. Science 2010, 327, 1000. (b) Nguyen, D. P.; Garcia Alai, M. M.; Kapadnis, P. B.; Neumann, H.; Chin, J. W. J. Am. Chem. Soc. 2009, 131, 14194. (c) Fekner, T.; Li, X.; Lee, M. M.; Chan, M. K. Angew. Chem., Int. Ed. 2009, 48, 1633. (d) Li, W. T.; Mahapatra, A.; Longstaff, D. G.; Bechtel, J.; Zhao, G.; Kang, P. T.; Chan, M. K.; Krzycki, J. A. J. Mol. Biol. 2009, 385, 1156. (e) Li, X.; Fekner, T.; Ottesen, J. J.; Chan, M. K. Angew. Chem., Int. Ed. 2009, 48, 9184. (f) Yanagisawa, T.; Ishii, R.; Fukunaga, R.; Kobayashi, T.; Sakamoto, K.; Yokoyama, S. Chem. Biol. 2008, 15, 1187. (5) Nguyen, D. P.; Lusic, H.; Neumann, H.; Kapadnis, P. B.; Deiters, A.; Chin, J. W. J. Am. Chem. Soc. 2009, 131, 8720. (6) Chen, P. R.; Groff, D.; Guo, J.; Ou, W.; Cellitti, S.; Geierstanger, B. H.; Schultz, P. G. Angew. Chem., Int. Ed. 2009, 48, 4052. 10.1021/ja104609m  2010 American Chemical Society and mammalian cells.2,6,7 Several unnatural amino acids have been site-speciﬁcally incorporated into proteins in mammalian cells by evolving the synthetase/tRNA pair in E. coli and subsequently transferring it to mammalian cells. This approach has the advantage of bypassing the requirement to evolve the amino acid speciﬁcity of the synthetase directly in a eukaryotic host.8-10 Many biological processes are more effectively addressed in the yeast Saccharomyces cereVisiae than in mammalian cells. Yeast has a rapid doubling time, bar-coded libraries of gene knockouts exist, protein interaction and transcriptome data is most complete, tap-tagged strains are readily available and powerful genetic approaches can be simply implemented. However, the requirement to evolve the current orthogonal pairs directly in yeast has limited the scope of unnatural amino acids that have been incorporated in yeast. Preliminary work by Yokoyama and co-workers introduced a PylRS/tRNACPyUlA pair into yeast and reported very weak phenotypes consistent with poor incorporation of Nε-tert-butyl- oxycarbonyl-L-lysine,7 but a properly characterized system for incorporating amino acids using PylRS/tRNACPyUlA pairs has not been reported. Here we report the creation and characterization of a functional and orthogonal PylRS/tRNAPyl CUA pair in yeast (7) Mukai, T.; Kobayashi, T.; Hino, N.; Yanagisawa, T.; Sakamoto, K.; Yokoyama, S. Biochem. Biophys. Res. Commun. 2008, 371, 818. (8) Chin, J. W.; Cropp, T. A.; Anderson, J. C.; Mukherji, M.; Zhang, Z.; Schultz, P. G. Science 2003, 301, 964. (9) Chin, J. W.; Cropp, T. A.; Chu, S.; Meggers, E.; Schultz, P. G. Chem. Biol. 2003, 10, 511. (10) Wu, N.; Deiters, A.; Cropp, T. A.; King, D.; Schultz, P. G. J. Am. Chem. Soc. 2004, 126, 14306. J. AM. CHEM. SOC. 2010, 132, 14819–14824 9 14819