title: Liposome-Based in Vitro Evolution of Aminoacyl-tRNA Synthetase for Enhanced Pyrrolysine Derivative Incorporation.
abstract: Methanosarcina species pyrrolysyl-tRNA synthetase (PylRS) attaches Pyl to its cognate amber suppressor tRNA. The introduction of two mutations (Y384F and Y306A) into PylRS was previously shown to generate a mutant, designated LysZ-RS, that was able to attach N-benzyloxycarbonyl-L-lysine (LysZ) to its cognate tRNA. Despite the potential of LysZ derivatives, further LysZ-RS engineering has not been performed; consequently, we aimed to generate LysZ-RS mutants with improved LysZ incorporation activity through in vitro directed evolution. Using a liposome-based in vitro compartmentalization (IVC) approach, we screened a randomly mutagenized gene library of LysZ-RS and obtained a mutant that showed increased LysZ incorporation activity both in vitro and in vivo. The ease and high flexibility of liposome-based IVC should enable the evolution of not only LysZ-RS that can attach various LysZ derivatives but also of other enzymes involved in protein translation.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871 (Japan).
authors.forename: Atsuko
authors.initials: A
authors.lastname: Uyeda
authors.affiliation: School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa, 923-1292 (Japan).
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Watanabe
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871 (Japan).
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Kato
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871 (Japan).
authors.forename: Hajime
authors.initials: H
authors.lastname: Watanabe
authors.affiliation: Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, 565-0871 (Japan).|Dynamical Microscale Reaction Environment Project, ERATO (Japan) Science and Technology Agency, 1-5 Yamadaoka, Suita, Osaka 565-0871 (Japan).|Graduate School of Frontier Biosciences, Osaka University, 1-5 Yamadaoka, Suita, Osaka, 565-0871 (Japan).
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Yomo
authors.affiliation: School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa, 923-1292 (Japan). hohsaka@jaist.ac.jp.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Hohsaka
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871 (Japan). matsuura_tomoaki@bio.eng.osaka-u.ac.jp.|Dynamical Microscale Reaction Environment Project, ERATO (Japan) Science and Technology Agency, 1-5 Yamadaoka, Suita, Osaka 565-0871 (Japan). matsuura_tomoaki@bio.eng.osaka-u.ac.jp.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Matsuura
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D019143:Evolution, Molecular; D015723:Gene Library; D008081:Liposomes / Q000378:metabolism*; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry; D008958:Models, Molecular; D017421:Sequence Analysis
chemical_list: D008081:Liposomes; D000604:Amino Acyl-tRNA Synthetases; C456839:pyrrolysine; D008239:Lysine
keywords: aminoacyl-tRNA synthetase; in vitro evolution; liposomes; protein engineering; unnatural amino acid