title: Design of S-Allylcysteine in Situ Production and Incorporation Based on a Novel Pyrrolysyl-tRNA Synthetase Variant.
abstract: The noncanonical amino acid S-allyl cysteine (Sac) is one of the major compounds of garlic extract and exhibits a range of biological activities. It is also a small bioorthogonal alkene tag capable of undergoing controlled chemical modifications, such as photoinduced thiol-ene coupling or Pd-mediated deprotection. Its small size guarantees minimal interference with protein structure and function. Here, we report a simple protocol efficiently to couple in-situ semisynthetic biosynthesis of Sac and its incorporation into proteins in response to amber (UAG) stop codons. We exploited the exceptional malleability of pyrrolysyl-tRNA synthetase (PylRS) and evolved an S-allylcysteinyl-tRNA synthetase (SacRS) capable of specifically accepting the small, polar amino acid instead of its long and bulky aliphatic natural substrate. We succeeded in generating a novel and inexpensive strategy for the incorporation of a functionally versatile amino acid. This will help in the conversion of orthogonal translation from a standard technique in academic research to industrial biotechnology.
authors.affiliation: Institut für Chemie, Technische Universität Berlin, Müller-Breslau-Strasse 10, 10623, Berlin, Germany.
authors.forename: Matthias P
authors.initials: MP
authors.lastname: Exner
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, Mattenstrasse 26, 4058, Basel, Switzerland.
authors.forename: Tilmann
authors.initials: T
authors.lastname: Kuenzl
authors.affiliation: Institut für Chemie, Technische Universität Berlin, Müller-Breslau-Strasse 10, 10623, Berlin, Germany.
authors.forename: Tuyet Mai T
authors.initials: TM
authors.lastname: To
authors.affiliation: Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 124, 10623, Berlin, Germany.
authors.forename: Zhaofei
authors.initials: Z
authors.lastname: Ouyang
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Roessle-Strasse 10, 13125, BerlinBuch, Germany.|Humboldt Universität zu Berlin, Department Chemie, Brook-Taylor-Strasse 2, 12489, Berlin, Germany.
authors.forename: Sergej
authors.initials: S
authors.lastname: Schwagerus
authors.affiliation: Institut für Chemie, Technische Universität Berlin, Müller-Breslau-Strasse 10, 10623, Berlin, Germany.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Hoesl
authors.affiliation: Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Roessle-Strasse 10, 13125, BerlinBuch, Germany.|Humboldt Universität zu Berlin, Department Chemie, Brook-Taylor-Strasse 2, 12489, Berlin, Germany.
authors.forename: Christian P R
authors.initials: CP
authors.lastname: Hackenberger
authors.affiliation: Institut für Chemie, Technische Universität Berlin, Strasse des 17. Juni 124, 10623, Berlin, Germany.
authors.forename: Marga C
authors.initials: MC
authors.lastname: Lensen
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zürich, Mattenstrasse 26, 4058, Basel, Switzerland.
authors.forename: Sven
authors.initials: S
authors.lastname: Panke
authors.affiliation: Institut für Chemie, Technische Universität Berlin, Müller-Breslau-Strasse 10, 10623, Berlin, Germany.
authors.forename: Nediljko
authors.initials: N
authors.lastname: Budisa
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D003547:Cysteine Synthase / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D017020:Methanosarcina / Q000201:enzymology; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C065299:S-allylcysteine; D003547:Cysteine Synthase; D000604:Amino Acyl-tRNA Synthetases; D003545:Cysteine
keywords: acetylserine sulfhydrylase; allylcysteine; gene expression; genetic code expansion; protein design; pyrrolysyl-tRNA synthetase