title: Overcoming the membrane barrier: Recruitment of γ-glutamyl transferase for intracellular release of metabolic cargo from peptide vectors.
abstract: Semipermeable membranes of cells frequently pose an obstacle in metabolic engineering by limiting uptake of substrates, intermediates, or xenobiotics. Previous attempts to overcome this barrier relied on the promiscuous nature of peptide transport systems, but often suffered from low versatility or chemical instability. Here, we present an alternative strategy to transport cargo molecules across the inner membrane of Escherichia coli based on chemical synthesis of a stable cargo-peptide vector construct, transport through the peptide import system, and efficient intracellular release of the cargo by the promiscuous enzyme γ-glutamyl transferase (GGT). Retaining the otherwise periplasmic GGT in the cytoplasm was critical for the functionality of the system, as was fine-tuning its expression in order to minimize toxic effects associated to cytoplasmic GGT expression. Given the established protocols of peptide synthesis and the flexibility of peptide transport and GGT, the system is expected to be suitable for a broad range of cargoes.
authors.affiliation: Bioprocess Laboratory, Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, 4058 Basel, Switzerland.
authors.forename: Tilmann
authors.initials: T
authors.lastname: Kuenzl
authors.affiliation: Bioprocess Laboratory, Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, 4058 Basel, Switzerland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Sroka
authors.affiliation: Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium.
authors.forename: Puneet
authors.initials: P
authors.lastname: Srivastava
authors.affiliation: Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium.
authors.forename: Piet
authors.initials: P
authors.lastname: Herdewijn
authors.affiliation: Institute of Systems & Synthetic Biology, Génopole, 5 rue Henri Desbruères, 91030 Evry Cedex, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Marlière
authors.affiliation: Bioprocess Laboratory, Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, 4058 Basel, Switzerland. Electronic address: sven.panke@bsse.ethz.ch.
authors.forename: Sven
authors.initials: S
authors.lastname: Panke
mesh_terms: D001693:Biological Transport, Active / Q000502:physiology; D053898:Biosynthetic Pathways / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D024861:Genetic Enhancement / Q000379:methods; D007424:Intracellular Fluid / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000502:physiology; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D005723:gamma-Glutamyltransferase / Q000235:genetics / Q000378:metabolism*
chemical_list: D026901:Membrane Transport Proteins; D010455:Peptides; C043912:peptide permease; D005723:gamma-Glutamyltransferase
keywords: Gamma-glutamyl transferase; Peptide transport; Portage transport; Retrobiosynthesis; Xenobiotic import