Original Research ArticleOvercoming the membrane barrier: Recruitment of γ-glutamyl transferase for intracellular release of metabolic cargo from peptide vectors

•A synthetic transport system to overcome the membrane barrier of E. coli.•Intracellular release of cargo molecules using γ-glutamyl transferase (GGT).•Expression of GGT in the cytoplasm of E. coli leads to toxic effects.•Toxic effects can be mitigated through ribosome binding site engineering.

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.

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