Overcoming the Membrane Barrier: Recruitment of ?-Glutamyl Transferase for Intracellular Release of Cargo Molecules

Semipermeable membranes of cells frequently pose an obstacle for metabolic engineering and synthetic biology approaches by limiting the uptake of potentially interesting molecules, and thus interfere with a broad variety of possible enzyme engineering or metabolic reconstruction strategies. Previous attempts to overcome this barrier relied on the unspecific 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 the bacterium Escherichia coli. It is composed of the following steps: (1) attachment of cargo molecules to the glutamate side chain of a dipeptide by chemical synthesis to form a stable cargo-peptide vector construct, (2) transport of this construct into the cell via the promiscuous dipeptide permease (Dpp) system, and (3) efficient enzymatic release of the cargo molecule in the cytoplasm. The functional key to successful implementation of this system is the efficient intracellular release of the cargo molecule from the peptide vector. This is achieved by recruiting the periplasmic enzyme γ-glutamyl transferase (GGT), which hydrolyzes a broad range of γ-glutamyl amides and esters.

We demonstrate that GGT can be retained in the cytoplasm of E. coli, and that it can be employed to specifically cleave off cargo molecules from the γ-carboxyl group of a glutamate residue in a dipeptide after uptake by the cell. Fine-tuning of GGT expression is critical for the functionality of the system, presumably in order to minimize toxic effects associated with cytoplasmic expression of GGT. We illustrate the functionality and versatility of the system with different natural and non-natural cargo molecules. In addition, we identify mutations in the periplasmic binding protein DppA that lead to improved uptake of dipeptides containing γ-substituted glutamate residues. Given the established protocols of peptide synthesis, the flexibility of cellular peptide transport, and the broad substrate specificity of GGT, this system offers the potential to overcome transport problems in a variety of metabolic engineering and synthetic biology applications.