Heterologous production of Escherichia coli penicillin G acylase in Pseudomonas aeruginosa.
Penicillin G acylase (PGA) is a widely studied bacterial enzyme of great industrial importance. Since its overproduction in the original organisms is mostly limited to the intracellular bacterial spaces which may lead to aggregation and cell toxicity, we have set out to explore the host organism Pseudomonas aeruginosa that possesses the Xcp machinery for secretion of folded proteins to the extracellular medium. We have made fusion proteins, consisting of Pseudomonas Sec- or Tat-specific signal peptides, the elastase propeptide and the mature penicillin G acylase. With all constructs we obtained production of PGA in P. aeruginosa, but we observed that processing of the PGA was temperature dependent and that the active enzyme could only be found after growth at 25 degrees C or lower temperatures. Remarkably, the mature protein, expressed from a TatProPGA hybrid, was not only found in the extracellular medium and the periplasm, but also in the cytoplasm as assessed by comparison to the reporter beta-lactamase protein. The unusual cytoplasmic localization of the mature protein strongly suggests that processing of PGA can also occur in the cytoplasm of P. aeruginosa. The extracellular localization of the TatProPGA hybrid was found not to be dependent on the tatABC-genes. The elastase signal sequence/propeptide combination appeared to be an inadequate carrier for transporting penicillin G acylase across the outer membrane of P. aeruginosa.
Krzeslak
Joanna
J
Department of Pharmaceutical Biology, University Center of Pharmacy, Groningen University Institute for Drug Exploration (GUIDE), Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Braun
Peter
P
Voulhoux
Rome
R
Cool
Robbert H
RH
Quax
Wim J
WJ
D001426:Bacterial Proteins; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics; D026901:Membrane Transport Proteins; D010196:Pancreatic Elastase / Q000235:genetics; D010399:Penicillin Amidase / Q000096:biosynthesis* / Q000235:genetics; D019897:Periplasm / Q000737:chemistry; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C089840:XcpP protein, Pseudomonas aeruginosa; D010196:Pancreatic Elastase; D010399:Penicillin Amidase