Improvement of posttranslational bottlenecks in the production of penicillin amidase in recombinant Escherichia coli strains.
Using periplasmic penicillin amidase (PA) from Escherichia coli ATCC 11105 as a model recombinant protein, we reviewed the posttranslational bottlenecks in its overexpression and undertook attempts to enhance its production in different recombinant E. coli expression hosts. Intracellular proteolytic degradation of the newly synthesized PA precursor and translocation through the plasma membrane were determined to be the main posttranslational processes limiting enzyme production. Rate constants for both intracellular proteolytic breakdown (k(d)) and transport (k(t)) were used as quantitative tools for selection of the appropriate host system and cultivation medium. The production of mature active PA was increased up to 10-fold when the protease-deficient strain E. coli BL21(DE3) was cultivated in medium without a proteinaceous substrate, as confirmed by a decrease in the sum of the constants k(d) and k(t). The original signal sequence of pre-pro-PA was exchanged with the OmpT signal peptide sequence in order to increase translocation efficiency; the effects of this change varied in the different E. coli host strains. Furthermore, we established that simultaneous coexpression of the OmpT pac gene with some proteins of the Sec export machinery of the cell resulted in up to threefold-enhanced PA production. In parallel, we made efforts to increase PA flux via coexpression with the kil gene (killing protein). The primary effects of the kil gene were the release of PA into the extracellular medium and an approximately threefold increase in the total amount of PA produced per liter of bacterial culture.
Ignatova
Z
Z
Institut für Biotecnologie II, Technische Universität Hamburg-Harburg, 21073 Hamburg, Germany. ignatova@tuhh.de
Mahsunah
A
A
Georgieva
M
M
Kasche
V
V
D001426:Bacterial Proteins; D001709:Biotechnology / Q000379:methods; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D010399:Penicillin Amidase / Q000235:genetics* / Q000378:metabolism*; D019897:Periplasm / Q000201:enzymology; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D003470:Culture Media; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C069003:kil protein, E coli; D012697:Serine Endopeptidases; C057840:omptin outer membrane protease; D010399:Penicillin Amidase