This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ignatova, Z.
Right arrow Articles by Kasche, V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ignatova, Z.
Right arrow Articles by Kasche, V.
Agricola
Right arrow Articles by Ignatova, Z.
Right arrow Articles by Kasche, V.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, February 2003, p. 1237-1245, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.1237-1245.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Improvement of Posttranslational Bottlenecks in the Production of Penicillin Amidase in Recombinant Escherichia coli Strains

Z. Ignatova,* A. Mahsunah, M. Georgieva, and V. Kasche

Institut für Biotecnologie II, Technische Universität Hamburg-Harburg, 21073 Hamburg, Germany

Received 25 June 2002/ Accepted 12 November 2002

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 (kd) and transport (kt) 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 kd and kt. 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.

* Corresponding author. Mailing address: Institut für Biotecnologie II, Technische Universität Hamburg-Harburg, Denickestr. 15, 21073 Hamburg, Germany. Phone: 49 40 42878 2204. Fax: 49 40 42878 2127. E-mail: ignatova{at}tuhh.de.

Applied and Environmental Microbiology, February 2003, p. 1237-1245, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.1237-1245.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Monroy-Lagos, O., Soberon, X., Gaytan, P., Osuna, J. (2006). Improvement of an unusual twin-arginine transporter leader Peptide by a codon-based randomization approach.. Appl. Environ. Microbiol. 72: 3797-3801 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»