Applied Microbiology and Biotechnology

, Volume 86, Issue 6, pp 1877–1885 | Cite as

Improvement of Sec-dependent secretion of a heterologous model protein in Bacillus subtilis by saturation mutagenesis of the N-domain of the AmyE signal peptide

  • Michael Caspers
  • Ulf Brockmeier
  • Christian Degering
  • Thorsten Eggert
  • Roland Freudl
Applied Genetics and Molecular Biotechnology


Due to the lack of an outer membrane, Gram-positive bacteria (e.g., Bacillus species) are considered as promising host organisms for the secretory production of biotechnologically relevant heterologous proteins. However, the yields of the desired target proteins were often reported to be disappointingly low. Here, we used saturation mutagenesis of the positively charged N-domain (positions 2–7) of the signal peptide of the Bacillus subtilis α-amylase (AmyE) as a novel approach for the improvement of the secretion of a heterologous model protein, cutinase from Fusarium solani pisi, by the general secretory pathway of B. subtilis. Automated high-throughput screening of the resulting signal peptide libraries allowed for the identification of four single point mutations that resulted in significantly increased cutinase amounts, three of which surprisingly reduced the net charge of the N-domain from +3 to +2. Characterization of the effects of the identified mutations on protein synthesis and export kinetics by pulse-chase analyses indicates that an optimal balance between biosynthesis and the flow of the target protein through all stages of the B. subtilis secretion pathway is of crucial importance with respect to yield and quality of secreted heterologous proteins.


Heterologous protein secretion Signal peptide Saturation mutagenesis Bacillus subtilis 



We are very grateful to H. Sahm, K.-E. Jaeger, and M. Bott for their support. M.C. was in part funded by the Graduiertenkolleg GRK 57/3-03 “Molekulare Physiologie: Stoff- und Energieumwandlung”. U.B was a recipient of a scholarship from the European Graduate College 795 entitled “Regulatory Circuits in Cellular Systems: Fundamentals and Biotechnological Applications” funded by the Deutsche Forschungsgemeinschaft (DFG).


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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Michael Caspers
    • 1
    • 4
  • Ulf Brockmeier
    • 2
    • 5
  • Christian Degering
    • 2
    • 3
  • Thorsten Eggert
    • 2
    • 3
  • Roland Freudl
    • 1
  1. 1.Institut für Biotechnologie 1Forschungszentrum Jülich GmbHJülichGermany
  2. 2.Institut für Molekulare EnzymtechnologieHeinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich GmbHJülichGermany
  3. 3.evocatal GmbH, Merowingerplatz 1aDüsseldorfGermany
  4. 4.Sanofi-Aventis Deutschland GmbHIndustriepark HöchstFrankfurt am MainGermany
  5. 5.Institut für PhysiologieUniversität Duisburg-EssenEssenGermany

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