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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

  • Applied Genetics and Molecular Biotechnology
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Abstract

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.

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Acknowledgments

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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Author notes

  1. Michael Caspers

    Present address: Sanofi-Aventis Deutschland GmbH, Industriepark Höchst, 65926, Frankfurt am Main, Germany

  2. Ulf Brockmeier

    Present address: Institut für Physiologie, Universität Duisburg-Essen, Hufelandstrasse 55, 45122, Essen, Germany

Authors and Affiliations

  1. Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany

    Michael Caspers & Roland Freudl

  2. Institut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich GmbH, 52426, Jülich, Germany

    Ulf Brockmeier, Christian Degering & Thorsten Eggert

  3. evocatal GmbH, Merowingerplatz 1a, 40225, Düsseldorf, Germany

    Christian Degering & Thorsten Eggert

Authors
  1. Michael Caspers
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  2. Ulf Brockmeier
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  3. Christian Degering
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  4. Thorsten Eggert
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  5. Roland Freudl
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Corresponding author

Correspondence to Roland Freudl.

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Michael Caspers and Ulf Brockmeier contributed equally to this work.

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Caspers, M., Brockmeier, U., Degering, C. et al. 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. Appl Microbiol Biotechnol 86, 1877–1885 (2010). https://doi.org/10.1007/s00253-009-2405-x

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  • DOI: https://doi.org/10.1007/s00253-009-2405-x

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