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

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.

Keywords

Heterologous protein secretion Signal peptide Saturation mutagenesis Bacillus subtilis 

Notes

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).

References

  1. Airaksinen A, Hovi T (1998) Modified base compositions at degenerate positions of a mutagenic oligonucleotide enhance randomness in site-saturation mutagenesis. Nucleic Acids Res 26:576–581CrossRefGoogle Scholar
  2. Akita M, Sasaki S, Matsuyama SI, Mizushima S (1990) SecA interacts with secretory proteins by recognizing the positive charge at the amino terminus of the signal peptide in Escherichia coli. J Biol Chem 265:8164–8169Google Scholar
  3. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: signalP 3.0. J Mol Biol 340:783–795CrossRefGoogle Scholar
  4. Borchert TV, Nagarajan V (1991) Effect of signal sequence alterations on export of levansucrase in Bacillus subtilis. J Bacteriol 173:276–282Google Scholar
  5. Brockmeier U, Caspers M, Freudl R, Jockwer A, Noll T, Eggert T (2006) Systematic screening of all signal peptides from Bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in Gram-positive bacteria. J Mol Biol 362:393–402CrossRefGoogle Scholar
  6. Carvalho CML, Aires-Barros MR, Cabral JM (1999) Cutinase: from molecular level to bioprocess development. Biotechnol Bioeng 66:17–34CrossRefGoogle Scholar
  7. Chen M, Nagarajan V (1994) Effect of alteration of charged residues at the N-termini of signal peptides on protein export in Bacillus subtilis. J Bacteriol 176:5796–5801Google Scholar
  8. Darmon E, Noone D, Masson A, Bron S, Kuipers OP, Devine KM, van Dijl JM (2002) A novel class of heat and secretion stress-responsive genes is controlled by the autoregulated CssRS two-component system of Bacillus subtilis. J Bacteriol 184:5661–5671CrossRefGoogle Scholar
  9. Eggert T, Brockmeier U, Dröge MJ, Quax WJ, Jaeger KE (2003) Extracellular lipases from Bacillus subtilis: regulation of gene expression and enzyme activity by amino acid supply and external pH. FEMS Microbiol Lett 225:319–324CrossRefGoogle Scholar
  10. Faß SH, Engels JW (1996) Influence of specific signal peptide mutations on the expression and secretion of the alpha-amylase inhibitor tendamistat in Streptomyces lividans. J Biol Chem 271:15244–15252Google Scholar
  11. Harwood CR, Cranenburgh R (2008) Bacillus protein secretion: an unfolding story. Trends Microbiol 16:73–79Google Scholar
  12. Hemilä H, Pakkanen R, Heikinheimo R, Palva ET, Palva I (1992) Expression of the Erwinia carotovora polygalacturonase-encoding gene in Bacillus subtilis: role of the signal peptide fusions on production of a heterologous protein. Gene 116:27–33CrossRefGoogle Scholar
  13. Jaeger KE, Eggert T, Eippner A, Reetz MT (2001) Directed evolution and the creation of enantioselective biocatalysts. Appl Microbiol Biotechnol 55:519–530CrossRefGoogle Scholar
  14. Kebir MO, Kendall DA (2002) SecA specificity for different signal peptides. Biochemistry 41:5573–5580CrossRefGoogle Scholar
  15. Kontinen VP, Sarvas M (1993) The PrsA lipoprotein is essential for protein secretion in Bacillus subtilis and sets a limit for high-level secretion. Mol Microbiol 8:727–737CrossRefGoogle Scholar
  16. Kunst F, Rapoport G (1995) Salt stress is an environmental signal affecting degradative enzyme synthesis in Bacillus subtilis. J Bacteriol 177:2403–2407Google Scholar
  17. Lehnhardt S, Pollitt S, Inouye M (1987) The differential effect on two hybrid proteins of deletion mutations within the hydrophobic region of the Escherichia coli OmpA signal peptide. J Biol Chem 262:1716–1719Google Scholar
  18. Lehnhardt S, Pollit NS, Goldstein J, Inouye M (1988) Modulation of the effects of mutations in the basic region of the OmpA signal peptide by the mature portion of the protein. J Biol Chem 263:10300–10303Google Scholar
  19. Leloup L, Driessen AJM, Freudl R, Chambert R, Petit-Glatron MF (1999) Differential dependence of levansucrase and α-amylase secretion on SecA (Div) during the exponential phase of growth of Bacillus subtilis. J Bacteriol 181:1820–1826Google Scholar
  20. Martinez C, De Geus P, Lauwereys M, Matthyssens G, Cambillau C (1992) Fusarium solani cutinase is a lipolytic enzyme with a catalytic serine accessible to solvent. Nature 356:615–618CrossRefGoogle Scholar
  21. Mathiesen G, Sveen A, Piard JC, Axelsson L, Eijsink VG (2008) Heterologous protein secretion by Lactobacillus plantarum using homologous signal peptides. J Appl Microbiol 105:215–226CrossRefGoogle Scholar
  22. Mathiesen G, Sveen A, Brurberg MB, Fredriksen L, Axelsson L, Eijsink VGH (2009) Genome-wide analysis of signal peptide functionality in Lactobacillus plantarum WCFSI. BMC Genomics 10:425CrossRefGoogle Scholar
  23. 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 72:3797–3801CrossRefGoogle Scholar
  24. Nakamura K, Itoh Y, Yamane K (1988) Enhanced secretion of ß-lactamase on structural modification of the Bacillus subtilis alpha-amylase signal peptide. J Biochem (Tokyo) 104:265–269Google Scholar
  25. Nakamura K, Fujita Y, Itoh Y, Yamane K (1989) Modification of length, hydrophobic properties and electric charge of Bacillus subtilis α-amylase signal peptide and their different effects on the production of secretory proteins in B. subtilis and Escherichia coli. Mol Gen Genet 216:1–9CrossRefGoogle Scholar
  26. Natale P, Brüser T, Driessen AJM (2008) Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane—distinct translocases and mechanisms. Biochim Biophys Acta 1778:1735–1756CrossRefGoogle Scholar
  27. Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6CrossRefGoogle Scholar
  28. Peterson JH, Woolhead CA, Bernstein HD (2003) Basic amino acids in a distinct subset of signal peptides promote interaction with the signal recognition particle. J Biol Chem 278:46155–46162CrossRefGoogle Scholar
  29. Saunders CW, Pedroni JA, Monahan PM (1991) Optimization of the signal sequence cleavage site for secretion from Bacillus subtilis of a 34-amino acid fragment of human parathyroid hormone. Gene 102:277–282CrossRefGoogle Scholar
  30. Schallmey M, Singh A, Ward OP (2004) Developments in the use of Bacillus species for industrial production. Can J Microbiol 50:1–17CrossRefGoogle Scholar
  31. van Dijl JM, de Jong A, Smith H, Bron S, Venema G (1991) Non-functional expression of Escherichia coli signal peptidase I in Bacillus subtilis. J Gen Microbiol 137:2073–2083Google Scholar
  32. van Wely KHM, Swaving J, Freudl R, Driessen AJM (2001) Translocation of proteins across the cell envelope of Gram-positive bacteria. FEMS Microbiol Rev 25:437–454CrossRefGoogle Scholar
  33. Vasantha N, Balakrishnan R, Kaur S, Jayaraman K (1980) Biosynthesis of polymyxin by Bacillus polymyxa. I. The status of the biosynthetic multienzyme complex during active antibiotic synthesis and sporulation. Arch Biochem Biophys 200:40–44CrossRefGoogle Scholar
  34. von Heijne G (1990) The signal peptide. J Membr Biol 115:195–201CrossRefGoogle Scholar
  35. Watanabe K, Tsuchida Y, Okibe N, Teramoto H, Suzuki N, Inui M, Yukawa H (2009) Scanning the Corynebacterium glutamicum R genome for high-efficiency secretion signal sequences. Microbiology (UK) 155:741–750CrossRefGoogle Scholar
  36. Winkler UK, Stuckmann M (1979) Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. J Bacteriol 138:663–670Google Scholar
  37. Zanen G, Houben EN, Meima R, Tjalsma H, Jongbloed JD, Westers H, Oudega B, Luirink J, van Dijl JM, Quax WJ (2005) Signal peptide hydrophobicity is critical for early stages in protein export by Bacillus subtilis. FEBS J 272:4617–4630CrossRefGoogle Scholar

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

Personalised recommendations