Systematic Screening of All Signal Peptides from Bacillus subtilis: A Powerful Strategy in Optimizing Heterologous Protein Secretion in Gram-positive Bacteria Ulf Brockmeier 1 , Michael Caspers 2 , Roland Freudl 2 Alexander Jockwer 3 , Thomas Noll 3 and Thorsten Eggert 1 ⁎ 1 Institut für Molekulare Enzymtechnologie, Heinrich- Heine-Universität Düsseldorf im Forschungszentrum Jülich D-52426 Jülich, Germany 2 Institut für Biotechnologie 1 Forschungszentrum Jülich GmbH, D-52425 Jülich Germany 3 Institut für Biotechnologie 2 Forschungszentrum Jülich GmbH, D-52425 Jülich Germany Efficient protein secretion is very important in biotechnology as it provides active and stable enzymes, which are an essential prerequisite for successful biocatalysis. Therefore, optimizing enzyme-producing bacterial strains is a major challenge in the field of biotechnology and protein production. In this study, the Gram-positive model bacterium Bacillus subtilis was optimized for heterologous protein secretion using a novel approach. Two lipolytic enzymes, cutinase from Fusarium solani pisi and a cytoplasmatic esterase of metagenomic origin, were chosen as reporters for heterologous protein secretion. In a systematic screening approach, all naturally occurring (non- lipoprotein) Sec-type signal peptides (SPs) from B. subtilis were character- ized for their potential in heterologous protein secretion. Surprisingly, optimal SPs in cutinase secretion were inefficient in esterase secretion and vice versa, indicating the importance of an optimal fit between the SP and the respective mature part of the desired secretion target proteins. These results highlight the need for individually optimal signal peptides for every heterologous secretion target. Therefore, the SP library generated in this study represents a powerful tool for secretion optimization in Gram- positive expression hosts. © 2006 Elsevier Ltd. All rights reserved. *Corresponding author Keywords: protein secretion; signal peptides; secretion efficiency; cutinase; Bacillus subtilis Introduction Efficient protein secretion is very important in biotechnology as it provides active and stable enzymes, which are an essential prerequisite for successful biocatalysis. Therefore, optimizing enzyme-producing bacterial strains is a major challenge in the field of industrial biotechnology, also known as White Biotechnology. As one of the most important industrial fermentation hosts, the Gram-positive bacterium Bacillus subtilis has been investigated intensively over the past few decades with respect to its potential for the secretion of heterologous proteins. In addition to the compo- nents of the secretion apparatus, the signal peptides (SPs) that channel the export proteins into the secretion machinery play a key role in the transloca- tion across the membrane. Therefore, SPs were investigated in detail with respect to their amino acid composition and to their role in membrane translocation of exported proteins. 1–5 Signal peptides share some common characteristic features, conserved in different organisms. Most SPs are composed of three distinct regions: (i) the positively charged N domain; (ii) the hydrophobic core region, the so-called H domain; and (iii) the hydrophilic signal peptidase (SPase) recognition site, termed the C domain. Based on these criteria, many signal peptide prediction tools have been developed 6 with SignalP being the most popular and user-friendly programm. SignalP is freely Present addresses: A. Jockwer, Roche Diagnostics GmbH, Penzberg, Germany; T. Noll, Cell Culture Technology, Technical Faculty, University of Bielefeld, DQ33501 Bielefeld, Germany. Abbreviation used: SP, signal peptide. E-mail address of the corresponding author: t.eggert@fz-juelich.de doi:10.1016/j.jmb.2006.07.034 J. Mol. Biol. (2006) 362, 393–402 0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.