This Article
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 Wu, X C
Right arrow Articles by Wong, S L
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wu, X C
Right arrow Articles by Wong, S L

 Previous Article  |  Next Article 

J Bacteriol. 1991 August; 173(16): 4952-4958

research-article

Engineering a Bacillus subtilis expression-secretion system with a strain deficient in six extracellular proteases.

X C Wu, W Lee, L Tran and S L Wong

Department of Biological Sciences, University of Calgary, Alberta, Canada.

ABSTRACT

We describe the development of an expression-secretion system in Bacillus subtilis to improve the quality and quantity of the secreted foreign proteins. This system consists of a strain (WB600) deficient in six extracellular proteases and a set of sacB-based expression vectors. With the inactivation of all six chromosomal genes encoding neutral protease A, subtilisin, extracellular protease, metalloprotease, bacillopeptidase F, and neutral protease B, WB600 showed only 0.32% of the wild-type extracellular protease activity. No residual protease activity could be detected when WB600 was cultured in the presence of 2 mM phenylmethylsulfonyl fluoride. By using TEM beta-lactamase as a model, we showed that WB600 can significantly improve the stability of the secreted enzyme. To further increase the production level we constructed an expression cassette carrying sacY, a sacB-specific regulatory gene. This gene was placed under the control of a strong, constitutively expressed promoter, P43. With this cassette in the expression vector, an 18-fold enhancement in beta-lactamase production was observed. An artificial operon, P43-sacY-degQ, was also constructed. However, only a partial additive enhancement effect (24-fold enhancement) was observed. Although degQ can stimulate the production of beta-lactamase in the system, its ability to increase the residual extracellular protease activity from WB600 limits its application. The use of the P43-sacY cassette and WB600 would be a better combination for producing intact foreign proteins in high yield.

J Bacteriol. 1991 August; 173(16): 4952-4958




This article has been cited by other articles:

  • Chen, X., Zhu, F., Cao, Y., Qiao, S. (2009). Novel Expression Vector for Secretion of Cecropin AD in Bacillus subtilis with Enhanced Antimicrobial Activity. Antimicrob. Agents Chemother. 53: 3683-3689 [Abstract] [Full Text]  
  • Kouwen, T. R. H. M., Dubois, J.-Y. F., Freudl, R., Quax, W. J., van Dijl, J. M. (2008). Modulation of Thiol-Disulfide Oxidoreductases for Increased Production of Disulfide-Bond-Containing Proteins in Bacillus subtilis. Appl. Environ. Microbiol. 74: 7536-7545 [Abstract] [Full Text]  
  • Matsuoka, S., Yukawa, H., Inui, M., Doi, R. H. (2007). Synergistic Interaction of Clostridium cellulovorans Cellulosomal Cellulases and HbpA. J. Bacteriol. 189: 7190-7194 [Abstract] [Full Text]  
  • Hong, B., Wang, L., Lammertyn, E., Geukens, N., Van Mellaert, L., Li, Y., Anne, J. (2005). Inactivation of the 20S proteasome in Streptomyces lividans and its influence on the production of heterologous proteins. Microbiology 151: 3137-3145 [Abstract] [Full Text]  
  • Westers, H., Braun, P. G., Westers, L., Antelmann, H., Hecker, M., Jongbloed, J. D. H., Yoshikawa, H., Tanaka, T., van Dijl, J. M., Quax, W. J. (2005). Genes Involved in SkfA Killing Factor Production Protect a Bacillus subtilis Lipase against Proteolysis. Appl. Environ. Microbiol. 71: 1899-1908 [Abstract] [Full Text]  
  • Cho, H.-Y., Yukawa, H., Inui, M., Doi, R. H., Wong, S.-L. (2004). Production of Minicellulosomes from Clostridium cellulovorans in Bacillus subtilis WB800. Appl. Environ. Microbiol. 70: 5704-5707 [Abstract] [Full Text]  
  • Tjalsma, H., Antelmann, H., Jongbloed, J. D.H., Braun, P. G., Darmon, E., Dorenbos, R., Dubois, J.-Y. F., Westers, H., Zanen, G., Quax, W. J., Kuipers, O. P., Bron, S., Hecker, M., van Dijl, J. M. (2004). Proteomics of Protein Secretion by Bacillus subtilis: Separating the "Secrets" of the Secretome. Microbiol. Mol. Biol. Rev. 68: 207-233 [Abstract] [Full Text]  
  • Vitikainen, M., Lappalainen, I., Seppala, R., Antelmann, H., Boer, H., Taira, S., Savilahti, H., Hecker, M., Vihinen, M., Sarvas, M., Kontinen, V. P. (2004). Structure-Function Analysis of PrsA Reveals Roles for the Parvulin-like and Flanking N- and C-terminal Domains in Protein Folding and Secretion in Bacillus subtilis. J. Biol. Chem. 279: 19302-19314 [Abstract] [Full Text]  
  • Ber, R., Mamroud, E., Aftalion, M., Tidhar, A., Gur, D., Flashner, Y., Cohen, S. (2003). Development of an Improved Selective Agar Medium for Isolation of Yersinia pestis. Appl. Environ. Microbiol. 69: 5787-5792 [Abstract] [Full Text]  
  • Lian, Q., Szarka, S. J., Ng, K. K. S., Wong, S.-L. (2003). Engineering of a Staphylokinase-based Fibrinolytic Agent with Antithrombotic Activity and Targeting Capability toward Thrombin-rich Fibrin and Plasma Clots. J. Biol. Chem. 278: 26677-26686 [Abstract] [Full Text]  
  • Williams, R. C., Rees, M. L., Jacobs, M. F., Pragai, Z., Thwaite, J. E., Baillie, L. W. J., Emmerson, P. T., Harwood, C. R. (2003). Production of Bacillus anthracis Protective Antigen Is Dependent on the Extracellular Chaperone, PrsA. J. Biol. Chem. 278: 18056-18062 [Abstract] [Full Text]  
  • Wu, S.-C., Castellino, F. J., Wong, S.-L. (2003). A Fast-acting, Modular-structured Staphylokinase Fusion with Kringle-1 from Human Plasminogen as the Fibrin-targeting Domain Offers Improved Clot Lysis Efficacy. J. Biol. Chem. 278: 18199-18206 [Abstract] [Full Text]  
  • Airaksinen, U., Penttila, T., Wahlstrom, E., Vuola, J. M., Puolakkainen, M., Sarvas, M. (2003). Production of Chlamydia pneumoniae Proteins in Bacillus subtilis and Their Use in Characterizing Immune Responses in the Experimental Infection Model. CVI 10: 367-375 [Abstract] [Full Text]  
  • Gat, O., Inbar, I., Aloni-Grinstein, R., Zahavy, E., Kronman, C., Mendelson, I., Cohen, S., Velan, B., Shafferman, A. (2003). Use of a Promoter Trap System in Bacillus anthracis and Bacillus subtilis for the Development of Recombinant Protective Antigen-Based Vaccines. Infect. Immun. 71: 801-813 [Abstract] [Full Text]  
  • Wu, S.-C., Yeung, J. C., Duan, Y., Ye, R., Szarka, S. J., Habibi, H. R., Wong, S.-L. (2002). Functional Production and Characterization of a Fibrin-Specific Single-Chain Antibody Fragment from Bacillus subtilis: Effects of Molecular Chaperones and a Wall-Bound Protease on Antibody Fragment Production. Appl. Environ. Microbiol. 68: 3261-3269 [Abstract] [Full Text]  
  • Murashima, K., Chen, C.-L., Kosugi, A., Tamaru, Y., Doi, R. H., Wong, S.-L. (2002). Heterologous Production of Clostridium cellulovorans engB, Using Protease-Deficient Bacillus subtilis, and Preparation of Active Recombinant Cellulosomes. J. Bacteriol. 184: 76-81 [Abstract] [Full Text]  
  • Thwaite, J. E., Baillie, L. W. J., Carter, N. M., Stephenson, K., Rees, M., Harwood, C. R., Emmerson, P. T. (2002). Optimization of the Cell Wall Microenvironment Allows Increased Production of Recombinant Bacillus anthracis Protective Antigen from B. subtilis. Appl. Environ. Microbiol. 68: 227-234 [Abstract] [Full Text]  
  • Hudson, K. D., Corfe, B. M., Kemp, E. H., Feavers, I. M., Coote, P. J., Moir, A. (2001). Localization of GerAA and GerAC Germination Proteins in the Bacillus subtilis Spore. J. Bacteriol. 183: 4317-4322 [Abstract] [Full Text]  
  • van Roosmalen, M. L., Jongbloed, J. D. H., Jong, A. d., van Eerden, J., Venema, G., Bron, S., Maarten van Dijl, J. (2001). Detergent-independent in vitro activity of a truncated Bacillus signal peptidase. Microbiology 147: 909-917 [Abstract] [Full Text]  
  • Jensen, C. L., Stephenson, K., Jørgensen, S. T., Harwood, C. (2000). Cell-associated degradation affects the yield of secreted engineered and heterologous proteins in the Bacillus subtilis expression system. Microbiology 146: 2583-2594 [Abstract] [Full Text]  
  • Tjalsma, H., Bolhuis, A., Jongbloed, J. D. H., Bron, S., van Dijl, J. M. (2000). Signal Peptide-Dependent Protein Transport in Bacillus subtilis: a Genome-Based Survey of the Secretome. Microbiol. Mol. Biol. Rev. 64: 515-547 [Abstract] [Full Text]  
  • Cohen, S., Mendelson, I., Altboum, Z., Kobiler, D., Elhanany, E., Bino, T., Leitner, M., Inbar, I., Rosenberg, H., Gozes, Y., Barak, R., Fisher, M., Kronman, C., Velan, B., Shafferman, A. (2000). Attenuated Nontoxinogenic and Nonencapsulated Recombinant Bacillus anthracis Spore Vaccines Protect against Anthrax. Infect. Immun. 68: 4549-4558 [Abstract] [Full Text]  
  • Lee, S. J., Kim, D. M., Bae, K. H., Byun, S. M., Chung, J. H. (2000). Enhancement of Secretion and Extracellular Stability of Staphylokinase in Bacillus subtilis by wprA Gene Disruption. Appl. Environ. Microbiol. 66: 476-480 [Abstract] [Full Text]  
  • Bolhuis, A., Tjalsma, H., Smith, H. E., de Jong, A., Meima, R., Venema, G., Bron, S., van Dijl, J. M. (1999). Evaluation of Bottlenecks in the Late Stages of Protein Secretion in Bacillus subtilis. Appl. Environ. Microbiol. 65: 2934-2941 [Abstract] [Full Text]  
  • Stephenson, K., Harwood, C. R. (1998). Influence of a Cell-Wall-Associated Protease on Production of alpha -Amylase by Bacillus subtilis. Appl. Environ. Microbiol. 64: 2875-2881 [Abstract] [Full Text]  
  • Wu, S.-C., Ye, R., Wu, X.-C., Ng, S.-C., Wong, S.-L. (1998). Enhanced Secretory Production of a Single-Chain Antibody Fragment from Bacillus subtilis by Coproduction of Molecular Chaperones. J. Bacteriol. 180: 2830-2835 [Abstract] [Full Text]  
  • Wu, X.-C., Ye, R., Duan, Y., Wong, S.-L. (1998). Engineering of Plasmin-Resistant Forms of Streptokinase and Their Production in Bacillus subtilis: Streptokinase with Longer Functional Half-Life. Appl. Environ. Microbiol. 64: 824-829 [Abstract] [Full Text]  
  • Farchaus, J. W., Ribot, W. J., Jendrek, S., Little, S. F. (1998). Fermentation, Purification, and Characterization of Protective Antigen from a Recombinant, Avirulent Strain of Bacillus anthracis. Appl. Environ. Microbiol. 64: 982-991 [Abstract] [Full Text]  
  • Poon, K. K. H., Chen, C.-L., Wong, S.-L. (2001). Roles of Glucitol in the GutR-mediated Transcription Activation Process in Bacillus subtilis. TIGHT BINDING OF GutR TO ITS BINDING SITE. J. Biol. Chem. 276: 9620-9625 [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»