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Applied and Environmental Microbiology, July 1999, p. 2934-2941, Vol. 65, No. 7
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Evaluation of Bottlenecks in the Late Stages of Protein Secretion in Bacillus subtilis

Albert Bolhuis, Harold Tjalsma, Hilde E. Smith,dagger Anne de Jong, Rob Meima,Dagger Gerard Venema, Sierd Bron, and Jan Maarten van Dijl*

Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751 NN Haren, The Netherlands

Received 8 February 1999/Accepted 13 April 1999

Despite a high capacity for secretion of homologous proteins, the secretion of heterologous proteins by Bacillus subtilis is frequently inefficient. In the present studies, we have investigated and compared bottlenecks in the secretion of four heterologous proteins: Bacillus lichenifomis alpha -amylase (AmyL), Escherichia coli TEM beta -lactamase (Bla), human pancreatic alpha -amylase (HPA), and a lysozyme-specific single-chain antibody. The same expression and secretion signals were used for all four of these proteins. Notably, all identified bottlenecks relate to late stages in secretion, following translocation of the preproteins across the cytoplasmic membrane. These bottlenecks include processing by signal peptidase, passage through the cell wall, and degradation in the wall and growth medium. Strikingly, all translocated HPA was misfolded, its stability depending on the formation of disulfide bonds. This suggests that the disulfide bond oxidoreductases of B. subtilis cannot form the disulfide bonds in HPA correctly. As the secretion bottlenecks differed for each heterologous protein tested, it is anticipated that the efficient secretion of particular groups of heterologous proteins with the same secretion bottlenecks will require the engineering of specifically optimized host strains.

* Corresponding author. Present address: Department of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands. Phone: 31 50 3633079. Fax: 31 50 3632348. E-mail: J.M.van.Dijl{at}farm.rug.nl.

dagger Present address: ID-DLO, 8200 AB, Lelystad, The Netherlands.

Dagger Present address: Department of Chemical Engineering, University of Washington, Seattle WA 98195-1750.

Applied and Environmental Microbiology, July 1999, p. 2934-2941, Vol. 65, No. 7
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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