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Applied and Environmental Microbiology, December 2008, p. 7536-7545, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.00894-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Modulation of Thiol-Disulfide Oxidoreductases for Increased Production of Disulfide-Bond-Containing Proteins in Bacillus subtilis{triangledown}

Thijs R. H. M. Kouwen,1 Jean-Yves F. Dubois,1 Roland Freudl,2 Wim J. Quax,3 and Jan Maarten van Dijl1*

Department of Medical Microbiology, University Medical Center Groningen and University of Groningen, Hanzeplein 1, P.O. Box 30001, 9700 RB Groningen, The Netherlands,1 Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany,2 Department of Pharmaceutical Biology, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands3

Received 18 April 2008/ Accepted 15 October 2008

Disulfide bonds are important for the correct folding, structural integrity, and activity of many biotechnologically relevant proteins. For synthesis and subsequent secretion of these proteins in bacteria, such as the well-known "cell factory" Bacillus subtilis, it is often the correct formation of disulfide bonds that is the greatest bottleneck. Degradation of inefficiently or incorrectly oxidized proteins and the requirement for costly and time-consuming reduction and oxidation steps in the downstream processing of the proteins still are major limitations for full exploitation of B. subtilis for biopharmaceutical production. Therefore, the present study was aimed at developing a novel in vivo strategy for improved production of secreted disulfide-bond-containing proteins. Three approaches were tested: depletion of the major cytoplasmic reductase TrxA; introduction of the heterologous oxidase DsbA from Staphylococcus carnosus; and addition of redox-active compounds to the growth medium. As shown using the disulfide-bond-containing molecule Escherichia coli PhoA as a model protein, combined use of these three approaches resulted in secretion of amounts of active PhoA that were ~3.5-fold larger than the amounts secreted by the parental strain B. subtilis 168. Our findings indicate that Bacillus strains with improved oxidizing properties can be engineered for biotechnological production of heterologous high-value proteins containing disulfide bonds.

* Corresponding author. Mailing address: Department of Medical Microbiology, University Medical Center Groningen and University of Groningen, Hanzeplein 1, P.O. Box 30001, 9700 RB Groningen, The Netherlands. Phone: 31-50-3615187. Fax: 31-50-3619105. E-mail: J.M.van.Dijl{at}med.umcg.nl

{triangledown} Published ahead of print on 24 October 2008.

Applied and Environmental Microbiology, December 2008, p. 7536-7545, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.00894-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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