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Appl. Environ. Microbiol. doi:10.1128/AEM.02356-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Changing a single amino acid in Clostridium perfringens -toxin affects efficiency of heterologous secretion by Bacillus subtilis

Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands

^* To whom correspondence should be addressed. Email: o.p.kuipers{at}rug.nl.

	Abstract

Efficient heterologous protein production and secretion by Bacillus subtilis is an attractive prospect, although often disappointingly low yields are reached. The expression of detoxified Clostridium perfringens -toxin (-toxoid) is exemplary for this. Although -toxin can efficiently be expressed and secreted by Bacillus subtilis, the genetically detoxified, and industrially interesting, -toxoid variant is difficult to obtain in high amounts. To optimize the expression of this putative vaccine component, we studied the differences in global gene regulation response of B. subtilis to overproduction of either -toxin or -toxoid by transcriptomics. A clear difference was the upregulation of the CssRS regulon, known to be induced upon secretion stress, when -toxoid was produced. YkoJ, a protein of unknown function, was also upregulated, and we show that its expression is dependent on cssS. We then focused on the heterologous protein itself and found that the major secretion bottleneck can be traced back to a single amino acid substitution between the -toxin and the -toxoid, which results in rapid degradation of -toxoid following secretion across the cytoplasm membrane. In contrast to -toxin, -toxoid protein is more prone to degradation directly after secretion, most likely due to poor folding characteristics introduced with the point mutations. Our results show that, although the host can be adapted in many ways, the intrinsic properties of a heterologous protein can play a decisive role when optimizing heterologous protein production.