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Changing a Single Amino Acid in Clostridium perfringens �-Toxin Affects the Efficiency of Heterologous Secretion by Bacillus subtilis �

By Reindert Nijl, René Heerlien, Leendert W. Hamoen and Oscar P. Kuipers

Abstract

Achieving 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 be efficiently 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 the global gene regulation responses 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 is 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 the rapid degradation of �-toxoid following secretion across the cytoplasmic membrane. In contrast to �-toxin, �-toxoid protein is more prone to degradation directly after secretion, most likely due to poor folding characteristics introduced with 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. Bacillus subtilis is widely used for protein production an

Year: 2006
OAI identifier: oai:CiteSeerX.psu:10.1.1.319.8153
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