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

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.

Comparative transcriptional analysis of Bacillus subtilis cells overproducing either secreted proteins, lipoproteins or membrane proteins

“…At the late stages, which include removal of the signal peptide, release from the translocase, folding and passing the cell wall, deficiency in signal peptidases, foldases, chaperones and presence of extracellular proteases resulting in incorrect folding of proteins and protein’s instability may also set limits to the secretion efficiency [1,3]. The focus on identification and later manipulation of factors involved in protein secretion have led to the improvement of B. subtilis as a production host, for example by deletion of extracellular and/or intracellular proteases [4-6], use of strong or inducible promoters [7-9], overproduction of chaperones [10,11] or signal peptidases [12,13], modification of the cell wall [14,15], protein modification [16,17] and deletion of stress responsive systems [18]. …”

Towards the development of Bacillus subtilis as a cell factory for membrane proteins and protein complexes

“…Besides engineering the host, also the expression system used to produce the protein can be modified in order to improve production and/or secretion, for example by the use of strong or inducible promoters [ 30 - 32 ]. Another strategy is to modify the protein that is being produced itself, for example by selecting an optimal signal peptide [ 33 , 34 ], or by rendering the protein less sensitive for degradation through site-specific mutagenesis [ 35 ]. The latter protein modification approach has the disadvantage that it can affect the functionality and folding of the protein.…”

Genetic or chemical protease inhibition causes significant changes in the Bacillus subtilis exoproteome

“…The CssS sensor senses "secretion stress" most probably by detection of malfolded protein accumulation at the membrane-cell wall interface [20]. Judged by its similarity with other known two-component systems, CssS autophosphorylates upon stimulation, and transfers the phosphate group to CssR, which then regulates the transcription of htrA, htrB, ykoJ, and the cssRS operon itself [24,25].…”