Changing a Single Amino Acid in Clostridium perfringens -Toxin Affects the 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.
Applied and Environmental Microbiology (Impact Factor: 3.67). 04/2007; 73(5):1586-93. DOI: 10.1128/AEM.02356-06
Source: PubMed


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 β-toxin (β-toxoid) is exemplary for this. Although β-toxin can be efficiently expressed and secreted by Bacillus subtilis, the genetically detoxified, and industrially interesting, β-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 β-toxin or β-toxoid by transcriptomics. A clear difference was the upregulation of the CssRS
regulon, known to be induced upon secretion stress, when β-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 β-toxin and the β-toxoid, which results in the rapid degradation of β-toxoid following
secretion across the cytoplasmic membrane. In contrast to β-toxin, β-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.

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    • "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]. "
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    ABSTRACT: Background Bacillus subtilis is a favorable host for the production of industrially relevant proteins because of its capacity of secreting proteins into the medium to high levels, its GRAS (Generally Recognized As Safe) status, its genetic accessibility and its capacity to grow in large fermentations. However, production of heterologous proteins still faces limitations. Results This study aimed at the identification of bottlenecks in secretory protein production by analyzing the response of B. subtilis at the transcriptome level to overproduction of eight secretory proteins of endogenous and heterologous origin and with different subcellular or extracellular destination: secreted proteins (NprE and XynA of B. subtilis, Usp45 of Lactococcus lactis, TEM-1 β-lactamase of Escherichia coli), membrane proteins (LmrA of L. lactis and XylP of Lactobacillus pentosus) and lipoproteins (MntA and YcdH of B. subtilis). Responses specific for proteins with a common localization as well as more general stress responses were observed. The latter include upregulation of genes encoding intracellular stress proteins (groES/EL, CtsR regulated genes). Specific responses include upregulation of the liaIHGFSR operon under Usp45 and TEM-1 β-lactamase overproduction; cssRS, htrA and htrB under all secreted proteins overproduction; sigW and SigW-regulated genes mainly under membrane proteins overproduction; and ykrL (encoding an HtpX homologue) specifically under membrane proteins overproduction. Conclusions The results give better insights into B. subtilis responses to protein overproduction stress and provide potential targets for genetic engineering in order to further improve B. subtilis as a protein production host.
    Full-text · Article · May 2012 · Microbial Cell Factories
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    • "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. "
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    ABSTRACT: Background The Gram-positive bacterium Bacillus subtilis is an important producer of high quality industrial enzymes and a few eukaryotic proteins. Most of these proteins are secreted into the growth medium, but successful examples of cytoplasmic protein production are also known. Therefore, one may anticipate that the high protein production potential of B. subtilis can be exploited for protein complexes and membrane proteins to facilitate their functional and structural analysis. The high quality of proteins produced with B. subtilis results from the action of cellular quality control systems that efficiently remove misfolded or incompletely synthesized proteins. Paradoxically, cellular quality control systems also represent bottlenecks for the production of various heterologous proteins at significant concentrations. Conclusion While inactivation of quality control systems has the potential to improve protein production yields, this could be achieved at the expense of product quality. Mechanisms underlying degradation of secretory proteins are nowadays well understood and often controllable. It will therefore be a major challenge for future research to identify and modulate quality control systems of B. subtilis that limit the production of high quality protein complexes and membrane proteins, and to enhance those systems that facilitate assembly of these proteins.
    Full-text · Article · Feb 2008 · Microbial Cell Factories
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    ABSTRACT: Transcriptome analysis was used to investigate the global stress response of the gram-positive bacterium Bacillus subtilis caused by overproduction of the well-secreted AmyQ α-amylase from Bacillus amyloliquefaciens. Analyses of the control and overproducing strains were carried out at the end of exponential growth and in stationary phase, when protein secretion from B. subtilis is optimal. Among the genes that showed increased expression were htrA and htrB, which are part of the CssRS regulon, which responds to high-level protein secretion and heat stress. The analysis of the transcriptome profiles of a cssS mutant compared to the wild type, under identical secretion stress conditions, revealed several genes with altered transcription in a CssRS-dependent manner, for example, citM, ylxF, yloA, ykoJ, and several genes of the flgB operon. However, high-affinity CssR binding was observed only for htrA, htrB, and, possibly, citM. In addition, the DNA macroarray approach revealed that several genes of the sporulation pathway are downregulated by AmyQ overexpression and that a group of motility-specific (σD-dependent) transcripts were clearly upregulated. Subsequent flow-cytometric analyses demonstrate that, upon overproduction of AmyQ as well as of a nonsecretable variant of the α-amylase, the process of sporulation is severely inhibited. Similar experiments were performed to investigate the expression levels of the hag promoter, a well-established reporter for σD-dependent gene expression. This approach confirmed the observations based on our DNA macroarray analyses and led us to conclude that expression levels of several genes involved in motility are maintained at high levels under all conditions of α-amylase overproduction.
    Full-text · Article · Sep 2007 · Applied and Environmental Microbiology
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