Contributions of the Pre- and Pro-Regions of a
            <i>Staphylococcus hyicus</i>
            Lipase to Secretion of a Heterologous Protein by
            <i>Bacillus subtilis</i>

Kouwen, Thijs R. H. M.; Nielsen, Allan K.; Denham, Emma L.; Dubois, Jean-Yves F.; Dorenbos, Ronald; Rasmussen, Michael; Quax, Wim J.; Freudl, Roland; Dijl, Jan Maarten van

doi:10.1128/aem.01671-09

Cited by 8 publications

(6 citation statements)

References 66 publications

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“…AmyQ was used as a control, because it was previously shown that its high-level expression from plasmid pKTH10 [ 33 ] causes strong induction of the P( htrA/htrB ) promoters [ 19 ]. Importantly, strain 168 Hm C5 contained a transcriptional fusion of the P(htrB) promoter with the gfpmut3 reporter gene [ 35 ]. To construct this fusion, the promoter region of htrB (~300 bp) plus an optimized ribosome-binding site (RBS) were fused directly upstream of the gfpmut3 gene, and the resulting construct was integrated into the genome by single cross-over recombination.…”

Section: Resultsmentioning

confidence: 99%

Homogeneity and heterogeneity in amylase production by Bacillus subtilis under different growth conditions

et al. 2016

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BackgroundBacillus subtilis is an important cell factory for the biotechnological industry due to its ability to secrete commercially relevant proteins in large amounts directly into the growth medium. However, hyper-secretion of proteins, such as α-amylases, leads to induction of the secretion stress-responsive CssR-CssS regulatory system, resulting in up-regulation of the HtrA and HtrB proteases. These proteases degrade misfolded proteins secreted via the Sec pathway, resulting in a loss of product. The aim of this study was to investigate the secretion stress response in B. subtilis 168 cells overproducing the industrially relevant α-amylase AmyM from Geobacillus stearothermophilus, which was expressed from the strong promoter P(amyQ)-M.ResultsHere we show that activity of the htrB promoter as induced by overproduction of AmyM was “noisy”, which is indicative for heterogeneous activation of the secretion stress pathway. Plasmids were constructed to allow real-time analysis of P(amyQ)-M promoter activity and AmyM production by, respectively, transcriptional and out-of-frame translationally coupled fusions with gfpmut3. Our results show the emergence of distinct sub-populations of high- and low-level AmyM-producing cells, reflecting heterogeneity in the activity of P(amyQ)-M. This most likely explains the heterogeneous secretion stress response. Importantly, more homogenous cell populations with regard to P(amyQ)-M activity were observed for the B. subtilis mutant strain 168degUhy32, and the wild-type strain 168 under optimized growth conditions.ConclusionExpression heterogeneity of secretory proteins in B. subtilis can be suppressed by degU mutation and optimized growth conditions. Further, the out-of-frame translational fusion of a gene for a secreted target protein and gfp represents a versatile tool for real-time monitoring of protein production and opens novel avenues for Bacillus production strain improvement.

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Section: Resultsmentioning

confidence: 99%

Homogeneity and heterogeneity in amylase production by Bacillus subtilis under different growth conditions

et al. 2016

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“…A previous study indicated that the pro-peptide of S. hyicus lipase could improve the secretion of the heterologous protein PhoA of E. coli in B. subtilis [37]. According to these previous studies, lipase MgehC was constructed to evaluate the secretion of GehC without the pro-peptide in B. subtilis.…”

Section: Production Of Pro-form and Mature Form Of Gehc Lipasementioning

confidence: 99%

Strategy for efficient production of recombinant Staphylococcus epidermidis lipase in Bacillus subtilis

Chen

Lin

et al. 2015

Biochemical Engineering Journal

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“…The propeptide of AmyE is the short type (with fewer than 60 residues) and is dispensable for secretion, folding, and stability, which differs from long propeptides (for example, the propeptide of Staphylococcus hyicus Lipase) [7,22,25,28]. hIFN-a2b is sensitive to extracellular protease, because it is a human protein.…”

Section: Discussionmentioning

confidence: 99%

“…Recent reports have discussed strategies for increasing the productivity of secreted proteins. Several methodological variations have been proposed, including the construction of protease-deficient strains, the coexpression of molecular chaperones, modifications of signal peptides, and the optimization of synthesis rates [3,[19][20][21][22][23][24]. Many useful methods relate to the late stage in secretion following translocation of the preproteins across the cytoplasmic membrane [3,7,19,21,25].…”

Section: Discussionmentioning

confidence: 99%

Enhanced Extracellular Production of Heterologous Proteins in Bacillus subtilis by Deleting the C-terminal Region of the SecA Secretory Machinery

et al. 2010

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In this study, we examined the effects of modifying the C-terminal region of the SecA protein on the production of heterologous proteins in Bacillus subtilis. SecA was selected as a candidate among the components of the Sec system due to its ability to interact directly with both the precursors and membrane translocases. A phylogenetic comparison demonstrated that the C-terminal region is not well conserved among eubacterial SecA proteins. The deletion of the 61 amino acids at the C-terminal region led to an 83% increase in extracellular alkaliphilic Bacillus sp. thermostable alkaline cellulase (Egl-237) activity. Moreover, the productivity of human interferon α (hIFN-α2b) was increased by 2.2-fold compared to the wild-type SecA, by deletion of these 61 amino acids. We indicated that the deletion of the C-terminal domain (CTD) of SecA enhanced the secretion of two different heterologous protein, Egl-237 and hIFN-α2b. This study provides a useful method to enhance the extracellular production of heterologous proteins in B. subtilis.

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Contributions of the Pre- and Pro-Regions of a Staphylococcus hyicus Lipase to Secretion of a Heterologous Protein by Bacillus subtilis

Cited by 8 publications

References 66 publications

Homogeneity and heterogeneity in amylase production by Bacillus subtilis under different growth conditions

Homogeneity and heterogeneity in amylase production by Bacillus subtilis under different growth conditions

Strategy for efficient production of recombinant Staphylococcus epidermidis lipase in Bacillus subtilis

Enhanced Extracellular Production of Heterologous Proteins in Bacillus subtilis by Deleting the C-terminal Region of the SecA Secretory Machinery

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