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

Abstract

Bacillus subtilis is a prolific producer of enzymes and biopharmaceuticals. However, the susceptibility of heterologous proteins to degradation by (extracellular) proteases is a major limitation for use of B. subtilis as a protein cell factory. An increase in protein production levels has previously been achieved by using either protease-deficient strains or addition of protease inhibitors to B. subtilis cultures. Notably, the effects of genetic and chemical inhibition of proteases have thus far not been compared in a systematic way. In the present studies, we therefore compared the exoproteomes of cells in which extracellular proteases were genetically or chemically inactivated. The results show substantial differences in the relative abundance of various extracellular proteins. Furthermore, a comparison of the effects of genetic and/or chemical protease inhibition on the stress response triggered by (over) production of secreted proteins showed that chemical protease inhibition provoked a genuine secretion stress response. From a physiological point of view, this suggests that the deletion of protease genes is a better way to prevent product degradation than the use of protease inhibitors. Importantly however, studies with human interleukin-3 show that chemical protease inhibition can result in improved production of protease-sensitive secreted proteins even in mutant strains lacking eight extracellular proteases.

Degradation of Extracytoplasmic Catalysts for Protein Folding in Bacillus subtilis

“…This is most likely due to the CssRS-dependent crossregulation of htrA and htrB (23,25). The deletion of only two extracytoplasmic proteases (NprB and AprE) in the BRB02 mutant caused an elevated level of HtrA in the growth medium, which is in line with the previous observation that the extracellular HtrA levels are increased in multiple-protease-mutant cells (19,34,36). The increased level of extracellular HtrA in the BRB02 mutant is not related to lysis, as evidenced by blotting for the cytoplasmic marker protein TrxA (Fig.…”

“…Together, these findings suggest that the elevated levels of secreted (heterologous) proteins in multiple protease mutant strains may be due not only to reduced degradation, but also to elevated levels of cell-associated protein-folding catalysts, in particular PrsA, and quality control factors like HtrA and HtrB. Accordingly, it is very conceivable that the improved capabilities for protein secretion of previously constructed multiple-protease-mutant B. subtilis strains (35)(36)(37)(41)(42)(43)(44), particularly the WB800 strain lacking wprA (44), can be attributed at least in part to elevated levels of PrsA, HtrA, and/or HtrB. On the other hand, presently, we cannot fully exclude the possibility that PrsA, HtrA, and HtrB are subject to proteolytic turnover, for example, upon loss of functionality.…”

“…Though this may ensure product quality control in some cases, it mostly leads to huge commercial losses. Multiple-protease-mutant strains of B. subtilis were therefore constructed, including the frequently used strains WB600, WB700, and WB800 (19,36,37). Indeed, these strains displayed increased levels of heterologous protein secretion, which was attributed to reduced proteolysis.…”

Comprehensive Characterization of Methicillin-resistant Staphylococcus aureus subsp. aureus COL Secretome by Two-dimensional Liquid Chromatography and Mass Spectrometry

“…This observation has been reported by several investigators, and it has been suggested that the secreted proteins are degraded by the action of their secreted proteases during culture and sample preparation (20,26,42,(65)(66)(67). Degradation of proteins by extracellular proteases has also been reported in other microorganisms; in B. subtilis, it has been demonstrated that mutants lacking proteases exhibit a substantial increase in the abundance of various extracellular proteins compared with the wild type (65)(66)(67). Degradation of extracellular proteins may be due to slow or incorrect post-translational folding of the proteins or to the presence of exposed protease recognition sequences in the folded protein (52,54).…”

The Twin-Arginine Signal Peptide of Bacillus subtilis YwbN Can Direct either Tat- or Sec-Dependent Secretion of Different Cargo Proteins: Secretion of Active Subtilisin via the B. subtilis Tat Pathway

“…This pathway is particularly effective in secreting homologous proteins and proteins from closely related gram-positive species, like other bacilli. Notably, proteins transported via the Sec pathway pass the membrane translocation channel in an unfolded state, which makes these proteins vulnerable to posttranslocational degradation (64,65,69). Indeed, posttranslocational folding defects set important limits on the yields of heterologous proteins secreted via the Sec pathway of B. subtilis (12,20,31,57).…”