Bacillus subtilis is a rod-shaped, Gram-positive soil bacterium that secretes numerous enzymes to degrade a variety of substrates, enabling the bacterium to survive in a continuously changing environment. These enzymes are produced commercially and this production represents about 60% of the industrial-enzyme market. Unfortunately, the secretion of heterologous proteins, originating from Gram-negative bacteria or from eukaryotes, is often severely hampered. Several bottlenecks in the B. subtilis secretion pathway, such as poor targeting to the translocase, degradation of the secretory protein, and incorrect folding, have been revealed. Nevertheless, research into the mechanisms and control of the secretion pathways will lead to improved Bacillus protein secretion systems and broaden the applications as industrial production host. This review focuses on studies that aimed at optimizing B. subtilis as cell factory for commercially interesting heterologous proteins.

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Bacillus species are valuable producers of industrial enzymes and biopharmaceuticals, because they can secrete large quantities of high‐quality proteins directly into the growth medium. This requires the concerted action of quality control factors, such as folding catalysts and ‘cleaning proteases’. The expression of two important cleaning proteases, HtrA and HtrB, of Bacillus subtilis is controlled by the CssRS two‐component regulatory system. The induced CssRS‐dependent expression of htrA and htrB has been defined as a protein secretion stress response, because it can be triggered by high‐level production of secreted α‐amylases. It was not known whether translocation of these α‐amylases across the membrane is required to trigger a secretion stress response or whether other secretory proteins can also activate this response. These studies show for the first time that the CssRS‐dependent response is a general secretion stress response which can be triggered by both homologous and heterologous secretory proteins. As demonstrated by high‐level production of a nontranslocated variant of the α‐amylase, AmyQ, membrane translocation of secretory proteins is required to elicit this general protein secretion stress response. Studies with two other secretory reporter proteins, lipase A of B. subtilis and human interleukin‐3, show that the intensity of the protein secretion stress response only partly reflects the production levels of the respective proteins. Importantly, degradation of human interleukin‐3 by extracellular proteases has a major impact on the production level, but only a minor effect on the intensity of the secretion stress response.

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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.