Optimization of Protease Secretion in Bacillus subtilis and Bacillus licheniformis by Screening of Homologous and Heterologous Signal Peptides

Abstract

Bacillus subtilis and Bacillus licheniformis are widely used for the large-scale industrial production of proteins. These strains can efficiently secrete proteins into the culture medium using the general secretion (Sec) pathway. A characteristic feature of all secreted proteins is their N-terminal signal peptides, which are recognized by the secretion machinery. Here, we have studied the production of an industrially important secreted protease, namely, subtilisin BPN from Bacillus amyloliquefaciens. One hundred seventy-three signal peptides originating from B. subtilis and 220 signal peptides from the B. licheniformis type strain were fused to this secretion target and expressed in B. subtilis, and the resulting library was analyzed by high-throughput screening for extracellular proteolytic activity. We have identified a number of signal peptides originating from both organisms which produced significantly increased yield of the secreted protease. Interestingly, we observed that levels of extracellular protease were improved not only in B. subtilis, which was used as the screening host, but also in two different B. licheniformis strains. To date, it is impossible to predict which signal peptide will result in better secretion and thus an improved yield of a given extracellular target protein. Our data show that screening a library consisting of homologous and heterologous signal peptides fused to a target protein can identify more-effective signal peptides, resulting in improved protein export not only in the original screening host but also in different production strains.Gram-positive bacteria of the genus Bacillus are industrially well-established microorganisms for the production of extracellular proteins. Due to the availability of relatively cheap large-scale production systems combined with the ability of bacteria to secrete up to 20 to 25 g/liter of a target protein into the growth medium, about 60% of commercially available enzymes are presently produced in Bacillus species (14, 28).The closely related species Bacillus subtilis and Bacillus licheniformis are widely used as production hosts on an industrial scale, and, in contrast to the well-known production species Escherichia coli, they are free of endotoxin and have GRAS (generally regarded as safe) status. The complete genome sequences of strains B. subtilis 168 (1, 18) and B. licheniformis DSM13 (isogenic to ATCC 14580) (26, 32) are available, greatly facilitating the construction of improved production strains.The Sec pathway constitutes the main secretion pathway in B. subtilis and B. licheniformis. Proteins secreted via the Sec pathway are initially synthesized with an N-terminal hydrophobic signal peptide (SP) consisting of a positively charged N domain followed by a longer, hydrophobic H domain and a C domain consisting of three amino acids which form the signal peptidase recognition site (35). Targeting of a secreted protein to the membrane, the translocation process itself, and subsequent processing by a signal peptidase represent the majo...

Efficient Expression of Antibody Fragments with the Brevibacillus Expression System

“…Protein productivity is markedly affected by the type of signal peptide used, as is evident in the Bacillus subtilis system [15,16]. We have examined the secretion efficiency of different signal sequences with a variety of proteins and found it to vary from one signal sequence to another.…”

Secretion of heterologous thermostable cellulases in Bacillus subtilis

“…Because different SPs mediated cellulase translocation across the cytoplasmic membrane by Sec translocase with different efficiencies as reported by Brockmeier et al (2006) and Degering et al (2010), the efficiency of SP cannot be predicted a priori. Previously, SPs of extracellular proteins secreted at high levels, such as SPs from NprE and AprE, have been intensively investigated (Fu et al, 2007;Nijland and Kuipers, 2008).…”

“…It is known that enzymes secreted through the Sec pathway are processed and folded into their active form only during or shortly after the translocation across the cytoplasmic membrane (Brockmeier et al, 2006;Degering et al, 2010). We did not determine the N-terminal residues of secreted cellulases, but the detected activities imply that they might be processed appropriately as designed.…”

“…However, to date, it is supposedly impossible to predict theoretically SP that would result in better secretion. It has even been suggested that screening a library consisting of different types of SP fused to the target protein may be the only way to identify an effective SP for improved secretion of target proteins (Degering et al, 2010). However, D-score is sometimes used to predict the probability of amino acid sequences functioning as a SP (Bendtsen et al, 2004).…”

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“…The Gram-positive bacterium Bacillus subtilis is a well-established host for the industrial-scale production of extracellular proteins. Different from E. coli, B. subtilis is endotoxin free and is generally regarded as safe (Degering et al 2010).…”