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.

FIG. 1.

Strategy used for secretion optimization in different Bacillus hosts. Three hundred ninety-three SPs originating from B. subtilis and B. licheniformis (A) were cloned in front of the gene encoding subtilisin BPN′ from B. amyloliquefaciens with its propeptide (PP) (B), which was used as a heterologous secretion target protein. Each SP was amplified with an artificial ribosome binding site (RBS), followed by a spacer region and ATG as the standardized start codon. (C) B. subtilis was used for screening of the signal peptide library, and two B. licheniformis strains were used to assess protease secretion levels for the best-performing SP-BPN′ fusions identified previously. HT, high throughput.

FIG. 2.

SPs which produced the most efficient secretion of BPN′, identified by screening in B. subtilis TEB1030. SPs originating from B. licheniformis DSM13 are labeled with the prefix “d,” and SPs from B. subtilis 168 are labeled with “s.” The extracellular enzyme activity obtained with the wild-type SP (wtSP) of BPN′ was defined as the benchmark (100%, corresponding to 0.83 units per ml). Error bars indicate standard deviations between proteolytic activities detected for each construct in at least three independent cultivations.

FIG. 3.

High-cell-density cultivation of B. subtilis TEB1030 carrying BPN′ fused to SPs wtSP and dBli00338. Cells were grown in fed-batch mode in a fermentor (culture volume: 3 liters), and culture supernatants were analyzed for extracellular protease activity (A) and amount of extracellular protease protein (B and C) by SDS-PAGE and subsequent staining with Coomassie brilliant blue (B) and Western blotting using antibodies against subtilisin BPN′ (C). The protein bands correspond to a molecular mass of 28 kDa, as deduced from positions of molecular weight standards (New England Biolabs [NEB]; broad range, 2 to 212 kDa, not shown) and thus correspond to the theoretical molecular mass of 27.6 kDa of mature BPN′.

FIG. 4.

Extracellular activities (A) and amount of protein (B) determined by Western blotting of subtilisin BPN′ in culture supernatants of screening host B. subtilis TEB1030 and expression host B. licheniformis H402. The protein bands correspond to a molecular mass of 28 kDa, as deduced from positions of molecular weight standards (NEB; broad range, 2 to 212 kDa, not shown) corresponding to the theoretical molecular mass of 27.6 kDa of mature BPN′. A B. subtilis strain with the vector encoding BPN′ without SP served as a control. Error bars indicate the standard deviations of proteolytic activities detected for each construct in at least three independent cultivations.

FIG. 5.

Extracellular enzyme activities of SP-BPN′ fusions identified by screening in B. subtilis and additionally expressed in B. licheniformis H402 and B. licheniformis MW3. (A) Protease activities in culture supernatants of SP-BPN′ constructs expressed in three different Bacillus strains were compared to the benchmark construct. (B) Secretion efficiencies in B. licheniformis H402 and MW3 shown as percent increases or decreases compared to that in B. subtilis. The boldface line represents the secretion efficiency in B. subtilis, which is defined as 0% deviation.