Improvement of Sec-dependent secretion of a heterologous model protein in Bacillus subtilis by saturation mutagenesis of the N-domain of the AmyE signal peptide

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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...

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confidence: 99%

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confidence: 99%

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

Degering

Eggert²,

Puls³

et al. 2010

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158

111

“…Because the Sec pathway is essential for bacterial survival (3,5), we could not analyze PhoD SP -GFP expression and secretion from strains containing deletions of Sec components. Instead, for comparison, we fused GFP to the Sec signal peptide derived from ␣-amylase AmyE and expressed the construct from the IPTGinducible P hyspank promoter (called AmyE SP -GFP) (72). After growing cells overnight in HPDM supplemented with IPTG, we detected very small amounts of intracellular AmyE SP -GFP expression by Western blotting (Fig.…”

Section: Resultsmentioning

confidence: 99%

The Canonical Twin-Arginine Translocase Components Are Not Required for Secretion of Folded Green Fluorescent Protein from the Ancestral Strain of Bacillus subtilis

Snyder

Mukherjee

Glass

et al. 2014

Cellular processes, such as the digestion of macromolecules, phosphate acquisition, and cell motility, require bacterial secretion systems. In Bacillus subtilis, the predominant protein export pathways are Sec (generalized secretory pathway) and Tat (twinarginine translocase). Unlike Sec, which secretes unfolded proteins, the Tat machinery secretes fully folded proteins across the plasma membrane and into the medium. Proteins are directed for Tat-dependent export by N-terminal signal peptides that contain a conserved twin-arginine motif. Thus, utilizing the Tat secretion system by fusing a Tat signal peptide is an attractive strategy for the production and export of heterologous proteins. As a proof of concept, we expressed green fluorescent protein (GFP) fused to the PhoD Tat signal peptide in the laboratory and ancestral strains of B. subtilis. Secretion of the Tat-GFP construct, as well as secretion of proteins in general, was substantially increased in the ancestral strain. Furthermore, our results show that secreted, fluorescent GFP could be purified directly from the extracellular medium. Nonetheless, export was not dependent on the known Tat secretion components or the signal peptide twin-arginine motif. We propose that the ancestral strain contains additional Tat components and/or secretion regulators that were abrogated following domestication.

“…A different approach was applied by Caspers et al (16) on the amyE signal peptide. By using saturation mutagenesis on the sequence encoding the N domain (amino acids 2 to 7) of amyE, they were able to increase the production yield of their reporter cutinase ϳ3-fold (16). We report here the application of combinatorial mutagenesis and the selection of CSP to develop new and improved signal sequences useful for high expression and translocation of heterologous proteins in E. coli.…”

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confidence: 99%

“…There seems to be a rather flexible consensus, both at the DNA level and at the protein level, for signal sequences targeted to the Sec translocation pathway in E. coli (for reviews, see references 12, 13, and 14) and the choice of signal sequence to ensure effective secretion should therefore be individually tested for each new protein (15)(16)(17). Typically, signal sequences are composed of a short, positively charged amino-terminal domain with 1 to 3 basic amino acid residues, followed by a hydrophobic core of 7 to 15 hydrophobic and neutral amino acids (13,18).…”

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confidence: 99%

Combinatorial Mutagenesis and Selection of Improved Signal Sequences and Their Application for High-Level Production of Translocated Heterologous Proteins in Escherichia coli

Heggeset

Kucharova

Nærdal

et al. 2013

bWe previously designed the consensus signal peptide (CSP) and demonstrated that it can be used to strongly stimulate heterologous protein production in Escherichia coli. A comparative study using CSP and two bacterial signal sequences, pelB and ompA, showed that the effect of signal sequences on both expression level and translocation efficiency can be highly protein specific. We report here the generation of CSP mutant libraries by a combinatorial mutagenesis approach. Degenerated CSP oligonucleotides were cloned in frame with the 5= end of the bla gene, encoding the mature periplasmic ␤-lactamase released from its native signal sequence. This novel design allows for a direct selection of improved signal sequences that positively affect the expression level and/or translocation efficiency of ␤-lactamase, based on the ampicillin tolerance level of the E. coli host cells. By using this strategy, 61 different CSP mutants with up to 8-fold-increased ampicillin tolerance level and up to 5.5-fold-increased ␤-lactamase expression level were isolated and characterized genetically. A subset of the CSP mutants was then tested with the alternative reporter gene phoA, encoding periplasmic alkaline phosphatase (AP), resulting in an up to 8-fold-increased production level of active AP protein in E. coli. Moreover, it was demonstrated that the CSP mutants can improve the production of the medically important human interferon ␣2b under high-cell-density cultivations. Our results show that there is a clear potential for improving bacterial signal sequences by using combinatorial mutagenesis, and bioinformatics analyses indicated that the beneficial mutations could not be rationally predicted.