Use of a Gram− signal peptide for protein secretion by Gram+ hosts: basic protease of Dichelobacter nodosus is produced and secreted by Bacillus subtilis

Wang, Lin‐Fa; Kortt, Alexander A.

doi:10.1016/0378-1119(93)90675-s

Cited by 5 publications

(3 citation statements)

References 24 publications

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“…SPs from extracellular proteins known to be secreted at high levels were identified and modified, e.g., SPs from NprE and AprE (14,23). Additionally, homologous and heterologous SPs were tested in different hosts (8,36); however, only very few examples of heterologous SPs producing significant increases in secretion efficiency have been described (10).…”

Section: Discussionmentioning

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

Appl Environ Microbiol

163

117

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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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Section: Discussionmentioning

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

Appl Environ Microbiol

163

117

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“…The signal peptide from aprE may be used to direct the secretion of other recombinant proteins from C. glutamicum and to study the mechanisms of secretion in this bacterium. Expression of basic protease in the native host, D. nodosus, and three heterologous hosts, E. coli (11), B. subtilis (19), and C. glutamicum, provides an opportunity to compare the gram-negative secretion systems with those of two distinct gram-positive bacteria.…”

Section: Discussionmentioning

confidence: 99%

Expression and secretion of heterologous proteases by Corynebacterium glutamicum

Billman‐Jacobe¹,

Lf²,

Aa³

et al. 1995

Appl Environ Microbiol

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Genes encoding the basic protease of Dichelobacter nodosus (bprV) and the subtilisin of Bacillus subtilis (aprE) were cloned and expressed in Corynebacterium glutamicum. In each case, enzymatically active protein was detected in the supernatants of liquid cultures. While the secretion of subtilisin was directed by its own signal peptide, the natural signal peptide of the bprV basic protease did not facilitate secretion. A hybrid aprE-bprV gene in which the promoter and signal peptide coding sequences of subtilisin replaced those of bprV could be expressed, and basic protease was secreted by C. glutamicum. Expression of these proteases in C. glutamicum provides an opportunity to compare protein secretion from this gram-positive host with that from other gram-positive and gram-negative bacteria.

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“…However, pNC3 has limitations as it requires a protease‐deficient host strain ( Wu et al . 1991 ; Wang et al . 1993 ).…”

Section: Introductionmentioning

confidence: 99%

Improved shuttle vector for expression of chitinase gene in Bacillus thuringiensis

Lertcanawanichakul

Wiwat

2000

Lett Appl Microbiol

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A 6.96‐kbp plasmid vector pBCX was constructed from the plasmid pBC16 (4·4 kbp) and a 2·56‐kbp fragment of pBluescript II KS. The bifunctional plasmid pBCX conferred ampicillin and tetracycline resistance in Escherichia coli but only tetracycline resistance in Bacillus thuringiensis. It has unique sites for BamHI, SmaI, PstI, HindIII, SalI, XhoI, DraII, ApaI and KpnI derived from pBluescript II KS and was lost at a low rate in B. thuringiensis subsp. israelensis when cultured in Luria–Bertani broth without antibiotic. The chitinase gene from B. circulans number 4·1 (pCHIB1) was subcloned into the HindIII sites of this vector and designated as pBX43 (9·56 kbp). This plasmid produced three times as much chitinase in B. thuringiensis subsp. israelensis strain c4Q272 as pHYB43, which comprises the commercial shuttle vector pHY300PLK plus the chitinase gene.

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Use of a Gram− signal peptide for protein secretion by Gram+ hosts: basic protease of Dichelobacter nodosus is produced and secreted by Bacillus subtilis

Cited by 5 publications

References 24 publications

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

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

Expression and secretion of heterologous proteases by Corynebacterium glutamicum

Improved shuttle vector for expression of chitinase gene in Bacillus thuringiensis

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