A Bacillus subtilis fusion protein system to produce soybean Bowman–Birk protease inhibitor

Vogtentanz, Gudrun; Collier, Katherine D.; Bodó, Michael; Chang, Judy H.; Day, Anthony G.; Estell, David A.; Falcon, Brandy C.; Ganshaw, G.; Jarnagin, Alisha Stephens; Kellis, James T.; Kolkman, Marc A. B.; Lai, Cindy; Meneses, Renato; Miller, Jeffrey V.; Nobel, Hans de; Power, Scott D.; Weyler, Walter; Wong, David L.; Schmidt, Brian F.

doi:10.1016/j.pep.2007.05.001

Cited by 11 publications

(5 citation statements)

References 52 publications

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“…B. subtilis strains were grown at 37 °C, 280 rpm in Lysogeny Broth (LB; Oxoid Limited), MBU medium or 5SM12 medium. The MBU medium is similar to the MBD medium as described by Vogtentanz et al [28], but lacks soytone and instead of 7.5% glucose it contains 2.1% glucose and 3.5% maltodextrin DE13-17. The 5SM12 medium consists of 75 mM K 2 HPO 4 , 25 mM NaH 2 PO 4 , 12% maltodextrin, 5% Difco Bacto Soytone, 2 mM sodium citrate, 0.5 mM MgSO 4 , 0.2 mM MnCl 2 , 0.03 mM calcium chloride, and 0.0053% ferric ammonium citrate.…”

Section: Methodsmentioning

confidence: 99%

Intramembrane protease RasP boosts protein production in Bacillus

et al. 2017

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BackgroundThe microbial cell factory Bacillus subtilis is a popular industrial platform for high-level production of secreted technical enzymes. Nonetheless, the effective secretion of particular heterologous enzymes remains challenging. Over the past decades various studies have tackled this problem, and major improvements were achieved by optimizing signal peptides or removing proteases involved in product degradation. On the other hand, serious bottlenecks in the protein export process per se remained enigmatic, especially for protein secretion at commercially significant levels by cells grown to high density. The aim of our present study was to assess the relevance of the intramembrane protease RasP for high-level protein production in B. subtilis.ResultsDeletion of the rasP gene resulted in reduced precursor processing and extracellular levels of the overproduced α-amylases AmyE from B. subtilis and AmyL from Bacillus licheniformis. Further, secretion of the overproduced serine protease BPN’ from Bacillus amyloliquefaciens was severely impaired in the absence of RasP. Importantly, overexpression of rasP resulted in threefold increased production of a serine protease from Bacillus clausii, and 2.5- to 10-fold increased production of an AmyAc α-amylase from Paenibacillus curdlanolyticus, depending on the culture conditions. Of note, growth defects due to overproduction of the two latter enzymes were suppressed by rasP-overexpression.ConclusionHere we show that an intramembrane protease, RasP, sets a limit to high-level production of two secreted heterologous enzymes that are difficult to produce in the B. subtilis cell factory. This finding was unexpected and suggests that proteolytic membrane sanitation is key to effective enzyme production in Bacillus.

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Section: Methodsmentioning

confidence: 99%

Intramembrane protease RasP boosts protein production in Bacillus

et al. 2017

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“…Expression with fusion tags is a strategy to facilitate target protein secretion in B. subtilis . Previously, several fusion tags were developed, such as cellulase (34.2 kDa), [ 27 ] green fluorescent protein (26.8 kDa), [ 42 ] and SUMO3 (8.6 kDa). [ 37 ] Among these fusion tags tested, SUMO3 is an ideal candidate, since it is small and able to enhance expression, folding, and stability of target protein.…”

Section: Discussionmentioning

confidence: 99%

“…[16,20] B. subtilis is a dominant host for the secretion of industrial proteins, such as amylase, lipase, cellulase, and proteases. [21,22] To improve protein secretion, the strategies of signal peptide screening, [23,24] signal peptide mutation [25,26] or expression with fusion tags [14,27] are usually attempted. Recently, Quan et al identified a unique chaperon protein Spy, which is located in the periplasmic space of E. coli.…”

Section: Introductionmentioning

confidence: 99%

Efficient secretion of mussel adhesion proteins using a chaperone protein Spy as fusion tag in Bacillus subtilis

Qing

Liu

et al. 2023

Biotechnology Journal

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BackgroundMussel foot proteins (Mfps) are considered as remarkable materials due to their extraordinary adhesive capability. Recombinant expression is an ideal way to synthesis these proteins at large scale. However, secretory expression of Mfps into culture medium has not been achieved in a heterologous host.Methods and ResultsHere, to realize the secretion of Mfp3 and Mfp5 in Bacillus subtilis, signal peptide screening was first performed. Minimal Mfp3‐6×His was targeted into the growth medium with AmyE signal peptide. We found that a small chaperone protein Spy was secreted efficiently in B. subtilis, and the fusion proteins Spy‐Mfp3‐6×His and Spy‐Mfp5‐6×His could also be delivered into growth medium well. The yield of Spy‐Mfp3‐6×His and Spy‐Mfp5‐6×His reached 255 and 119 mg L−1 at shake flask conditions, respectively. Mfp3‐6×His and Mfp5‐6×His were finally purified via TEV protease cleavage and NTA affinity chromatography.ConclusionMfp3‐6×His and Mfp5‐6×His could be efficiently secreted using a chaperone protein Spy as fusion tag in B. subtilis.

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“…The 34.2‐kDa BCE103 cellulase from an alkalophilic Bacillus sp. as a fusion protein led to the efficient secretion of the fused soybean Bowman‐Birk protease inhibitor at a level of 198‐276 mg/L (Vogtentanz et al, 2007). However, the Bowman‐Birk protease inhibitor was a peptide with a molecular weight of only 7.9 kDa.…”

Section: Discussionmentioning

confidence: 99%

Efficient secretory production of large‐size heterologous enzymes in Bacillus subtilis: A secretory partner and directed evolution

Liu

Wang

Zhu

et al. 2020

Biotech & Bioengineering

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Secretory production of recombinant proteins provides a simple approach to the production and purification of target proteins in the enzyme industry. We developed a combined strategy for the secretory production of three large‐size heterologous enzymes with a special focus on 83‐kDa isoamylase (IA) from an archaeon Sulfolobus tokodaii in a bacterium Bacillus subtilis. First, a secretory protein of the B. subtilis family 5 glycoside hydrolase endoglucanase (Cel5) was used as a fusion partner, along with the NprB signal peptide, to facilitate secretory production of IA. This secretory partner strategy was effective for the secretion of two other large enzymes: family 9 glycoside hydrolase from Clostridium phytofermentas and cellodextrin phosphorylase from Clostridium thermocellum. Second, the secretion of Cel5‐IA was improved by directed evolution with two novel double‐layer Petri‐dish‐based high‐throughput screening (HTS) methods. The high‐sensitivity HTS relied on the detection of high‐activity Cel5 on the carboxymethylcellulose/Congo‐red assay. The second modest‐sensitivity HTS focused on the detection of low‐activity IA on the amylodextrin‐I2 assay. After six rounds of HTS, a secretory Cel5‐IA level was increased to 234 mg/L, 155 times the wild‐type IA with the NprB signal peptide only. This combinatory strategy could be useful to enhance the secretory production of large‐size heterologous proteins in B. subtilis.

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A Bacillus subtilis fusion protein system to produce soybean Bowman–Birk protease inhibitor

Cited by 11 publications

References 52 publications

Intramembrane protease RasP boosts protein production in Bacillus

Intramembrane protease RasP boosts protein production in Bacillus

Efficient secretion of mussel adhesion proteins using a chaperone protein Spy as fusion tag in Bacillus subtilis

Efficient secretory production of large‐size heterologous enzymes in Bacillus subtilis: A secretory partner and directed evolution

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