scholarly journals Approaches for Improving Protein Production in Multiple Protease-Deficient Bacillus subtilis Host Strains

10.5772/30092 ◽  
2012 ◽  
Author(s):  
Takeko Kodama ◽  
Kenji Manabe ◽  
Yasushi Kageyama ◽  
Shenghao Liu ◽  
Katsutoshi Ara ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Protein Production ◽  
Protease Deficient ◽  
Host Strains

scholarly journals Complete Genome Sequence of Bacillus subtilis Strain WB800N, an Extracellular Protease-Deficient Derivative of Strain 168

2018 ◽  
Vol 7 (18) ◽  
Author(s):  
Haeyoung Jeong ◽  
Da-Eun Jeong ◽  
Seung-Hwan Park ◽  
Seong Joo Kim ◽  
Soo-Keun Choi
Keyword(s):  
Bacillus Subtilis ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Genetically Engineered ◽  
Subtilis Strain ◽  
Secretory Proteins ◽  
Extracellular Proteases ◽  
Content Type ◽  
Protease Deficient

Bacillus subtilis WB800N is a genetically engineered variant of B. subtilis 168, such that all extracellular proteases are disrupted, which enables WB800N to be widely used for the expression of secretory proteins. Here, we report the 4.2-Mb complete genome sequence of WB800N and present all of the disrupted gene structure.

scholarly journals Developing rapid growing Bacillus subtilis for improved biochemical and recombinant protein production

2020 ◽  
Vol 11 ◽  
pp. e00141
Author(s):  
Yanfeng Liu ◽  
Anqi Su ◽  
Rongzhen Tian ◽  
Jianghua Li ◽  
Long Liu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production

scholarly journals Genes Involved in SkfA Killing Factor Production Protect a Bacillus subtilis Lipase against Proteolysis

2005 ◽  
Vol 71 (4) ◽  
pp. 1899-1908 ◽  
Author(s):  
Helga Westers ◽  
Peter G. Braun ◽  
Lidia Westers ◽  
Haike Antelmann ◽  
Michael Hecker ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Protease Inhibitor ◽  
Bactericidal Activity ◽  
Industrial Applications ◽  
High Potential ◽  
Proteolytic Degradation ◽  
Wild Type ◽  
Factor Production ◽  
Protease Deficient ◽  
High Level

ABSTRACT Small lipases of Bacillus species, such as LipA from Bacillus subtilis, have a high potential for industrial applications. Recent studies showed that deletion of six AT-rich islands from the B. subtilis genome results in reduced amounts of extracellular LipA. Here we demonstrate that the reduced LipA levels are due to the absence of four genes, skfABCD, located in the prophage 1 region. Intact skfABCD genes are required not only for LipA production at wild-type levels by B. subtilis 168 but also under conditions of LipA overproduction. Notably, SkfA has bactericidal activity and, probably, requires the SkfB to SkfD proteins for its production. The present results show that LipA is more prone to proteolytic degradation in the absence of SkfA and that high-level LipA production can be improved significantly by employing multiple protease-deficient B. subtilis strains. In conclusion, our findings imply that SkfA protects LipA, directly or indirectly, against proteolytic degradation. Conceivably, SkfA could act as a modulator in LipA folding or as a protease inhibitor.

scholarly journals Cymbopogon nardus Essential Oil as Protein Inducer in Bacillus subtilis ATCC21332

2018 ◽  
Vol 1 (1) ◽  
Author(s):  
Hairul Shahril Muhamad ◽  
Ismatul Nurul Asyikin Ismail ◽  
Nabilah Ahmad Alhadi ◽  
Salina Mat Radzi ◽  
Maryam Mohamed Rehan ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Bacillus Subtilis ◽  
Essential Oil ◽  
Protein Production ◽  
Antimicrobial Agents ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extracellular Proteins ◽  
Specific Chemical ◽  
Cymbopogon Nardus

Protein production by bacteria might be increased in stressful conditions such as in the presence of antimicrobial agents. Many studies have proven that antibiotics or antimicrobial agents at low concentration are able to activate or repress gene transcription process in bacteria. However, there have been comparatively few studies on the potential of natural compounds in nature as a specific chemical signal that can trigger a variety of biological functions. An attempt was made to study the effect of essential oil from Cymbopogon nardus in regulating protein production by Bacillus subtilis ATCC21332. The bacterial cells were further exposed to the C. nardus essential oil at concentration of 0.02 % for 48 h at 37°C. The intracellular proteins were then isolated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins profile showed that a band with approximate size of 180 kDa appeared for the treated bacteria with C. nardus essential oil. An alignment of peptide sequences to the NCBI BLAST database revealed that B. subtilis ATCC21332 in stressful condition tend to produce intracellular protein recognized as respiratory nitrate reductase ? subunit enzyme. Besides, the extracellular proteins secreted by B. subtilis ATCC21332 after being subjected to 0.02% of C. nardus essential oil for 48 and 72 h at 30°C, were further analyzed on antimicrobial activity. The extracellular proteins secreted by B. subtilis ATCC21332 prior to enhancing with 0.02 % C. nardus essential oil at 30°C for 72 h exhibited antimicrobial activity towards two strains of bacteria, which are Bacillus cereus and Escherichia coli.

scholarly journals Engineering of Plasmin-Resistant Forms of Streptokinase and Their Production in Bacillus subtilis: Streptokinase with Longer Functional Half-Life

1998 ◽  
Vol 64 (3) ◽  
pp. 824-829 ◽  
Author(s):  
Xu-Chu Wu ◽  
Ruiqiong Ye ◽  
Yanjun Duan ◽  
Sui-Lam Wong
Keyword(s):  
Bacillus Subtilis ◽  
Half Life ◽  
Blood Clot ◽  
Active Form ◽  
Molar Ratio ◽  
Plasminogen Activation ◽  
Kinetics Parameters ◽  
Protease Deficient

ABSTRACT The short in vivo half-life of streptokinase limits its efficacy as an efficient blood clot-dissolving agent. During the clot-dissolving process, streptokinase is processed to smaller intermediates by plasmin. Two of the major processing sites are Lys59 and Lys386. We engineered two versions of streptokinase with either one of the lysine residues changed to glutamine and a third version with both mutations. These mutant streptokinase proteins (muteins) were produced by secretion with the protease-deficient Bacillus subtilisWB600 as the host. The purified muteins retained comparable kinetics parameters in plasminogen activation and showed different degrees of resistance to plasmin depending on the nature of the mutation. Muteins with double mutations had half-lives that were extended 21-fold when assayed in a 1:1 molar ratio with plasminogen in vitro and showed better plasminogen activation activity with time in the radial caseinolysis assay. This study indicates that plasmin-mediated processing leads to the inactivation of streptokinase and is not required to convert streptokinase to its active form. Plasmin-resistant forms of streptokinase can be engineered without affecting their activity, and blockage of the N-terminal cleavage site is essential to generate engineered streptokinase with a longer in vitro functional half-life.

scholarly journals The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Patrick Lenz ◽  
Fabienne Hilgers ◽  
Alina Burmeister ◽  
Leonie Zimmermann ◽  
Kristina Volkenborn ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Recombinant Protein ◽  
Single Cell ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Online Monitoring ◽  
Protein Detection ◽  
Single Cell Level ◽  
Cell Level

Abstract Background Bacillus subtilis is one of the most important microorganisms for recombinant protein production. It possesses the GRAS (generally recognized as safe) status and a potent protein secretion capacity. Secretory protein production greatly facilitates downstream processing and thus significantly reduces costs. However, not all heterologous proteins are secreted and intracellular production poses difficulties for quantification. To tackle this problem, we have established a so-called intracellular split GFP (iSplit GFP) assay in B. subtilis as a tool for the in vivo protein detection during expression in batch cultures and at a single-cell level. For the iSplit GFP assay, the eleventh β-sheet of sfGFP is fused to a target protein and can complement a detector protein consisting of the respective truncated sfGFP (GFP1-10) to form fluorescent holo-GFP. Results As proof of concept, the GFP11-tag was fused C-terminally to the E. coli β-glucuronidase GUS, resulting in fusion protein GUS11. Variable GUS and GUS11 production levels in B. subtilis were achieved by varying the ribosome binding site via spacers of increasing lengths (4–12 nucleotides) for the GUS-encoding gene. Differences in intracellular enzyme accumulation were determined by measuring the GUS11 enzymatic activity and subsequently by adding the detector protein to respective cell extracts. Moreover, the detector protein was co-produced with the GUS11 using a two-plasmid system, which enabled the in vivo detection and online monitoring of glucuronidase production. Using this system in combination with flow cytometry and microfluidics, we were able to monitor protein production at a single-cell level thus yielding information about intracellular protein distribution and culture heterogeneity. Conclusion Our results demonstrate that the iSplit GFP assay is suitable for the detection, quantification and online monitoring of recombinant protein production in B. subtilis during cultivation as well as for analyzing production heterogeneity and intracellular localization at a single-cell level. Graphic abstract

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