Construction of a Shuttle Vector for Protein Secretory Expression in Bacillus subtilis and the Application of the Mannanase Functional Heterologous Expression

Guo, Su; Tang, Jiajie; Wei, Dongzhi; Wei, Wei

doi:10.4014/jmb.1311.11009

Cited by 17 publications

(14 citation statements)

References 19 publications

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“…Compared to E. coli which was extensively used for protein expression, B. subtilis displayed significant advantages in efficient secretion capacity of functional extracellular proteins directly into culture medium, which made downstream separation and purification processes much more simple [ 5 , 44 ]. In this study, we used the B. subtilis WB600, a protease-deficient strain, for secreted expression of BcManA.…”

Section: Resultsmentioning

confidence: 99%

“…However, the yield of β-mannanases produced from native microbes, especially from extremophilic microbes, is usually low and hard to be improved, which led to the increasing attention on the heterologous production of β-mannanases in recent years [ 33 ]. Many β-mannanase genes originated from different microbes have been cloned and heterologously expressed in the frequently used protein expression systems such as Escherichia coli [ 37 – 39 ], Pichia pastoris [ 33 , 40 – 42 ] and Bacillus subtilis [ 5 , 43 , 44 ] in the past decade. Compared to E. coli , P. pastoris and B. subtilis can secreted the enzyme directly into the culture medium, which making the downstream separation and purification much easier.…”

Section: Introductionmentioning

confidence: 99%

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Characterization and high-efficiency secreted expression in Bacillus subtilis of a thermo-alkaline β-mannanase from an alkaliphilic Bacillus clausii strain S10

Zhou

Xue

2018

Microb Cell Fact

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Backgroundβ-Mannanase catalyzes the cleavage of β-1,4-linked internal linkages of mannan backbone randomly to produce new chain ends. Alkaline and thermostable β-mannanases provide obvious advantages for many applications in biobleaching of pulp and paper, detergent industry, oil grilling operation and enzymatic production of mannooligosaccharides. However, only a few of them are commercially exploited as wild or recombinant enzymes, and none heterologous and secretory expression of alkaline β-mannanase in Bacillus subtilis expression system was reported. Alkaliphilic Bacillus clausii S10 showed high β-mannanase activity at alkaline condition. In this study, this β-mannanase was cloned, purified and characterized. The high-level secretory expression in B. subtilis was also studied.ResultsA thermo-alkaline β-mannanase (BcManA) gene encoding a 317-amino acid protein from alkaliphilic Bacillus clausii strain was cloned and expressed in Escherichia coli. The purified mature BcManA exhibited maximum activity at pH 9.5 and 75 °C with good stability at pH 7.0–11.5 and below 80 °C. BcManA demonstrated high cleavage capability on polysaccharides containing β-1,4-mannosidic linkages, such as konjac glucomannan, locust bean gum, guar gum and sesbania gum. The highest specific activity of 2366.2 U mg−1 was observed on konjac glucomannan with the Km and kcat value of 0.62 g l−1 and 1238.9 s−1, respectively. The hydrolysis products were mainly oligosaccharides with a higher degree of polymerization than biose. BcManA also cleaved manno-oligosaccharides with polymerization degree more than 3 without transglycosylation. Furthermore, six signal peptides and two strong promoters were used for efficiently secreted expression optimization in B. subtilis WB600 and the highest extracellular activity of 2374 U ml−1 with secretory rate of 98.5% was obtained using SPlipA and P43 after 72 h cultivation in 2 × SR medium. By medium optimization using cheap nitrogen and carbon source of peanut meal and glucose, the extracellular activity reached 6041 U ml−1 after 72 h cultivation with 6% inoculum size by shake flask fermentation.ConclusionsThe thermo-alkaline β-mannanase BcManA showed good thermal and pH stability and high catalytic efficiency towards konjac glucomannan and locust bean gum, which distinguished from other reported β-mannanases and was a promising thermo-alkaline β-mannanase for potential industrial application. The extracellular BcManA yield of 6041 U ml−1, which was to date the highest reported yield by flask shake, was obtained in B. subtilis with constitutive expression vector. This is the first report for secretory expression of alkaline β-mannanase in B. subtilis protein expression system, which would significantly cut down the production cost of this enzyme. Also this research would be helpful for secretory expression of other β-mannanases in B. subtilis.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0973-0) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Characterization and high-efficiency secreted expression in Bacillus subtilis of a thermo-alkaline β-mannanase from an alkaliphilic Bacillus clausii strain S10

Zhou

Xue

2018

Microb Cell Fact

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“…There were also some constitutive plasmids originated from pUB110, e.g. pBNS2 and pBSG, which have shown potentials in foreign expressions [49,50]. However, further developments of these shuttle plasmids were restricted by the lack of systematical analysis about plasmids constructions.…”

Section: Discussionmentioning

confidence: 99%

High copy number and highly stable Escherichia coli–Bacillus subtilis shuttle plasmids based on pWB980

Zhao

Tan

et al. 2020

Microb Cell Fact

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Background: pWB980 derived from pUB110 is a promising expression vector in Bacillus for its high copy number and high stability. However, the low transformation rate of recombinant plasmids to the wild cells limited the application of it. On the basis of pWB980, constructing an E. coli-B. subtilis shuttle plasmid could facilitate the transformation rate to Bacillus cells. Because the insertion site for E. coli replication origin sequence (ori) is not unique in pWB980, in order to investigate the best insertion site, eight shuttle plasmids (pUC980-1 ~ pUC980-8) containing all possible insertion sites and directions were constructed. Results: The results showed that all the selected insertion sites could be used to construct shuttle plasmid but some sites required a specific direction. And different insertion sites led to different properties of the shuttle plasmids. The best shuttle plasmids pUC980-1 and pUC980-2, which showed copies more than 450 per cell and segregational stabilities up to 98%, were selected for heterologous expressions of an alkaline pectate lyase gene pelN, an alkaline protease spro1 and a pullulanase gene pulA11, respectively. The highest extracellular activities of PelN, Spro1 and PulA11 were up to 5200 U/mL, 21,537 U/mL and 504 U/mL correspondingly after 54 h, 60 h and 48 h fermentation in a 10 L fermentor. Notably, PelN and Spro1 showed remarkably higher yields in Bacillus than previous reports. Conclusion: The optimum ori insertion site was the upstream region of BA3-1 in pWB980 which resulted in shuttle plasmids with higher copy numbers and higher stabilities. The novel shuttle plasmids pUC980-1 and pUC980-2 will be promising expression vectors in B. subtilis. Moreover, the ori insertion mechanism revealed in this work could provide theoretical guidance for further studies of pWB980 and constructions of other shuttle plasmids.

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“…Zhang et al 2013). There are several publications demonstrating the effective transport capacity of the signal peptide of AmyQ from Bacillus amyloliquefaciens in B. subtilis (Guo et al 2014;Phan et al 2006).…”

Section: Crosslinking Of Spimentioning

confidence: 99%

Heterologous signal peptides-directing secretion of Streptomyces mobaraensis transglutaminase by Bacillus subtilis

Qiao

et al. 2018

Appl Microbiol Biotechnol

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Construction of a Shuttle Vector for Protein Secretory Expression in Bacillus subtilis and the Application of the Mannanase Functional Heterologous Expression

Cited by 17 publications

References 19 publications

Characterization and high-efficiency secreted expression in Bacillus subtilis of a thermo-alkaline β-mannanase from an alkaliphilic Bacillus clausii strain S10

Characterization and high-efficiency secreted expression in Bacillus subtilis of a thermo-alkaline β-mannanase from an alkaliphilic Bacillus clausii strain S10

High copy number and highly stable Escherichia coli–Bacillus subtilis shuttle plasmids based on pWB980

Heterologous signal peptides-directing secretion of Streptomyces mobaraensis transglutaminase by Bacillus subtilis

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