Efficient expression of nattokinase in <i>Bacillus licheniformis</i>: host strain construction and signal peptide optimization

Wei, Xuetuan; Zhou, Yinhua; Chen, Jingbang; Cai, Dongbo; Wang, Dan; Qi, Gaofu; Chen, Shouwen

doi:10.1007/s10295-014-1559-4

Cited by 73 publications

(64 citation statements)

References 31 publications

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“…In this study, both nattokinase and α-amylase were selected as prototype target proteins for secretion to evaluate the function of SppA and TepA in B. licheniformis BL10, derived from the native strain B. licheniformis WX-02 [7]. Our results identified and confirmed that SppA is the main SPP for protein secretion, and the concentrations of total extracellular proteins and target proteins increased significantly for a strain overexpressing sppA .…”

Section: Introductionmentioning

confidence: 58%

“…Nattokinase expression vector pP43SacCNK was made in a previous study [7], and the α-amylase expression vector pHY-amyL harboring P43 promoter (K02174.1), amyL (FJ556804.1) coding for α-amylase (containing its own signal peptide and terminator) was verified by DNA sequence. The pP43SacCNK and pHY-amyL plasmids were individually electro-transferred into the parental strain B. licheniformis BL10, as well as the SPP gene deficient strains BL10S and BL10T.…”

Section: Resultsmentioning

confidence: 99%

“…The nattokinase expression vector used in this study was obtained from our previously reported research [7], and the α-amylase expression vector was constructed according to the following method. P43 promoter was from B. subtilis 168.…”

Section: Methodsmentioning

confidence: 99%

“…Compared to other expression systems, Bacillus species are regarded as promising host strains with numerous advantages including: non-toxicity, convenience for gene modification and high yields of target proteins [4–6]. In particular, Bacillus licheniformis has been shown to be an effective host strain for protein production in previous studies [7, 8]. …”

Section: Introductionmentioning

confidence: 99%

“…For example, B. licheniformis BL10 was constructed as a highly efficient host strain for protein production by knocking out ten genes coding for extracellular proteases (Mpr, Vpr, AprX, Epr, Bpr, WprA, AprE, BprA), flagellin and amylase in B. licheniformis WX-02. Using this engineered B. licheniformis BL10, a previous study has demonstrated that nattokinase activity could be increased by 39% [7]. Also, the translocation elements (SecA, SecDF etc.…”

Section: Introductionmentioning

confidence: 99%

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A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis

Cai

Wang

et al. 2017

Microb Cell Fact

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BackgroundSignal peptide peptidases play an important role in the removal of remnant signal peptides in the cell membrane, a critical step for extracellular protein production. Although these proteins are likely a central component for extracellular protein production, there has been a lack of research on whether protein secretion could be enhanced via overexpression of signal peptide peptidases.ResultsIn this study, both nattokinase and α-amylase were employed as prototypical secreted target proteins to evaluate the function of putative signal peptide peptidases (SppA and TepA) in Bacillus licheniformis. We observed dramatic decreases in the concentrations of both target proteins (45 and 49%, respectively) in a sppA deficient strain, while the extracellular protein yields of nattokinase and α-amylase were increased by 30 and 67% respectively in a strain overexpressing SppA. In addition, biomass, specific enzyme activities and the relative gene transcriptional levels were also enhanced due to the overexpression of sppA, while altering the expression levels of tepA had no effect on the concentrations of the secreted target proteins.ConclusionsOur results confirm that SppA, but not TepA, plays an important functional role for protein secretion in B. licheniformis. Our results indicate that the sppA overexpression strain, B. licheniformis BL10GS, could be used as a promising host strain for the industrial production of heterologous secreted proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0688-7) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 58%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis

Cai

Wang

et al. 2017

Microb Cell Fact

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Research progress on the fibrinolytic enzymes produced from traditional fermented foods

Wang

Peng

Xie

et al. 2023

Food Science & Nutrition

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Cardiovascular diseases (CVDs) are a global health problem and leading cause of death worldwide. Thrombus formation, one of the CVDs, is essentially the formation of fibrin clots. The existing thrombolytic agents have the disadvantages of high price, short half‐life, and high bleeding risk; hence, there is an urgent need to find the alternative thrombolytic agents. In recent years, traditional fermented foods have been widely investigated for their outstanding effects in the prevention and treatment of thrombus formation. In this review, we have focused on fibrinolytic enzymes produced by microorganisms during the fermentation of traditional fermented foods and their potential use for treating CVDs. First, we discussed about the sources of fibrinolytic enzymes and microbial strains that produce those enzymes followed by the optimization of fermentation process, purification, and physicochemical properties of fibrinolytic enzymes. Finally, we have summarized the thrombolytic effects of fibrinolytic enzymes in humans and mice. Fibrinolytic enzymes produced by microorganisms during the fermentation of traditional fermented foods not only lyse thrombi but also acts as anti‐atherosclerotic, anti‐hyperlipidemia, and neuroprotection agents. Therefore, fibrinolytic enzymes from traditional fermented foods have great potential for the prevention and treatment of CVDs.

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Improvement of levan production in Bacillus amyloliquefaciens through metabolic optimization of regulatory elements

Zheng

Feng

et al. 2017

Appl Microbiol Biotechnol

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Levan is a functional homopolymer of fructose with considerable applications in food, pharmaceutical and cosmetic industries. To improve the levan production in Bacillus amyloliquefaciens, the regulatory elements of sacB (encoding levansucrase) expression and levansucrase secretion were optimized. Four heterologous promoters were evaluated for sacB expression, and the Pgrac promoter led to the highest level for both sacB transcription and levansucrase enzyme activity. The levan production in the corresponding recombinant strain ΔLP-pHTPgrac reached 30.5 g/L, which was 114% higher than that of the control strain NK-ΔLP. In a further step, eight signal peptides were investigated (with Pgrac as the promoter for sacB expression) for their effects on the levansucrase secretion and levan production. The signal peptide yncM was identified as the optimal one, with a secretion efficiency of approximately 90%, and the levan production in the corresponding recombinant strain ΔLP-Y reached 37.4 g/L, which was 161% higher when compared with the control strains NK-ΔLP. Finally, fed-batch fermentation was carried out in 5-L bioreactors for levan production using the recombinant strain ΔLP-Y. A final levan concentration of 102 g/L was achieved, which is very close to the ever reported highest levan production level from the literature. To our best knowledge, this is the first report of the improvement of levan production through metabolic optimization for sacB expression and levansucrase secretion. The results from this study provided essential insights for systematically metabolic engineering of microbial cell factories for enhanced biochemical production.

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Efficient expression of nattokinase in Bacillus licheniformis: host strain construction and signal peptide optimization

Cited by 73 publications

References 31 publications

A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis

A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis

Research progress on the fibrinolytic enzymes produced from traditional fermented foods

Improvement of levan production in Bacillus amyloliquefaciens through metabolic optimization of regulatory elements

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