Abstract
Nattokinase (NK) possesses the potential for prevention and treatment of thrombus-related diseases. In this study, high-level expression of nattokinase was achieved in Bacillus licheniformis WX-02 via host strain construction and signal peptides optimization. First, ten genes (mpr, vpr, aprX, epr, bpr, wprA, aprE, bprA, hag, amyl) encoding for eight extracellular proteases, a flagellin and an amylase were deleted to obtain B. licheniformis BL10, which showed no extracellular proteases activity in gelatin zymography. Second, the gene fragments of P43 promoter, Svpr, nattokinase and TamyL were combined into pHY300PLK to form the expression vector pP43SNT. In BL10 (pP43SNT), the fermentation activity and product activity per unit of biomass of nattokinase reached 14.33 FU/mL and 2,187.71 FU/g respectively, which increased by 39 and 156 % compared to WX-02 (pP43SNT). Last, Svpr was replaced with SsacC and SbprA, and the maximum fermentation activity (33.83 FU/mL) was achieved using SsacC, which was 229 % higher than that of WX-02 (pP43SNT). The maximum NK fermentation activity in this study reaches the commercial production level of solid state fermentation, and this study provides a promising engineered strain for industrial production of nattokinase, as well as a potential platform host for expression of other target proteins.
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Abbreviations
- NK:
-
Nattokinase
- aprN :
-
The gene of nattokinase
- mpr :
-
The gene of glutamyl endopeptidase Mpr
- vpr :
-
The gene of extracellular serine protease Vpr
- aprX :
-
The gene of serine protease AprX
- epr :
-
The gene of minor extracellular protease Epr
- bpr :
-
The gene of bacillopeptidase Bpr
- wprA :
-
The gene of protease WprA
- aprE :
-
The gene of serine protease AprE
- bprA :
-
The gene of bacillopeptidase BprA
- hag :
-
The gene of flagellin
- amyl :
-
The gene of α-amylase
- Svpr:
-
The signal peptide of Vpr
- SsacC:
-
The signal peptide of SacC
- SbprA:
-
The signal peptide of BprA
- TamyL:
-
The terminator of α-amylase gene
- pHY300PLK:
-
E. coli and B. subtilis shuttle vector
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Acknowledgments
This work was funded by the National Science & Technology Pillar Program during the Twelfth Five-year Plan Period (2013AA102801-52), National Key Basic Research Program of China (2015CB150505), the Natural Science Foundation of Hubei Province (2014CFB943), the Opening Project of State Key Laboratory of Agricultural Microbiology (AMLKF201403), and the National Natural Science Foundation of China (Grant 21106191).
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Wei, X., Zhou, Y., Chen, J. et al. Efficient expression of nattokinase in Bacillus licheniformis: host strain construction and signal peptide optimization. J Ind Microbiol Biotechnol 42, 287–295 (2015). https://doi.org/10.1007/s10295-014-1559-4
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DOI: https://doi.org/10.1007/s10295-014-1559-4