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

  • Genetics and Molecular Biology of Industrial Organisms
  • Published:
Journal of Industrial Microbiology & Biotechnology

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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Authors and Affiliations

  1. State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District, Wuhan, 430070, People’s Republic of China

    Xuetuan Wei, Yinhua Zhou, Jingbang Chen, Dongbo Cai, Gaofu Qi & Shouwen Chen

  2. College of Food Science and Technology, Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District, Wuhan, 430070, People’s Republic of China

    Xuetuan Wei

  3. Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, College of Life Sciences, Hubei University, Wuhan, 430062, People’s Republic of China

    Shouwen Chen

  4. College of Chemistry and Chemical Engineering, Chongqing University, Chongqing, 400044, People’s Republic of China

    Dan Wang

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  1. Xuetuan Wei
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Correspondence to Shouwen Chen.

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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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