caa a22 4500 606213139 CHVBK 20210128101046.0 cr unu---uuuuu 210128e20150201xx s 000 0 eng 10.1007/s10295-014-1559-4 doi (NATIONALLICENCE)springer-10.1007/s10295-014-1559-4 Efficient expression of nattokinase in Bacillus licheniformis [Elektronische Daten] host strain construction and signal peptide optimization [Xuetuan Wei, Yinhua Zhou, Jingbang Chen, Dongbo Cai, Dan Wang, Gaofu Qi, Shouwen Chen] 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 14.33FU/mL and 2,187.71FU/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 (33.83FU/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. Society for Industrial Microbiology and Biotechnology, 2014 Nattokinase nationallicence B. licheniformis nationallicence Recombinant expression nationallicence Host construction nationallicence Signal peptides optimization nationallicence NK : Nattokinase nationallicence aprN : The gene of nattokinase nationallicence mpr : The gene of glutamyl endopeptidase Mpr nationallicence vpr : The gene of extracellular serine protease Vpr nationallicence aprX : The gene of serine protease AprX nationallicence epr : The gene of minor extracellular protease Epr nationallicence bpr : The gene of bacillopeptidase Bpr nationallicence wprA : The gene of protease WprA nationallicence aprE : The gene of serine protease AprE nationallicence bprA : The gene of bacillopeptidase BprA nationallicence hag : The gene of flagellin nationallicence amyl : The gene of α-amylase nationallicence Svpr : The signal peptide of Vpr nationallicence SsacC : The signal peptide of SacC nationallicence SbprA : The signal peptide of BprA nationallicence TamyL : The terminator of α-amylase gene nationallicence pHY300PLK : E. coli and B. subtilis shuttle vector nationallicence Wei Xuetuan State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District, 430070, Wuhan, People's Republic of China aut Zhou Yinhua State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District, 430070, Wuhan, People's Republic of China aut Chen Jingbang State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District, 430070, Wuhan, People's Republic of China aut Cai Dongbo State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District, 430070, Wuhan, People's Republic of China aut Wang Dan College of Chemistry and Chemical Engineering, Chongqing University, 400044, Chongqing, People's Republic of China aut Qi Gaofu State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District, 430070, Wuhan, People's Republic of China aut Chen Shouwen State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District, 430070, Wuhan, People's Republic of China aut Journal of Industrial Microbiology & Biotechnology Springer Berlin Heidelberg 42/2(2015-02-01), 287-295 1367-5435 42:2<287 2015 42 10295 https://doi.org/10.1007/s10295-014-1559-4 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s10295-014-1559-4 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Wei Xuetuan State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District, 430070, Wuhan, People's Republic of China aut NATIONALLICENCE 700 1- Zhou Yinhua State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District, 430070, Wuhan, People's Republic of China aut NATIONALLICENCE 700 1- Chen Jingbang State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District, 430070, Wuhan, People's Republic of China aut NATIONALLICENCE 700 1- Cai Dongbo State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District, 430070, Wuhan, People's Republic of China aut NATIONALLICENCE 700 1- Wang Dan College of Chemistry and Chemical Engineering, Chongqing University, 400044, Chongqing, People's Republic of China aut NATIONALLICENCE 700 1- Qi Gaofu State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District, 430070, Wuhan, People's Republic of China aut NATIONALLICENCE 700 1- Chen Shouwen State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District, 430070, Wuhan, People's Republic of China aut NATIONALLICENCE 773 0- Journal of Industrial Microbiology & Biotechnology Springer Berlin Heidelberg 42/2(2015-02-01), 287-295 1367-5435 42:2<287 2015 42 10295