title: Efficient expression of nattokinase in Bacillus licheniformis: host strain construction and signal peptide optimization.
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.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, No. 1 Shizishan Street, Hongshan District, Wuhan, 430070, People's Republic of China, weixuetuan@mail.hzau.edu.cn.
authors.forename: Xuetuan
authors.initials: X
authors.lastname: Wei
authors.forename: Yinhua
authors.initials: Y
authors.lastname: Zhou
authors.forename: Jingbang
authors.initials: J
authors.lastname: Chen
authors.forename: Dongbo
authors.initials: D
authors.lastname: Cai
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.forename: Gaofu
authors.initials: G
authors.lastname: Qi
authors.forename: Shouwen
authors.initials: S
authors.lastname: Chen
mesh_terms: D000681:Amylases / Q000235:genetics; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D005285:Fermentation; D005408:Flagellin / Q000235:genetics; D017353:Gene Deletion; D055786:Gene Knockout Techniques; D005822:Genetic Vectors / Q000235:genetics; D010447:Peptide Hydrolases / Q000235:genetics; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013381:Subtilisins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D005408:Flagellin; D000681:Amylases; D010447:Peptide Hydrolases; D013381:Subtilisins; C053771:nattokinase