High-level extracellular production of recombinant nattokinase in Bacillus subtilis WB800 by multiple tandem promoters
High-level extracellular production of recombinant nattokinase in Bacillus subtilis WB800 by multiple tandem promoters
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DOI：
10.1186/s12866-019-1461-3
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发表时间：
2019-05-07
期刊：
影响因子：
4.2
通讯作者：
Zhou, Zhemin
中科院分区：
文献类型：
--
作者：
Liu, Zhongmei;Zheng, Wenhui;Zhou, Zhemin
纳豆激酶（NK）是枯草菌素家族的一员，是一种有效的纤溶酶，可能对血栓治疗有用。人们已经做了大量的工作来改善食品工业的生产。本研究的目的是通过串联启动子提高枯草芽孢杆菌WB800的NK产量。结果共构建了6株含有不同启动子质粒的重组菌株（P-P43、P-HpaII、P-BcaprE、P-gsiB、P-yxiE和P-luxS），其纤溶活性分析表明，P-P43和P-HpaII的表达活性高于其他菌株。P-P43和P-HpaII介导的NK产量分别达到140.53.9 FU/ml和110.8 +/- 3.6 FU/ml。这些启动子串联排列可以提高NK的表达水平，我们的研究结果表明，启动子串联排列对NK的表达水平有内在的影响。随着重复P-P43或P-HpaII数量的增加，NK的表达水平增加至三启动子，但不是无条件增加。此外，P-P43或P-HpaII的重复核心区可以有效地促进NK的产生。构建了8个不同顺序的P-P43和P-HpaII三启动子，最终NK的最高产率达到264.2 +/- 7.0 FU/ml，由启动子P-HpaII-P-HpaII-P-P43介导。在5-L发酵罐中也进行了p - hpai - p - hpai - p - p43促进NK的规模化生产，NK活性达到816.7 +/- 30.0 FU/mL。结论我们的研究表明NK在枯草芽孢杆菌中通过串联启动子高效过量产生。P-HpaII-P-HpaII-P-P43促进的纤溶活性最高，远高于以往研究报道。这些多重串联启动子成功地控制了NK的表达，可能有助于提高其他基因的表达水平。
BackgroundNattokinase (NK), which is a member of the subtilisin family, is a potent fibrinolytic enzyme that might be useful for thrombosis therapy. Extensive work has been done to improve its production for the food industry. The aim of our study was to enhance NK production by tandem promoters in Bacillus subtilis WB800.ResultsSix recombinant strains harboring different plasmids with a single promoter (P-P43, P-HpaII, P-BcaprE, P-gsiB, P-yxiE or P-luxS) were constructed, and the analysis of the fibrinolytic activity showed that P-P43 and P-HpaII exhibited a higher expression activity than that of the others. The NK yield that was mediated by P-P43 and P-HpaII reached 140.53.9 FU/ml and 110.8 +/- 3.6 FU/ml, respectively. These promoters were arranged in tandem to enhance the expression level of NK, and our results indicated that the arrangement of promoters in tandem has intrinsic effects on the NK expression level. As the number of repetitive P-P43 or P-HpaII increased, the expression level of NK was enhanced up to the triple-promoter, but did not increase unconditionally. In addition, the repetitive core region of P-P43 or P-HpaII could effectively enhance NK production. Eight triple-promoters with P-P43 and P-HpaII in different orders were constructed, and the highest yield of NK finally reached 264.2 +/- 7.0 FU/ml, which was mediated by the promoter P-HpaII-P-HpaII-P-P43. The scale-up production of NK that was promoted by P-HpaII-P-HpaII-P-P43 was also carried out in a 5-L fermenter, and the NK activity reached 816.7 +/- 30.0 FU/mL.Conclusions Our studies demonstrated that NK was efficiently overproduced by tandem promoters in Bacillus subtilis. The highest fibrinolytic activity was promoted by P-HpaII-P-HpaII-P-P43, which was much higher than that had been reported in previous studies. These multiple tandem promoters were used successfully to control NK expression and might be useful for improving the expression level of the other genes.