School of Biotechnology, Key Laboratory of Industrial Biotechnology (Ministry of Education), Jiangnan University, Wuxi, Jiangsu, 214122, China.
Microb Biotechnol. 2018 Sep;11(5):930-942. doi: 10.1111/1751-7915.13298. Epub 2018 Jul 8.
Nattokinase (NK) is an important serine-protease with direct fibrinolytic activity involving the prevention of cardiovascular disease as an antithrombotic agent. Dozens of studies have focused on the characterization of intrinsic novel promoters and signal peptides to the secretory production of recombinant proteins in Bacillus subtilis. However, intrinsic genetic elements have several drawbacks, which cannot mediate the production of NK to the desired level. In this study, the genetic elements, which were used to overproduce the recombinant secretory NK, were rationally modified in B. subtilis in a stepwise manner. The first step was to select a suitable signal peptide for the highly efficient secretion of NK. By comparison of the secretory levels mediated by two different signal peptides, which were encoded by the genes of a minor extracellular protease epr (SP ) and cell-wall associated protease wapA (SP ), respectively, SP was verified as the superior secretory element. Second, P04, which was a synthetic promoter screened from an array of mutants based on the promoter cloned from the operon of a quorum-sensing associated gene srfA (P ), was paired to SP The secretory level of NK was obviously augmented by the combination of these two genetic elements. Third, the cis-acting element CodY-binding sequence positioned at the 5'UTR was deleted (yielding P08), and thus the secretory level was significantly elevated. The activity of NK, which was defined as fibrinolytic units (FU), reached to a level of 270 FU ml . Finally, the superior genetic element composed of P08 and SP was utilized to overproduce NK in the host B. subtilis WB800, which was able to produce the secretory NK at 292 FU ml . The strategy established in this study can not only be used to overproduce NK in B. subtilis but also might be a promising pipeline to modify the genetic element for the synthetic secretory system.
纳豆激酶（NK）是一种重要的丝氨酸蛋白酶，具有直接的纤溶活性，可作为抗血栓药物预防心血管疾病。数十项研究集中于内在新型启动子和信号肽的特征描述，以实现枯草芽孢杆菌中重组蛋白的分泌生产。然而，内在遗传元件有几个缺点，不能将 NK 的生产调节到所需的水平。在这项研究中，逐步在枯草芽孢杆菌中对用于过表达重组分泌型 NK 的遗传元件进行了合理修饰。第一步是选择合适的信号肽，以实现 NK 的高效分泌。通过比较由两个不同信号肽编码的基因（epr 编码的次要细胞外蛋白酶的信号肽（SP）和细胞壁相关蛋白酶 wapA 编码的信号肽（SP）介导的分泌水平，证实 SP 是优越的分泌元件。其次，P04 是一种基于克隆自群体感应相关基因 srfA（P）操纵子的启动子的突变体阵列筛选的合成启动子，与 SP 配对。NK 的分泌水平通过这两个遗传元件的组合明显增加。第三，删除了位于 5'UTR 的顺式作用元件 CodY 结合序列（产生 P08），从而显著提高了分泌水平。NK 的活性，定义为纤溶单位（FU），达到 270 FU/ml 的水平。最后，利用由 P08 和 SP 组成的优良遗传元件在宿主枯草芽孢杆菌 WB800 中过表达 NK，能够生产 292 FU/ml 的分泌型 NK。本研究建立的策略不仅可用于枯草芽孢杆菌中 NK 的过表达，也可能是修饰合成分泌系统遗传元件的有前途的途径。
