Development of an efficient autoinducible expression system by promoter engineering in Bacillus subtilis.
Development of an efficient autoinducible expression system by promoter engineering in Bacillus subtilis.
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通过枯草芽孢杆菌启动子工程开发高效的自诱导表达系统
DOI：
10.1186/s12934-016-0464-0
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发表时间：
2016-04-25
影响因子：
6.4
通讯作者：
Zhou Z
中科院分区：
文献类型：
--
作者：
Guan C;Cui W;Cheng J;Zhou L;Liu Z;Zhou Z
背景枯草芽孢杆菌是一种革兰氏阳性生物，由于其突出的生物学特性，已被开发为一种有吸引力的表达平台，可产生分泌蛋白和细胞质蛋白。我们之前开发了一种包含 srfA 启动子 (PsrfA) 的自诱导表达系统，该启动子由作用于群体感应途径的信号分子激活以获得能力。 PsrfA启动子表现出与细胞密度密切相关的无诱导剂活性的独特特性。结果为了将PsrfA介导的表达系统改进为无法形成孢子的枯草芽孢杆菌突变株用于工业生产的高细胞密度发酵，开发了孢子突变株BSG1682，并通过启动子工程增强了PsrfA启动子。使用绿色荧光蛋白 (GFP) 作为报告基因，在质粒或染色体表达的 BSG1682 中观察到比野生型枯草芽孢杆菌 168 更高的荧光强度。此后，对 PsrfA 进行了改造，产生了 GFP 表达强度不同的 PsrfA 衍生物文库。 P23启动子表现出最好的性能，几乎比PsrfA强两倍。两种异源蛋白，氨肽酶（AP）和纳豆激酶（NK），在BSG1682中在P23的控制下成功过量产生。最后，在5L发酵罐中的分批发酵中证明了表达系统的能力。结论表达系统表现出自诱导启动子活性的突出。通过将相应基因整合到菌株BSG1682的质粒或染色体上的启动子下游，可以高效、稳定地产生所需的蛋白质。该表达系统有利于BSG1682高细胞密度发酵中药物和异源蛋白的工业化生产。
BackgroundBacillus subtilis, a Gram-positive organism, has been developed to be an attractive expression platform to produce both secreted and cytoplasmic proteins owing to its prominent biological characteristics. We previously developed an auto-inducible expression system containing the srfA promoter (PsrfA) which was activated by the signal molecules acting in the quorum-sensing pathway for competence. The PsrfA promoter exhibited the unique property of inducer-free activity that is closely correlated with cell density.ResultsTo improve the PsrfA-mediated expression system to the high-cell-density fermentation for industrial production in the B. subtilis mutant strain that is unable to sporulate, a spore mutant strain BSG1682 was developed, and the PsrfA promoter was enhanced by promoter engineering. Using green fluorescent protein (GFP) as the reporter, higher fluorescent intensity was observed in BSG1682 with expression from either plasmid or chromosome than that of the wild type B. subtilis 168. Thereafter, the PsrfA was engineered, yielding a library of PsrfA derivatives varied in the strength of GFP expression. The P23 promoter exhibited the best performance, almost twofold stronger than that of PsrfA. Two heterologous proteins, aminopeptidase (AP) and nattokinase (NK), were successfully overproduced under the control of P23 in BSG1682. Finally, the capacity of the expression system was demonstrated in batch fermentation in a 5-L fermenter.ConclusionsThe expression system demonstrates prominence in the activity of the auto-inducible promoter. Desired proteins could be highly and stably produced by integrating the corresponding genes downstream of the promoter on the plasmid or the chromosome in strain BSG1682. The expression system is conducive to the industrial production of pharmaceuticals and heterologous proteins in high-cell-density fermentation in BSG1682.