High-level extracellular protein production in Bacillus subtilis using an optimized dual-promoter expression system.
High-level extracellular protein production in Bacillus subtilis using an optimized dual-promoter expression system.
复制标题
使用优化的双启动子表达系统在枯草芽孢杆菌中产生高水平的细胞外蛋白
DOI：
10.1186/s12934-017-0649-1
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发表时间：
2017-02-20
影响因子：
6.4
通讯作者：
Wu J
中科院分区：
文献类型：
--
作者：
Zhang K;Su L;Duan X;Liu L;Wu J
背景我们最近构建了一株枯草芽孢杆菌菌株（CCTCC M 2016536），其中我们删除了srfC、spoIIAC、nprE、aprE和amyE基因。该菌株能够稳健地生产重组蛋白，并适于高细胞密度发酵。因为启动子是影响靶蛋白产生的因素之一，所以优化初始启动子，来自解淀粉芽孢杆菌的PamyQ，应该改善使用该菌株的蛋白表达。本研究旨在建立一种新的、高效的B细胞表达系统。结果以β-环糊精糖基转移酶（β-CGTase）为报告蛋白，以B. subtilis CCTCC M 2016536作为宿主，使用摇瓶培养筛选9个配备有单启动子的质粒。含有PamyQ′启动子的质粒产生最大的胞外β-CGTase活性，为24.1 U/mL。随后，使用相同的方法构建并评价了六个配备有双启动子的质粒。含有双启动子PHpaII-PamyQ '的质粒产生最高的细胞外β-CGTase活性（30.5 U/mL），并且葡萄糖相对受到抑制。双启动子PHpaII-PamyQ′在摇瓶培养过程中也介导了大量的胞外普鲁兰酶（90.7 U/mL）和α-CGTase（9.5 U/mL）的表达，证明了该系统的普遍适用性。最后，在3L发酵罐中考察了双启动子PHpaII-PamyQ′系统对β-CGT酶的产酶情况。结论PHpaII-PamyQ′双启动子系统可上级表达β-CGTase，表达量为571.2U/mL（2.5mg/mL），具有工业化应用的潜力。枯草芽孢杆菌CCTCC M 2016536。这一系统似乎普遍适用，并可扩大规模。
BackgroundWe recently constructed a Bacillus subtilis strain (CCTCC M 2016536) from which we had deleted the srfC, spoIIAC, nprE, aprE and amyE genes. This strain is capable of robust recombinant protein production and amenable to high-cell-density fermentation. Because the promoter is among the factors that influence the production of target proteins, optimization of the initial promoter, PamyQ from Bacillus amyloliquefaciens, should improve protein expression using this strain. This study was undertaken to develop a new, high-level expression system in B. subtilis CCTCC M 2016536.ResultsUsing the enzyme β-cyclodextrin glycosyltransferase (β-CGTase) as a reporter protein and B. subtilis CCTCC M 2016536 as the host, nine plasmids equipped with single promoters were screened using shake-flask cultivation. The plasmid containing the PamyQ′ promoter produced the greatest extracellular β-CGTase activity; 24.1 U/mL. Subsequently, six plasmids equipped with dual promoters were constructed and evaluated using this same method. The plasmid containing the dual promoter PHpaII–PamyQ′ produced the highest extracellular β-CGTase activity (30.5 U/mL) and was relatively glucose repressed. The dual promoter PHpaII–PamyQ′ also mediated substantial extracellular pullulanase (90.7 U/mL) and α-CGTase expression (9.5 U/mL) during shake-flask cultivation, demonstrating the general applicability of this system. Finally, the production of β-CGTase using the dual-promoter PHpaII–PamyQ′ system was investigated in a 3-L fermenter. Extracellular expression of β-CGTase reached 571.2 U/mL (2.5 mg/mL), demonstrating the potential of this system for use in industrial applications.ConclusionsThe dual-promoter PHpaII–PamyQ′ system was found to support superior expression of extracellular proteins in B. subtilis CCTCC M 2016536. This system appears generally applicable and is amenable to scale-up.