Development of a Bacillus subtilis expression system using the improved Pglv promoter.
Development of a Bacillus subtilis expression system using the improved Pglv promoter.
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DOI：
10.1186/1475-2859-9-55
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发表时间：
2010-07-10
影响因子：
6.4
通讯作者：
Sheng GY
中科院分区：
文献类型：
--
作者：
Ming YM;Wei ZW;Lin CY;Sheng GY
B。枯草芽孢杆菌是生物技术应用中的重要微生物。高效表达系统是在B中生产重组基因产物所需的。枯草杆菌。最近，我们在B中开发了一种新的诱导表达系统。枯草芽孢杆菌（B. subtilis），该菌株由B.枯草麦芽糖利用操纵子启动子Pglv.该系统证明了在B中靶蛋白的高水平表达。枯草芽孢杆菌。然而，该系统显着抑制葡萄糖。这限制了该系统作为一种高效表达工具在生物技术领域的应用。本研究的目的是进一步完善Pglv启动子系统，提高其表达强度。在此，促进定点诱变以增强Pglv的表达强度。四个突变体的转录水平增加。突变体的β-Gal产量是野生型启动子的1.8倍。在5%麦芽糖诱导下，两个突变体的β-Gal产量分别达到14.3U/ml和13.8U/ml，比野生型启动子（8.8U/ml）提高了63.5%和57.5%。因此，定点突变缓解了葡萄糖的抑制，提高了表达活性。为了进一步完善启动子系统，将B. subtilis表达宿主，其中B.枯草杆菌的组成型启动子P43取代了B中glv操纵子的启动子。枯草杆菌染色体通过双交换事件。与原系统相比，改良系统的β-半乳糖苷酶产量（21.1 U/mL）有所提高。同时，葡萄糖引起的阻遏作用也得到进一步缓解。在本研究中，我们通过定点突变获得了一个突变的启动子Pglv-M1，它表现出高表达强度，并减轻了葡萄糖引起的阻遏。此外，我们通过重建B减轻了Pglv-M1启动子系统的抑制并增强了其表达活性。枯草杆菌宿主因此，我们提供了一个有价值的表达系统在B。枯草芽孢杆菌
B. subtilis is an important organism in the biotechnological application. The efficient expression system is desirable in production of recombinant gene products in B. subtilis. Recently, we developed a new inducible expression system in B. subtilis, which directed by B. subtilis maltose utilization operon promoter Pglv. The system demonstrated high-level expression for target proteins in B. subtilis when induced by maltose. However, the system was markedly repressed by glucose. This limited the application of the system as a high-expression tool in biotechnology field. The aim of this study was to further improve the Pglv promoter system and enhance its expression strength. Here, site-directed mutagenesis was facilitated to enhance the expression strength of Pglv. The transcription level from four mutants was increased. Production of β-Gal from the mutants reached the maximum 1.8 times as high as that of wildtype promoter. When induced by 5% maltose, the production of β-Gal from two mutants reached 14.3 U/ml and 13.8 U/ml, 63.5% and 57.5% higher than wildtype promoter (8.8 U/ml) respectively. Thus, site-directed mutagenesis alleviated the repression of glucose and improved the expression activity. To further improve the promoter system, the B. subtilis expression host was reconstructed, in which B. subtilis well-characterized constitutive promoter P43 replaced the promoter of the glv operon in B. subtilis chromosome through a double crossover event. The β-galactosidase production from the improved system (21.1 U/mL) increased compared to that from origin system. Meanwhile, the repression caused by glucose was further alleviated. In this study, we obtained a mutated promoter Pglv-M1 through site-directed mutagenesis, which demonstrated high expression strength and alleviated the repression caused by glucose. Moreover, we alleviated the repression and enhanced the expression activity of the Pglv-M1 promoter system via reconstruction of the B. subtilis host. Thus, we provided a valuable expression system in B. subtilis.
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影响因子：
2.6
作者：
Nguyen, HD;Nguyen, QA;Schumann, W
通讯作者：
Schumann, W
影响因子：
3.5
作者：
GueroutFleury, AM;Frandsen, N;Stragier, P
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Stragier, P
影响因子：
1.6
作者：
Phan, TTP;Nguyen, HD;Schumann, W
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Schumann, W
影响因子：
4.4
作者：
CAVARI, BZ;PHELPS, G
通讯作者：
PHELPS, G
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4.8
作者：
Thompson, J;Pikis, A;Sekiguchi, J
通讯作者：
Sekiguchi, J