Stable high level expression of the violacein indolocarbazole anti-tumour gene cluster and the Streptomyces lividans amyA gene in E. coli K12
Stable high level expression of the violacein indolocarbazole anti-tumour gene cluster and the Streptomyces lividans amyA gene in E. coli K12
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DOI：
10.1016/j.plasmid.2009.11.004
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发表时间：
2010-03-01
期刊：
影响因子：
2.6
通讯作者：
Pemberton, John M.
中科院分区：
文献类型：
--
作者：
Ahmetagic, Adnan;Pemberton, John M.
先前的研究表明，当pPSX-vioABCDE用于转化大肠杆菌K12 DH5 α时，即使在100代的非选择生长后，菌株仍保留了质粒，但产生了低水平的抗肿瘤抗生素violacein。大肠杆菌K12 DH5 α pUC18-vioABCDE和鞘氨单胞菌sp. JMP4092 pPSX-vioABCDE的紫色素合成水平显著提高。不幸的是，无论是否选择抗生素来保留质粒，这两种菌株都非常不稳定。目前的研究是为了确定是否可以分离出稳定地过量生产紫罗兰素的大肠杆菌K12菌株。当用pPSX-vioABCDE转化一系列大肠杆菌K12菌株时，大多数菌株产生少量紫罗兰素。而少数相关菌株大肠杆菌K12 JM101、JM105和JM109不仅过量生产紫罗兰素，而且保持了较高的稳定性。此外，大肠杆菌102 JM109强烈表达了来自lividans链霉菌的α淀粉酶基因（amyA），表明lividans amyA启动子在大肠杆菌K12 JM109中具有高活性。在另一组实验中，分离出质粒pPSX-vioABCDE的紫色素过量突变（opv-1），使大肠杆菌K12 DH5 α过量产生紫色素，同时保持高稳定性。质粒pPSX-vioABCDE opv-1在violacein操纵子的启动子区域有一个碱基对缺失。将超产菌株大肠杆菌K12 JM109与超产质粒pPSX-vioABCDE opv-1结合，构建了稳定的超产菌株大肠杆菌K12 JM109 pPSX-vioABCDE opv-1。最后，构建了两个稳定载体pPSX10和pPSX20，用于亚克隆和功能分析研究。(C) 2009爱思唯尔公司版权所有。
Previous studies showed that when pPSX-vioABCDE was used to transform E. coli K12 DH5 alpha the strain retained the plasmid even after 100 generations of unselected growth but produced a low level of the anti-tumour antibiotic violacein. Markedly higher levels of violacein synthesis were obtained from E. coli K12 DH5 alpha pUC18-vioABCDE and Sphingomonas sp. JMP4092 pPSX-vioABCDE. Unfortunately, both strains were extremely unstable regardless of presence or absence of antibiotic selection to retain the plasmid. The current study was undertaken to determine if strains of E coli K12 could be isolated which stably over produce violacein. When a range of E. coli K12 strains were transformed with pPSX-vioABCDE, most produced small amounts of violacein. However, a small number of related strains of E. coli K12 JM101, JM105 and JM109 not only over-produced violacein, but also maintained the high stability. In addition, E. coli 102 JM109 strongly expressed an alpha amylase gene (amyA) from Streptomyces lividans indicating that the S. lividans amyA promoter is highly active in E. coli K12 JM109. In another set of experiments, a violacein overproduction mutation (opv-1) of the plasmid pPSX-vioABCDE was isolated which enabled E. coli K12 DH5 alpha to overproduce violacein while retaining high stability. The plasmid pPSX-vioABCDE opv-1 possesses a single base pair deletion in the promoter region of the violacein operon. By combining the over producing strain E coli K 12 JM109 and the over producing plasmid pPSX-vioABCDE opv-1, a stable hyper producing strain (F. coli K12 JM109 pPSX-vioABCDE opv-1) was constructed. Finally, two additional stable vectors, pPSX10 and pPSX20, were constructed to facilitate subcloning and functional analysis Studies. (C) 2009 Elsevier Inc. All rights reserved.