School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane 4072, Australia.
Plasmid. 2010 Mar;63(2):79-85. doi: 10.1016/j.plasmid.2009.11.004. Epub 2009 Dec 3.
Previous studies showed that when pPSX-vioABCDE was used to transform E. coli K12 DH5alpha 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 DH5alpha 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 K12 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 DH5alpha to overproduce violacein while retaining high stability. The plasmid pPSX-vioABCDEopv-1 possesses a single base pair deletion in the promoter region of the violacein operon. By combining the over producing strain E. coli K12 JM109 and the over producing plasmid pPSX-vioABCDEopv-1, a stable hyper producing strain (E. coli K12 JM109 pPSX-vioABCDEopv-1) was constructed. Finally, two additional stable vectors, pPSX10 and pPSX20, were constructed to facilitate subcloning and functional analysis studies.
先前的研究表明，当使用 pPSX-vioABCDE 转化大肠杆菌 K12 DH5alpha 时，该菌株即使在未经选择生长 100 代后仍保留质粒，但产生低水平的抗肿瘤抗生素紫霉素。从大肠杆菌 K12 DH5alpha pUC18-vioABCDE 和鞘氨醇单胞菌 sp. JMP4092 pPSX-vioABCDE 中获得了明显更高水平的紫霉素合成。不幸的是，无论是否存在抗生素选择来保留质粒，两种菌株都极不稳定。目前的研究旨在确定是否可以分离出能够稳定过量生产紫霉素的大肠杆菌 K12 菌株。当一系列大肠杆菌 K12 菌株用 pPSX-vioABCDE 转化时，大多数菌株产生少量的紫霉素。然而，少数相关的大肠杆菌 K12 JM101、JM105 和 JM109 菌株不仅过量生产紫霉素，而且还保持了高稳定性。此外，大肠杆菌 K12 JM109 强烈表达了来自链霉菌的α-淀粉酶基因（amyA），表明链霉菌 amyA 启动子在大肠杆菌 K12 JM109 中高度活跃。在另一组实验中，分离出质粒 pPSX-vioABCDE 的紫霉素过表达突变（opv-1），使大肠杆菌 K12 DH5alpha 能够过量生产紫霉素，同时保持高稳定性。质粒 pPSX-vioABCDEopv-1 在紫霉素操纵子的启动子区域有一个单个碱基对缺失。通过组合过表达菌株大肠杆菌 K12 JM109 和过表达质粒 pPSX-vioABCDEopv-1，构建了一个稳定的超产菌株（大肠杆菌 K12 JM109 pPSX-vioABCDEopv-1）。最后，构建了两个额外的稳定载体 pPSX10 和 pPSX20，以方便亚克隆和功能分析研究。
