BACKGROUND pWB980 derived from pUB110 is a promising expression vector in Bacillus for its high copy number and high stability. However, the low transformation rate of recombinant plasmids to the wild cells limited the application of it. On the basis of pWB980, constructing an E. coli-B. subtilis shuttle plasmid could facilitate the transformation rate to Bacillus cells. Because the insertion site for E. coli replication origin sequence (ori) is not unique in pWB980, in order to investigate the best insertion site, eight shuttle plasmids (pUC980-1 ~ pUC980-8) containing all possible insertion sites and directions were constructed. RESULTS The results showed that all the selected insertion sites could be used to construct shuttle plasmid but some sites required a specific direction. And different insertion sites led to different properties of the shuttle plasmids. The best shuttle plasmids pUC980-1 and pUC980-2, which showed copies more than 450 per cell and segregational stabilities up to 98%, were selected for heterologous expressions of an alkaline pectate lyase gene pelN, an alkaline protease spro1 and a pullulanase gene pulA11, respectively. The highest extracellular activities of PelN, Spro1 and PulA11 were up to 5200 U/mL, 21,537 U/mL and 504 U/mL correspondingly after 54 h, 60 h and 48 h fermentation in a 10 L fermentor. Notably, PelN and Spro1 showed remarkably higher yields in Bacillus than previous reports. CONCLUSION The optimum ori insertion site was the upstream region of BA3-1 in pWB980 which resulted in shuttle plasmids with higher copy numbers and higher stabilities. The novel shuttle plasmids pUC980-1 and pUC980-2 will be promising expression vectors in B. subtilis. Moreover, the ori insertion mechanism revealed in this work could provide theoretical guidance for further studies of pWB980 and constructions of other shuttle plasmids.

中文翻译：

---

基于pWB980的高拷贝数和高度稳定的枯草芽孢杆菌穿梭质粒。

背景技术源自pUB110的pWB980由于其高拷贝数和高稳定性而在芽孢杆菌中是有希望的表达载体。然而，重组质粒向野生细胞的低转化率限制了其应用。在pWB980的基础上，构建大肠杆菌B。枯草杆菌穿梭质粒可以促进芽孢杆菌细胞的转化。由于pWB980中大肠杆菌复制起点序列（ori）的插入位点不是唯一的，因此为了研究最佳插入位点，构建了包含所有可能的插入位点和方向的八个穿梭质粒（pUC980-1〜pUC980-8） 。结果结果表明，所有选择的插入位点均可用于构建穿梭质粒，但有些位点需要特定的方向。并且不同的插入位点导致穿梭质粒的特性不同。选择了最好的穿梭质粒pUC980-1和pUC980-2，每个细胞显示拷贝数超过450，分离稳定性高达98％，用于碱性果胶酸裂合酶基因pelN，碱性蛋白酶spro1和支链淀粉酶基因pulA11的异源表达。 ， 分别。在10 L发酵罐中发酵54 h，60 h和48 h后，PelN，Spro1和PulA11的最高细胞外活性分别高达5200 U / mL，21,537 U / mL和504 U / mL。值得注意的是，芽孢杆菌中PelN和Spro1的产量明显高于以前的报道。结论最佳的ori插入位点是pWB980中BA3-1的上游区域，这导致穿梭质粒具有更高的拷贝数和更高的稳定性。新的穿梭质粒pUC980-1和pUC980-2将是在枯草芽孢杆菌中有希望的表达载体。此外，这项工作揭示的ori插入机制可以为进一步研究pWB980和其他穿梭质粒的构建提供理论指导。