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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.

High copy number and highly stable Escherichia coli–Bacillus subtilis shuttle plasmids based on pWB980