pWB980 vector

pWB980 derived from pUB110 is a promising expression vector in Bacillus because of its high copy number and high stability. However, the transformation rate of recombinant plasmids on wild-type cells is low, which limits its application. On the basis of pWB980, constructing E.coli–B. Bacillus subtilis shuttle plasmid can improve the transformation rate of Bacillus cells. Since the insertion site of the E. coli replication origin sequence (ori) is not unique in pWB980, in order to study the optimal insertion site, 8 shuttle plasmids (pUC980-1 ~ pUC980-8) containing all possible insertion sites and directions were constructed.

Select the best shuttle plasmids pUC980-1 and pUC980-2, which have more than 450 copies per cell and an isolation stability of up to 98%, for heterologous expression of an alkaline pectate lyase gene pelN, an alkaline protease spro1 and a pullulanase gene pulA11, respectively. After fermentation for 54 h, 60 h and 48 h in a 10 L fermentor, the highest extracellular activities of PelN, Spro1 and PulA11 reached 5200 U/mL, 21,537 U/mL and 504 U/mL respectively.

Figure 1. Pullulanase productions in B. subtilis WB600. a The activity of pullulanase expressed from different plasmids was measured in WB600 supernatant. b The percentages of PulA11 protein in the supernatant of the recombinant strains containing plasmids pWB980-pulA11, pWB980-DB-pulA11, pUC980-1-pulA11, and pUC980-2-pulA11 after fermentation for 48 hours were 6%, 8%, 27%, and 30% respectively. (Zhao, XingYa, et al., 2020)