启动子替代构建克雷伯氏菌普鲁兰酶高产菌株

Construction of high pullulanase producing strain of Klebsiella by promoter swapping

应用启动子替代技术将普鲁兰酶产生菌克雷伯氏菌（Klebsiellasp．）HN9的普鲁兰酶基因的启动子替换为枯草芽孢杆菌（Bacillussubtilis）的P43启动子，构建高产酶菌株。通过融合PCR，将由3个DNA片段融合得到启动子替代同源重组片段，该3个DNA片段分别是带有克雷伯氏菌普鲁兰酶基因启动子上游部分同源序列的四环素抗性基因（Tet）的上游片段Tet1、带有枯草芽孢杆菌P43启动子部分上游同源序列的Tet下游片段Tet2和带有克雷伯氏菌普鲁兰酶基因上游同源序列的P43启动子部分序列的Pro。经电转化将同源重组片段导入克雷伯氏菌中，转化子的普鲁兰酶基因表达水平比出发菌株提高10．23～11．45倍，摇瓶发酵酶活力提高8．97～10．52倍。结果表明，应用高效组成型启动子P43替代原始菌株的普鲁兰酶基因的启动子能够显著提高普鲁兰酶基因的表达量和发酵酶活力。

The high pullulanase producing strains of Klebsiella sp. HN9 were constructed through the replacement of HN9 pullulanase gene promoter by constitutive promoter P43 derived from Bacillus subtilis via promoter swapping technique. The homologous recombination fragment for promoter swapping was obtained by overlap extension PCR to fused the three DNA fragments, which were Tetl （comprised of the upstream of HN9 pullulanase gene promoter and the upstream in tetracycline resistance gene） , Tet2 （comprised of the downstream in tetracycline resistance gene and the upstream in P43） , and Pro （comprised of the downstream in tetracycline resistance gene and the downstream in P43）. The homologous recombination fragment was introduced into HN9 by electroporation transformation. The pullu- lanase gene expression levels and fermentation enzyme activities of the transformants were respectively 10.23 - 11.45 times and 8.97 - 10.52 times higher than those of wild type strain. The results showed that use of the constitutive promoter P43 to drive the expression of pullulanase gene may be an effective approach for improving the pullulanase gene expression and fermentation enzyme activities.