Gene amplification induced by a tunicamycin-resistant mutation in Bacillus subtilis and its application to protein production.

本研究的目的是诱导设计的枯草芽孢杆菌染色体基因扩增及其在蛋白质生产中的应用。我们已经阐明了在枯草芽孢杆菌中诱导基因扩增的转化DNA的基本结构。重复单元的结点结构对于主动变换是必不可少的。如果是这样，我们可以在体外通过连接两个独立的DNA片段来形成所设计的重复单元的结合点来构建一个转化DNA。从枯草芽孢杆菌的染色体DNA中，我们克隆了一个5.1 kb的EcoRI片段，位于amyE上游，以及一个0.8 kb的Hind <III> -ClaI片段，位于aroI附近。将两个DNA片段在体外连接以构建目标重复单元（22 kb）的连接点。用10微克构建的DNA胜任转化，获得10个耐衣霉素转化子。其中4株具有< α > -淀粉酶高产性。我们选择了两个超应变进行进一步分析。对限制性内切酶酶切的染色体dna进行琼脂糖凝胶电泳和Southern杂交。所有数据均证实了所设计的重复单元为22 kb的基因扩增的存在。amyE和tmrB基因同时扩增导致了α - > -淀粉酶高产和tunicamycin耐药。与aroI扩增结果一致，莽草激酶活性提高了4倍。

The aim of this study is to induce a designed gene ampliciation on the chromosome of Bacillus subtilis and its application to protein production. We have already elucidated the essential structure of a transforming DNA which can induce gene amplifcation in B. subtilis. The juction point structure of the repeating unit is essential for the active transformation. If so, we can construct in in vitro a transforming DNA by ligating two independent DNA fragments to form a juction point of the designed repeating unit. From the chromosomal DNA of B. subtilis, we cloned a 5.1 kb EcoRI fragment which is located at the upstream of amyE and also a 0.8 kb Hind <III> -ClaI fgrament which is located near aroI. The two DNA fragments were ligated in in vitro to construct a juction point of the aimed repeating unit (22 kb). By competence transformation with 10 ug of the constructed DNA, 10 tunicamycin-resistant transformants were obtained. Among them four strains showed hyper productivity of <alpha> -amylase. We selected two of the hyper strains for the further analyses. Restriction endonuclease-digested chromosomal DNAs were subjected to an agarose gel electrophoresis and Southern hybridization with several appropriate probes. All the data confirmed the occurrence of the designed gene amplification having a 22 kb repeating unit. <alpha> -Amylase hyper productivity and tunicamycin-resistance were brought about by the simulteneous amplification of amyE and tmrB genes. Shikimate kinase activity was also enhanced by 4 folds in accordance with the amplification of aroI.