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

枯草芽孢杆菌衣霉素抗性突变诱导的基因扩增及其在蛋白质生产中的应用。

• 批准号: 60470129
• 负责人: YAMASAKI Makari
• 金额: $ 3.71万
• 依托单位: Faculty of Agriculture, The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1985
• 资助国家: 日本
• 起止时间: 1985 至 1986
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-60470129/
• 关键词: Bacillus subtilis; Tunicamycin-resistance; Gene amplification; <alpha> -Amylase; シキミ酸キナーゼ

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.

本研究旨在诱导枯草芽孢杆菌染色体上的基因扩增，并将其应用于蛋白质生产。我们已经阐明了能诱导B中基因扩增的转化DNA的基本结构。枯草杆菌。重复单元的临界点结构对于活性转化是必不可少的。如果是这样的话，我们可以通过连接两个独立的DNA片段以形成所设计的重复单元的接合点来在体外构建转化DNA。从B的染色体DNA中。在枯草芽孢杆菌中，我们克隆了位于amyE上游的5.1kb EcoRI片段和位于aroI附近的0.8kb <III>Hind-ClaI片段。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 μ g构建的DNA进行感受态转化，获得10个衣霉素抗性转化子。其中有4个菌株表现出高产α<alpha>-淀粉酶的特性。我们选择了两个超级菌株进行进一步的分析。限制性内切酶消化的染色体DNA进行琼脂糖凝胶电泳和Southern杂交与几个适当的探针。所有数据证实了具有22 kb重复单元的设计基因扩增的发生。<alpha>- 淀粉酶高产和衣霉素抗性是由amyE和tmrB基因的同时扩增引起的。莽草酸激酶活性也随着aroI的扩增而增强4倍。

项目成果

M.Mori, K.Hashiguchi, K.Yoda and M.Yamasaki: "Designed gene amplification on Bacillus subtilis chromosome." J. Bacteriol.

M.Mori、K.Hashiguchi、K.Yoda 和 M.Yamasaki：“在枯草芽孢杆菌染色体上设计基因扩增。”

M.Mori;K.Hashiguchi;K.Yoda;M.Yamasaki: J.Bacteriol.,.

M.Mori；K.Hashiguchi；K.Yoda；M.Yamasaki：J.Bacteriol.，。