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。

基本信息

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

项目摘要

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.

这项研究的目的是诱导枯草芽孢杆菌及其在蛋白质生产中的染色体上设计的基因扩增。我们已经阐明了转化的DNA的基本结构，该结构可以诱导枯草芽孢杆菌中的基因扩增。重复单元的Juction点结构对于主动转换至关重要。如果是这样，我们可以通过连接两个独立的DNA片段在体外转化DNA中构建，以形成设计的重复单元的JUCTION点。从枯草芽孢杆菌的染色体DNA中，我们克隆了一个位于Amye上游的5.1 kb Ecori片段，以及位于Aroi附近的0.8 kb Hind <iiii> clai fgrament。将两个DNA片段在体外结扎，以构建瞄准重复单元（22 kb）的Juction点。通过具有10 ug构造的DNA的能力转化，获得了10种抗穿肌霉素的转化剂。其中四种菌株显示出<Alpha> - 淀粉酶的生产率过高。我们选择了两种超应菌株进行进一步分析。限制性核酸内切酶消化的染色体DNA经过琼脂糖凝胶电泳和南部杂交进行几种适当的探针。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> - 淀粉酶的生产率过高和女衣霉素的抗性是通过AMYE和TMRB基因的同时扩增引起的。根据AROI的扩增，还通过4倍倍增强了光滑的激酶活性。