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Development of Rhodococcus promoter for utilization in actinomycetes

开发用于放线菌的红球菌启动子

基本信息

批准号：
13556011
负责人：
KOBAYASHI Michihiko
金额：
$ 8.9万
依托单位：
University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13556011/
关键词：
Promoter Gene Expression Rhodococcus

项目摘要

The microbial degradation of nitrites proceeds through two enzymatic pathways : nitrite hydratase (NHase} catalyzes hydration of nitrite to amide, whereas nitrilase catalyzes hydrolysis of nitrite to acid and ammonium. Nitrilase is strongly induced by the addition of ε-caprolactam or isovaleronitrile to the culture medium ; the formed nitrilase corresponds to 35ーIo of aft soluble protein in Rhodococcus rhodochrous J1. On the other hand, NHase is strongly induced by the addition of urea to the culture medium ; the formed NHase corresponds to 50% of all soluble protein in this strain. At first, we trimmed the DNA region containing the nitrilase gene cluster, and constructed several plasmids where the upstream or downstream region of the gene cluster was deleted. We transformed each plasmid into Escherichia coli, and examined the nitrilase formation in the resultant transformants. SDS-PAGE and enzyme assay by HPLC revealed that the Rhodococcus nitrilase expression system was not able to function in E. coli. Next, we tried to investigate whether the nitrilase regulatory system is active or not in other Rhodococcus actinomycetes, and found that the nitrilase expression system functioned, indicating the development of a hyper-expression vector. We also obtained similar results for the NHase expression system. Furtheremore, we tried to construct a Rhodococcus host-vector system, considering the useful characteristics of this genus. We bought more than 100 Rhodococcus strains from microorganism stock centers, and searched for Rhodococcus strains carrying cryptic plasmids. We found three strains. with a cryptic plasmid, and succeeded in the construction of a Rhodococcus-E.coli shuttle-vector using one of their plasmids

微生物对亚硝酸盐的降解通过两条酶途径进行：亚硝酸水合酶(NHase)催化亚硝酸根水合成酰胺，而腈酶催化亚硝酸根水合成酸和铵。在培养基中加入ε-己内酰胺或异戊腈可强烈诱导产生氰化酶，其产量相当于红球菌J1中35ーIo的AFT可溶性蛋白。另一方面，在培养基中加入尿素可以强烈地诱导NHase，所形成的NHase相当于该菌株所有可溶性蛋白质的50%。首先，我们对含腈酶基因簇的DNA区域进行了剪裁，并在基因簇的上游或下游区域缺失的地方构建了几个质粒。我们将每个质粒都转化到了大肠杆菌中，并检测了转化子中的腈酶形成情况。SDS-PAGE和高效液相色谱酶分析表明，该表达系统不能在大肠杆菌中发挥作用。接下来，我们试图研究在其他红球菌放线菌中是否存在氰化酶调控系统，发现该氰化酶表达系统发挥了作用，这表明我们已经开发出了一种高效表达载体。对于NHase表达系统，我们也得到了类似的结果。此外，考虑到红球菌属的有用特性，我们还尝试构建了红球菌宿主-载体系统。我们从微生物储备中心购买了100多株红球菌，并寻找携带隐质粒的红球菌菌株。我们发现了三个菌株。并成功构建了红球菌-大肠杆菌穿梭载体。